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In skeletal muscle, the size of the capillary bed is adapted to the type of muscle metabolism and can be altered by adaptation to different environments or increased activity. Muscle fibres with high aerobic metabolism have more capillaries, and an increase in aerobic metabolism is usually followed by capillary growth. It is assumed that local hypoxia -created by increased demand for oxygen during growth, cold exposure or increased activity -can stimulate proliferation of capillaries. Capillary density is reduced in parallel with enhanced glycolytic metabolism.
The size of the capillary bed can also increase without any apparent change in the oxidative metabolism (e.g. in the early stages of chronic electrical muscle stimulation or as a result of long-term administration of vasodilating drugs), and it is argued that the growth of capillaries in these cases may be due to various mechanical factors connected with increased blood flow.
It has been assumed since the pioneering work of Krogh ( 1919a,b) that the capillary bed in skeletal muscle serves mainly to supply oxygen. This idea is supported by the observation that red muscle, characterized by a highly aerobic metabolism, has a much more dense capillary network than white muscle, with high glycolytic metabo-lism. However, capillaries are also needed to remove different metabolites from muscles, one of the most important being lactate, and to reduce the heat produced mainly during muscle contraction. It is still not clear what induces the development and adaptation of the capillary bed which differs greatly between different muscles and different species.
Adaptation of the capillary bed according to oxygen demand or consumption has been disputed by Maxwell, White & Faulkner (1980), who found a very poor corre-lation between capillary density and chemically estimated succinate dehydrogenase in a variety of muscles and animals. On the other hand, there is a good correlation between citrate synthase and capillary density both in different muscles and in the heart (Hudlická, 1982), and an excellent correlation between capillary density and volume density of mitochondria in various muscles of 20 different mammalian species (Hoppeler et al. 1981b). Moreover, it has been shown that changes in muscle oxidative metabolism, induced either as a consequence of altered activity during cross-innervation or by administration of thyroxine, produce an adaptation in the vascular bed with more numerous capillaries around highly oxidative fibres and lower density around the glycolytic ones (Romanul & Pollock, 1969), although Capo & Sillau (1983) described increased capillary density without any change in the oxidative enzymes after administration of thyroid hormones.
Comparisons of the size of the capillary bed are usually made either on the basis of capillaries mm−2 (capillary density, CD), or on the number of capillaries per muscle fibre (capillary/fibre ratio, C/F). Ideally, capillary supply should be described in terms of capillary surface area or capillary length per volume of tissue. Since capillaries in most muscles are arranged parallel to the fibre axis, it is possible to use cross-sections for the evaluation of the number of profiles. Mathieu, Cruz-Orive, Hoppeler & Weibel (1983) showed that the contribution of tortuosity towards the total capillary length represents only 12 % in cat soleus — a muscle with the most tortuous capillaries — and it is thus justifiable to compare and describe capillary supply under most circumstances either as CD or C/F.
Capillary density is inevitably dependent on the size of fibres — muscle composed of larger fibres having lower capillary density (Plyley & Groom, 1975; Ripoll, Sillau & Banchero, 1979; Aquin, Sillau, Lechner & Banchero, 1980). It is therefore better to compare the extent of capillarization in terms of C/F ratio which is relatively independent of fibre area (Fig. 1).
CAPILLARY SUPPLY TO VARIOUS MUSCLES IN DIFFERENT SPECIES
Comparison of capillary supply to different types of fibre can be readily made in fish, most of which have highly oxidative red fibres in superficial muscle, and glycolytic white fibres in the deep part of the trunk. There is a C/F of 1·9-2·5 in red muscles and 0·16-0·3 in white muscles of several seawater teleosts (Mosse, 1978) and a similar situation in the juvenile European eel (Eggington & Johnston, 1982). In freshwater fish there is a higher C/F in white muscles (0·8 in tench, Johnston & Bernard, 1982; 1·4 in carp, Johnston, 1982). This difference can be explained by different activity, as higher teleosts can recruit white fibres over a wide range of swimming speed.
Birds also have a higher proportion of capillaries supplying red rather than white muscles. In one study of chicken flight muscles, the red anterior latissimus dorsi (ALD) were found to have a C/F of 1·77 while the white posterior latissimus dorsi (PLD) had a value of 1-34 (Cotter, 1975). In another study, a C/F of 1·11 was reported for the red muscle, and 1·08 for the white (Gray, McDonagh & Gore, 1983).
In mammals, the highest capillary density is in the diaphragm and in the masseter muscle (Hoppeler et al. 1981a). The values in these two muscles are rather similar in different species of greatly different body weight (Schmidt-Nielsen & Pennycuick, 1961). The C/F ratio of muscles involved in locomotion increases with the proportion of oxidative fibres (Fig. 2) and a higher ratio is found in the same muscle (e.g. soleus or gastrocnemius) in wild animals than in domesticated ones (Wachtlová & Pafízková, 1972).
Even in mixed muscles (most animal muscles and all human muscles are mixed) fibres with a high oxidative capacity are surrounded by a greater number of capillaries than those with a high content of glycolytic enzymes (Romanul, 1965; Gray & Renkin, 1978).
The data for capillary density in man are very variable (Table 1 ). This is partly due to different methods (depicting the capillaries either by histochemical staining or by electron microscopy) or to different sampling, but is obviously mainly due to inter-individual variability, because even data published by the same group of authors show great differences. The emerging pattern shows a higher C/F ratio in the soleus, a lower C/F in women in most muscles that have been studied, and decreasing C/F ratio in old age, when fibre diameters are smaller. The last observation is in agreement with studies of animals during development (Ripoll et al. 1979; Aquin et al. 1980).
DEVELOPMENT OF THE VASCULAR BED
There are surprisingly few data on the development of the microvascular bed. The capillary network of rats on the 17th day of gestation is formed by polygonal loops which gradually stretch under the tension of the growing muscle fibres (Wolff, Goerz, Bar & Goldnerf, 1975). There are quite a number of capillary sprouts representing up to 50% of the capillary network; these gradually disappear by 21 days postpartum (Welt, Scheller, Schippel & Schippel, 1975).
Capillary growth gradually slows although fibre growth continues ; CD decreases while C/F increases and is linearly related to fibre size in different muscles in chicken (Cotter, 1975), guinea-pigs (Aquin et al. 1980), rats (Ripoll et al. 1979) and hamsters (Sarelius, Damon & Duling, 1981). The C/F ratio increases equally during develop-ment in dogs, but in this species capillary density remains constant irrespective of the more than 70-fold increase in fibre area. Thus many more smaller fibres in a 6-day-old dog are supplied by the same number of capillaries as relatively few fibres per mm2 in adults. This can probably be explained by the observation that all fibres in puppy leg muscles are glycolytic, and they are replaced by an entirely oxidative population in adult dogs (Aquin & Banchero, 1981).
