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Around the NFL: If Michael Vick departs, who backs up Nick Foles?
It has been assumed that Michael Vick won't return to the Eagles so he can seek a starting job elsewhere. What hasn't been assumed is who will replace Vick as Nick Foles' backup at quarterback.
This is a position Eagles coach Chip Kelly takes very seriously.
"I think you can never have enough quarterbacks," Kelly said Jan. 6. "I've said that no matter where I was. In this league, we were fortunate to have had two this year, and that was a huge benefit to us. We'll continue to always look at quarterbacks, and I think anybody who's any good in this league always does that."
The speculation is that the backup quarterback for 2014 might be a veteran signed as a free agent, or perhaps someone taken in the draft. But Kelly gave every indication that it could be Matt Barkley, who just completed his rookie season.
Barkley, the former Southern Cal star, played in three games this past season, and none were very impressive. He replaced Foles against the Dallas Cowboys on Oct. 20 when Foles left with a concussion and threw three interceptions in the fourth quarter.
The next week, Barkley replaced Vick at halftime after Vick reinjured his hamstring and threw another interception. The Eagles didn't score an offensive touchdown in either game. Barkley's final appearance of the season came on Nov. 3 in the final minutes of the fourth after Foles had already thrown seven touchdown passes against the Oakland Raiders, tying an NFL record.
But Kelly said there was a lot more to Barkley than what he showed on the field. The Eagles thought highly enough at the draft to have him ranked in their top 50. So when Barkley was still available to start the fourth round, they traded up to get him 98th overall.
"Matt was awesome," Kelly said. "I'm excited to get a full offseason with Matt in here to really get a chance to work with him because I think he's got a skill set that's exciting when you see him, how the ball comes out of his hands, the decisions that he makes, and that's part of this whole deal."
Everything this season was new to Barkley. He had started all four of his seasons in high school and all four years at USC only to find himself third on the Eagles' depth chart. Barkley now has a chance to move up to No. 2. He said upon leaving for the offseason that the experience he received – albeit brief and not very successful – will help him going into next season.
"It helped me understand the speed of the NFL," Barkley said. "I didn't think it was that drastic of a change, but the little things here and there that you're used to in college – being able to elude guys or fit balls into a window. That's different now. The little bit of experience I got helped, but I obviously want more of that."
Unlike Vick, Barkley won't be a free agent, so he can't canvas NFL teams for a chance to start. But he can compete for the backup role with the Eagles, knowing full well, as Kelly said: "You're a chin strap away from playing in this league."
That's what happened to Foles when Vick got hurt last October, and he went on to challenge several records. Barkley hopes to put himself in position to do the same thing.
Eagles' connections in title games
Seattle safety Earl Thomas has never played for the Eagles, but he's considered linked to them for the wrong reasons.
In the 2010 draft, the Eagles traded up to the No. 13 pick, and many draft experts figured it was to select Thomas. Instead, the Eagles took defensive end Brandon Graham, who is now an outside linebacker. The Seahawks took Thomas with the next pick.
Graham has yet to establish himself as a starter, while Thomas recently made the Pro Bowl for the third time.
Seattle also has former Eagle Chris Clemons on the defensive line. Clemons had seven sacks total during two disappointing seasons with the Eagles in 2008 and 2009. He went to Seattle and had three straight seasons with at least 11 sacks before getting 4.5 this season.
The 49ers had former Eagles cornerback Nnamdi Asomugha, whom they released in November. They do have tight end Garrett Celek, the younger brother of Eagles tight end Brent Celek. Garrett Celek has two catches this season.
As for the AFC teams, the New England Patriots have Isaac Sopoaga at defensive tackle after trading for him in October. Sopoaga was inactive the last two games of the regular season and in last week's playoff game against Indianapolis.
Denver has former Eagles cornerback Dominique Rodgers-Cromartie, who had three interceptions (one returned for a touchdown) this year. The Broncos also have offensive lineman Winston Justice, who played in four games this season, and linebacker Stewart Bradley, who's been on injured reserve all season.
Contact Martin Frank at email@example.com.


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To reach the last step, you must always start with first. The same goes for yoga also. It is not some exercise that you can start from any step. It is a type of exercise in a peaceful manner that you have to begin from the start to develop your body so as to match with the rhythm of its every pose. Although there are few poses of yoga that look easy to perform but are not meant for beginners. Therefore, if you are thinking of practicing yoga for your health then you must have the knowledge of the poses you should start from. These introducing poses might or might not be so vigorous but will definitely give you a head start in advancing. Only when you will be able to do them correctly, you will head towards the difficult steps. Therefore, down here are given 9 yoga poses along with benefits for beginners.
9 Yoga Poses For Beginners
1. Mountain Pose
To begin with yoga, you must start with tadasana. Only when your mind is clear of thoughts and your body posture allows you to head ahead, you can perform yoga. This pose is also a solid breathing exercise. In this pose, simply stand up straight and keep your hands erect at the shoulder distance in the air. You can also join hands in the air. Bring your neck in alignment with your spine.
2. Downward Facing Pose
Also known as adho mukha svanasana, this yoga pose encourages blood circulation in full body and also stretches your calves and heels. In this pose, you have to be like a mountain with the support of your feet and hands. Try to set your heels on the flour for stronger stretch.
3. Warrior Pose
This pose has another name as virbhadrasana. The warrior pose develops an amazing symphony by strengthening and stretching your ankles and legs. Stand up and separate your legs 3-4 feet away in the same line. Keeping your backward foot straight, bend your forward foot at 90 degree from the knee. Now open your hands straight at your shoulder distance. Keep one hand straight ahead and the other straight behind. Aim your focus on your fingers for as long as you can.
4. Tree Pose
This pose is also known as vriksasana. This pose not just gives numerous benefits like improving balance, strengthening your thighs, calves, ankles and spine, but is also very simple to perform. The key step of this pose is shifting your balance on one feet. Stand straight and take the feet on one leg from other leg’s ankle to shin and finally thighs. As you are a beginner in this pose, therefore you can start from ankle or shin also. Keep your body posture straight and finally join the hands like you are praying on one leg.
5. Bridge Pose
In Sanskrit, this pose is also called as setu bhanda asana. By giving strength to your chest, neck and spine, this pose is a warm up for further extreme backbends. Keeping your arms at your sides, lie straight on the floor. Now bend your knees facing them upwards. Make a fist and keep under your lower back. In the final step, lift your body from neck till knees up in the air so that your chin touches your chest. Try to raise the body parallel to the floor.
6. Triangle Pose
This pose has other name as trikonasana. From thighs, till knees, and ankles, this pose allows full body stretch. It also relieves your backache. Not just for the beginners but pregnant ladies can also perform this. Stand straight and try to extend both of your legs in the opposite direction. Bend to one side and touch your right feet with right hand’s fingers. Keep your other hand high in the air as if you are touching the ceiling. Look toward the ceiling and repeat the same pose with other hand also.
7. Upward Facing Dog
Also called as urdhva mukha svanasana, this pose strengthens and stretches your spine, arms and wrists. Lie face down on the mat and try to raise your front portion up with the support of your hands. Try to stretch as much as you can. When tired, relax for few second and repeat again.
8. Crow Pose
Crow pose or bakasana, aims to strengthen your wrist, arms, and abs. Although, it is a bit of a challenging pose but a great trick to perform all other poses with ease. Sit down on your feet and keep your arms inside your thighs. Keeping the legs bent, lift your entire frame solely on the support of your hands. Raise your body to the extent that your feet face toward the ceiling. Aim to do it without falling.
9. Child’s Pose
This pose is also named as balasana. It provides relaxation, brings stretch in your body parts and relieves any kind of body pain. Sit on folded legs and raising arms high in the air, bring them lowly to the floor. Keeping your back straight, try to touch your nose on the floor.

