text:
Management of Combined CHF and CRF Ri 王薏茜 2003-06-23 CRF ↔ CHF (1) Average SCr of CHF patient : 1.5 mg/dl Mortality of CHF patients 40% sudden death 40% worsening CHF 20% others Cancer, COPD, infection… CRF ↔ CHF (2) CV disease in CRF patients At starting diaslysis 30-70% HTN 60% IHD 18-20% LVH 31-34% CHF CVD mortality: 5-50x(10-30x) more than in normal population Account for >50% of ESRD patient mortality CRF ↔ CHF (3) incidence CAD LVH CHF General population 5-12 20 5 Dialysis 40 75 40 Renal transplant 15 50 - CRF - 25-50 - No/1000pts/year 20-44 45-64 >65 CV death 43 76 179 After transplant 3.1 7.7 - Risk factors for CV diseases Normal population Related to uremia Related to dialysis Old age, male, race Hyper/hypo tension Hyper/hypo tension Hyperlipidemia Anemia Hypertension, DM Low HDL, High LDL Malnutrition homocysteinemia Hypertriglyceridemia Hypoalbuminemia Physical inactivity Lp(a) Low body mass index Family history Hyperparathyoidism Na water retention Menopause Ca X P Socioeconomic Uremic toxins status Oxidative stress Smoking Impaired gibrinosysis Infectous agents Insulin resistance Hymocysteine Thrombogenic factors Endothelialdysfunction Chronic inflammation Carbonyl stress Sleep apnea HTN in ESRD Strongest risk factor of LV hypertrophy For SCr = 3.3 ± 1.1 mg/dl Optimal BP: 3% High normal: 9% Stage 1 HTN (140-160): 34.4% Stage 2/3 HTN (160-200): 52.5% Mortality vs hypertension: J-shaped HTN in ESRD: mechanism Total sodium increase Plasma renin activity increase Noradrenergic hyperactivity Na/water retention AV fistula Anemia Hypotention in ESRD? BP<110: 4x increase in mortality Now suggested as a marker of ventricular systolic/diastolic dysfunction IHD in ESRD At starting dialysis: 18-20% with IHD Presentation Infarction: 56% Angina:82% CABG: 14% Angioplasyt: 1% With IHD Without IHD Progression to heart failure 24m 55m Mean survival time 44m 56m IHD in ESRD: risk factors Older age DM HTN Dyslipidemia Hypoalbuminemia Hyperhomocysteinemia: 83% of patients having levels higher than 90th percentile Associate with 7x increase in mortality Lp(a) LVH in ESRD Mechanism Re-expression of fetel Growth Factor/GFR Myocyte death, fibroblast growth (ESRD>DM, HTN) Interstitial fibrosis Diastolic dysfunction Intolerate to volume change (wall stiffness) Early reflection arrhythmia Independent prognostic factor for survival !! LVMI> 125 mg/m2: 25% (4-y) LVMI< 125 mg/m2: 55% LVEF<40%: odds ratio for mortality: 1.89 Survival in ESRD with/without LVH LVH in ESRD : prevalence In early renal dz (CCr>30ml/min) 65% eccentric hypertrophy 16% concentric hypertrophy In patients with CCr=10-30ml/min 26% concentric hypertrophy In dialysis pts (CCr<10ml/min) 44% eccentric 42-50% concentric LVH in ESRD: independent factors for LVH Hypertention BP ↑ 5mmHg: LVMI ↑10g/m2 Male gender BMI >25 Hb <10-12 Hb ↓ 0.5 mg/dl: LVMI ↑10g/m2 LVH in ESRD: hemodynamic mechanism Volume overload AV fistula Na/water retention Anemia Pressure overload Aotic wall/ventricular wall stiffness Atherosclerosis RAS overactivity: ACEI Dialysis: ∆ Ca(inotropic) and sympathetic tone LVH in ESRD: role of anemia When Hb<10-12 Reactive hemodynamic change Stroke volume ↑ Heart rate ↑ Odds ratio for CRF =1.32 / 0.5 Hb ↓ Odds ratio for ESRD = 1.46 / 1 Hb ↓ EPO? CHF in ESRD Epidemiology In starting dialysis 31% with CHF 25% develop CHF later Mortality 8.9% die of CHF/year Survival With CHF Without CHF 4-y survival 20% 60% Mean suvival 36 months (29m/45m) 62 months Survival in ESRD with/without CHF CHF in ESRD: factors Factors related to onset: Age DM Ischemic heart disease Factors related to recurrence: Ischemic heart disease Anemia Hypoalbuminemia hypertension D/D intrinsic myocardial dysfunction v.s. pure volume overload Echocardiography Radionuclide tecniques ANF and BNF(brain natriuretic factor): Stress receptor in atriumrelease of ANF, BNF Stress receptor in ventriclerelease of BNF NF receptors in kidney, adrenal glomerulose, vascular smooth muscle… Na excretion, vasodilatation, renin/aldosterone ↓,… ANF: associated more with volume overload BNF: associated more with ventricular dysfunction Management principles Preventive intervention should be initiated early in the first year of dialysis. Later treatment (CHF) has limited possibility of success. Management principles Major goal: treating underlying factors predispose to heart failure HTN, DM, hyperPTH, dyslipidemia, anemia Treatment of hemodynamic overload Pharmacologic therapy Diuretics Higher dose/ combine thiazide/ IF continuous use Monitor K+, regular supplement ACEI/Angiotensin Receptor Blockers: proven survival benefit, IHD↓,LVMI↓, GFR decline↓ If hyperkalemia/renal function↓: hydralizine + nitrate Side effect: anemia: EPO ↓, bone marrow ultilization of EPO↓ Beta-blocker: IHD, HTN, CHF Digoxin Cleared by kidney, NOT removed by dialysis Impact on symptom, functional capacity, hospitalized frequency, NOT on survival Management: aggressive correction of anemia CRA syndrome: cardio-renal-anemia Anemia CHF Damaged myocyte EPO production↓ Depress progenitor erythrocyte in bone marrow Interfere with RE system release of iron Management: aggressive correction of anemia 50% of CHF patients have Hb<12 66-80% of class IV CHF pts have Hb<12 Clinical trial in 2001 126 pts: anemic, CHF treatmtne-resistant, NYHA class 3-4 Target goal: keep Hb = 12.5-13 for 12.4 ± 8.2 m Mean: EPO 4000-5000 u if Hb<12.5 Keep serum ferritin>500ug/L, Sat>40% Management: aggressive correction of anemia 1-year Mortality in: Class 3-4 CHF patients: 30-50% This trial: 7.1% Management: intensive volume control Basis LVH accounts for large No of mortality in ESRD sBP elevation is the strongest risk factor for LVH Regression of LVH with BP control is well established Difficulty in controlling BP in ESRD pts, may be due to hidden volume expansion, which is out of reach of antihypertensive medications. Management: intensive volume control Effect of intensive hemodialysis on BP control Mean: 12h H/D per week, without antihypertensive drug As much UF as possible, without excess BP drop Dietary salt restriction 3 months of intensive volume control 12 months of follow up Management: intensive volume control sPB dBP BW CTI LA EDD ESD LVMI % Htc LVH Pre-HD 168 97 63.3 48 - - - - - 29.5 Post-HD 127 78 60.3 46 24.3 29.3 18.8 164 63 33.7 (3ms) 6ms 120 75 62.4 44 22.6 26.7 17.3 121 32 34.2 12ms 118 73 65.4 43 22.6 26.4 18.0 112 18 33.2 Avoid rapid volume shift Maintaining a low dry weight Regression of LVH, LVD, LV stiffness Conclusion CRF patients have a very high risk of develop CVD: HTN, LVH, IHD, CHF Account for more than 50% of ESRD patient mortality Management: risk reduction: anemia, BP control, volume management, medication toward symptoms: diuretics, digoxin, ACEI/ARBs, beta-blockers, correct dyslipidemia Proper dialysis Early intervention! Reference Seminars in dialysis 2003 vol 16(2):85-94 J of Nephrology 2002 15:655-60 Clinical nephrology 2002 vol 58 (supple1):s37-45 Ame J of Kidney diseases 2001 vol 38(4, supple1):s38-46 Peritoneal dialysis international. 2001 Vol 21(S3):s236-9 Seminars in nephrology. 2001 vol 21 (1):3-12 Thank you for your attention !
Pages to are hidden for
"Management of Combined CHF and C"Please download to view full document

domain: medical


text:
The Container Examination facility is located at the Curtain Brothers’ Dock Yard, outside Port Moresby.
It took the national government almost seven years to build, at a cost of K50 million.
PNG Customs Commissioner, Ray Paul, said the facility will cut down the amount of time customs officers take to do manual checks. Consequently, he is expecting the facility to process at least 40 containers in a day, but challenged customs officers to look after the facility.
The Examination Facility contains inspection rooms, and check stations where scanning of containers can be done, with the click of button.
However, if customs officers find prohibited items, they have the powers to conduct thorough checks on the entire container.
Minister Pruaitch, and Finance Minister, James Marape, were given a tour of the facility during its launch today.
After formal speeches, a ribbon was cut to mark the official opening of the facility.
PNG Customs has over the last five years started using new technologies, to monitor the transportation of items that come in, or go out of the country, at international airports and wharves.
The launching of this container examination facility is a benchmark set by PNG Customs to examine all containers that come into Papua New Guinea.
A similar facility is expected to be built in Lae.