Capillaries in younger animals are more tortuous (Cotter, 1975; Sarelius et al. 1981) and gradually straighten, elongate and become more parallel to muscle fibres. The decrease in capillary density in chickens and hamsters is accompanied by decreased blood flow (Hudlická, 1969; Sareliuset al. 1981). It is also accompanied by a decrease in oxidative capacity during development in chickens (Hudlická, Pette & Staudte, 1973), rabbits and kittens (Cotter, 1975). However, the increase in C/F in kittens is faster in the soleus than in the gastrocnemius even if the increase in the activity of oxidative enzymes is similar in both muscles (Fig. 3). It is possible that the high glycolytic activity in the gastrocnemius (Fig. 3) suppresses capillary develop-ment in this muscle.
ADAPTATION OF THE CAPILLARY BED TO INCREASED ACTIVITY
The discovery that contracting muscles had a capillary density up to ten times higher than resting muscles (Krogh, 19196) initiated a great number of investigations to find out whether the capillary bed enlarges with training (for review see Hudlická, 1977). However, most of the studies showing an increase in the number of perfused capillaries were not really indicative of capillary growth, as capillaries were visualized either by dye injection or by counting capillaries filled with erythrocytes. It was only recently, when either histochemical staining or electron microscopy was used, that capillary growth has been demonstrated to result from training. Andersen & Henriksson (1977) found a significant increase in both cytochrome oxidase and succinate dehydrogenase (SDH) together with an increase in capillary density in biopsies from human vastus lateralis after 8 weeks of endurance training. The C/F increased earlier, after only 5 weeks, a result of muscle fibre hypertrophy. There is a very close correlation between C/F ratio and maximal (Ingjer, 1978; Zumstein, Mathieu, Howald & Hoppeler, 1983) or volume density of mitochondria (Zumstein et al. 1983). There is a higher capillary density, as well as higher C/F ratio in vastus lateralis and deltoid muscle in female swimmers, with a greater increase in relation to oxidative fibres (Nygaard, 1982). The C/F ratio in the vastus lateralis has been found to increase with heavy resistance training in two studies (Schantz, 1982, 1983) but not in another (Tesch, Daniels & Sharp, 1984). The C/F ratio is inversely correlated with both muscle and blood lactate concentration during cycling (Tesch, Sharp & Daniels,1982). The increased capillary density in the vastus lateralis of endurance-trained men and women is accompanied by very little increase in fibre diameters thus indicating real capillary growth (Brodal, Ingjer & Hermansen, 1977; Ingjer & Brodal, 1978). The increase is also higher around slow oxidative fibres than around fast glycolytic fibres (Ingjer, 1979). Similar findings have been made in guinea-pigs (Mai, Edgerton & Barnard, 1970) and rats (Zika, Lojda & KuCera, 1973). In humans, there was a considerably greater increase in the number of subsarcolemmal mitochondria in slow oxidative than in fast glycolytic fibres, and it appears that the increase in capillary density was related more closely to the mitochondrial content than to fibre types (Ingjer, 1979). All these findings can be explained by the fact that submaximal work performed during endurance training activates predominantly slow oxidative fibres whereas fast glycolytic fibres are only active during maximal or supramaximal contraction of a very short duration (e.g. Gollnick, Piehl & Saltin, 1974). In trained rat soleus (84 % slow oxidative) in which the area of subsarcolemmal mitochondria is increased, there is hardly any change in C/F (Pafízková, Wachtlová & Soukupová, 1972; Müller, 1976). This suggests that the amount of activity superimposed on normally very active soleus is not great enough to produce an increase in capillary density, although it is sufficient to produce an increase in aerobic capacity. Unfortunately, the experiments were not carried out for a sufficiently long period to show whether any capillary growth would have been found later. Increased capillary density has also been described in young, but not in adult, rats trained by swimming (Adolfsson, Ljungqvist, Tornling & Unge (1981). There were no changes in the incorporation of [3H] thymidine into capillary endothelium in skeletal muscles of adult rats under similar conditions even though there was capillary proliferation in the heart (Ljungqvist & Unge, 1977).
Exercise thus seems to produce an increase in capillary density that can be explained by capillary growth in young animals, and, if performed for a sufficiently long time (minimum 8 weeks), in adults. This growth appears in the vicinity of permanently active slow oxidative fibres and may perhaps be preceded by an increase in the oxidative capacity.
Another situation when the capillary bed is adjusted to increased activity can be achieved by long-term electrical stimulation, when the increase in activity is similar in all muscle fibres. When fast rabbit muscles were indirectly stimulated by implanted electrodes for 8 h day−1, at a frequency naturally occurring in nerves to slow muscles (10Hz), the activity of oxidative enzymes was increased and that of glycolytic decreased (Pette, Smith, Staudte & Vrbová, 1973), and C/F was almost doubled within 28days (Cotter, Hudlická & Vrbová, 1973b; Brown, Cotter, Hudlická & Vrbová, 1976). The muscles did not hypertrophy as during exercise. The average fibre diameter in extensor digitorum longus (EDL) stimulated for 28 days decreased from 72 ± 1·25 μm to 65 ± 1·0μm and was accompanied by the transformation of large fast glycolytic into small oxidative fibres. Consequently, C/F increased to the value normally found in slow soleus muscle. A small increase in both CD and C/F appeared after only 4 days of such stimulation ( Fig. 4). This preceded the increase in the activity of oxidative enzymes estimated chemically in muscle homogenates (Cotter et al. The doubling of capillary density after 28 days of stimulation could only be explained by capillary growth and this was confirmed by identification of sprouts in rat extensor hallucis proprius and rabbit tenuissimus muscles stimulated for 7-12 days (Myrhage & Hudlická, 1978): both sac-like and tapering sprouts, similar in appearance to previously observed sprouts (e.g. Cliff, 1963) were found in muscles stimulated for 7 days, and their incidence increased to four sprouts per 10 capillaries (a percentage similar to that found for C/F) after 12-14 days of stimulation. The sprouts were more numerous on the venular side of the capillaries, which were also wider and more tortuous. In these studies the growth of sprouts invariably started from a point at which the pre-existing capillary was bent. Sprouts made connection to other capillaries whose lumens were too narrow for erythrocytes to pass through initially but which eventually widened and resembled normal capillaries. No signs of capillary growth were found in control or sham-operated muscles.