domain: hobby


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Cognitive impairment is a well-known complication of diabetes mellitus (DM). Microvascular compromise was described one DM complication. Recently we showed blood brain barrier (BBB) permeability and memory loss are associated with diminution of tight junctions (TJ) in brain endothelium and pericyte coverage and inflammation in cerebral microvessels and brain tissue paralleling hyperglycemia in mice of both DM types. The current study demonstrates that exposure of brain microvessels to hyperglycemic conditions or advanced glycation end products (AGEs) ex vivo resulted in significant abnormalities in membranous distribution of TJ proteins. We found significant increase in the amount of extracellular vesicles (EVs) isolated from DM mice and enhanced presence of TJ proteins, occludin and claudin-5, on EVs. Exposure of BMVECs to high glucose and AGEs led to significant augmentation of ICAM and VCAM expression, elevated leukocyte adhesion to and migration across BMVEC monolayers, and increased BBB permeability in vitro. Pericytes exposed to hyperglycemia and AGEs displayed diminished expression of integrin α1, PDGF-R1β and connexin-43. Our findings indicate BBB compromise in DM ex vivo, in vitro and in vivo models in association with BMVEC/pericyte dysfunction and inflammation. Prevention of BBB injury may be a new therapeutic approach to avert cognitive demise in DM.
In recent years, significant efforts have been made to define underlying causes of dementia (cognitive impairment) and its association with vascular pathology. A number of mechanisms have been suggested, including genetic defects in Parkinson’s disease and Alzheimer’s disease (AD), metabolic abnormalities [Diabetes Mellitus (DM)], toxic effects of drugs and environmental factors, etc.), chronic neuroinflammatory conditions (autoimmune or infectious origin), hypertension, stroke and traumatic brain injury. Several of these conditions congregate on defects in the brain microvasculature1 and its coupling to neuronal functions causing cerebral hypo-perfusion and subsequent neuronal demise2,3 leading to vascular cognitive impairment and dementia (VCID). DM is a metabolic disease related either to the failure to generate insulin for proper utilization of glucose (type 1) or insulin resistance when the produced hormone is not able to interact with its receptors (type 2). Both DM types result in high glucose levels, enhanced generation of reactive oxygen species (ROS) and end organ injury stemming from microvascular abnormalities4. Our recent study3 presented a strong association between hyperglycemia, enhanced BBB permeability and cognitive dysfunction in animal models of both DM types. Hyperglycemic animals showed a pro-inflammatory phenotype both in brain microvessels (BMVs) and signs of neuroinflammation in brain tissue. We found that disruption of the BBB was associated with cognitive decline in animals, similar to recently demonstrated BBB dysfunction linked to memory impairment in DM patients5. Immunohistochemical evaluation showed a significant decrease in pericyte coverage in BMVs and an increase in ICAM-1 expression in DM mice3. DM patients are assumed to develop an abnormal endothelial phenotype due to the high levels of circulating inflammatory markers and ICAM-16,7. Previously, reduction in pericyte presence has been shown during DM both in the blood retinal barrier and BBB8. Pericytes provide functional support to the brain endothelium and their loss leads to enhanced permeability and tissue injury in DM9 and neuroinflammatory conditions10.
Membrane-bound extracellular vesicles (EVs) (diameter ~30–400 nm) are important mediators of intercellular communication among different tissues and organs in a wide spectrum of biological functions11. EVs are detected in most bodily fluids, and contain proteins, RNA, and lipids and can be categorized into at least one of three groups (exosomes, microvesicles and apoptosis bodies). The first group is released via the fusion of multivesicular bodies with the plasma membrane. The second group is released by budding of the plasma membrane, and the third group of EVs is shed from dying cells11. While EVs have been studied in the context of many diseases, little is known about EVs in the context of human diabetes12,13 and their relationship to vascular pathology. Recent studies in the db/db DM type 2 mouse model have been shown that EVs isolated from adipose tissue can activate macrophages and promote inflammatory signals13. Other reports have shown that platelet-derived EVs expressed different patterns in expression and procoagulant activity in patients with both DM types14.
In this report, we show that EVs isolated from DM mice express high levels of TJ proteins, occludin and claudin-5. BMVs, subjected to hyperglycemic conditions or AGEs in ex vivo settings, exhibited abnormal occludin and claudin-5 membrane TJ localization. Using our in vitro model of BBB, utilizing primary human brain microvascular endothelial cells (BMVEC) and primary human pericytes, we demonstrate defective barrier function by transendothelial electrical resistance (TEER) in hyperglycemic conditions. BMVECs displayed increased expression of adhesion molecules such as VCAM and ICAM when exposed to high glucose (HG) or AGEs, which resulted in augmented leukocyte adhesion to and crossing of the endothelial layer. RhoA and Rac1 GTPases have shown a significant increase in their activation in BMVEC stimulated with HG and AGE treatments. Since RhoA and Rac1 are small GTPases that control cytoskeleton, TJ and adhesion molecule expression in BMVEC and endothelial cells15,16,17, their activation in DM environment might explain barrier dysfunction.
Expression of integrin α1 [a key molecule guaranteeing adhesion to basement membrane (BM) matrix on pericytes] was altered in hyperglycemic conditions in vitro. mRNAs of BM proteins such as fibronectin, nidogen were also found to be down-regulated in a hyperglycemic environment in pericytes. Pericytes supporting BBB function also displayed a decrease in expression of the crucial pericyte receptor, PDGF-Rβ, which assures proper pericyte function and barrier integrity10. Pericytes, along with brain endothelium, synthesize the BM and its abnormalities are known to be present in a number of neurodegenerative and neurovascular diseases18. This report offers evidence from in vitro models and ex vivo treated BMVs and DM serum-isolated EVs for the causes of BBB dysfunction, and might lead to development of future therapeutics to reduce its burden.
Materials and methods
Glyoxal (GO) and methylglyoxal (mGO) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Lipopolysaccharides from Escherichia coli O111:B4 (LPS) and streptozotocin (STZ) were from Sigma/Aldrich (St. Louis, MO). Monocyte chemotactic protein-1 (MCP-1) was from R&D Systems (Minneapolis, MN). Rho inhibitor CTO4 and Rac activator CN04 were from Cytoskeleton (Denver, CO). Human tumor necrosis factor alpha (TNFα) was from Peprotech (Rocky Hill, NJ). ROS inhibitor, Trolox, and caspase inhibitor, Z-VAD-FMK, were purchased from Selleck Chemicals (Houston, TX).
Animals and induction of diabetes
C57BL/6 mice (10-week old male) were acquired from the Jackson Laboratory (Bar Harbor, ME). To achieve statistical significance in each experiment, mice were divided into groups of 6 to 10 animals (exact numbers for each experiment are indicated in figure legends). All in vivo experiments were approved by the Temple University Institutional Animal Care and Use Committee in accordance with guidelines based on the National Institutes of Health (NIH) guide for care and use of laboratory animals and ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines (www.nc3rs.org.uk/arrive-guidelines). Diabetes type 1 was induced as described3. In short, C57BL/6 mice (25-30 g body weight) were randomly divided into groups. One group received once daily intraperitoneal (i.p.) injection of streptozotocin (STZ) for five consecutive days (50 mg/kg in citrate buffer, pH 4.5, freshly made every day). Control group mice received citrate buffer only. The first day of STZ injection was assigned as the starting time for diabetes. Serum glucose concentrations were monitored on 7 days, 4, 8 and 12 weeks after the start. Blood glucose levels (BGL) were determined by glucose analyzer (Bayer Contour, Ascensia Diabetes Care, Parsippany, NJ), according to manufacturer’s instructions.
Brain microvessel isolation and ex vivo treatment
Mouse brain microvessels (BMVs) were isolated using a modified protocol based on previously published studies19,20,21. In short, mice were overdosed with CO2 and their brains harvested. All following steps were carried out on ice (or at 4 °C). Following a wash in phosphate-buffered saline, the brains were homogenized using a Dounce homogenizer (0.25 mm clearance) (whole brain is defined as the S0 fraction; the nomenclature of S0, S1, and S5 describes the BMVs fractionation steps consistent with the BMVs isolation procedure as described below and previously by Yousif20. Overall, 15 mL of 30% Ficoll was added to 10 mL of the homogenate and mixed thoroughly. The resulting density gradient was centrifuged at 5,800 × g for 20 minutes (the pellet is defined as the S1 fraction). The pellet was resuspended in 1 mL phosphate-buffered saline with 1% bovine serum albumin and passed through a glass bead column, with 100 μm nylon mesh filter on the top and a 40-μm nylon mesh filter at the bottom. The glass beads were gently agitated in phosphate-buffered saline with 1% bovine serum albumin to obtain BMVs. The resulting sample (defined as the S5 fraction) was washed with bovine serum albumin-free phosphate-buffered saline and resuspended in complete RPMI media with 10% fetal bovine serum and 1% penicillin-streptomycin. BMVs were deposited on 8-well chamber slides (Thermo Fisher, Waltham, MA) which had been coated with 0.01% poly-L-lysine (Sigma/Aldrich) and allowed to settle for 1 hour at 37 °C prior to addition of treatments for 48 hours at the concentrations shown.
At conclusion of ex vivo treatment, BMVs were fixed for 10 minutes at 25 °C by 4% formaldehyde. After 3-4 PBS washes, BMVs were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes, then washed 3-4X in PBS and blocked in 5% BSA for 30 minutes. Incubation with primary antibodies for either claudin-5 (1:25, Cat 35-2500, Invitrogen, Carlsbad, CA) or occludin (1:25, Cat 71-1500, Invitrogen) occurred overnight at 4 °C. Following 3-4 washes in 25 °C PBS, a 5% BSA block was performed for 30 minutes and incubation with AlexaFluor 488-conjugated secondary antibodies for either goat α-mouse (1:500, A28175, Thermo Fisher) or goat α-rabbit (1:500, A11034, Thermo Fisher) was performed for 45 minutes. BMVs were then incubated with DyLight 594 labelled Lycopersicon esculentum lectin (1:200, DL-1177, Vector Laboratories, Burlingame, CA) for 1 hour and washed 3-4 X with PBS. A solution of DAPI (1:2500) in PBS was added for 30 minutes and then 4-5 washes in PBS were performed before coverslip mounting with ProLong Gold Antifade reagent (ThermoFisher). Microscopic examination of brains used a standardized protocol with sections from the neocortex (frontal and parietal), basal ganglia, hippocampus, midbrain, pons, medulla, and cerebellum. Paraffin sections (5 μm) were stained with hematoxylin-eosin. One section from the frontal cortical lobe was used for the evaluation of neuroinflammation and BBB structure by immunohistochemistry using ZO-1 antibodies (1:200, clone ZMD.437, ThermoFisher). Primary antibodies were detected by Dako Envision Kit (Dako, Carpinteria, CA) with DAB chromogen. Samples were imaged under 400x objective magnification using a DS-Fi1c camera (Nikon Corporation, Tokyo) configured to an upright microscope (Eclipse 55i, Nikon). For each animal, 4–6 randomly selected fields in the cortex and basal ganglia were analyzed, as described3. Immunostaining for ZO-1 was evaluated in blinded fashion using semi-quantitative scoring: 0 – no staining, 1 – weak staining (<10% of capillaries), 2 – moderate, variable labeling (10-75% capillaries), 3 – strong, variable staining (>75% capillaries) and 4 – strong, uniform staining (95%).
Imaging and analysis
All BMVs images were captured on a Nikon A1R confocal microscope using a 60x oil-immersion lens and NIS-Elements software (Nikon, Tokyo, Japan). Z-series images were acquired through the full depth of 7 vessels in each well. Gain and laser settings were adjusted for the antigen being imaged (claudin-5, occludin) and then kept constant across imaging of all treatment groups. The 3D Deconvolution tool was applied to all images and analysis of tight junction fluorescent intensity was carried out using the NIS-Elements General Analysis 3 package. All representative images are maximum intensity projections of the entire z-series viewed across the XY plane and are shown with identical LUT settings applied across all treatment groups and include only original captured channels, unaltered by the analysis process described below. Due to a significant incidence of background fluorescence outside of the BMVs within claudin-5 and occludin channels, a binary rendering of the vessel was created from the lectin channel and utilized to crop out fluorescence occurring outside the vessel. The binary rendering was generated by using the Threshold3D module (range 451 to 65535, Smooth x5, Clean x2, Fill Holes ON) and then FilterObjects3D module to remove objects with a volume less than 20 µm3. The binary vessel rendering was then input to MaskImage module, generating a new channel which included only claudin-5 or occludin fluorescence within the vessel space. All subsequent analysis functions utilized this channel as an input. In order to identify regions where tight junctional (TJ) complexes were located, several image pre-processing modules were combined to boost the intensity of linear staining patterns versus those which were more punctate or diffuse. The resulting image was used to create and refine a binary rendering of the TJs, which was then applied to the original vessel-masked image to determine the intensity of fluorescence occurring within TJ complexes. Specifically, the Local Contrast (degree 100%, radius 0.50 µm), Auto Contrast (low 1%, high 0.1%), and Smooth (count 3) modules were applied in sequence prior to use of the Threshold3D module (range 23000 to 65535, Smooth x3, Clean x2) to create a binary rendering. FilterObjects3D module was applied to remove objects with volume less than 0.4 µm3 or elongation index less than 1.5. The rendering was skeletonized using Skeleton3D module, then Dilate3D (count 4) and Smooth3D (radius 0.250 µm) modules were sequentially applied (Supplemental Fig. 1). To determine the ratio of fluorescent intensity occurring within the TJ versus other cellular locations, the sum fluorescent intensity of TJ versus non-TJ regions were quantified and divided by volume of their respective regions to arrive at mean fluorescent intensity (MFI) for each region. The MFI within TJ was divided by non-TJ MFI to generate the ratio shown. Of note, the non-TJ intensity and volume were calculated indirectly through subtraction of TJ values from total vessel values (Supplemental Fig. 2). In order to account for background autofluorescence occurring within the vessel space, the fluorescence within vessels incubated with only secondary antibody was measured. The average background value was subtracted from the vessel-masked images across all treatment groups before fluorescent measurements were made.