domain: economics


text:
Q-DDL released a large collection of free 3D Color LUTs in 4 categories. Go check it out!
Take a look at the new strand-based hair and fur system in Unreal Engine 4 that works in real-time.
Check out a new add-on for Blender that will ease up the scattering workflow in it.
Raul Aparicio shared his approach to creating chippies in Houdini. Check it out!
Creating realistic digital humans for real-time 3D animation is now possible with Reallusion’s new Digital Human Shader.
James Jacobs, Co-founder and CEO of Ziva Dynamics, discussed the company's tool, Ziva VFX, talked about its capacities and shared the workflow in it.
Muhammad Etman released an update of the Maya/Clarisse bridge tool. Check it out!
Lou Cortes shared some insights into the production of real-time effects for Ghost Recon Breakpoint: smoke, lava surface, lightning, and more.
Spider-Man System for UnrealThanks for the share !·
Eureka: Sculpting Stylized Cloth & PBR TexturingFun character concept and great execution! Thanks for the overview. I...·
Procedural Forest Terrain Production with Houdini and UE4This is gold! Thanks. Tried Houdini a year or so back and got scared,...·
Resident Evil 7: The Use of Photogrammetry for VRWondermouse technology is top notch Windows App Development agency Ind...·
Join thousands of game designers and keep track of your subscriptions

domain: news


Could Lamar Jackson Be the NFL's Shohei Ohtani?
Shohei Ohtani's exceptional skills as a two-way player have been on full display the past two nights at Major League Baseball's Home Run Derby and All-Star Game.
So with the Los Angeles Angels' pitcher/designated hitter the talk of the sports world, NFL Network analysts discussed which current NFL player could excel on both sides of the ball.
For Michael Robinson, that player is Lamar Jackson.
"He's really good in short areas. He has very good short area quickness," Robinson said. "He can do it all. I know you're thinking, 'Hey, he's a quarterback; that's one of the least physical positions on the field.' Well, have you seen Lamar Jackson run the football? He runs the football like a tailback.
"So I think he could play defensive back. I think he can backpedal and catch the football. He can read defenses because he does that [with] that quarterback background. And I don't think this kid is afraid to stick his nose and his face right on the football and make the tackle."
The Ravens quarterback actually did play some safety in high school and laid some big hits in his day.
Robinson isn't the first former player to say that Jackson has what it takes to be a defensive back in the NFL. Hall of Fame safety Ed Reed said last year that the Ravens quarterback "has the heart of a defender."
"He's the ultimate athlete," Reed said. "I think he could've played safety."
Jackson flashed his ability as a two-way player over the weekend at his annual "Fun Day with LJ" event in his native South Florida. Videos of Jackson playing pickup football with kids showed him looking like a lock-down cornerback (as well as a wide receiver with good hands who can get separation).
On a side note, it was a little disappointing that the NFL Network analysts didn't show any love for Pro Bowl fullback Patrick Ricard, a defensive lineman in college who actually has played on both sides of the ball in the NFL.
Does AFC North Have the Best Starting Quarterbacks?
NFL Network's Lorenzo Alexander said NFL.com’s Adam Schein got it wrong in his rankings of the eight divisions by quarterback.
The AFC North (Jackson, Joe Burrow, Baker Mayfield, Ben Roethlisberger) was No. 3, behind the NFC West (Kyler Murry, Matthew Stafford, Jimmy Garoppolo/Trey Lance, Russell Wilson) and AFC West (Teddy Bridgewater/Drew Lock, Patrick Mahomes, Derek Carr, Justin Herbert), in Schein's rankings. Alexander said the AFC North should've been No. 1.
"Lamar Jackson — MVP. Joe Burrow, future MVP the way he played last year as a rookie, real dominant. Baker Mayfield helped the Cleveland team win their first playoff game in who knows how many years. Big Ben was balling out last year. Everybody forgets how they started the season," Alexander said.
Schein and Alexander were in agreement on one thing: When talking about quarterbacks in the AFC North, the conversation begins with Jackson.
"Jackson is a flat-out stud and the best quarterback in this division, breaking ankles, throwing darts and generally taking breaths away," Schein wrote. "He's a former MVP who will be in the mix for the award every year for the foreseeable future."
Schein said he considered putting the AFC North at No. 2 over the AFC West, noting that Denver's situation (Bridgewater, Lock) "is the glaring 'Yeah, but …" Ultimately, Schein went with the AFC West because "Mahomes raised the bar and Roethlisberger lowered it."
"Big Ben is pushing 40 and his arm shows it," Schein wrote. "Obviously, he's a Hall of Famer, but remember: This ranking is based on my expectations for the 2021 season. And those aren't too high when it comes to Roethlisberger, especially behind Pittsburgh's patchwork offensive line. In 2021, Ben's the fourth-best quarterback in his own division."
Assessing Where the Ravens Got Better and Got Worse
With the start of training camp less than two weeks away, The Athletic's Jeff Zrebiec analyzed the Ravens roster and assessed where they are better, worse and about the same as last year.
Here's a look at two of the four areas where Zrebiec believes the team has improved and the only two where he thinks they've taken a step back:
Better: Interior offensive line
"An experienced and accomplished [Kevin] Zeitler should stabilize the right guard spot, which was a problem area for much of the 2020 season. [Bradley] Bozeman is also a more formidable option at center, assuming he's able to flawlessly get snaps back to the quarterback."
Better: Wide receiver
"The talent has been upgraded significantly. The addition of [Rashod] Bateman, [Tylan] Wallace and [Sammy] Watkins, coupled by another year of development for Marquise Brown and the team's returning young receivers, gives the Ravens one of the better receiving corps that they've had in recent years."
Worse: Offensive tackle
"The Ravens will be OK if [Ronnie] Stanley is back to himself and [Alejandro] Villanueva benefits from a change of scenery and position. However, there are still too many 'ifs' for the organization to feel all that comfortable at offensive tackle. It's certainly a position to watch this summer. Improvement from Tyre Phillips, one of the top swing tackle candidates, would help."
Worse: Outside linebacker
"[Matthew] Judon and [Yannick] Ngakoue didn't have big sack numbers last year, but they are accomplished pass rushers with big-play capabilities and other teams had to account for them. The Ravens didn't replace either one with a veteran. Unless [Jaylon] Ferguson or [Tyus] Bowser breaks out as pass rushers or [Odafe] Oweh is ready to make a major impact immediately, the roster lacks an 8-10 sack-a-year kind of guy."
Three Players the Ravens Should Build Around
Pro Football Focus’ Ben Linsey identified three players to build around for all 32 teams based on production, age and positional value.
For the Ravens, Linsey went with Jackson, Stanley and cornerback Marlon Humphrey. It's hard to argue with those choices. All three are All-Pros between the ages of 24 and 27.
"Baltimore's offense — which has been built entirely around Jackson's unique skill set as a runner — ranks behind only the Chiefs in expected points added per play since 2019. That's worth building around," Linsey wrote. "Stanley is the other major cornerstone on offense, ranking in the 88th percentile of all tackles in pass-blocking grade since 2016.
"Humphrey would be one of the top defenders to build around in the league. He just turned 25 years old and is the only qualifying cornerback in the NFL to record coverage grades of at least 80.0 in the slot and out wide since 2017."


Four suspects were arrested by Sri Lanka Navy with 01kg and 21mg of Ice (Crystal Methamphetamine).
They were arrested during a operation led by North Central Naval Command in the Urumalai beach in Talaimannar on March 3 while trying to smuggle drugs in to the country via sea.
The street value of the drugs believed to be around 6 million rupees
Narcotic drugs which were package concealed in a fishing net was taken in to naval custody and later handed over to the Talaimannar Police for further leagal actions.
Also Police Spokesman (DIG) Ajith Rohana states today after a special anti-narcotics operations conducted in Weligama, Imaduwa and Midigama areas as the police has identified as as the hub for the distribution of narcotics in south province.


text:
Healthy Heart Quiz
This quiz is not designed to diagnose heart disease. It is only an indicator of your overall risk. Only your doctor can evaluate your actual risk and devise a treatment plan to reduce your risk. If you have more questions about your risk for heart disease, contact Dr. Shapiro and have your physician fill out the Physician Referral Form.


text:
invocation for pain in the feet: for pain in the head: an invocation for headache: an innovation for migraine: an invocation for pain in the eye
Edema, also spelled oedema or œdema, is an abnormal accumulation of fluid in the interstitium, located beneath the skin and in the cavities …
SINUSITIS ACUTE SINUSITIS. Sinusitis is an inflammation, thickening, and swelling of the normal tissue called mucosa, which lines all the sinuses.
Jaw swelling is often caused by an underlying issue with the teeth. Find out if your situation is caused by a cyst, abscess or wisdom tooth infection.
Explore some of the key signs and symptoms of cancer. If you notice a persistent change in your body, tell your doctor.
List of 31 disease causes of Mouth swelling, patient stories, diagnostic guides, 25 drug side effect causes. Diagnostic checklist, medical tests, doctor questions, and related signs or symptoms for Mouth swelling.
What is mouth inflammation? Inflammation (also known as swelling) is a normal reaction your body may have to an injury. It can affect many parts of your body, including your mouth.
EDEMA OVERVIEW. Edema is the medical term for swelling caused by a collection of fluid in the spaces that surround the body’s tissues and organs.
How to Reduce Abdominal Swelling After Tummy Tuck Surgery. First off, swelling is going to happen. It is a fact of life after tummy tuck surgery.
Arnica is a toxic herb, but in highly diluted homeopathic doses, it is used for blood clots, diarrhea, swelling, and pain. WebMD tells you more.


Electric Husqvarna E-01 scooter and E-Pilen bike in the works, to be manufactured in India
Electric is the buzz word, as almost every automobile manufacturer in the world is developing an EV. This has a lot of relevance to India as the Government has set a deadline of 2030 where it wants 40 per cent of the vehicles sold to be electric. Swedish brand Husqvarna is working on two electric offerings, that will go on production in 2022. This update has been released in an investor presentation. What makes it an important update is that both these electric Husqvarnas will be manufactured at the Bajaj Auto plant in Chakan, Maharashtra.
Husqvarna E-Pilen electric bike
The details that have surfaced in the investor document reveals the E-Pilen naming. Going by the company's naming nomenclature of dividing the cafe racer and the urban scrambler into Vitpilen and Svartpilen, the pilen (Swedish for arrow) suffix will be seen on all the series production offerings. The E-Pilen, which will translate to an electric arrow will mark the third vertical of two-wheelers. The document also mentions the two powertrains that will be offered on the E-Pilen, a 4kW electric motor and a 10kW electric motor, which will power the entry-spec and the premium variants of the EV. This platform will also be shared with KTM and Bajaj.
In terms of styling, a sketch of the E-Pilen in the document highlights the combination of different elements from both Svartpilen and the Vitpilen, which include knuckle guards, rear-set footpegs, luggage rack on the tank and the compact tank design, which could hold the battery unit. Distinguishing itself from its IC siblings will be the fluorescent highlights on the entire design. The suspension setup could be from WP with USD forks in the front and mono-shock in the rear, both of which will be manufactured in India. The underpinnings will be a Trellis frame, which should ensure lightweight construction and engaging handling. Here's our detailed first ride review of the Husqvarna Svartpilen and Vitpilen 250.
Husqvarna E-01 electric scooter
The upcoming Husqvarna E-01 electric scooter will be based on the Bajaj Chetak. This is an obvious fact as Rajiv Bajaj, during the official unveiling of the Chetak electric scooter, had mentioned how eager Stefan Pierer was about the Chetak's platform, that will also take shape of an electric KTM scooter as well. Going by the sketch illustrated in the investor document, the Husqvarna E-01 could carry the edgy design language that has been on its production motorcycles.
The basic frame and the battery position of being nestled between the frame for better weight management in the Bajaj Chetak electric could be carried over in the same form on the E-01. The image also shows a similar arrangement of trailing link front suspension and a single-sided unit for the rear, exposing the wheels. The electric scooter will be powered by a 4kW unit only. Going by Chetak's claimed full-charge riding range of 95km, the E-01's claim could be in a similar ballpark. However, what could change in the E-01 is the features list, which could include more modern instrumentation and dual-channel ABS. Here's a quick look at our detailed Bajaj Chetak electric first ride experience.
Considering that toth these offerings will be manufactured at the Bajaj Auto's plant in Chakan, both the electric offerings will be sold in India as well. The time frame for that could be announced in these two years. In addition to catering to the domestic markets, both these products will be exported to the European markets which have a strong demand for EVs
Starts Rs 1,00,000