Long-term stimulation of rabbit fast muscles at a pattern of frequency more similar to that occurring naturally in their supplying nerves (intermittent bursts of tetani) did not change capillary density or C/F even after 28 days when contractions of 40 Hz were applied for 5 s every 100 s for 8hday−1 (Brown et al. 1976). When the total number of impulses was increased to give the same number of stimuli per hour as the stimulation at 10 Hz (contractions at 40 Hz for 5 s, three times per minute), no change in CD or C/F was found after 4 days, but prolonged stimulation for 14 and 28 days had a similar effect as stimulation at 10 Hz (Fig. 4). At the same time, the muscles change from a mixed population of glycolytic and oxidative fibres to an apparently uniform population of highly oxidative fibres (Hudlická & Tyler, 1984). Any type of electrical stimulation -provided it lasts long enough and is sufficiently intensive -thus produces capillary growth in fast muscles (Fig. 4), and it is therefore the total amount of activity which is important for capillary growth.
However, the adaptation in the capillary bed varies with different frequencies of stimulation in the early stages of stimulation, and it also varies in fast muscles with the different proportion of oxidative and glycolytic fibres: stimulation at 10Hz induces very early on -after only 4 days -an increase in the proportion of oxidative fibres (Hudlická, Dodd, Renkin & Gray, 1982) and increases SDH activity in IIb fibres in tibialis anterior (TA) which has a higher percentage of oxidative fibres than EDL. Muscles stimulated at 40 Hz show a gradual increase in SDH up to 14 days (Pette & Tyler, 1983). In EDL, qualitative histochemistry shows no changes in the proportion of glycolytic or oxidative fibres in muscles stimulated for 4 days at 10 Hz (Hudlická et al. 1982), and a lower proportion of glycolytic fibres in muscles stimulated at 40 Hz (Hudlická & Tyler, 1984). In this muscle C/F increases first in the vicinity of predominantly glycolytic fibres with both types of stimulation. Why the two muscles show slightly different changes in response to long-term stimulation is open to speculation: rabbit TA has a slightly lower proportion of glycolytic fibres than EDL, and it is possible that the increased metabolic demands during the increased activity could be met in the first place by increased blood supply to the oxidative fibres with their already high capillary supply and the possibility of higher oxygen extraction. On the other hand, this increased activity could produce local hypoxia in EDL which has a higher proportion of glycolytic fibres not adapted to long-lasting contractions. Activity of oxidative enzymes has previously been shown to increase in muscles with decreased blood supply (Holm, Bjömtorp & Scherstén, 1972). When the stimulation was discontinued, capillary density remained high for as long as 4 weeks and gradually returned to control values 6 weeks after the cessation of stimulation (Cotter, 1975). Similar detraining effects were described by Houston, Betzen & Larsen (1979), Klausen, Andersen & Pelle (1981) and Saltin & Rowell (1981) in endurance-trained subjects.
FACTORS INVOLVED IN THE ADAPTATION OF THE VASCULAR BED
Possible factors responsible for capillary growth -whether during normal development, changes in environment or increased activity -could be either metabolic or mechanical. Changes in the capillary bed during development, exercise or chronic electrical stimulation are almost always accompanied by changes in the activity of oxidative enzymes and by changes in the volume density of mitochondria, with an increase or decrease in the oxidative capacity usually preceding the increase or decrease in the size of the vascular bed. Ashton (1961) suggested that endothelial cells are in some way directly sensitive to oxygen, multiplying at low oxygen levels, resting at normal, and (dying at high oxygen concentrations. In muscles stimulated chronically at 10 Hz, capillary growth appeared to be induced by local hypoxia (Hudlická & Schroeder, 1978; Hudlická et al. 1982), but in muscles stimulated at 40 Hz, was found to increase after 4 or 7 days (Kanabus, Hudlická & Tyler, 1980). However, there is a greater accumulation of lactate in rat muscles stimulated for 7 days at 40Hz than at 10Hz (Cotter & Hudlická, 1979). It was also shown that in a predominantly glycolytic muscle -cat gracilis -the size of the capillary bed bears no relationship to the volume density of mitochondria, but is related to the lactate output (Hoppeler & Hudlická, 1984).
Increased blood flow and factors connected with it were suggested as being important for vessel growth during normal development by Thoma (1911) and Clark (1918). Whereas the former thought that capillaries grow when blood through the terminal vascular bed is increased because of its mechanical expansion, the latter supposed that capillary growth is induced by mechanical friction connected with the increased amount of filtered fluid.
Exercise, as well as long-term stimulation, produces a long-lasting increase in blood flow (see Hudlická, 1977), connected with an increased proportion of perfused capillaries (e.g. Petrén, Sjöstrand & Sylvén, 1937; Cotter & Hudlická, 1979). Increased capillary density was found during endurance exercise around oxidative fibres, while it appeared first around glycolytic fibres in chronically stimulated muscles. Blood flow during exercise is increased much more in muscles, or parts of muscles, composed of oxidative fibres which are recruited first (Laughlin & Armstrong, 1982). Even in chronic stimulation, which activates all fibres at the same time, there is a greater and more consistent increase in blood flow to glycolytic rather than oxidative fibres (Dawson, Hudlická & Tyler, 1983).
Different patterns of blood flow can perhaps also explain the different onset of capillary growth with different patterns of stimulation : 10 Hz continuous contractions lead to a long-lasting increase, intermittent 40 Hz stimulation not only causes a short period without blood flow at the peak of each tetanus, but the total increase in blood flow per minute is not well maintained over a long period of isotonic contractions during the first hours of stimulation (Aitman, Hudlická & Tyler, 1979). These differences disappear with continuing stimulation, and both resting flow and functional hyperaemia are very similar in muscles stimulated at either frequency for 14 days (Hudlická, Tyler & Aitman, 1980). At this time, the increase in both capillary density and capillary/fibre ratio is virtually the same with either frequency.
The factors connected with increased blood flow that would stimulate capillary proliferation are not quite clear. Since capillary sprouts in chronically stimulated muscles are always found at bends of pre-existing capillaries (Myrhage & Hudlická, 1978) it is quite possible that mechanical factors of a nature different even from those suggested by Thoma (1911) or Clark (1918) can be involved: slight damage to endothelial cells exposed to more frequently moving erythrocytes could induce their proliferation ; proliferation of damaged endothelial cells has been found in the aorta (Payling-Wright, 1972) and DNA synthesis is increased in tissue culture in areas with damaged cell surfaces (Gimbrone, Cotran & Folkman, 1974). Brånemark (1965) suggested that pulsatory movements of erythrocytes during the first phases of vascularization are of primary importance in capillary growth. If any of the factors connected with increased blood flow are involved in capillary growth, it should be possible to elicit growth by long-lasting vasodilation, and this has been reported in rats treated with dipyridamole (Tornling, Adolfsson, Unge & Ljungqvist, 1980), in rats and rabbits treated with axanthine derivate (Wright, Hudlická, Tyler & Ziada, 1981), in rabbits on long-term administration of adenosine (Hudlická, Tyler, Wright & Ziada, 1983) and in rats treated with prazosin (Tyler & Ziada, 1983).
The support of the MRC of Great Britain is gratefully acknowledged.