Primary BMVEC, isolated from vessels from brain resection tissue (showing no abnormalities) of patients undergoing surgery for treatment of intractable epilepsy, were supplied by Michael Bernas and Dr. Marlys Witte (University of Arizona, Tucson, AZ) and maintained as described22,23. Primary human monocytes were obtained from the University of Nebraska Medical Center Cell Core (Omaha, NE), maintained as described24, and used within 24 hr of isolation. Procedures were approved by the Institutional Review Board (IRB) of Temple University School of Medicine according to the ethical guidelines of the Helsinki Declaration of 1975 (and as revised in 1983). Primary human brain vascular pericytes were purchased from ScienCell Research Laboratories (Carlsbad, CA). We established pericyte culture conditions providing a quiescent, non-proliferating phenotype similar to ones present in the CNS under physiologic conditions. Use of growth medium containing 5% of growth factors slowed pericyte proliferation rate, with a majority of cells in the G1 phase of the cell cycle25. These culture conditions had no effect on pericyte viability (data not shown). Pericyte cultures were free of glial or endothelial cell contamination (Supplemental Material, Fig. 1). BBB models were assembled as previously described24 with BMVEC alone, pericytes alone, or BMVEC co-cultured over pericytes (5:1 seeding ratio of BMVEC:pericytes).
Monocyte adhesion and transmigration assays
Adhesion assays were performed as described21,22,23. BMVEC were pretreated overnight with DM relevant stimuli, high glucose, glyoxal (GO) or methylglyoxal (mGO), followed by stimulation with TNFα (75 ng/ml) for 1 hr. Treatments were removed prior to monocyte introduction. Fluorescently-labeled monocytes (2.5 × 105 cells/well) were added to the endothelial monolayers for 15 min at 37 °C. After adhesion, monolayers were washed and relative fluorescence of the attached monocytes was acquired on a fluorescence plate reader (Synergy 2 plate reader, BioTek, Winooski, VT). Results are presented as the mean ±SEM fold adhesion (number of adherent monocytes for each experimental condition divided by the basal adhesion of the untreated control). Transendothelial migration assays were done as previously described24. BMVEC or BMVEC/pericytes were plated on rat-tail collagen type I coated FluoroBlok tinted tissue culture plates (3 μm pores, BD Biosciences, Franklin Lakes, NJ) at a density of 2.5 × 104 BMVEC/insert and 0.5 × 104 pericytes/insert one week prior to use for migration assays. Medium was replaced, cell monolayers were washed and monocyte chemotactic protein type 1 (MCP-1/CCL2, 30 ng/ml, R&D Systems) was added to the lower chamber. Monocytes were labeled with calcein-AM as described for adhesion assays, washed, placed in the upper chamber and allowed to migrate for 2 hr at 37 °C. The number of migrated monocytes was determined with ImageJ software, version 1.43 (NIH, Bethesda, MD) and is expressed for each experimental condition as the mean of triplicate determinations calculated as the number of migrated monocytes divided by the number of migrated monocytes in untreated, no chemoattractant control. Data are presented as percent of input defined as the number of monocytes that migrated to the lower chamber over the total monocytes initially placed in the upper chamber x100.
Transendothelial electrical resistance (TEER)
BMVEC alone or in co-culture with pericytes were plated on collagen type I coated 96W20idf electrode arrays (Applied Biophysics, Troy, NY) and were treated with 25 mM glucose or AGEs for 30 hr. To inhibit RhoA or Rac1 GTPase activity, cells were pretreated with specific inhibitors, 1 μg/ml CT04 (Cytoskeleton) or 75 μM NSC23766 (EMD-Millipore, Burlington, MA), respectively. To inhibit ROS or apoptosis, cells were pretreated with specific inhibitors, 25 μM Trolox or 100 μM Z-VAD-FMK (both from Selleck Chemicals), respectively. TEER measurements were performed using the 1600R ECIS System (Applied Biophysics, Troy, NY) as described21,22,23. The results are presented as the average percent change from baseline TEER (expressed as average ± SEM) from at least three independent experiments consisting of four to six replicates each.
PCR array and qPCR
Total RNA was extracted from cells using the PARIS RNA Isolation Kit (Thermo Fisher). Total RNA (200 ng) was converted to cDNA using the RT² cDNA Synthesis Kit (Qiagen, Hilden, Germany). Specific primers and probes for nidogen, fibrinogen, PDGFRβ and Cx-43 genes were obtained from Thermo Fisher and analyses were executed using the QuantStudio S3 real-time PCR system (Thermo Fisher). Amplification was examined using the ΔΔCt method, using a web-based data investigation tool (SABiosciences/Qiagen) by normalization to housekeeping genes and fold-change calculated from the difference between experimental condition and untreated control, as described26. Data are presented from two independent experiments in triplicate for each gene. To eliminate biased data, two people performed analysis in blinded fashion.
RhoA and Rac1 guanosine triphosphatase (GTPase) activity assay
RhoA and Rac1 GTPase activity was measured in cell lysates prepared from primary BMVEC after stimulation with high glucose or AGEs. For a positive control, cells were stimulated with the GTPase activator CN04 (1 μg/ml) (Cytoskeleton Inc, Denver, CO). To inhibit RhoA or Rac1 GTPase activity, cells were pretreated with specific inhibitors, 1 μg/ml CT04 (Cytoskeleton) or 75 μM NSC23766 (EMD-Millipore), respectively, as described27. To measure RhoA and Rac1 GTPase activity, G-LISA RhoA and G-LISA Rac1 Activation Assay kits (Cytoskeleton) were used according to the manufacturer’s instructions.
ICAM, VCAM and Integrin-α1 expression quantification by flow cytometry
Analysis of surface expression of adhesion molecules was performed using the conjugated antibodies ICAM-1-APC and vascular cell adhesion molecule 1 (VCAM-1)-FITC (BD Biosciences). Integrin-α1 protein was stained with anti- Integrin-α1 Alexa-Fluor 405 (Novus Biologicals, LLC., Centennial CO). Following staining, data were acquired with a FACS BD Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo software v9.9.6 (Tree Star, Inc., Ashland, OR). Data were collected from at least 20,000 events and repeated twice with BMVEC or pericytes each time from different donors. The mean fluorescent intensity (MFI) of stain was calculated in a cell population and presented as average ± SD.
Extracellular vesicle isolation and staining
EVs were isolated from 50 μl of mouse serum utilizing Exo-Quick solution (System Biosciences LLC., (SBI), Palo Alto, CA), according to the manufacturer’s instructions. Size and concentration of the EVs were analyzed on the NanoSight NS300 (Malvern Instruments Ltd., Westborough, MA). Isolated EVs were captured with CD63 Exo-Flow Capturing Kit (SBI) and stained with anti-claudin-5 Alexa 488 or anti-occludin Alexa 488 (both from Thermo Fisher). Following staining, data were acquired with a FACS BD Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo software v9.9.6 (Tree Star).
Reactive oxygen species (ROS) measurement
BMVEC or pericytes were treated with diabetes conditions and Reactive Oxygen Species (ROS) measurement was performed utilizing the cell permeant reagent, 2′,7′–dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA or DCFH-DA), a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity within the cell according to the manufacturer’s instructions (Abcam, Cambridge, MA). Fluorescence was measured by plate reader and ROS production is presented as arbitrary fluorescence units for triplicate measurements from at least two donors as mean ± SD.
Results are expressed as the mean ± SEM of experiments conducted multiple times. Multiple group comparisons were performed by one-way analysis of variance with Dunnett’s posthoc tests, as described21,26,27. Statistical analyses were performed utilizing Prism v8.3.0 software (GraphPad Software Inc., La Jolla, CA). Differences were considered significant at P values <0.05.
Hyperglycemia leads to disruption of TJ complexes in ex vivo treated cerebral microvessles
DM is a metabolic disorder characterized by hyperglycemia leading to end-organ injury in various organs due to microvascular compromise (cardiovascular disease, nephropathy and retinopathy) and inflammation. BBB breakdown has been suggested as one of the causes of dementia in DM and AD28. Recently we have shown in animal models of DM types 1 and 2 decrease BBB integrity and promote a pro-inflammatory phenotype of brain endothelium (resulting in low level inflammation) that further exacerbates barrier injury3. In spite of our assumption that BBB integrity in DM would be strongly associated with down-regulation in TJ proteins, we found that expression of occludin was prominently upregulated in isolated BMVs from animals with DM. We hypothesized that BBB integrity might be compromised due to improper folding or incorporation in cell membranes that may reflect a compensatory phenomenon in DM. To check this hypothesis, we subjected BMVs to DM conditions ex vivo and assessed TJ localization (membrane incorporation). BMVs were isolated from mice, as described21, plated on EZ slides (Millipore), treated ex-vivo with HG or AGEs and analyzed for expression of TJ complexes (Fig. 1). Isolated vessels were labeled with Lycopersicon esculentum lectin (LEL, red), DAPI (blue), and immunostained for either claudin-5 (a-f) or occludin (g-l) (green) to allow vessel imaging with a Nikon A1R confocal microscope (Nikon, Tokyo, Japan). To rigorously evaluate TJ expression, gain and laser settings were adjusted for the antigen being imaged (claudin-5, occludin) and then kept constant across imaging of all treatment groups. The 3D Deconvolution tool was applied to all images and analysis of tight junction fluorescent intensity was carried out using the NIS-Elements General Analysis 3 package (Nikon). All representative images are maximum intensity projections of the entire z-series viewed across the XY plane. To determine the ratio of fluorescent intensity occurring within the TJ versus other cellular locations, the sum fluorescent intensity of TJ versus non-TJ regions was quantified and divided by the volume of their respective regions to arrive at mean fluorescent intensity (MFI) for each region (Supplemental Fig. 2). The MFI within TJ was divided by non-TJ MFI to generate the ratio shown (Fig. 1). Treatment with glyoxal (GO) caused significant reduction ~23% and ~18% in TJ expression for claudin-5 and occludin, respectively (Fig. 1). Methylglyoxal (MGO) presence resulted in significant diminution of ~20% in TJ staining for claudin-5 (Fig. 1d,f). Similar reductions in TJ localization for both occludin and claudin-5 were noticed in LPS ex vivo-treated BMVs (Fig. 1b,f,h,l) suggesting commonality of the hyperglycemic DM environment and pro-inflammatory conditions (LPS).
Diabetes leads to increased presence of TJ proteins in extracellular microvesicles
In recent years, several groups reported that EVs could play important roles in the pathogenesis and/or complications of DM12,13,14. Some found EV involvement in atherosclerosis progression in DM14, others stated EV’s role as antigen presentation to the immune system or playing a messenger role between cells during inflammation12,13. Since we saw increased BBB permeability in type 1 and 2 animal models of DM, we hypothesized that this phenomenon might be attributed to the shedding of TJ proteins in EVs, thus affecting proper TJ presence in the endothelial membranes. To test this hypothesis, we isolated EVs from the serum of STZ-induced DM type 1 mice3 as described in Methods section. We used NanoSight’s nanoparticle tracking analysis (NTA) tool to characterize both EV size and concentration. The size of the EVs increased by 23% after 8 weeks since DM type induction by STZ-injection in mice, and 4 weeks later the size of the EVs in this group of mice was further boosted by 16% (Fig. 2a). Interestingly, we noticed that there was a significant rise of 1.4-fold in size of the EVs both in the control group of mice and STZ-treated mice when compared 8 and 12 weeks after the start of the experiment, while there was no further size increase when compared to STZ-treated group and control group at the 12-week time point (Fig. 2a). NTA revealed that upon development of DM there was a significant 1.6-1.8-fold increase in the amount of EVs in the serum (Fig. 2b). Isolated EVs were pulled-down utilizing CD63 Exo capturing magnetic beads, as described in methods, and stained for TJ proteins with anti-claudin-5 or occludin antibodies, and subjected to flow cytometry analysis. FACS analysis demonstrated that there was up to a 40% boost of occludin positive EVs in mouse serum at the 12-week time point (Fig. 2c), whereas there was substantial increase of claudin-5 positive EVs at both 8-week and 12- week time points (Fig. 2e). When analyzing protein expression levels on the EVs, we discovered that despite of difference in amount of occludin positive EVs only at 12-week time point, expression of occludin was significantly amplified at both 8- and 12-week points (Fig. 2d). Claudin-5 expression on EVs was increased by 1.28 ± 0.9-fold in mouse serum after 8 weeks after DM development that eventually decreased toward the end of the experiment (Fig. 2f). Elevated levels of expression of TJ proteins on EVs demonstrate another factor that might lead to increased BBB permeability due to the shedding of TJs from the endothelium.