text:
Robin Thicke has already conquered the summer with his hit single “Blurred Lines” and album of the same title.
The R&B singer broke the records for the longest streak on Billboard’s top 100 list with the Blurred Lines single and the new album scored Thicke his first No. 1 album on the Billboard charts. All of this has most critics declaring him the “king of summer.”
However Robin is done just yet. He’s back with a new video “Give it 2 U,” featuring 2 Chainz and Kendrick Lamar that is equally sex-appealing as his last video.
According to mtv.com:
“Sexy female football players, mini-monster trucks, barely-there bikinis, giant jars of caviar, a cotton-candy-covered dancer and verses from Kendrick Lamar and 2 Chainz. Oh, and Robin Thicke taking his shirt off for a change.
“If you thought, Blurred Lines was a party out of control, well, Thicke has definitely upped his game for the sequel. Imagine the sexiest halftime show ever and you’ll get a flavor of what Thicke and director Diane Martel have cooked up for an encore.”
The song is the second single from Thicke’s Blurred Lines album. The song’s hard-hitting pop beat sounds very much like the work of Thicke’s buddy, Pharrell, but it’s actually produced by Dr. Luke and Cirkut.
Thicke is set to perform tomorrow to kick off MTV’s Video Music Awards, along with Big Sean.
Check out the video above and the behind-the-scenes look below.