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We are focused on providing you with the best results and service in the industry. We listen carefully to understand your real estate goals and work hard to create solutions that make sense for you. Whether you are new to the market or an experienced investor, we have the expertise, proven track record, and resources to help you achieve your real estate goals.
As is often said, real estate is about location, location, location. We have extensive knowledge of the Tyler, TX area and can help you find the right home for you or the right buyer for your home.
Whether you are moving down the street or relocating, we have the local expertise and resources to help you find the perfect home or condo for you and your family. We work closely with our clients to understand your real estate needs and goals to help you find the best home at the best possible price.View Buyer Information
We are experts in the local market and will guide you through the entire process of selling your property from listing to sale. Our marketing plans assure your property maximum exposure and we pride ourselves on personal service. We encourage you to contact our office with your questions and to discuss your individual needs.View Seller Information
We encourage you to check out our website for local neighborhood and school details, mortgage information, interactive maps, listings of homes for sale, and many more features. Please feel free to contact us if you have any questions about the Tyler real estate market, or buying or selling a home in Tyler, TX.Search Local Listings

domain: geography


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Tollywood stars are unstoppable when it comes to signing new projects. Young actor Naga Chaitanya completed the shoot of Love Story and the film is expected to release soon. Vikram Kumar’s Thank You is in the final stages of the shoot and Naga Chaitanya commenced the shoot of Aamir Khan’s Laal Singh Chaddha yesterday. The actor will complete the portions of the shoot before the end of this month. Naga Chaitanya also signed a web series for Amazon Prime and it will be directed by Vikram Kumar. Chaitanya allocated three months dates for the project.
As per the current update, Naga Chaitanya gave his nod for one more new film. He gave his nod for a romantic entertainer that will be directed by Kishore Tirumala. DVV Danayya will bankroll this prestigious project and the shoot commences next year. Kishore Tirumala is busy with Sharwanand’s Aadavallu Meeku Joharlu that will start shooting in August. Kishore will complete his work for the film after which Naga Chaitanya’s film will roll.