DM conditions increase expression of adhesion molecules on BMVEC and subsequently lead to enhanced leukocyte adhesion to and migration across endothelial monolayer.
To mimic BBB injury in DM conditions in vitro, we modeled it in the previously established in vitro BBB model, primary human BMVEC21,22,24,29,30 from our laboratory using a combination of high glucose (HG) or AGEs. Several groups have shown increased levels of TNFα in blood of DM patients31,32. We treated BMVEC with TNFα under physiologic or hyperglycemic conditions and investigated whether these conditions would lead to altered leukocyte adhesion to and/or migration across the endothelial monolayer. Our results demonstrate that TNFα treatment led to a 2.7-fold increase in leukocyte adhesion to endothelium in physiological glucose concentration or 2.2-, 2.3- and 1.65-fold increases in HG, GO or mGO treatments, respectively (Fig. 3a). HG or AGEs escalated leukocyte adhesion even without TNFα addition to the media. A similar boost in leukocyte migration across the monolayer was noticed under DM conditions (Fig. 3b). Since we saw increased adhesion of primary monocytes to the brain endothelial monolayer, we decided to assess whether HG or AGEs would increase expression of adhesion molecules in BMVEC. Indeed, AGEs upregulated ICAM and VCAM expression (Fig. 3c), whereas HG did not affect levels of adhesion molecules. 1.6-fold in RhoA-GTP (Figs. 4a) and 1.21-fold in Rac1-GTP (Fig. 4b) in BMVEC stimulated with HG. Activity of both GTPases was augmented by AGEs treatment. Several groups demonstrated that hyperglycemic conditions and AGEs might result in oxidative stress responses33,34. Indeed, we found that AGEs treatment resulted in a significant increase of ROS production in endothelial cells (Fig. 4c).
To assess barrier function in diabetes, we measured TEER in BMVEC monolayers or their co-culture with pericytes. Exposure of BMVEC alone to HG resulted in an immediate drop of TEER and HG conditions showed further decline in BBB tightness (up to 40% of control after 24 h, Fig. 5a). Next, we investigated whether AGEs would have any influence on barrier function and found a dose-dependent effect with GO treatment (500 μM and 1 mM, light and dark blue lines), were higher concentration lead to 50% loss in TEER values. Interestingly, in BMVEC co-cultured with pericytes, only GO influenced barrier tightness in a dose-dependent manner. Neither HG nor mGO had any effect on two-cell BBB layer tightness (Fig. 5b). Next, we checked whether inhibition of small GTPases, ROS or apoptosis processes would rescue the loss in tightness caused by GO. Interestingly, Rac1 GTPase inhibitor (NSC) and ROS inhibitor (Trolox) were able to significantly alleviate GO-induced TEER drop (Fig. 5c). Neither RhoA small GTPase inhibitor (CT04) nor caspase inhibitor (Z-VAD-FMK) had any effect on BBB tightness decrease caused by GO (Fig. 5d). We performed viability test and did not see any cytotoxic effects (data not shown). Therefore, we conclude that hyperglycemic conditions lead to small GTPase activation and ROS production and potentially affect the actin cytoskeleton rearrangements and BBB tightness.
Hyperglycemic conditions result in pericyte dysfunction
Lastly, we evaluated the effects of diabetic conditions on pericyte functions. We demonstrated that expression levels of integrin α1 (a key molecule assuring adhesion to basement membrane matrix) on pericytes was reduced by 19% (HG), whereas mGO and GO diminished it by 51% and 32%, respectively (Fig. 6A). Treatments with TNFα or H2O2 (to replicate inflammatory or oxidative stress conditions) diminished integrin α1 expression on pericytes as well. GO treatment led to a ~5.2-fold reduction in expression of gap junction protein Cx-43 (key for communication between BMVEC and pericytes). Similarly, exposure to AGEs resulted in a 20% ± 6% diminution in PDGF-Rβ expression (important for proper function of pericytes at the BBB). Basal membrane protein genes, fibronectin and nidogen, showed 4.1- and 2-fold downregulation, respectively, in pericytes challenged with GO (Fig. 6B). Next, we determined that AGEs treatment resulted in significant increase of ROS production in pericytes (Fig. 6C). In summary, using primary human brain endothelial cells and pericytes, we were able to demonstrate a pro-inflammatory phenotype and dysfunction of cellular elements of the neurovascular unit under diabetic conditions.
BBB dysfunction is often found in a wide range of VCID syndromes35,36. The BBB serves as a discriminating barrier preserving the homeostasis in the CNS by controlling ion balance, assisting in nutritional transport, and blocking influx of potentially neurotoxic molecules from the circulation at the level of the cerebral microvascular endothelium35,36. DM is a metabolic disorder depicted by hyperglycemia leading to end-organ injury in different organs due to microvascular compromise (cardiovascular disease, nephropathy and retinopathy) and inflammation. Learning disabilities and memory deficits have been documented in DM type 1 and type 2 patients37,38,39, which might be due to cerebral vascular dysfunction. Connection of microvascular changes to cognitive deterioration in DM has not been substantiated until very recently, with defects noted in neuronal function, metabolic function, white matter microstructure, and blood perfusion40,41.
It has been implied in the literature, that BBB breakdown as one of the grounds of dementia in DM and AD28. However, the precise mechanisms of injury, association amongst enhanced permeability and memory loss, DM and therapeutic potential of BBB protective approaches in cognitive decline are presently unknown. Here, we scrutinized the idea that DM settings decrease BBB integrity directly (via effects on brain endothelium and pericytes, separately and together) and stimulate a pro-inflammatory phenotype of brain endothelium (resulting in a low-level inflammation) that further aggravates harm of the barrier. We postulated that BBB integrity might be affected in DM due to inopportune folding or incorporation in cell membranes. In the current study, we checked this hypothesis by assessing TJ localization (membrane incorporation) in BMVs which were subjected to DM conditions ex vivo. Treatments with AGEs, such as GO, caused significant reduction of both claudin-5 and occludin protein expression, while MGO treatment resulted in significant diminution of claudin-5 in TJ staining. Similar reduction in TJ expression for both occludin and claudin-5 were noticed in LPS ex vivo-treated BMVs. These results suggest the commonality of the hyperglycemic DM environment and pro-inflammatory conditions, in this case caused by LPS.
In recent years, there have been major advances in the understanding of the biology of extracellular vesicles. Clinical and experimental studies have shown that EVs play a vital role in the pathophysiology of inflammation-associated disorders. A cardinal feature of these disorders is an enhanced generation of platelet-, endothelial-, and leukocyte-derived EVs42. Here in our study we determined that hyperglycemic conditions led to a significant increase of EVs in serum of DM type 1 mice. Interestingly, DM progression also led to an increase in size of the EVs, from 110 ± 12 nm to 138 ± 15.3 nm. Microvesicles are generally larger than exosomes (100–1000 nm vs. 30-120 nm, respectively). Microvesicles are formed by direct budding of the plasma membrane, whereas exosomes are formed within endosomal compartments and secreted by the fusion of multivesicular bodies with the plasma membrane. Apoptotic bodies are released upon programmed cell death by membrane blebbing and can be from 50 nm to 5 μm in diameter. However, due to a significant overlap in size, similarities in composition and lack of specific markers, it is very difficult to assign individual EVs to one of the biogenesis pathways. Recently, small GTPases were shown to contribute EV production and secretion43,44. Further studies are required to determine what type of EVs were found in our study, and which mechanism was involved. Additionally, EVs isolated from serum from DM type 1 mice had a significantly high content of the endothelial TJ proteins, claudin-5 and occludin compared to normal glycemic mice. Presence of the TJ on EVs could potentially explain why the BBB in DM mice was compromised and why these mice shown higher significant reduction in TJ occludin and claudin-5 protein expression in immunohistochemical examination3 and ZO-1 protein (Supplemental Fig. 3). Previously we also identified that at least occludin mRNA levels were highly increased in microvessels isolated from DM mice, implying either a compensatory phenomenon in DM to compensate leakage of the TJ protein via EVs or improper incorporation in cell membranes. Further studies will be required to test whether ZO-1 protein is shed by EVs or its expression is affected on transcriptional/translational levels. Additionally, presence of the TJ proteins on the EVs might serve as a potential biomarker of BBB damage. Further studies are required to see whether TJ presence on EVs would correlate with dementia progression.
To simulate BBB injury in DM conditions in vitro, we used the previously established in our lab in vitro BBB model, primary human BMVEC21,22,24,29,30 using combination of HG or AGEs. Since some research groups31,32 have shown increased levels of TNFα in blood of DM patients, we treated BMVEC with TNFα under normal or hyperglycemic conditions and investigated to see whether these conditions would lead to altered leukocyte adhesion to and/or migration across the endothelial monolayer. Indeed, our results demonstrate that TNFα treatment led to an increase in leukocyte adhesion to endothelium under physiological glucose concentration and hyperglycemic conditions. HG or AGEs caused enhanced leukocyte adhesion even without TNFα addition to the media. A boost in leukocyte migration across the monolayer was noticed under DM conditions. Because we saw increased adhesion of primary monocytes to the brain endothelial monolayer, we decided to evaluate whether HG or AGEs would increase expression of adhesion molecules in BMVEC. Indeed, AGEs upregulated ICAM and VCAM expression, whereas HG did not affect adhesion molecule expression levels. Altannavch and colleagues45 have shown detectable effects of HG on the adhesion molecules (VCAM and ICAM) and TNFα cytokine expression in human umbilical vein endothelial cells (HUVEC) after 24 hours. Another group shown that longer exposure to HG is required for microvascular endothelial cells isolated from bovine or aortic aortas or skin to elevate VCAM expression46. Others demonstrated that intermittent exposure of HUVEC to HG also elevates adhesion molecule expression47, which can be explained by the difference in response of endothelial cells of different origin.
A critical role in determining junctional integrity between endothelial cells and permeability regulation of the endothelium is assigned to the actin cytoskeleton machinery16,48,49. The microvascular endothelium controls discriminatory permeability of the BBB to fluids and solutes. Since small GTPases, RhoA and Rac1, control cytoskeleton, TJ and adhesion molecule expression in BMVEC and endothelial cells of other organs15,16,17, we examined the ability of DM conditions to affect their activation. Indeed, both RhoA and Rac1 GTPases show a significant increase in their activation in BMVEC stimulated with HG. Activity of both GTPases was augmented by AGE treatments as well. Exploiting our BBB in vitro model, we plated BMVEC alone or in co-culture with pericytes and measured endothelial barrier function utilizing TEER. Exposure of BMVEC alone to HG resulted in immediate drop of TEER and HG conditions showed further decline in BBB tightness, and exposure to AGEs (GO) resulted in a dose-dependent effect the endothelial barrier function. Interestingly, when BMVEC were co-cultured with pericytes, only GO influenced barrier tightness (also in a dose-dependent manner); however, neither HG nor mGO had any effect on two-cell BBB layer tightness. Intriguingly, Rac1 GTPase inhibitor (NSC) and ROS inhibitor (Trolox) were able to significantly improve GO-induced TEER decline. However, neither RhoA small GTPase inhibitor (CT04) nor caspase inhibitor (Z-VAD-FMK) had any effect on GO-caused BBB layer tightness diminution Therefore, we conclude that hyperglycemic conditions lead to small GTPase activation and ROS production and potentially affect the actin cytoskeleton rearrangements and BBB tightness.
Lastly, we evaluated the effects of diabetic conditions on pericyte functions. We and others have shown that integrin α1 levels are affected in different pro-inflammatory conditions10,25,35,36. HG and AGEs exposure resulted in significant reduction of integrin α1 protein level. Treatment with TNFα or H2O2 (to replicate inflammatory or oxidative stress conditions) diminished integrin α1 expression on pericytes as well. Since integrin α1 is a key molecule assuring adhesion to the basement membrane (BM) matrix on pericytes, such alteration may lead to defective attachment to BM, impaired microvessel stability and hemorrhage50. Down-regulation of BM protein mRNAs, such as fibronectin and nidogen, were found to be down-regulated in hyperglycemic conditions in pericytes. Pericytes, along with brain endothelium, synthesize the BM and its abnormalities are known to be present in a number of neurodegenerative and neurovascular diseases18. Hyperglycemic conditions resulted in significant reduction in mRNA levels of PDGF-R1β and Cx-43 which are important for pericyte function25,51,52,53. Our results indicate that both BMVEC and pericytes produce augmented amounts of ROS. Oxidative stress in hyperglycemic conditions could be very harmful for proper endothelial cell and/or pericyte function33,34,54. In summary, using primary human brain endothelial cells and pericytes, we were able to demonstrate a pro-inflammatory phenotype and dysfunction of cellular elements of the neurovascular unit under diabetic conditions.
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This work was supported in part by NIH research grants R01AA015913 (YP) and R01MH115786 (YP).
The authors declare no competing interests.
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Rom, S., Heldt, N.A., Gajghate, S. et al. Hyperglycemia and advanced glycation end products disrupt BBB and promote occludin and claudin-5 protein secretion on extracellular microvesicles. Sci Rep 10, 7274 (2020). https://doi.org/10.1038/s41598-020-64349-x
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domain: health