domain: artistic


text:
US 8101187 B2
Novel proteins from Streptococcus pneumoniae are described, together with nucleic acid sequences encoding them. Their use in vaccines and in screening methods is also described.
1. A composition that comprises a Streptococcus pneumoniae protein or polypeptide, wherein said protein or polypeptide comprises one of the sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, and wherein said composition is substantially free of other Streptococcus pneumoniae proteins.
2. A composition that comprises a Streptococcus pneumoniae protein or polypeptide wherein said protein or polypeptide comprises a sequence that has at least 80% sequence identity to one of the sequences selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:3, and SEQ ID NO:5, and wherein said composition is substantially free of other Streptococcus pneumoniae proteins.
3. A method for eliciting an anti-Streptococcal immune response in a subject comprising administering to the subject an antigenic composition, said composition comprising a protein or polypeptide comprising one of the sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, as an antigen.
4. An antigenic composition comprising one or more proteins or polypeptides that comprise one or more of the sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5, wherein said composition is substantially free of other Streptococcus pneumoniae proteins.
5. The composition of
6. The composition of
7. The composition of
The present invention relates to proteins derived from Streptococcus pneumoniae, nucleic acid molecules encoding such proteins, the use of the nucleic acid and/or proteins as antigens/immunogens and in detection/diagnosis, as well as methods for screening the proteins/nucleic acid sequences as potential anti-microbial targets.
Streptococcus pneumoniae, commonly referred to as the pneumococcus, is an important pathogenic organism. The continuing significance of Streptococcus pneumoniae infections in relation to human disease in developing and developed countries has been authoritatively reviewed (Fiber, G. R., Science, 265:1385-1387 (1994)). That indicates that on a global scale this organism is believed to be the most common bacterial cause of acute respiratory infections, and is estimated to result in one million childhood deaths each year, mostly in developing countries (Stansfield, S. K., Pediatr. Infect. Dis., 6:622 (1987)). In the USA it has been suggested (Breiman et al, Arch. Intern. Med., 150:1401 (1990)) that the pneumococcus is still the most common cause of bacterial pneumonia, and that disease rates are particularly high in young children, in the elderly, and in patients with predisposing conditions such as asplenia, heart, lung and kidney disease, diabetes, alcoholism, or with immunosupressive disorders, especially AIDS. These groups are at higher risk of pneumococcal septicaemia and hence meningitis and therefore have a greater risk of dying from pneumococcal infection. Over 50,000 cases of invasive pneumococcal disease (meningitis and bacteraemia) are believed to occur annually in the United States. The pneumococcus is also the leading cause of otitis media and sinusitis, which remain prevalent infections in children in developed countries, and which incur substantial costs. S. pneumoniae is responsible for approximately seven million cases of middle ear infections in children under two years of age in the United States alone.
The need for effective preventative strategies against pneumococcal infection is highlighted by the recent emergence of penicillin-resistant pneumococci. It has been reported that 6.6% of pneumoccal isolates in 13 US hospitals in 12 states were found to be resistant to penicillin and some isolates were also resistant to other antibiotics including third generation cyclosporins (Schappert, S. M., Vital and Health Statistics of the Centres for Disease Control/National Centre for Health Statistics, 214:1 (1992)). The rates of penicillin resistance can be higher (up to 20%) in some hospitals (Breiman et al, J. Am. Med. Assoc., 271:1831 (1994)). Since the development of penicillin resistance among pneumococci is both recent and sudden, coming after decades during which penicillin remained an effective treatment, these findings are regarded as alarming.
For the reasons given above, there are therefore compelling grounds for considering improvements in the means of preventing, controlling, diagnosing or treating pneumococcal diseases.
Various approaches have been taken in order to provide vaccines for the prevention of pneumococcal infections. Difficulties arise for instance in view of the variety of serotypes (at least 90) based on the structure of the polysaccharide capsule surrounding the organism. Vaccines against individual serotypes are not effective against other serotypes and this means that vaccines must include polysaccharide antigens from a whole range of serotypes in order to be effective in a majority of cases. An additional problem arises because it has been found that the capsular polysaccharides (each of which determines the serotype and is the major protective antigen) when purified and used as a vaccine do not reliably induce protective antibody responses in children under two years of age, the age group which suffers the highest incidence of invasive pneumococcal infection and meningitis.
A modification of the approach using capsule antigens relies on conjugating the polysaccharide to a protein in order to derive an enhanced immune response, particularly by giving the response T-cell dependent characteristics. This approach has been used in the development of a vaccine against Haemophilus influenzae, for instance. There are, however, issues of cost concerning both the multi-polysaccharide vaccines and those based on conjugates. In addition, the composition of the conjugate vaccines preferably requires to be varied to accommodate different geographical and demographical populations as the serotype coverage that they offer is limited. There may also be problems with conjugate carrier-induced suppression or overload due to the relatively large total dose of carrier protein administered.
A third approach is to look for other antigenic components which offer the potential to be vaccine candidates. This is the basis of the present invention. Using a specially developed bacterial expression system, we have been able to identify a group of protein antigens from pneomococcus which are associated with the bacterial envelope or which are secreted.
Thus, in a first aspect the present invention provides a Streptococcus pneumoniae protein or polypeptide having a sequence selected from those shown in Table 1.
A protein or polypeptide of the present invention may be provided in substantially pure form. For example, it may be provided in a form which is substantially free of other proteins.
As discussed herein, the proteins and polypeptides of the invention are useful as antigenic material. Such material can be “antigenic” and/or “immunogenic”. Generally, “antigenic” is taken to mean that the protein or polypeptide is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject. “Immunogenic” is taken to mean that the protein or polypeptide is capable of eliciting a protective immune response in a subject. Thus, in the latter case, the protein or polypeptide may be capable of not only generating an antibody response but, in addition, a non-antibody based immune response.
The skilled person will appreciate that homologues or derivatives of the proteins or polypeptides of the invention will also find use in the context of the present invention, ie as antigenic/immunogenic material. Thus, for instance proteins or polypeptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar “type”. For instance replacing one hydrophobic amino acid with another.
One can use a program such as the CLUSTAL™ program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
In the case of homologues and derivatives, the degree of identity with a protein or polypeptide as described herein is less important than that the homologue or derivative should retain the antigenicity or immunogenicity of the original protein or polypeptide. However, suitably, homologues or derivatives having at least 60% similarity (as discussed above) with the proteins or polypeptides described herein are provided. Preferably, homologues or derivatives having at least 70% similarity, more preferably at least 80% similarity are provided. Most preferably, homologues or derivatives having at least 90% or even 95% similarity are provided.
In an alternative approach, the homologues or derivatives could be fusion proteins, incorporating moieties which render purification easier, for example by effectively tagging the desired protein or polypeptide. It may be necessary to remove the “tag” or it may be the case that the fusion protein itself retains sufficient antigenicity to be useful.
In an additional aspect of the invention there are provided antigenic/immunogenic fragments of the proteins or polypeptides of the invention, or of homologues or derivatives thereof.
For fragments of the proteins or polypeptides described herein, or of homologues or derivatives thereof, the situation is slightly different. It is well known that is possible to screen an antigenic protein or polypeptide to identify epitopic regions, ie those regions which are responsible for the protein or polypeptide's antigenicity or immunogenicity. Methods for carrying out such screening are well known in the art. Thus, the fragments of the present invention should include one or more such epitopic regions or be sufficiently similar to such regions to retain their antigenic/immunogenic properties. Thus, for fragments according to the present invention the degree of identity is perhaps irrelevant, since they may be 100% identical to a particular part of a protein or polypeptide, homologue or derivative as described herein. The key issue, once again, is that the fragment retains the antigenic/immunogenic properties.
Thus, what is important for homologues, derivatives and fragments is that they possess at least a degree of the antigenicity/immunogenicity of the protein or polypeptide from which they are derived.
Gene cloning techniques may be used to provide a protein of the invention in substantially pure form. These techniques are disclosed, for example, in J. Sambrook et al Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989). Thus, in a second aspect, the present invention provides a nucleic acid molecule comprising or consisting of a sequence which is:
The nucleic acid molecules of the invention may include a plurality of such sequences, and/or fragments. The skilled person will appreciate that the present invention can include novel variants of those particular novel nucleic acid molecules which are exemplified herein. Such variants are encompassed by the present invention. These may occur in nature, for example because of strain variation. For example, additions, substitutions and/or deletions are included. In addition, and particularly when utilising microbial expression systems, one may wish to engineer the nucleic acid sequence by making use of known preferred codon usage in the particular organism being used for expression. Thus, synthetic or non-naturally occurring variants are also included within the scope of the invention.
The term “RNA equivalent” when used above indicates that a given RNA molecule has a sequence which is complementary to that of a given DNA molecule (allowing for the fact that in RNA “U” replaces “T” in the genetic code).
When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package) BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length.
Preferably, sequences which have substantial identity have at least 50% sequence identity, desirably at least 75% sequence identity and more desirably at least 90 or at least 95% sequence identity with said sequences. In some cases the sequence identity may be 99% or above.
Desirably, the term “substantial identity” indicates that said sequence has a greater degree of identity with any of the sequences described herein than with prior art nucleic acid sequences.
It should however be noted that where a nucleic acid sequence of the present invention codes for at least part of a novel gene product the present invention includes within its scope all possible sequence coding for the gene product or for a novel part thereof.
The nucleic acid molecule may be in isolated or recombinant form. It may be incorporated into a vector and the vector may be incorporated into a host. Such vectors and suitable hosts form yet further aspects of the present invention.
Therefore, for example, by using probes based upon the nucleic acid sequences provided herein, genes in Streptococcus pneumoniae can be identified. They can then be excised using restriction enzymes and cloned into a vector. The vector can be introduced into a suitable host for expression.
Nucleic acid molecules of the present invention may be obtained from S. pneumoniae by the use of appropriate probes complementary to part of the sequences of the nucleic acid molecules. Restriction enzymes or sonication techniques can be used to obtain appropriately sized fragments for probing.
Alternatively PCR techniques may be used to amplify a desired nucleic acid sequence. Thus the sequence data provided herein can be used to design two primers for use in PCR so that a desired sequence, including whole genes or fragments thereof, can be targeted and then amplified to a high degree.
Typically primers will be at least 15-25 nucleotides long.
As a further alternative chemical synthesis may be used. This may be automated. Relatively short sequences may be chemically synthesised and ligated together to provide a longer sequence.
There is another group of proteins from S. pneumoniae which have been identified using the bacterial expression system described herein. These are known proteins from S. pneumoniae, which have not previously been identified as antigenic proteins. The amino acid sequences of this group of proteins, together with DNA sequences coding for them are shown in Table 2. These proteins, or homologues, derivatives and/or fragments thereof also find use as antigens/immunogens.
A further group of proteins have been identified from recently published S. pnuemoniae genomes that have a degree of homology with ID-304L1 which all possess the following highly conserved sequence of 23 amino acids either at or near the N-terminus:
MELVLPNNYVV(D,A)I(L)D(E)E(Q)EEMMYLDGG(E) (SEQ ID NO:45) where the bracketed residues represent alternatives to the preceding amino acid. Amino acid sequences for these homologues, and the DNA sequences encoding them are given in Table 3.
Thus, in a further aspect the present invention provides a Streptococcus pneumoniae protein which has the N terminal sequence
MELVLPNNYVV(D,A)I(L)D(E)E(Q)EEMMYLDGG(E) (SEQ ID NO:45) or fragment or homologue or derivative thereof.
In another aspect the present invention provides the use of a protein or polypeptide having a sequence selected from those shown in Tables 1 to 3, or homologues, derivatives and/or fragments thereof, as an immunogen/antigen.
In yet a further aspect the present invention provides an immunogenic/antigenic composition comprising one or more proteins or polypeptides selected from those whose sequences are shown in Tables 1 to 3, or homologues or derivatives thereof, and/or fragments of any of these. In preferred embodiments, the immunogenic/antigenic composition is a vaccine or is for use in a diagnostic assay.
In the case of vaccines, suitable additional excipients, diluents, adjuvants or the like may be included. Numerous examples of these are well known in the art.