domain: celebrity


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Today is International Day of Persons with Disabilities, a day set up and championed by the United Nations since 1992 to celebrate disabled people’s achievements across the world. But if you’re a disabled person in this country you might not feel like celebrating because so many of us are losing vital benefits, being pushed into debt, and having our independence and quality of life eroded by the policies of the current government. A day celebrating disabled people’s achievements feels empty when it comes in the midst of such relentless cuts and daily vilification in the media.
But there’s one reason for disabled people to celebrate and I was reminded of this when I caught a bit of ITV’s morning show, Daybreak, earlier. Francesca Martinez was being interviewed about the WOW (War on Welfare) petition.
The WOW petition, created and driven forward by disabled people, calls for a Cumulative Impact Assessment of all cuts and changes affecting sick and disabled people, their families and carers. The WOW team have worked tirelessly to get to this point and I can’t think of a better was to celebrate International Day of Persons with Disabilities than to sign this petition. It hit 100,000 signatures last week, which means it now has to be debated in the House of Commons.
Francesca said it best in her very powerful interview this morning: “More signatures means more pressure.”
She went on…
“Let’s fight to create a society that values human wellbeing above profit and ensures that everyone has an equal right to happiness regardless of income or ability – that’s one worth fighting for.”
There’s not long left to sign the petition so please do it now, and then share it as widely as you can.
“Christmas Tree Correction Centre.”

domain: sociology


So, the 43rd Tokyo Motor Show is opening its doors on November 22 at the Tokyo Big Sight convention center in Odaiba. Needless to say, Honda Motor Company will be among the major Japanese carmakers looking to make a lot of noise at the auto show. Honda\'s booth theme this year will be, \"Live outside the box.\"
Honda intends to follow its own advice by exhibiting a compact open-top sports car called the S660 concept, as you see in these photos. For now, not much has been revealed about the car apart from this short official description: \"A next-generation open-top sports-type mini vehicle that features a dynamic and advanced styling design, as well as various new initiatives such as a Super cockpit interior that thoroughly pursues the ultimate space for the driver.\"
We\'re tempted to think this is the successor to the now-defunct S2000, which bowed out of production in 2009. But the S660 could also have hybrid propulsion. Or maybe not. Best to attend the actual unveiling. We can\'t wait to inspect this car on the motor-show floor.


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Minutes of the Previous Overview and Scrutiny Committee
Decision status: Recommendations Approved
Is Key decision?: No
Is subject to call in?: No
Councillors raised issue that part of their discussion was missing. It was to do with inviting someone in to talk about the committee systems. It was advised that this discussion was recorded in the Scrutiny Steering Group minutes and was part of the work programme that would be later discussed.
RESOLVED – that the minutes of the meeting held on 13th June 2019 be
approved by the Chair as a true record.
Publication date: 09/08/2019
Date of decision: 11/07/2019
Decided at meeting: 11/07/2019 - Overview and Scrutiny Committee

domain: politics


This LMT 14.5” Chrome Lined 5.56×45 1:7” RH barrel is designed to match the LMT MRP or MRP CQB quick-change barrel systems. This barrel is perfect for M4-length carbines, balancing compactness with ballistics.
- 14.5” Chrome-lined 1:7” RH Barrel for MRP Monolithic Upper Receivers
- 5.56×45 Caliber
- Cryogenically treated for maximum durability
- Low profile gas block
- Carbine Length straight gas tube
- A2 Birdcage Pre-Installed
LMT’s quality engineering is well respected in the industry, and their barrels are no exception. Chrome lined and cryogenically treated, this barrel has all the durability-enhancing features that one would expect from a high-grade carbine. Combine incredible durability with LMT’s reputation for accuracy, and you have a versatile duty rifle that can keep up in any environment.
The gas system has already been installed to match your MRP upper receiver. To install this barrel, use the factory torque wrench to release the retaining bolts at the base of your handguard, swap this barrel in, and tighten the bolts down to LMT’s spec. If you want to change calibers or barrel lengths, we offer several other MRP barrels to match your needs.
Comes with an A2 birdcage flash hider pre-installed!
Note: This flash hider is not pinned, so the true barrel length is 14.5”. Installation of this barrel should be completed with a full understanding of SBR laws.


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Call to men in Barnsley to put their health first
South West Yorkshire Partnership NHS Foundation Trust is encouraging men in Barnsley to be more health aware ahead of Men’s Health Week (11-17 June).
Research repeatedly highlights men’s reluctance to visit their doctor compared with women. Men are less likely than women to acknowledge health problems – to themselves or others – are less aware of their symptoms than women and delay going to the doctor. This can result in men being diagnosed with conditions at a stage when their symptoms are more advanced and harder to treat.
George Smith, assistant director of nursing for the Trust, said, "Men may think they don’t have time to look after their health, or that it’s an inconvenience, but if conditions are not diagnosed and treated early they will take up a lot more time.
"If you notice something that doesn’t seem right to you, or you’re feeling different and don’t know why, there are lots of local health services which can help – a simple phone call now could save weeks of unnecessary worry."
In line with the key messages of Men’s Health Week, the Trust has expanded its intranet to include a new resource dedicated to encouraging men to keep an eye on their health and, more importantly, seek help when needed.
Alan Davis, director of human resources for the Trust, said, "South West Yorkshire Partnership NHS Foundation Trust is committed to improving staff health and wellbeing. With more than a thousand male staff in the Trust, any steps we can take to promote greater self-awareness have the potential to make a real difference to the organisation’s overall health and wellbeing.
"As a health trust, it is particularly important that we don’t forget our staff’s own health needs: a mentally and physically fit workforce enables us to provide the best possible services for the people we care for."
Health information on a range of topics is also available on the Trust’s website: http://www.southwestyorkshire.nhs.uk/your-wellbeing/