EU Emission Trading System (EU ETS)
<1 Min. Read Time
The EU ETS is the oldest emissions cap-and-trade program, first launched in 2005. It covers ~40% of the total EU emissions, including activities from the power sector, manufacturing industry, and aviation (including flights from the EU to the United Kingdom). In 2020, Switzerland linked with the EU ETS. Since inception, stationary installations have seen an emission reduction of around 43%.
Year in Review:
In 2021, the EU ETS entered its fourth trading phase. New policy reforms were introduced under the “European Climate Law,” which set new regional climate targets including a 55% cut in GHG emissions compared to 1990 levels by 2030 and climate neutrality by 2050. The European Commission’s proposed reform package, known as Fit for 55, that calls for several revisions to the EU ETS. The package is not yet finalized as it is currently under review by the EU legislature.
Key changes included in the package are: an increased linear reduction factor from 2.2% to 4.2%, and a one-off reduction to the cap; a phasing-out of free allocation to the aviation sector; introduction of a carbon border adjustment mechanism (CBAM); updated MSR parameters such a lower price spike trigger mechanism and an extension of the current intake rate of 24% beyond 2023; inclusion of the maritime sector and a separate fuel-based ETS for buildings and road transport; and, greater regulation on revenue investments.
Source: International Carbon Action Partnership, “EU Emissions Trading System (EU ETS) Factsheet,” retrieved 12/31/2022