It is also possible to utilise the nucleic acid sequences shown in Tables 1 and 2 in the preparation of so-called DNA vaccines. Thus, the invention also provides a vaccine composition comprising one or more nucleic acid sequences as defined herein. DNA vaccines are described in the art (see for instance, Donnelly et al, Ann. Rev. Immunol., 15:617-648 (1997)) and the skilled person can use such art described techniques to produce and use DNA vaccines according to the present invention.
As already discussed herein the proteins or polypeptides described herein, their homologues or derivatives, and/or fragments of any of these, can be used in methods of detecting/diagnosing S. pneumoniae. Such methods can be based on the detection of antibodies against such proteins which may be present in a subject. Therefore the present invention provides a method for the detection/diagnosis of S. pneumoniae which comprises the step of bringing into contact a sample to be tested with at least one protein, or homologue, derivative or fragment thereof, as described herein. Suitably, the sample is a biological sample, such as a tissue sample or a sample of blood or saliva obtained from a subject to be tested.
In an alternative approach, the proteins described herein, or homologues, derivatives and/or fragments thereof, can be used to raise antibodies, which in turn can be used to detect the antigens, and hence S. pneumoniae. Such antibodies form another aspect of the invention. Antibodies within the scope of the present invention may be monoclonal or polyclonal.
Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a protein as described herein, or a homologue, derivative or fragment thereof, is injected into the animal. If desired, an adjuvant may be administered together with the protein. Well-known adjuvants include Freund's adjuvant (complete and incomplete) and aluminium hydroxide. The antibodies can then be purified by virtue of their binding to a protein as described herein.
Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique (Nature 256 (1975)) or subsequent variations upon this technique can be used.
Techniques for producing monoclonal and polyclonal antibodies that bind to a particular polypeptide/protein are now well developed in the art. They are discussed in standard immunology textbooks, for example in Roitt et al, Immunology second edition (1989), Churchill Livingstone, London.
In addition to whole antibodies, the present invention includes derivatives thereof which are capable of binding to proteins etc as described herein. Thus the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtec, 12:372-379 (September 1994).
Antibody fragments include, for example, Fab, F(ab′)2 and Fv fragments. Fab fragments are discussed in Roitt et al [supra]. Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining Vh and Vl, regions, which contributes to the stability of the molecule. Other synthetic constructs that can be used include Complementarity Determining Regions (CDR) peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains.
Synthetic constructs include chimaeric molecules. Thus, for example, humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimaeric antibodies are discussed for example by Morrison et al in PNAS, 81:6851-6855 (1984) and by Takeda et al in Nature. 314:452-454 (1985).
Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label). Alternatively, it may be a pharmaceutically active agent.
Antibodies, or derivatives thereof, find use in detection/diagnosis of S. pneumoniae. Thus, in another aspect the present invention provides a method for the detection/diagnosis of S. pneumoniae which comprises the step of bringing into contact a sample to be tested and antibodies capable of binding to one or more proteins described herein, or to homologues, derivatives and/or fragments thereof.
In addition, so-called “Affibodies” may be utilised. These are binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain (Nord et al in Nature Biotechnology, 15:772-7 (1997)). Thus, small protein domains, capable of specific binding to different target proteins can be selected using combinatorial approaches.
It will also be clear that the nucleic acid sequences described herein may be used to detect/diagnose S. pneumoniae. Thus, in yet a further aspect, the present invention provides a method for the detection/diagnosis of S. pneumoniae which comprises the step of bringing into contact a sample to be tested with at least one nucleic acid sequence as described herein. Suitably, the sample is a biological sample, such as a tissue sample or a sample of blood or saliva obtained from a subject to be tested. Such samples may be pre-treated before being used in the methods of the invention. Thus, for example, a sample may be treated to extract DNA. Then, DNA probes based on the nucleic acid sequences described herein (ie usually fragments of such sequences) may be used to detect nucleic acid from S. pneumoniae.
In additional aspects, the present invention provides:
(a) a method of vaccinating a subject against S. pneumoniae which comprises the step of administering to a subject a protein or polypeptide of the invention, or a derivative, homologue or fragment thereof, or an immunogenic composition of the invention;
(b) a method of vaccinating a subject against S. pneumoniae which comprises the step of administering to a subject a nucleic acid molecule as defined herein;
(c) a method for the prophylaxis or treatment of S. pneumoniae infection which comprises the step of administering to a subject a protein or polypeptide of the invention, or a derivative, homologue or fragment thereof, or an immunogenic composition of the invention;
(d) a method for the prophylaxis or treatment of S. pneumoniae infection which comprises the step of administering to a subject a nucleic acid molecule as defined herein;
(e) a kit for use in detecting/diagnosing S. pneumoniae infection comprising one or more proteins or polypeptides of the invention, or homologues, derivatives or fragments thereof, or an antigenic composition of the invention; and
(f) a kit for use in detecting/diagnosing S. pneumoniae infection comprising one or more nucleic acid molecules as defined herein.
Given that we have identified a group of important proteins, such proteins are potential targets for anti-microbial therapy. It is necessary, however, to determine whether each individual protein is essential for the organism's viability. Thus, the present invention also provides a method of determining whether a protein or polypeptide as described herein represents a potential anti-microbial target which comprises antagonising, inhibiting or otherwise interfering with the function or expression of said protein and determining whether S. pneumoniae is still viable.
A suitable method for inactivating the protein is to effect selected gene knockouts, ie prevent expression of the protein and determine whether this results in a lethal change. Suitable methods for carrying out such gene knockouts are described in Li et al, P.N.A.S., 94:13251-13256 (1997) and Kolkman et al, J. Bacteriol., 178:3736-3741 (1996).
In a final aspect the present invention provides the use of an agent capable of antagonising, inhibiting or otherwise interfering with the function or expression of a protein or polypeptide of the invention in the manufacture of a medicament for use in the treatment or prophylaxis of S. pneumoniae infection.
As mentioned above, we have used a bacterial expression system as a means of identifying those proteins which are surface associated, secreted or exported and thus, would find use as antigens or antimicrobial targets.
The information necessary for the secretion/export of proteins has been extensively studied in bacteria. In the majority of cases, protein export requires a signal peptide to be present at the N-terminus of the precursor protein so that it becomes directed to the translocation machinery on the cytoplasmic membrane. During or after translocation, the signal peptide is removed by a membrane associated signal peptidase. Ultimately the localization of the protein (i.e. whether it be secreted, an integral membrane protein or attached to the cell wall) is determined by sequences other than the leader peptide itself.
We are specifically interested in surface located or exported proteins as these are likely to be antigens for use in vaccines, as diagnostic reagents or as targets for therapy with novel chemical entities. We have therefore developed a screening vector-system in Lactococcus lactis that permits genes encoding exported proteins to be identified and isolated. We provide below a representative example showing how given novel surface associated proteins from Streptococcus pneumoniae have been identified and characterized. The screening vector incorporates the staphylococcal nuclease gene nuc lacking its own export signal as a secretion reporter. Staphylococcal nuclease is a naturally secreted heat-stable, monomeric enzyme which has been efficiently expressed and secreted in a range of Gram positive bacteria (Shortle, Gene, 22:181-189 (1983); Kovacevic et al., J. Bacteriol., 162:521-528 (1985); Miller et al., J. Bacteriol., 169:3508-3514 (1987); Liebl et al., J. Bacteriol., 174:1854-1861 (1992); Le Loir et al., J. Bacteriol., 176:5135-5139 (1994); Poquet et al., J. Bacteriol., 180:1904-1912 (1998)).
Recently, Poquet et al. ((1998), supra) have described a screening vector incorporating the nuc gene lacking its own signal leader as a reporter to identify exported proteins in Gram positive bacteria, and have applied it to L. lactis. This vector (PFUN) contains the PAMβ1 replicon which functions in a broad host range of Gram-positive bacteria in addition to the ColE1 replicon that promotes replication in Escherichia coli and certain other Gram negative bacteria. Unique cloning sites present in the vector can be used to generate transcriptional and translational fusions between cloned genomic DNA fragments and the open reading frame of the truncated nuc gene devoid of its own signal secretion leader. The nuc gene makes an ideal reporter gene because the secretion of nuclease can readily be detected using a simple and sensitive plate test; recombinant colonies secreting the nuclease develop a pink halo whereas control colonies remain white (Shortle, (1983), supra; Le Loir et al., (1994), supra).
Thus, the invention will now be described with reference to the following representative example, which provides details of how the proteins, polypeptides and nucleic acid sequences described herein identified as antigenic targets.
We describe herein the construction of three reporter vectors and their use in L. lactis to identify and isolate genomic DNA fragments from Streptococcus pneumoniae encoding secreted or surface associated proteins. Furthermore, Southern blot hybridisation experiments have been conducted to demonstrate the presence of a vaccine candidate gene in a range of Streptococcus pneumoniae strains. The invention will now be described with reference to the examples, which should not be construed as in any way limiting the invention.
(i) Construction of the pTREP1-nuc Series of Reporter Vectors
(a) Construction of Expression Plasmid pTREP1
The pTREPI plasmid is a high-copy number (40-80 per cell) theta-replicating gram positive plasmid, which is a derivative of the pTREX plasmid which is itself a derivative of the previously published pIL253 plasmid. pIL253 incorporates the broad Gram-positive host range replicon of pAMβ1 (Simon and Chopin, Biochimie, 70:559-567 (1988)) and is non-mobilisable by the L. lactis sex-factor. pIL253 also lacks the tra function which is necessary for transfer or efficient mobilisation by conjugative parent plasmids exemplified by pIL501. The Enterococcal pAMβ1 replicon has previously been transferred to various species including Streptococcus, Lactobacillus and Bacillus species as well as Clostridium acetobutylicum, (Oultram and Klaenhammer, FEMS Microbiological Letters, 27:129-134 (1985); Gibson et al., (1979); LeBlanc et al., Proceedings of the National Academy of Science USA, 75:3484-3487 (1978)) indicating the potential broad host range utility. The pTREP1 plasmid represents a constitutive transcription vector.
The pTREX vector was constructed as follows. An artificial DNA fragment containing a putative RNA stabilising sequence, a translation initiation region (TIR), a multiple cloning site for insertion of the target genes and a transcription terminator was created by annealing two complementary oligonucleotides and extending with Tfl DNA polymerase. The sense and anti-sense oligonucleotides contained the recognition sites for NheI and BamHI at their 5′ ends respectively to facilitate cloning. This fragment was cloned between the XbaI and BamHI sites in pUC19NT7, a derivative of pUC19 which contains the T7 expression cassette from pLET1 (Wells et al, J. Appl. Bacteriol., 74:629-636 (1993)) cloned between the EcoRI and HindIII sites. The resulting construct was designated pUCLEX. The complete expression cassette of pUCLEX was then removed by cutting with HindIII and blunting followed by cutting with EcoRI before cloning into EcoRI and SacI (blunted) sites of pIL253 to generate the vector pTREX (Wells and Schofield, In Current advances in metabolism, genetics and applications-NATO ASI Series, H 98:37-62 (1996)). The putative RNA stabilising sequence and TIR are derived from the Escherichia coli T7 bacteriophage sequence and modified at one nucleotide position to enhance the complementarity of the Shine Dalgarno (SD) motif to the ribosomal 16s RNA of Lactococcus lactis (Schofield et al. pers. coms. University of Cambridge Dept. Pathology).
A Lactococcus lactis MG1363 chromosomal DNA fragment exhibiting promoter activity which was subsequently designated P7 was cloned between the EcoRI and BglII sites present in the expression cassette, creating pTREX7. This active promoter region had been previously isolated using the promoter probe vector pSB292 (Waterfield et al, Gene, 165:9-15 (1995)). The promoter fragment was amplified by PCR using the Vent DNA polymerase according to the manufacturer.
The pTREPI vector was then constructed as follows. An artificial DNA fragment which included a transcription terminator, the forward pUC sequencing primer, a promoter multiple-cloning site region and a universal translation stop sequence was created by annealing two overlapping partially complementary synthetic oligonucleotides together and extending with sequenase according to the manufacturer's instructions. The sense and anti-sense (pTREPF and pTREPR) oligonucleotides contained the recognition sites for EcoRV and BamHI at their 5′ ends respectively to facilitate cloning into pTREX7. The transcription terminator was that of the Bacillus penicillinase gene, which has been shown to be effective in Lactococcus (Jos et al., Applied and Environmental Microbiology, 50:540-542 (1985)). This was considered necessary as expression of target genes in the pTREX vectors was observed to be leaky and is thought to be the result of cryptic promoter activity in the origin region (Schofield et al. pers. coms. University of Cambridge Dept. Pathology). The forward pUC primer sequencing was included to enable direct sequencing of cloned DNA fragments. The translation stop sequence which encodes a stop codon in 3 different frames was included to prevent translational fusions between vector genes and cloned DNA fragments. The pTREX7 vector was first digested with EcoRI and blunted using the 5′-3′ polymerase activity of T4 DNA polymerase (NEB) according to manufacturer's instructions. The EcoRI digested and blunt ended pTREX7 vector was then digested with Bgl II thus removing the P7 promoter. The artificial DNA fragment derived from the annealed synthetic oligonucleotides was then digested with EcoRV and Bam HI and cloned into the EcoRI (blunted)-Bgl II digested pTREX7 vector to generate pTREP. A Lactococcus lactis MG1363 chromosomal promoter designated P1 was then cloned between the EcoRI and BglII sites present in the pTREP expression cassette forming pTREP1. This promoter was also isolated using the promoter probe vector pSB292 and characterised by Waterfield et al., (1995), supra. The P1 promoter fragment was originally amplified by PCR using VENT® DNA polymerase according to the manufacturer's instructions and cloned into the pTREX as an EcoRI-BglII DNA fragment. The EcoRI-BglII P1 promoter containing fragment was removed from pTREX1 by restriction enzyme digestion and used for cloning into pTREP (Schofield et al. pers. coms. University of Cambridge, Dept. Pathology).