domain: health


Farm diversification can seem like a daunting process for those new to trialling new projects, but many surveys show the huge benefits on offer for those who achieve successful farm diversification. Check out our blog on planning your farm marketing strategy here
Ingenuity, drive, skill and hard work are all clear to see in the results of Farmers Weekly’s diversification survey, carried out in association with Carter Jonas. A vast range of alternative ventures are run by the 250 people who completed the survey, with almost 100 different enterprises listed.
More than two-thirds (68%) have diversified, into areas ranging from the familiar and the popular such as commercial and residential developments, renewable energy and tourism, to a flying school, plumbing, dog training, quarrying and boatyards.
We have a full range of blogs on farm diversification planning. We cover diversification ideas for many farm types such as our dairy diversification blog, sheep farm diversification and pork farm diversification.
A huge 97% scored their alternative enterprise a success in profitability terms, with about half saying that it was profitable to a great extent and one-third seeing a profit in the first, second and third years of operation.
Eight in 10 believed it was an environmental success too.
More than half of those already diversifying plan to expand these interests in the next five years.
Areas that farmers find the most challenging are securing planning permission, funding, and correctly budgeting for a new enterprise.
Only one in five had any grant funding to help with the new venture.
Among those planning to diversify, most are looking at commercial lets such as workshops, offices and storage, alongside residential developments for tourism and the domestic rental market.
There is also a notable trend to bolt extra offerings on to existing diversifications – for example, farm shops and cafes are increasingly introducing function or meeting rooms, bringing them not only the hire income for the room, but also the chance to sell to or cater for those using the facilities.
Enterprises with an educational aspect were noticeable, both in existing and planned businesses.
This is usually strongly linked to a desire to educate both children and the wider public about farming and food production, as well as providing additional income.
One farmer is planning to have an Open Farm Sunday-type event every fortnight.
While some saw diversification as essential, helping them to stay in farming, others were critical of the fact they had felt forced to go down the alternative enterprise route when they would prefer that farming provided a decent income on its own.
Others warned of the potential effect on the farming business if too much management time or cash was diverted to the new venture.
It was important to be flexible, said one farmer, who had developed a wedding barn venue in an area that was now overrun with such offerings
“There was one when we did ours, now there are eight – take care and be flexible!”
High planning fees, including one example of £90,000 to convert a barn, came in for criticism too, alongside the complex and sometimes impenetrable grants offerings.
Most respondents said they would not change anything, but where they would, the most common comments centred on starting at a larger scale and starting the new enterprise earlier.
Others said they would have:
Survey respondents are running a huge range of alternative enterprises:
Article taken from Farming Weekly