text:
Natural cycle IVF is IVF treatment without the use of stimulation drugs. During the natural cycle IVF, only the follicles naturally formed during the menstrual cycle are used for treatment. In most cases, one follicle forms, and, therefore, one egg is used for treatment.
Any woman who ovulates and has regular cycles between 24 and 33 days can have natural cycle IVF. This method of treatment is particularly suitable for women ages 39 and over who are known to have a low ovarian reserve. This would include women with an Antral follicle count of less than 5 or an anti-Mullerian hormone (AMH) of less than 3.
Natural cycle IVF would also be suitable for women who have had a previous poor response to ovarian stimulation or those women who are unable to tolerate stimulation drugs.
You will need to contact TFP on day one of your period in the cycle that you would like to have treatment. We will monitor your cycle by performing bloods tests to measure your hormone levels and vaginal ultrasound scans to monitor your follicle growth.
We may recommend the use of medication to control ovulation. When a dominant follicle has formed a trans-vaginal egg collection will be performed. This procedure will be carried out under sedation and will take approximately 15 minutes. If an egg is retrieved, it will be inseminated using either standard IVF or intra cytollasmic sperm injection (ICSI), depending on the quality of the sperm. The resulting embryo(s) will be transferred back to the uterus two or three days later.
No side effects from stimulation drugs
Reduced cost as no stimulation drugs needed
Reduced risk of developing OHSS (ovarian hyperstimulation syndrome)
Reduced risk of multiple pregnancy
No break needed between cycles, so cycles can be performed back to back
Approximately 40% of cycles do not result in the formation of an embryo, either because an egg is not retrieved or because the egg did not fertilise.
Clinical pregnancy rates per cycle started are approximately 7% in patients with low ovarian reserve. This is the same pregnancy rate that is achieved with stimulated IVF in patients with low ovarian reserve.
These are considerably reduced when compared to a full IVF cycle. You will be given a costed treatment plan following your initial consultation.
For more details, please see the price list for the clinic you attend.
Should your cycle be cancelled prior to theatre you will only be liable for the monitoring costs. On the day of theatre, you will be scanned prior to the egg retrieval. If there is no follicle seen on the scan, your egg retrieval will be cancelled and a refund of £1000.00 will be paid. If there is no egg retrieved following egg collection, your cycle will count as a complete cycle and no refund will be made. If you were to withdraw from treatment, this would count as a complete cycle and no refund would be given.
2 cycle package:
If you fall pregnant on cycle 1: £2000.00
3 cycle package:
If you fall pregnant on cycle 1: £4200.00
If you fall pregnant on cycle 2: £2000.00
Many women want to avoid the use of fertility drugs, often due to an existing medical condition, and thus undergo a different type of fertility treatment. We offer the widest range of treatments, from mild IVF to in vitro maturation (IVM). We can work with you to advise which is the best option for your needs.


text:
With the new packaging release and range expansion, Batiste wanted a bright new website that displayed this new character and colour whilst also clearly displaying the full range. They also had an existing CMS template that they wanted the new design to fit into.
I like to push boundaries. And so I pushed the boundaries on a set CMS to create something that looked free flowing and new. By use of angles, the fun and creative feel was added to simple imagery and life is given to buttons/CTAs. A new menu system was also devised that took a once complicated function, to be smooth and seemless. The site is responsive for the best and easiest transition between devices.
- SkillsVisual Design

domain: artistic


text:
1866 Brownsville Rd
Hong Luck Restaurant
2221 Veterans Hwy Levittown, NY
China Garden Inn Restaurant
1007 Easton Rd. Willow Grove, PA
Phoenix Chinese Restaurant is right near my best friend's house and the food is really good and the prices are just as good!
May 06, 2010
Phoenix Chinese Restaurant
If you've been to or used
Phoenix Chinese Restaurant, leave a review.
It's easy, only takes a couple of minutes and you'll help thousands make an informed decision.