(b) PCR Amplification of the S. aureus nuc Gene.
The nucleotide sequence of the S. aureus nuc gene (EMBL database accession number V01281) was used to design synthetic oligonucleotide primers for PCR amplification. The primers were designed to amplify the mature form of the nuc gene designated nucA which is generated by proteolytic cleavage of the N-terminal 19 to 21 amino acids of the secreted propeptide designated Snase B (Shortle, (1983), supra). Three sense primers (nucS1, nucS2 and nucS3, Appendix 1) were designed, each one having a blunt-ended restriction endonuclease cleavage site for EcoRV or SmaI in a different reading frame with respect to the nuc gene. Additionally BglII and BamHI were incorporated at the 5′ ends of the sense and anti-sense primers respectively to facilitate cloning into BamHI and BglII cut pTREP1. The sequences of all the primers are given in Appendix 1. Three nuc gene DNA fragments encoding the mature form of the nuclease gene (NucA) were amplified by PCR using each of the sense primers combined with the anti-sense primer described above. The nuc gene fragments were amplified by PCR using S. aureus genomic DNA template, VENT® DNA Polymerase (NEB) and the conditions recommended by the manufacturer. An initial denaturation step at 93° C. for 2 min was followed by 30 cycles of denaturation at 93° C. for 45 sec, annealing at 50° C. for 45 seconds, and extension at 73° C. for 1 minute and then a final 5 min extension step at 73° C. The PCR amplified products were purified using a Wizard® clean up column (Promega) to remove unincorporated nucleotides and primers.
(c) Construction of the pTREPI -nuc Vectors
The purified nuc gene fragments described in section (b) were digested with Bgl II and BamHI using standard conditions and ligated to BamHI and BglII cut and dephosphorylated pTREPI to generate the pTREP1-nuc1, pTREPI-nuc2 and pTREP1-nuc3 series of reporter vectors. General molecular biology techniques were carried out using the reagents and buffer supplied by the manufacturer or using standard conditions (Sambrook and Maniatis, (1989), supra). In each of the pTREP1-nuc vectors the expression cassette comprises a transcription terminator, lactococcal promoter P1, unique cloning sites (BglII, EcoRV or SmaI) followed by the mature form of the nuc gene and a second transcription terminator. Note that the sequences required for translation and secretion of the nuc gene were deliberately excluded in this construction. Such elements can only be provided by appropriately digested foreign DNA fragments (representing the target bacterium) which can be cloned into the unique restriction sites present immediately upstream of the nuc gene.
In possessing a promoter, the pTREPI-nuc vectors differ from the pFUN vector described by Poquet et al. (1998), supra, which was used to identify L. lactis exported proteins by screening directly for Nuc activity directly in L. lactis. As the pFUN vector does not contain a promoter upstream of the nuc open reading frame the cloned genomic DNA fragment must also provide the signals for transcription in addition to those elements required for translation initiation and secretion of Nuc. This limitation may prevent the isolation of genes that are distant from a promoter, for example genes which are within polycistronic operons. Additionally there can be no guarantee that promoters derived from other species of bacteria will be recognised and functional in L. lactis. Certain promoters may be under stringent regulation in the natural host but not in L. lactis. In contrast, the presence of the P1 promoter in the pTREP1-nuc series of vectors ensures that promoterless DNA fragments (or DNA fragments containing promoter sequences not active in L. lactis) will still be transcribed.
(ii) Screening for S. pneumoniae Secreted Proteins
Genomic DNA isolated from S. pneumoniae was digested with the restriction enzyme Tru9I. This enzyme which recognises the sequence 5′-TTAA-3′ was used because it cuts A/T rich genomes efficiently and can generate random genomic DNA fragments within the preferred size range (usually averaging 0.5-1.0 kb). This size range was preferred because there is an increased probability that the P1 promoter can be utilised to transcribe a novel gene sequence. However, the P1 promoter may not be necessary in all cases as it is possible that many Streptococcal promoters are recognised in L. lactis. DNA fragments of different size ranges were purified from partial Tru9I digests of S. pneumoniae genomic DNA. As the Tru9I restriction enzyme generates staggered ends the DNA fragments had to be made blunt ended before ligation to the EcoRV or SmaI cut pTREP1-nuc vectors. This was achieved by the partial fill-in enzyme reaction using the 5′-3′ polymerase activity of Klenow enzyme. Briefly Tru9I digested DNA was dissolved in a solution (usually between 10-20 μl in total) supplemented with T4 DNA ligase buffer (New England Biolabs; NEB) (1×) and 33 μM of each of the required dNTPs, in this case dATP and dTTP. Klenow enzyme was added (1 unit Klenow enzyme (NEB) per μg of DNA) and the reaction incubated at 25° C. for 15 minutes. The reaction was stopped by incubating the mix at 75° C. for 20 minutes. EcoRV or SmaI digested pTREP-nuc plasmid DNA was then added (usually between 200-400 ng). The mix was then supplemented with 400 units of T4 DNA ligase (NEB) and T4 DNA ligase buffer (1×) and incubated overnight at 16° C. The ligation mix was precipitated directly in 100% Ethanol and 1/10 volume of 3M sodium acetate (pH 5.2) and used to transform L. lactis MG1363 (Gasson, 1983). Alternatively, the gene cloning site of the pTREP-nuc vectors also contains a BglII site which can be used to clone for example Sau3AI digested genomic DNA fragments.
L. lactis transformant colonies were grown on brain heart infusion agar and nuclease secreting (Nuc+) clones were detected by a toluidine blue-DNA-agar overlay (0.05 M Tris pH 9.0, 10 g of agar per liter, 10 g of NaCl per liter, 0.1 mM CaCl2, 0.03% wt/vol. salmon sperm DNA and 90 mg of Toluidine blue O dye) essentially as described by Shortle, 1983, supra and Le Loir et al., 1994, supra). The plates were then incubated at 37° C. for up to 2 hours. Nuclease secreting clones develop an easily identifiable pink halo. Plasmid DNA was isolated from Nuc+ recombinant L. lactis clones and DNA inserts were sequenced on one strand using the NucSeq sequencing primer described in Appendix 1, which sequences directly through the DNA insert.
(iii) Isolation of Genes Encoding Exported Proteins from S. pneumoniae
A large number of gene sequences putatively encoding exported proteins in S. pneumoniae have been identified using the nuclease screening system. These have now been further analysed to remove artifacts. The sequences identified using the screening system have been analysed using a number of parameters.
1. All putative surface proteins were analysed for leader/signal peptide sequences using the software programs SEQUENCHER™ (Gene Codes Corporation) and DNA Strider (Marck, Nucleic Acids Res., 16:1829-1836 (1988)). Bacterial signal peptide sequences share a common design. They are characterised by a short positively charged N-terminus (N region) immediately preceding a stretch of hydrophobic residues (central portion-h region) followed by a more polar C-terminal portion which contains the cleavage site (c-region). Computer software is available which allows hydropathy profiling of putative proteins and which can readily identify the very distinctive hydrophobic portion (h-region) typical of leader peptide sequences. In addition, the sequences were checked for the presence of or absence of a potential ribosomal binding site (Shine-Dalgarno motif) required for translation initiation of the putative nuc reporter fusion protein.
2. All putative surface protein sequences were also matched with all of the protein/DNA sequences using the publicly available databases [OWL-proteins inclusive of SwissProt and GenBank translations]. This allows us to identify sequences similar to known genes or homologues of genes for which some function has been ascribed. Hence it has been possible to predict a function for some of the genes identified using the LEEP system and to unequivocally establish that the system can be used to identify and isolate gene sequences of surface associated proteins. We should also be able to confirm that these proteins are indeed surface related and not artifacts. The LEEP system has been used to identify novel gene targets for vaccine and therapy.
3. Some of the genes identified proteins did not possess a typical leader peptide sequence and did not show homology with any DNA/protein sequences in the database. Indeed these proteins may indicate the primary advantage of our screening method, i.e. the isolation of atypical surface-related proteins, which may have been missed in all previously described screening protocols or approaches based on sequence homology searches.
In all cases, only partial gene sequences were initially obtained. Full length genes given in Table 2 were obtained in all cases by reference to the TIGR S. pneumoniae database (www.tigr.org). Thus, by matching the originally obtained partial sequences with the database, we were able to identify the full length gene sequences. Hence, as described herein, two groups of genes were clearly identified, ie a group of genes encoding previously unidentified S. pneumoniae proteins (Table 1), and a second group which encoded known S. pneumoniae proteins, which were, however, not known as antigens (Table 2).
Two further S. pneumoniae genomes have been recently sequenced and the information published and subsequently made available on the NCBI database. The “Annotated Draft Genomic Sequence from a Streptococcus pneumoniae Type 19F Clinical Isolate” was published in July 2001 by Dopazo et al. (Microbial Drug Resistance, Volume 7, pp 99-125). The “Genome of the Bacterium Streptococcus pneumoniae Strain R6 was published in October 2001 by Hoskins et al. (Journal of Bacteriology, Volume 183, pp 5709-5717). Through BLAST analysis, homologues of ID-304L1 have been identified in these genomes which all possess a highly conserved sequence of 23 amino acids either at or near their N-terminus:
MELVLPNNYVV(D,A)I(L)D(E)E(Q)EEMMYLDGG(E), (SEQ ID NO:45), where the bracketed residues represent alternatives to the preceding amino acid. Sequences for these homologues are given in Table 3.
Conservation and Variability of ID-304L Variants Among Different Isolates of Streptococcus pneumoniae
The presence of genes ID304L1 and ID305 in the S. pneumoniae serotype 3 strain ATCC 49619 was investigated. Oligonucleotide primers were designed based upon the known nucleic acid sequences given in Table 1 and these gene targets were amplified by PCR.
(i) Amplification and Labelling of Specific Target Genes as DNA Probes for Southern Blot Analysis
Oligonucleotide primers were designed to amplify corresponding gene-specific DNA probes (Appendix 2). Specific gene targets (ID304L1 and ID305) were amplified by PCR using PFUTURBO® DNA polymerase (Stratagene) according to the manufacturer's instructions. Typical reactions were carried out in a 50 μl volume containing 100 ng of template DNA, a one tenth volume of enzyme reaction buffer, 100 ng of each primer, 200 μM of each dNTP and 1.25 Units of PFUTURBO® DNA polymerase. A typical reaction contained an initial 3 minute denaturation at 95 C, followed by a single 60 second cycle at 94 C, followed by 30 cycles at 50 C for 60 seconds. A single cycle of 2 minutes at 72 C was then followed with a final extension period of 10 minutes at 72 C.
All PCR amplified products were purified using the QIAquick™ PCR Purification Kit (Qiagen). The presence of homologues to ID304L1 and ID305 in strain ATCC49619 was thereby confirmed.
For use as DNA probes, purified amplified gene DNA fragments from ID304L1 were labelled with digoxygenin using the DIG Nucleic Acid High Prime Labelling Kit (Roche) according to the manufacturer's instructions.
(ii) Southern Blot Hybridisation Analysis of Group B Streptococcal Genomic DNA
A Southern blot analysis was carried out to determine cross-serotype conservation of novel Streptococcus pneumoniae genes isolated using the LEEP system. The Streptococcus pneumoniae strains used in this analysis are given in the legend to
Genomic DNA isolated from strains of Streptococcus pneumoniae were investigated for conservation of ID 304L1 derived gene targets. Appropriate DNA concentrations were digested using HindIII restriction enzymes (Roche) according to the manufacturer's instructions and analysed by agarose gel electrophoresis. Following agarose gel electrophoresis of DNA samples, the gel was denatured in 0.5M NaOH-1.5M NaCl for 20 minutes, neutralised in 0.5M Tris HCl (pH 7.5)-1.5M NaCl for 40 minutes and DNA was transferred onto Hybond™ N+ membrane (Amersham) by overnight capillary blotting. The method is essentially as described in “The DIG System User's Guide for Filter Hybridization” (Boehringer Mannheim, 1995) using Whatman 3MM wicks on a platform over a reservoir of 20×SSC (salt sodium citrate). After transfer, the filter was washed briefly in 2×SSC and stored at −20 C.
Filters were pre-hybridised, hybridised with the digoxygenin labelled DNA probes and washed using conditions recommended by Boehringer Mannheim when using their DIG Nucleic Acid Detection Kit. Filters were pre-hybridised at 42 C for one hour in DIG “EasyHyb”. The digoxygenin labelled DNA probe was denatured at 100 C for 10 minutes and chilled on ice before being added to the hybridisation buffer (DIG“EasyHyb”). Hybridisation was allowed to proceed overnight in a rotating Hybaid tube in a Hybaid Mini-hybridisation oven. Unbound probe was removed by washing the filter twice with 2×SSC-0.1% SDS for 5 minutes at room temperature. For increased stringency, filters were then washed twice with 0.5×SSC-0.1% SDS for 15 minutes at 68 C. The DIG Nucleic Acid Detection Kit (Roche) was used to detect specifically bound digoxygenin labelled DNA probes immunologically.
The Southern blot hybridisation demonstrates the presence of an ID-304L1 homologue in the majority of the strains analysed. Lane 12, from which the probe was amplified, has only a very faint band, but this is due to a low level of DNA being applied to the gel in this case. This may also explain the absence of a band in Lane 5, where the background is significantly fainter than for the other lanes. In some strains two bands are observed which suggests that there may be more than one homologue present (as found in the G54 and R6 strains). The presence of genes for this protein in a wide number of clinically relevant strains indicates that this is a conserved protein that is a good vaccine candidate.
Appendix I—Oligonucleotide Primers for LEEP Screening
The primers were engineered to provide restriction enzyme sites for later use in cloning (given in bold type below). GGC clamps allowing the restriction enzymes greater binding capacity are underlined.