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Divorce proceedings enjoys a far more positive impact on people.
Women Can Be Healthier After Divorce Or Separation For Most Explanations, These 9 Especially
A report by Kingston college supposed to study the unfavorable influence of shock on gents and ladies came up with some shocking conclusions about divorce or separation and girls.
In research, scientists surveyed 10,000 people in the U.K. amongst the ages of sixteen and sixty. In the review players happened to be asked to level their unique delight pre and post their divorce proceedings. During a 20 12 months period, researchers learned that females comprise happier and satisfied with their particular life after breakup.
“from inside the learn we took under consideration the reality that separation and divorce can sometimes has a negative economic affect female, but even though it nevertheless means they are much happier than males,” Professor Yannis Georgellis, Director in the Center for study in jobs, abilities and Society (CRESS) at Kingston company college, mentioned.
How come lady deal with breakup better than guys? I’m sure that some argue that this is due to additional ladies declare breakup than males, that ladies are happier as they are getting what they need.
That argument merely keeps water should you explore the numerous factors girls file for divorce case. Many reasons exist females apply for a divorce or separation rather than all of those causes have actually anything to carry out with falling out in clumps of prefer or no further are happier in marriage.
4 grounds a female may file for breakup:
1. She has come left behind and left without recourse but to apply for a divorce and realize youngsters service through the household judge system. Something that is not taken into account as soon as we review research towards simple fact that most divorces were registered by female ‘s the reason they declare separation. In many circumstances, women file for breakup if they’ve become backed into a corner and feel they’ve got few other choice.
2. the girl partner suffers a midlife situation and endangers the girl economic safety, emotional safety and acts in a manner that try harmful to the girl along with her future welfare.
3. Her spouse try abusive and she has no recourse when safeguarding by herself besides to file for a divorce case and place length between herself together with abuser.
4. Her spouse features an extra-marital affair, moves from the marital house, in using additional lady and actually leaves the lady accountable for financial servicing of the house and families.
The reason for the separation and divorce just isn’t a consideration in how good a lady will heal and move forward along with her life when she’s divorced. Split up is a hard alternatives to create but as soon as really made a woman provides selections she will be able to make. She will be able to provide in to the shock associated with the divorce or separation or rebuild this lady lifetime and obtain on using the company of living. The majority of decide to can get on the with businesses of living.
9 explanations women are happier after split up:
1. Women are inclined than people to look for help for all the psychological traumatization caused by divorced from a therapist friend or, friend. Guys keep it close to the vest whenever working with emotional difficulty. Maybe not extend for support prolongs their own distress in addition to energy it will require to cure.
2. ladies are inclined than people to surround by themselves with an optimistic service program including relatives and buddies.
3. girls need different emotional coping tricks. While guys hunt outward whenever pursuing convenience from psychological soreness, women have a look inwards. They bring an inside supply associated with the role they played in the demise on the wedding, they work at getting their particular mental “ducks consecutively” and letting run of the past to allow them to focus on the potential future.
4. Women are less inclined to turn-to alcohol, drugs, brand new relationships and informal sex to disturb them through the upheaval of breakup.
5. Women are prone to find brand new encounters after breakup, activities it enhance their everyday lives and give all of them a feeling of a cure for the future.
6. Women are more likely to focus on their needs. They are going to place an endeavor into keeping physically healthier during stress of divorce proceedings. They have even more target consuming correctly and working call at an attempt to prevent problems and anxiety.
7. Women are no more powerful mentally than boys. They are doing but need different coping skill than males whenever handling mental shock and, based on the research, those abilities make it possible for women to maneuver on and become happier than men after breakup.
8. ladies have a substantial sense of persistence. Giving up is almost always the easy way out. Resilient group express the capability to stay glued to things to get them accomplished. It really is ladies who perform some BBW dating sex lion’s display of childcare after divorce or separation, it works outside of the house while, additionally having to keep your homes together. Women can be more likely to accept, gladly the difficulties of single motherhood and relish for the reason that part.
9. ladies are very likely to getting comfy in their own body. These include much more comfortable together with the concept of supposed they alone, selecting a program of motion they rely on and advancing. Most women don’t brain hanging out with by themselves and just have an awareness of importance of repairing after their unique divorce before jumping into a relationship. Their decreased enduring loneliness after divorce case permits them to check out enjoyable recreation often by yourself or even in the business of friends. They don’t head out straight away trying to find an upgraded product for the partner they simply separated.


Corsair H55 RGB CPU Liquid Cooler
- MODEL NUMBER: CW-9060052-WW
- SERIES: H55 RGB
- VOLTAGE: 5 Volts
- WARRANTY: 3 Year
₹5,999.00 Incl. GST
The CORSAIR H55 RGB all-in-one liquid CPU cooler delivers strong, dependable cooling and stunning lighting, with a CORSAIR SP120 RGB ELITE PWM fan, a 120mm radiator, and 21 bright RGB LEDs.
COOL CPU, COOLER LIGHTING All-in-one CPU cooling that illuminates your PC with dramatic color, thanks to 120mm CORSAIR SP RGB ELITE PWM fans generating great airflow and vibrant RGB lighting.
FLEXIBLE MOTHERBOARD DIGITAL +5V ARGB SUPPORT – Conveniently control your lighting through compatible motherboards.
CONCENTRATED COOLING WITH AIRGUIDE
HIGH-PERFORMANCE COLD PLATE AND PUMP
Equipped with a thermally optimized copper cold plate and low-noise pump design for quiet cooling.
EASY TO INSTALL
Includes a modular, tool-free mounting bracket for quick and easy installation on all modern CPU sockets, including LGA 1200,115X, 2066, and AM4.
THREE RADIATOR SIZES
H55 RGB fits in standard 120mm fan mount locations for optimal compatibility, while H100 RGB and H150 RGB offer 240mm and 360mm sizes, respectively, for increased cooling performance.
|Manufacturer||Corsair, Corsair (Hong Kong) Limited, Suite 2602-03, 26/F, Millennium City 5, 418 Kwun Tong Road, Kowloon, Hong Kong, Toll Free# 1800-425-5464, firstname.lastname@example.org|
|Item Height||15.4 Centimetres|
|Item Width||19.5 Centimetres|
|Product Dimensions||22.7 x 19.5 x 15.4 cm; 680 Grams|
|Item model number||CW-9060052-WW|
|Country of Origin||China|
|Item Weight||680 g|


Sheikh Sultan visits several museums, monasteries and other historical buildings in Madrid, Spain, on Thursday.
The gesture reflects the Sharjah Ruler’s keenness to promote locally-produced literature within the UAE’s book industry by supporting Emirati writers and intellectuals, and ultimately enhancing their presence in libraries.
Sharjah takes centre stage at The London Book Fair 2020, from March 10 – 12, at Olympia, with the leading publishing event’s ‘Market Focus’ title, to be held.
His Highness Dr Sheikh Sultan Bin Mohammed Al Qasimi, Supreme Council Member and Ruler of Sharjah, arrived on Monday in Madrid on an official visit that would last for several days.
Tens of thousands of protesters marched in support of Palestinians on Saturday in major European cities including London, Berlin, Madrid and Paris.
The big winner was from Egypt, Asmaa Al Awani, who won the grand prize, a brand-new BMW car, while 5 shoppers won shopping worth Dhs5,000, 10 winners won Dhs2,500 shopping vouchers, and 10 lucky shoppers won a cash prize worth Dhs1,000 for each.
Cases have fallen steadily in some Indian states hit by an initial surge in infections, such as the richest state of Maharashtra and the capital New Delhi, after they imposed stringent lockdowns. Testing rates also at their lowest since Sunday.
The NCOC late last month announced a new set of restrictions including a blanket ban on tourism and inter-provincial transport in an attempt to contain a further rise in already mounting coronavirus cases under the "Stay Home, Stay Safe” strategy.