domain: food


MATTERHORN – returns with new single
Trondheim rockers Matterhorn returns with new single The Brightest Light.
Matterhorn is the brainchild of songwriter and vocalist Tommy Sebastian Halseth, who in 2013 started to assemble various musical ideas which gradually turned into the project Matterhorn.
As time passed, the songs matured both musically and lyrically, as Tommy involved different musicians with broad backgrounds to fulfill the Matterhorn vision.
The band’s debut album titled Outside, via Apollon Records, in November 2020. And now they release a the new single «The Brightest Light» on Decmber 8th 2023. Matterhorn is now aiming for a more guitar-oriented soundscape.
The lyrics of the song are written around personal change. It refers to a spiritual and to an emotional change, associated with experienced panic anxiety and depression.
The main message of the song is that there is always hope!
“Let your heart decide, not your fear”
A crow represents change or transformation.
But much more than that, it refers more to a spiritual or emotional change.
These intelligent birds give us valuable insight, into situations around usand help us adapt as required
Tommy Sebastian Halseth: Voice
Tage Johnsen: Piano, Voice
Bendik Vidvei: Guitars
Terje Craig: Spoken Words
Jarl Ivar Andresen: Synth, Programing
Music and lyrics by Tommy Sebastian Halseth
Produced by Tage Johnsen
Recorded at TJ Studio
Mixed by Jarl Ivar Andresen
Mastering by Morten Stendahl
Artwork by Eh Toori


text:
Browse the CollectionBuy Now >>
50 in(127 cm x 127 cm)
In this piece there is a very small area of light. Yet the light pulsates with an energy I hadn’t really thought would be so strong when I planned the quilt. The small area of light floods the darkness, flowing forth, ebbing back, and flowing forth again, emanating hope.
Solar printed, machine pieced, hand quilted


text:
DIY: Back to school
Some kids look forward to heading back to school, while others may dread it. But some simple touches, such as decorating school supplies, might make the process a little more fun.
Make back-to-school time exciting with personalized pencils, recycled snack containers and bookmarks.
Back-to-school can also be an expensive time for parents, so making supplies at home can help save money.
Here are just a few ideas for useful and fun back-to-school crafts.
Milk jug lunch containers
• Plastic milk jug (gallon or half gallon)
• Black marker
• Hair tie, rubber band or other elastic material
• Button or similar
• Hot glue or sewing materials
• Optional — scrapbook paper, stickers or other embellishments
This relatively simple project from Crème de la Craft helps you recycle old materials, save money and personalize lunch containers all at the same time.
First, thoroughly wash the milk container and let dry.
Then draw four half-circles, one on each side of the milk jug, with a black marker. Make one side higher to serve as the lid of the new container.
Cut along the inside of the black lines. Discard the rest of the milk jug.
Bend down the flaps to form the container.
To create a container closure, sew or glue a button on the top of the lid. Then poke a hole just below the front flap on the container and put a hair tie or rubber band halfway through. Tie a knot on the inside of the container. The loop goes over the button to keep the container closed.
• Try to make the three shorter semi-circles that will form the sides of the container about the same size. The lid should be about twice as tall.
• If the container isn't folding properly after cutting off the extra milk jug material, try making deeper slits on the corners.
• If you decide to decorate the lid with scrapbook paper or anything else that will cover the entire top, do that before gluing on the button.
• Make sure the button is securely fastened to the lid. That is what will keep the container from popping open and spilling snacks in backpacks or lunchboxes.
• Plain paper
• Decorative scrapbook paper
• Shape punch
• Glue or tape
Personalized bookmarks are a simple way to mark your spot in textbooks, notebooks and planners. Plus, kids of all ages can enjoy the easy craft in the final days before school.
Cut a piece of thick plain paper to the desired size for the bookmark. Use the shaped punch down the paper.
Cut a piece of scrapbook paper to the same size as the plain sheet and glue or tape on the back. Use the punch on the decorative paper and glue or tape that piece to the front, if desired.
Poke a hole near the top of the book mark. Cut a piece of ribbon, fold over and slip the looped end through the hole. Pull the other end of the ribbon through.
• Use thick plain paper, such as cardstock or even cardboard, to make sure the bookmark is sturdy.
• Keep in mind paper punches with shapes designed to go a certain way may not reach to the bottom of the bookmark, so you might need to flip it upside down or only punch through the top.
Recycled pencil holders
• Cardboard box, such as from cereal or pop
• Hook-and-loop fastener (ex. Velcro)
• Clear tape
• Scoring tool, such as butter knife
• Template (optional)
• Scrapbook paper or fabric (optional)
Create a pencil box from your favorite cereal or soda. Make your own design or use the template found here: http://cdn.makezine.com/make/craft/2010/03/pencibox.pdf courtesy of Creative Itch.
Flatten the cardboard box into one layer.
Cut out the template shape, or whatever box size you designed.
Fold and tape with clear tape.
Place Velcro strips on the inside of the lid and the top of the container.
If you don't want the cardboard design to show, glue or tape on fabric or scrapbook paper.
• Use bigger boxes if you want more of the logo and less of the other information on the box.
• If you make your own template, be sure to measure the pencils or supplies you plan to store to make sure the box is wide and tall enough.
First day photo board
• Whiteboard or chalkboard
• Permanent marker/paint
Remember what your kids were like at the start of each school year by taking a photo by the first day board.
Simply write categories you want to highlight with permanent marker or paint on the board. Here are some examples:
• When I grow up I want to be:
• My favorite color is:
• My favorite food is:
Then each year, fill in the categories with wipe-off marker or chalk and take a photo of your child holding the board before they head off to school.
• You also can create a template and print out a new document each year.
• Pencils and pens
• Decorative tape, glue and scrapbook paper and/or paint
Make your school supplies your own by simply decorating them. Wrap decorative tape around pencils, paint with glue and cover in scrapbook paper or paint the pens and pencils.
• Decorate pencils before sharpening (if you're not using mechanical ones).
• The tape is easiest to sharpen through later, versus glue or fabric.


text:
My skills is getting rusty! I used to paint and draw, raked a lot of awards and was very passionate about it. Anyway, my grandma loves it 🙂 Thanks to my bes who gave me art materials from Muji.
Here’s what I used:
I am currently obsess with parang bagong gising look lang naman 🙂 I love to put volume on my hair, yes… more big hair pluez! I don’t need any rebond na 🙂 My hair is to manipis, kainis! jeje LOL will master the art of it, soon.
By the way, I love swarzkopf hair spray, it is really strong hold, no fuzz, no flakes and just a little spray… ta-da! gora na! great buy 🙂
Belo sun screen
MAC studio fix NC35
MAC pencil in Taupe
Carmex lip balm in tube
MAC wonderwoman in russian red


text:
this is a peaceful looking setting. Great work.
I really like the feel of the drawing.
It’s pleasant to look at. I think that you did a super good job coloring, and just overall it’s a weary great piece(drawing/painting) :D


- “I’m hyperactive and have ADHD (attention deficit hyperactivity disorder), so as soon as I drink cola or coffee I go mental.”
- The 24-year-old truck driver was hoping a striptease would win the judges.
- “I will miss my little girl if I get in, but it’s a chance for me to get started in a career,”
- Dubliner Catherine Mahon claimed she got the day off work for the audition by pretending she had a hospital appointment.
I wonder if its too late to build a fence around the RDS and trap them all there for the good of the nations gene pool.


text:
- Publish Date
- Friday, 21 February 2020, 2:40PM
On Sunday night, some of the biggest names in Australian music joined forces for a one-off benefit concert to raise money for Australian wildfire relief.
As an estimated 75,000 people gathered in Sydney's ANZ Stadium for the star-studded event, concert-goers were moved by national treasure John Farnham performance, who closed the show alongside Olivia Newton-John and Queen guitarist Brian May.
Belting out his iconic 1986 track 'You're The Voice,' the 70-year-old powerhouse singer was also joined by Indigenous artist Mitch Tambo, who sang the song's second verse in Gamilaraay as the sounds of a didgeridoo filled the stadium.
Joining the trio at the gig was also Icehouse, Alice Cooper, k.d lang, Delta Goodrem, Michael Bublé, Tina Arena, Guy Sebastian and many more.
The sold-out concert raised over $9 million.
The fires, fueled by high winds and scorching temperatures, have ravaged more than 25 million acres and killed 28 people since October.
NOTE: SKIP FORWARD TO THE 19 MINUTE, 30 SECOND MARK FOR 'YOU'RE THE VOICE.'
Take your Radio, Podcasts and Music with you