domain: biology


text:
The Surf Coast Century ultra marathon is gearing up for its 12th edition this Saturday, September 16, 2023 in Anglesea, Victoria. With an impressive 1300 participants, the event has firmly established itself as a must-do ultra trail marathon in Australia, drawing elite runners and relay teams from around the country.
650 competitors will be participating as individual runners in either the 100km or 50km courses; with the remaining field taking part in a relay team of two, three, or four runners over the 100km course, covering approximately 25km each.
The Surf Coast Century is said to be more than race – it’s a celebration of perseverance, camaraderie, and a course that encapsulates the breathtaking natural beauty of Victoria’s Great Ocean Road region.
Earning its’ reputation as one of Australia’s “bucket-list” ultra trail running events; it is mostly thanks to the event’s unique 100km ultra marathon course, ranking amongst the World’s most scenic!
The figure-8 course design course takes 100km competitors from Anglesea to Torquay along the beach, returning along the cliff top tracks and hinterland trails to Moggs Creek and Aireys Inlet; before heading back to Anglesea on the Surf Coast Walk trail, via Point Roadknight. Whilst the 50km course does the second-half of the 100km.
Among the individual participants is previous Surf Coast Century winner and 9-time participant Ross Hopkins, who believed Rapid Ascent put this type of race and distance on the ultra-trail running radar.
Relay Team “Dads of the Century” is headed up by Torquay resident Stuart Jones, who has lured a team of fellow dads into the event;
“I’ve always loved the idea of the 100km Surf Coast Century course and the stunning beauty along the coastal trails,” said Jones. “I was fortunate enough to find a team of other dads at the Torquay Coast Primary school who were also keen to do the run.”
Up for grabs at the Century is the $2,000 cash on offer for the first and fastest team to break the 7-hour relay team course record. Team “Dawn League” were ever so close in 2022 clocking in just 17 minutes over.
For those who prefer to watch, an extensive event expo at the Anglesea Green will accompany the race. There are also recommended viewing and cheering spots along the coastal tracks for spectators to enjoy the action (details HERE).
The celebration continues the day after the race at the Anglesea Surf Life Saving Club with presentations, recovery activities, BBQ, spectacle events and the Meli Kids Run (details HERE).
“We invite you to come and see just how good the ‘feel-good’ ultra is – whether as spectator or competitor!” said Maffett.
The event is supported by the Surf Coast Shire; with limited entries remaining for those up for a last minute challenge.
Entries and further event information can be found here.

domain: geography


text: This is a casting move that makes sense. Ousted UT men's basketball coach Bruce Pearl will be part of the next season of "Celebrity Apprentice," the NBC reality competition series starring Donald Trump. "I'm ready to take on Trump," Pearl is quoted as saying. Participants on "Apprentice" do the show to raise money for their charities. Other celebrities next season include Charlie Sheen and Lindsay Lohan. If you've not caught up so far, you're a sucka. It's April Fools Day, lady.

domain: celebrity


text:
- Subscribe NowLimited Time Offer
Sign Up for the Memphis Business Journal Afternoon Edition Newsletter
Send this story to a friend
Landlords less likely to respond to rental inquiries from gay couples
Landlords are less likely to respond to inquiries about rental units from same-sex couples than they are to inquires from heterosexual couples, according to a new study from the Department of Housing and Urban Development.
HUD conducted an experiment using email inquiries to listed rental units in 50 metropolitan areas. In the emails, the interested renters identified themselves as either a heterosexual couple, a male gay couple or a lesbian couple. HUD found that landlords were most likely to respond to heterosexual couples. Lesbians were next; gay guys were the least favorite potential renters.
In nearly 16 percent of the tests, heterosexual couples were favored over gay male couples; heterosexuals were favored over lesbian couples in 15.6 percent of the tests.
HUD speculates the actual level of discrimination against gay and lesbian couples is higher, since these email inquiries were "at the very threshold of the rental transaction."
Under the Fair Housing Act, it's illegal to discriminate against potential renters or home buyers on the basis of race, religion or sex, but the law doesn't address sexual orientation. But 20 states and more than 150 localities prohibit housing discrimination against lesbians, gays, bisexuals and transgender people.
The study, however, found that gays and lesbians were treated slightly worse in these states than they were in states without legislative protections.
HUD recently issued guidance that treats discrimination based on "gender nonconformity" or "sex stereotyping" as sex discrimination under the Fair Housing Act.
"As this study shows, we need to continue our efforts to ensure that everyone is treated the same when it comes to finding a home to call their own, regardless of their sexual orientation,” said HUD Secretary Shaun Donovan.
My only question about the survey is this: In the real world, how many people would define their relationship status when they inquire about a rental unit? Two people looking for an apartment could just be roommates -- it's no business of the landlord's as to whether they're in a relationship.
- Most popular
- Neely's Bar-B-Que moves to reopen in Memphis
- Another women's clothing chain in Memphis closing its doors
- Sharky's files for bankruptcy with debts near $1M
- Sources: Memphis in talks to bring Hard Rock Hotel to Beale
- Delta pulls $1.3M permit to consolidate airport offices
- Another GTx executive resigns
- Wright Medical files permit for Arlington expansion
- Facebook IPO fraud puts lien on Sharky's property
- Coldwater Creek to close Memphis stores
- High Cotton gets $50K grant for taproom renovation
- Director of Finance Woodland Presbyterian School | Memphis, TN
- Network Manager - Long Term Support Services (LTSS) - East & West Tennessee - #84415 National Government Services | Memphis, TN
- Preschool Photographers Lifetouch Preschool Portraits | Memphis, TN
- RETAIL MERCHANDISER – SALES TEAM - DELIVERY REPRESENTATIVE – CLASS A CDL – DELIVERY TEAM BUDWEISER OF MEMPHIS | Memphis, TN
- Sales Agents (m/f) GOLFINO AG | Memphis, TN

domain: sociology


text:
Changing Face of the RMBI
Now recognised as one of the foremost charities in the country for the provision of care for the elderly and for those with dementia, the Royal Masonic Benevolent Institution owes its existence to the persistence of a number of notable Freemasons from the middle of the 19th century, such as Dr Robert Crucefix, who fought against prejudice and the hierarchy to create an organisation designed to support aged and infirm Freemasons at a time when the only alternative was the Workhouse.
The story of the RMBI is a fascinating one; in its early years it's full of intrigue as the various factions sought to bring their vision of support to the fore. Once established, the organisation has faced numerous challenges fundraising, World Wars, the changing needs of society and of Masons, etc to ensure that it survives and continues to be relevant in the modern age.
In the first illustrated history of the RMBI for more than a century Dr John Reuther examines the origins of the organisation and how it has evolved over more than 150 years. Drawing upon a wide range of archive and other sources, the book allows the reader to appreciate fully how the early promoters helped set the first asylum up and how the organisation has grown and developed since the early days in Croydon.
168 Printed Pages
Beautifully illustrated throughout


text:
The film is doing okay in the B and C centres and has collected nearly 80 percent of the film’s budget. Sunil is of the opinion that audience reaction and response is more important than what a few reviews have to say about his film.
Now that his film is a safe bet for his makers, he has quickly moved on to his next. Meanwhile, Sunil will also be seen in a crucial role in Chiru’s 150th film which is currently on floors.
- NTR to host Bigg Boss’ second season?
- Quirky teaser of Mahanubhavudu unveiled
- Not so encouraging talk from Vivegam’s premieres
- Tamil star to produce Telugu movies
- Bellamkonda Sreenivas’ next to be shot in Pollachi
Articles that might interest you