text:
How Much Does Lead Generation Cost?
Trying to build a perfect marketing campaign may be a hard task. And if you ever spent a significant amount of money on marketing and later wondered if it was a reasonable use of your cash, you may dive a bit deeper into generating leads, answering an important question: “How much should lead generation cost?”
Figuring out a lead cost can give you more confidence in running lead generation campaigns. You will be able to determine whether your campaign was effective or not. And in the end, it can guarantee the sustainable development of your business.
Table of Contents
In simple words, lead generation is the process of attracting viewers to your website and converting them into someone interested in your product or service. Many think that there must be a conversion in lead generation. But this is not a complete truth. Even if a visitor comes to your site but doesn’t take any action, they can still count as a lead.
The important thing to learn is that there exist two types of leads because of the different characteristics of marketing and sales terms:
- Marketing Qualified Leads (MQLs) are the potential customers that your company managed to attract. These could be people who just visited your website.
- Sales Qualified Leads (SQLs) are customers that interact with your business by purchasing your product, downloading an ebook, and so on.
Most people who own businesses believe they waste a lot of money on leads and prefer to get them at zero cost. However, you want to consider reserving a part of your budget for lead generation if you want your marketing campaign to be successful. For example, you can use sales intelligence software. It will allow you to make the sales process faster as you won’t have actual people generating leads. But be attentive when choosing a provider as you will need verified data.
Another thing you can do is to buy a list of sales leads. It may look like a reasonable investment, but such leads are usually of very low quality. You will have to evaluate all the leads on the list manually. However, you can outsource it to a company that does lead generation. But you need to understand that marketers who do the calls on your behalf may not have enough information about your business. Eventually, it might lower conversion rates.
A good idea would be to pay for lead advertisements on social media. But it is quite expensive because a team of marketing specialists will run the campaigns and monitor the budget for online ads.
The best option for effective lead generation is to hire a team of marketing specialists and properly train them. In the end, you will get a team that is completely aware of your business which means the process of generating leads will be better and the quality of the leads will increase. Choose the best option for your business depending on how much money you are ready to invest in lead generation.
Lead generation is the key strategy that can assist you with converting a simple visitor into a paying customer. Many marketers agree on the fact that lead generation should be a top priority for any business.
But before you decide on how much you are able to spend on lead generation, you need to figure out your cost per lead so you won’t pay more than they actually cost.
Cost per lead or CPL is the cost of one lead or rather how much you need to pay to get a single lead. In order to calculate the cost of one lead, you need to know the total amount of money you spend on a marketing campaign and the total number of new leads. Then just use a simple formula:
Total Marketing Expenses/ Total New Leads = Cost Per Lead (CPL)
For example, if you invest $1,500 in your marketing campaign and you get 10 leads, your CPL is $150.
In 2021 the average cost per lead was $198. But this number may be useless for evaluating lead generation costs. All because lead costs vary significantly depending on the industry. In addition to that, the target market and the competition in your area of business can also influence the cost of leads.
For example, the most expensive leads are in the technology field, and the cheapest ones are in the field of travel and tourism.
Here are examples of an average lead cost depending on the industry:
Average cost per lead
Media & Publishing
Travel & Tourism
Every marketing manager’s goal is to make their campaigns as effective as possible. In order to do that, you need to understand the number of leads you actually need to reach the income goal of your business.
You need to conduct a thorough analysis, considering all the data elements of your business, such as:
- Targeted Revenue — your planned income you want to acquire at the end of a specific period;
- Marketing Driven Revenue — the part of total revenue that is received due to a marketing campaign;
- Average Sales Price (ASP) — Net Sales / Number of Sold Units;
- Marketing Driven Deals — Marketing Driven Revenue / Average Sales Price;
- Opportunity to Sale Ratio — how close you are to transform a single lead into a paying customer out of the total amount of leads (%);
- Qualified Lead to Opportunity Ratio — the number of leads required to transform a single lead into a paying customer (%);
- Lead to Qualified Lead Ratio — the necessary analysis of how you will transform one lead into a customer.
So let’s figure out how many leads you need in this example.
Let’s assume you want to get $100,000 income by the end of a current month while your marketing-driven income is $45,000 and the average price for sales is $5,000. You need to calculate Marketing Driven Deals — $45,000 / $5,000 = $9. The Opportunity to Sale Ratio is foreseen as 3, so you are able to transform one lead into a customer out of 3 leads.
Then, let’s calculate the number of opportunities you need: 3 x 9 = 27.
The Qualified Lead to Opportunity Ratio is predicted as 6, so you have a chance to transform one lead into a paying customer if you have 6 leads at least. And now you can calculate the number of leads you actually need for your business 6 x 27 = 162.
So now you know that you need 162 leads at least so you could get zero profit.
You can do it manually but consider using some lead calculators which will help you figure out the break-even point, make all the processes of lead generation faster, and eventually make your marketing campaign more effective and successful.
When you calculate the cost per lead for your business, and in case it is higher than you expected, you will have to try lowering lead generation costs. There are several options on how you can achieve that.
1. Widen your database of leads
It is one of the easiest options you may try to achieve your goals. For example, you may use Google Chrome extensions for LinkedIn. They will help with accessing and exporting email addresses so you will be able to contact your ideal customers directly. The advantage of such an extension is that you won’t have to create a contacts database manually anymore, so the process of getting valid B2B contact data will become easier and cheaper.
2. Pay attention to your landing page design
You need to pay close attention to your landing page, it should include an offer that is clear and understandable so you can optimize the conversion process. Especially if you pay for traffic on Facebook or other sources of leads. It’s a good idea to test your landing page before launching. You may just show it to someone and ask for their opinion to figure out if your offer is clearly visible and if they can understand it at first sight.
3. Monitor your target audience
Getting optimal CPL might be difficult if you don’t create a profile of an ideal customer for your company. You need to understand what type of customers or companies will need your products to improve the cost-effectiveness of all your efforts to optimize lead generation.
4. Use an inbound marketing strategy
Gathering bottom-of-the-funnel leads who will be later converted should look natural and organic so you can attract more leads. As an option, you may offer various types of content for every single stage of the funnel and nurture contacts through to the sale. It is a lot easier and more effective than, for example, paid advertising.
5. Get rid of poorly performing channels
Whatever method of collecting leads you choose, remember to always monitor the lead generation cost in every campaign. It will help you invest your resources in the most efficient strategies and eliminate those that unnecessarily increase the cost per lead.
As you already understood, there’s no simple answer to how much a lead should cost. However, some methods can help you calculate the cost of a single lead for your business. Just make sure you remember a couple of things:
- Adjust your marketing budget according to the strategy of lead generation you’ve chosen. And don’t think you will waste your money because an effective marketing campaign will eventually pay off inviting more customers and increasing your revenue.
- Always analyze your current and potential customers, their needs, and requirements so you can determine lead costs and better adjust your budget.
Now you know that in some industries, the average costs of leads are higher than in others, but you shouldn’t focus solely on the average lead cost in your industry. It is always better to calculate a cost for a single lead just for your business and therefore be able to adjust your budget according to the received data. Make sure you know your marketing strategy and all the risks you may face. In the end, a successful marketing campaign is key to increasing your company’s revenue.

domain: economics


text:
Russell Brand enjoying 'freedom'
Russell Brand is making the most of being single again.
The Rock Of Ages star's divorce from singer Katy Perry was finalised this week.
He told US radio host Howard Stern: "I'm kind of enjoying the freedom of it."
He joked: "If you've been on a diet of rice for a while and suddenly Willy Wonka asked you to take over the factory, you'd be kicking over barrels of candy and swimming in a chocolate river."
The British comedian said of his relationship with Katy that when they were dating "I thought 'I've just got to marry her, I love her so much'. When we were married, I thought 'OK, this isn't working'."
He added: "It was a lovely relationship. It mostly didn't work for practical reasons. We've gone our separate ways."
But Russell refused to divulge any details about their break-up, saying: "I don't want anything to hurt her."
He added: "She's younger than me, she's a young woman and she's beautiful and she's sensitive and I care about her deeply."
Russell, who split from the Teenage Dream singer last year after 14 months of marriage, went on to confirm he is now dating Isabella Brewster, the younger sister of Fast Five star Jordana Brewster.
"Some things in the newspaper aren't true," he told Howard when quizzed about their relationship. Howard asked: "That's not true at all?" and Russell replied: "That's true, actually."

domain: entertainment


text:
Matt Damon is goodwill hunting in India. Yes, the Hollywood actor and Oscar winner was in the city for a day and a half – Tuesday and Wednesday – before he caught a flight to Chennai. The 38-year-old actor has come to India for a water project in Chennai.
Damon has been actively involved in a lot of charity work – he’s the founder of the H2O Africa Foundation, which raises awareness about the lack of clean water in Africa. Damon plans to start something similar in India, and that’s why he’s headed to Chennai. "I’m here for a water project in Chennai. I’ll be there for a couple of days. This is my first trip for it. If need be, I’ll come back,” he said, when DT caught up with him, and added, “I’m very happy to be in India finally."
The interesting thing was that the actor was registered in his hotel as Matthew Damon. There was nothing starry about him, though, and he managed to garner quite a lot of goodwill in his day and a half in the city at the Radisson Hotel, Mahipalpur. In fact, when called sexy, he actually became a little embarrassed. Damon is in India with his foundation’s production partners Marc Joubert, Larry Tanz and Keith Quinn. Talk to Marc about Damon being the titled the sexiest man of 2007, and he says, “Oh come on, he’s such a putz (American slang for jerk). He and sexy? Nah. Look at him, he's’s ageing, man! Why'd women look at him? No, well, I'm just kidding. Right now, he's a man on a mission."
Damon will be in Chennai till Friday, and will return to Delhi just to take a flight back home.
On being recognized from Rounders
Matt: "It depends on what city I’m in. If I’m here in Las Vegas, it’s mostly 'Rounders.' If someone says, 'I loved you in' and it’s a guy between the ages of 18 and 40, I can guarantee you he’ll say ‘Rounders.'"

domain: news