text:
The electric blue acara has been very popular among beginner and advanced aquarists. The shiny and bright colors they have are completely mesmerizing. This fish will definitely brighten up the mood in your aquarium.
Besides the stunning colors they have, the blue acaras are quite peaceful and sociable by default. They are not as aggressive as other fish from Cichlid species, but they can get pretty territorial and aggressive during the breeding season. Don’t worry, you can just move them to another tank, and there will be no major problems.
Their peaceful and friendly behavior makes them suitable for a community tank. They can get along with other fish in your tank. You only need to make sure that the other inhabitants are about the same size.
One other thing that makes electric blue acaras fancied by many people is that they are easy to care for. If you’re planning to get some of these gorgeous fish, you can keep reading. You will find some care guides that might be useful during your fish-keeping journey.
Table of Contents
Electric Blue Acara Species Overview
|Common Names:||Electric Blue Acara, Blue Acara|
|Scientific Name:||Andinoacara pulcher|
|Size:||Up to 7 inches|
|Life Expectancy:||Up to 10 years|
|Origin:||Central and South America, Venezuela, and Trinidad|
|Temperament:||Peaceful, but can be territorial when spawning|
|Minimum Tank Size:||30 gallons|
|Water Temperature:||Tropical, 72–86 °F (22–30 °C )|
|Water Hardness:||2 – 20 dGH|
|Water pH Level:||6.5-8.0|
|Tank Conditions:||Freshwater, Uproots Plants, Sand and Gravel, Rocks and Caves|
Origin and Distributions
These stunning blue-colored fish are originally found in Central and South American areas such as Columbia, Venezuela, and Trinidad. They can be found swimming in shallow rivers with slow streams and calm water. They can survive in still lakes as well. In the wild, they usually feed on worms, crustaceans, or insects.
They are very adaptive, making a wide distribution range for them. They can survive in most Central and South American areas, where the water is calm and relatively warm.
Just by looking at its name, you may have guessed that this fish is electric blue-colored. And you’re right!
Young Electric Blue Acara’s body usually has a pale, shiny blue color, but as they grow older, their color becomes more vibrant.
Although the fish is called electric blue, there’s some variety in its colors besides just blue. Some electric blue acara have a rather metallic gray, bluish-green, or faded yellow color. You might also see stripes and dots on their bodies.
Also, don’t be surprised if you see their heads have different colors. Many electric blue acara has a gray or black colored head, but that’s totally normal.
As it’s been mentioned earlier, the blue acara has rich colors which you can enjoy. And it’s no exception for their fins. Most of the time, you can see the yellow or orange horizontal lines along the blue acaras’ fins.
Electric blue acara has rather pointy dorsal and anal fins. The fins are longer in male specimens than in females.
As for their build, they have typical cichlid bodies. The electric blue acaras are long and have an average thickness for their bodies.
The Electric Blue Acaras is incredibly famous among aquarists, not without reason. Besides having eccentric colors, they also behave nicely. The Electric Blue Acara is particularly different compared to other fish in the Cichlid family.
They are unaggressive, peaceful, and very active. It’s absolutely amusing to watch them swim around in the tank. They may follow your movement from outside the tank too!
Since they are very active, you will often see them swimming around to explore the tank and dig actively through the substrate. They like to search for some things to nibble on. If you put some plants in the tank, they might uproot it, so watch out!
When this fish is tired, you can find them stopping under the plants and rocks or caves, if you have any.
Size and Lifespan
Unlike other Cichlids, the electric blue acara is not very big in proportion. Their maximum size is 7 inches long when fully grown. They can be considered mid-range fish and are suitable for your average or large-sized tank.
When kept in aquariums, electric blue acara has a lifespan from 8 to 10 years on average. But it can be doubled if they’re in their natural habitat!
If you want to keep your blue acara for quite a while, make sure they are taken care of properly. A clean and well-maintained aquarium will be best for them. That will prevent stress and diseases that shorten their lifespan. Check the water condition regularly and their diet as well (don’t overfeed them!).
Tank and Environment
The electric blue acaras love to dig and hide. A soft and sandy substrate is advisable for you if you have blue acaras in your tank. What about gravel, then? Since gravel is pretty rough, it may cause harm to your blue acaras, so we don’t really recommend it.
If you’re feeling a bit extra, try to find dark-colored sandy substrate. It will complement blue acaras’ shiny, bright colors and make them really popped.
The blue acaras like to rest and settle in between dense plants or rocks. You can also provide them caves for them to hide when they’re stressed.
Lastly, you can put dried leaves in your tank. In the wild, their water is filled with leaves litter. Mimicking the fish’s natural habitat will help them to feel more lively and happy.
Tank size is one crucial matter if you want your fish to thrive. The electric blue acaras are not tiny fish, and they need sufficient room to swim freely. A tank size of 30 gallons (minimum) is recommended. If you want to keep any extra fish, you have to add 15 gallons per new fish. By doing this, you can hopefully keep your fish happy and healthy.
The rivers or lakes where the blue acaras live in their natural habitat are heavily vegetated and filled with leaves litter. This condition prevents bigger fish or predators to see and prey on them. In other words, the blue acaras feel safe and happy with many plants surrounding them.
Besides, the plants will also help to block too much light from entering the tank.
Other than plants, consider putting some rockwork and caves in the aquarium as well. As we discussed earlier, the blue acaras really like to hide. Therefore, providing them some hiding places can surely make them feel happy in lives the tank.
Make sure to also check the water condition regularly. A well-maintained and clean tank will support your fish to thrive and live optimally.
Plants and Decorations
Filling up your tank with many plants and decorations sounds like a good idea. That is because the blue acaras love to hide and settle in between plants or rocks when stressed. You can also put some driftwood or caves for their extra hiding and resting places.
The blue acaras are active diggers, so they might uproot the plants. In that case, potted or floating plants will suit them best. Some plants that may be perfect for your blue acaras are:
- Amazon Sword
- Anubis Nana
- Java Fern
- Java Moss
If you have other fish species in your tank that like to eat the plants, you must keep an eye on them. They might be nibbling together with your electric blue acara.
Maintaining the water condition much like the blue acaras’ natural environment will help them live happily. Originated from Central and South American areas, the blue acaras prefer warm tropical water. Water temperature from 72 to 86°F (22–30 °C) with a pH ranging from 6.5-8.0 will be suitable for these cichlids. As for water hardness level, it can range between 2 and 20 dGH.
Diet and Feeding
For your fish to thrive and be healthy, you need to maintain a rich and balanced diet. It’s best if you can duplicate their diet just like in their natural habitat as much as possible.
Electric blue acaras are omnivores, but they eat mostly a meaty-protein diet. In the wild, they feed on crustaceans, insects, or worms.
In your captivity, you can feed them live food. Bloodworms, brine shrimps, and earthworms will do. You can also give them small insects, just like in their natural environment.
To keep their diet balanced, it’s important to give them plant-based food as well. After all, the blue acaras are omnivores and they enjoy varieties in their food. Other foods you can feed them include flaked foods or pellets.
Keep in mind that you need to feed them twice or three times a day. The blue acaras are peaceful fish, but they are voracious eaters. You should give them just an adequate amount of food each time you feed them. Leftover food might lead to fighting with other fish, in case you put them in a community tank.
Electric Blue Acara Tank Mates
The electric blue acaras are peaceful, unaggressive, and quite sociable. For that reason, they can make great tankmates with various other fish species. Keep in mind that you need to put only fish around their size. That’s because bigger-sized fish may be aggressive and threatening towards your blue acaras. On the other hand, smaller fish or creatures like ghost shrimp may end up as dinner.
If you’re planning to make a community tank, here are several fish that are compatible with the blue acaras:
- Banded Cichlids
- Bristlenose plecos
- Corydoras catfish
- Pearl cichlids
Above are just suggestions on what you could get to accompany your blue acaras. It is still best to keep them in their own groups. Although keeping them with other fish species doesn’t sound like a bad idea at all. You only need to find the most compatible tankmates from your judgment and consideration.
Blue acaras are very hardy. There is no particular disease that happens exclusively to them. However, there are still some conditions or symptoms you can look out for. The most common diseases the blue acaras might get are ich and skin flukes, which also happen with any type of fish.
Ich (White Spot Diseases)
Ich is a skin disease caused by parasite ciliate, Ichthyophthirius multifiliis. If your fish suffer from this disease, it indicates dirty tank water. This parasite affects all freshwater fish as well.
There are some symptoms you will notice if your fish caught the disease. First, they will breathe more rapidly than usual. Secondly, you will see them flicking themselves against plants and rocks. Then, they’ll develop white patches or dots on their body.
The parasite stays in the water, so the best way to tackle it is to take care of the whole tank. You can change the water or put medication into the water. Raising the water temperature to 82°F will also help the fish recover.
Malawi bloat can be caused by various factors. That includes dirty tank condition, poor diet, and Clostridium difficile bacteria.
Common symptoms that recognizable are loss of appetite, discolored feces, and the appearance of ulcers and lesions. You can treat your infected fish by quarantining them in a separate tank and with antibiotics.
Skin flukes are tiny parasites that ingrain themselves in your fish. Infected fish will show symptoms such as excess mucus, itchiness (they can be seen rubbing themselves on tank decor), red skin, and loss of appetite. The correct treatment for this disease would be using an over-the-counter drug (as directed) and correct the water parameters.
Most blue acaras are ready to spawn when they reach the size of 4 inches. Another characteristic you may notice is also the change in their colors. Their colors intensity will increase and become more vibrant. The ready-to-mate blue acaras will get closer to each other more frequently. You will also see them spend more time on the bottom of the tank. They will also remain together after the spawning time!
Breeding Tank (water condition and parameter)
When you consider that your blue acaras are ready to mate, it’s recommended to put them in a separate tank. In terms of size, the breeding tank needs to be 20 gallons minimum. Water temperature from 73 to 79°F with a pH ranging from 6.5 to 7.0 will be best for blue acaras breeding. The water hardness should be between 3 and 12 dGH, which is soft to medium.
You can fill the bottom of the breeding tank with soft sandy substrate. Any color will do. There also need to be fewer plants and caves than in the main tank. What to note is, you need to put a sufficient amount of rocks with flat surfaces. These are the places where female blue acaras will deposit the eggs. The female lays up to 200 eggs on rocks surfaces.
The eggs will hatch, and you can see juvenile blue acaras within two or three days. During the first few weeks, the babies will stay close to their mother and be guarded by both parents.
It’s not difficult to breed them in a house tank. Healthy blue acaras can mate several times within a year.
We can agree that the electric blue acara is one attractive fish that can make your tank look more festive. On top of that, the gorgeous fish is also relatively easy to care for.
The blue acara is friendly, so they’re compatible to be tankmates for your fish.
You can also breed them in your house tank. You won’t need to spend so much effort on them, regardless of being a beginner or not.
Just keep in mind that they are no tiny fish. So medium or large-sized tanks suit them best since they will likely swim around actively. You can place some plants and decorations too. It will keep them happy.
Also, make sure to maintain their diet balanced. The blue acaras love varieties in food. You can feed them meaty food, as well as plant-based foods.
Now that you’ve been introduced to the electric blue acara, do you think they’re suitable for you?
We hope the tips and care guide we shared will help you care for your electric blue acara. If you have any other tips or interesting experiences with your blue acaras, let us know! We’d love to hear from you.

domain: pet


Marc Brew is an award-winning Australian performer, director and choreographer. Marc’s work has toured to critical acclaim across the world, his outdoor work (i)land being commissioned for the Glasgow 2014 Cultural programme and Without Walls.
Brew’s Unlimited Commission Fusional Fragments as part of the London 2012 Cultural Olympiad was a collaboration with world-renowned percussionist Dame Evelyn Glennie and toured the UK and Internationally.
Marc has also produced work for companies such as Scottish Ballet, Indepen-dance 4, AXIS Dance Theatre (USA), Candoco Dance Company, Scottish Dance Theatre, Touch Compass (NZ), Amy Seiwert’s Imagery (USA), The London Contemporary Dance School and YDance.
Marc has choreographed two productions for Ballet Cymru, Stuck In The Mud and Traces Imprinted.
Other Staff Members


text:
I just realized that I didn’t ever tell you guys… I finished my Moon River!
And it actually turned out pretty great! (Massive, but pretty great, none the less.)
I think the last time I talked about this project, I was crying about losing at yarn chicken. Well, I ripped back a significant portion, and re-knit it (a couple times actually… it was very frustrating), and eventually figured out that the only way I was going to make it to the end was if I changed the bind off I was doing.
I had been doing a 2-stitch picot edge, which made lovely, elegant points, but used up a ton of yarn. I had to change to a less yarn-intensive bind off. So I ripped back a couple feet (it’s a massive shawl, and I didn’t want to rip back the whole thing), and re-bound-off with a 1-stitch picot edge.You can see where I changed my bind-off technique here. It looks a little silly if you look too closely, but I figure that once I have it on, no one will notice.
But, I managed to weave in all the ends and get the shawl blocked, and I couldn’t be more pleased with the result.

domain: hobby