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NEW YORK (
CHANGE IN RATINGSDavita(DVA - Get Report) was downgraded at Goldman Sachs to sell from neutral. $110 price target. Company faces earnings headwinds, Goldman Sachs said.
Gogo(GOGO - Get Report) was initiated with an outperform rating at William Blair. Company is a market leader and can deliver solid growth, William Blair said.
Medley Capital(MCC) was upgraded to buy at TheStreet Ratings.
[Read: Book Profits Now on These 9 Sell-Rated Stocks]
PPL(PPL - Get Report) was initiated with an equal-weight rating at Morgan Stanley. $32 price target. Company is transitioning to a regulated utility, but consensus expectations appear too high, Morgan Stanley said.
Scholastic(SCHL) was upgraded to buy at TheStreet Ratings.
Tenneco(TEN - Get Report) was upgraded at Goldman Sachs to buy from neutral. Company has strong secular drivers and margin prospects, Goldman said.
[Read: Apple May Grab Kinect Technology: Report]
STOCK COMMENTS/EPS CHANGES
Bally Technologies(BYI) numbers were increased at Sterne Agee. Shares are now seen reaching $74. Estimates were also increased, as the company has good visibility and can expand margins, Sterne Agee said. Buy rating.
Citigroup(C) estimates, price target were increased at Sterne Agee. Estimates were raised through 2014. Company is posting better earnings quality, Sterne Agee said. Buy rating and new $61 price target.
Cintas(CTAS) estimates, price target were cut at Jefferies. Shares are now seen reaching $51. Estimates were also reduced, as organic rental growth is slowing, Jefferies said. Buy rating.
[Read: What You Can Do About That Brewing Debt Storm]
Dish Networks(DISH) numbers were boosted at Credit Suisse. Shares are now seen reaching $50. Estimates were also increased, as spectrum valuations are rising, Credit Suisse said. Outperform rating.
Ford(F) was cut from the Conviction Buy List at Goldman Sachs.
General Motors(GM) appears to have better near-term catalysts.
General Motors was added to Conviction Buy List at Goldman Sachs. Company has potential near-term catalysts, Goldman said.
Priceline.com(PCLN) numbers were increased at Jefferies. Estimates were boosted through 2014. European demand continues to improve, Jefferies said. Buy rating and new $1005 price target.
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domain: economics


text:
Hollywood/Western Station opened on June 12, 1999 and has a single entrance inside the plaza in front of its transit village at the SE corner Hollywood Blvd and Western Avenue. Here two escalators and a staircase lead down an intermediate landing before continuing to the main short corridor which leads to the station's turnstiles. Just before it there is a single street elevator that has its own enclosure and location a short was down Hollywood Blvd from the corner and staircase entrance. The stations turnstiles lead to an elevator and two sets of staircase/single escalators down to the middle of an island platform. The walls of the columns in the middle of the platform and the mezzanines are all covered in a checkerboard of different light colored tiles. These are all part of Untitled by May Sun. There are also photographs inside the mezzanine of the history of the station's location. Along the walls of the mezzanine facing one of the staircases down to the platform are replicas of the ends of the Red Line Streetcars that formerly served the station.
Photos 1-22 taken on 17 March, 2008, 23: 19 January, 2014


The general pharmacologist in the pharmaceutical industry is challenged to generate physiologically relevant data on possible safety liabilities or on secondary therapeutic uses as early as possible in drug development. This implies the need for efficient use of usually only small supplies of test article. For this reason, we have developed a new animal model combining various elements to provide a broad spectrum of data focussing on the so-called vital physiological functions: cardiovascular, respiratory, and central nervous system. This system uses rats chronically implanted with transmitters for the measurement of arterial pressure, ECG, and body temperature. Modification of the transmitters also allows for the simultaneous assessment of locomotor activity. Studies are performed with these rats in plethysmographs placed directly over the antenna units thus allowing for the additional assessment of respiratory function in the same studies. Using this system, we can generate simultaneously a wide range of relevant physiological parameters in conscious rats with a modest requirement for test article. Such an approach is highly useful for getting early safety readouts of potential drug development candidates as well as for detecting possible secondary therapeutic actions of a drug.


Sen. Marshall Introduces and Questions Kansan Shawn Tiffany at Hearing on Cattle Markets
(Washington, D.C., April 26, 2022) – Today, at Senate Agriculture Committee hearing, U.S. Senator Roger Marshall, M.D. introduced Shawn Tiffany from Herington, Kansas who is President-Elect of the Kansas Livestock Association and co-owner of Tiffany Cattle Company. The hearing focused on two pieces of legislation pertaining to cattle markets and Senator Marshall had a chance to question Shawn on the negative implications of moving forward with the bills. Senator Marshall believes that a bill mandating negotiated cash sales is not the answer and will only restrict the way Kansas cattle producers market their cattle. You may click HERE or on the image below to watch the Senator introduce and question Shawn.
Senator Marshall also spoke at the hearing about his concerns with consolidation of the industry. He believes the cause was overregulation and the solutions are eliminating burdensome regulations, more accurate labeling of beef, and allowing interstate shipments of state inspected meat. You may click HERE or on the image below to watch Senator Marshall discuss these concerns.
In July, Senators Marshall and Mike Rounds (SD), as well as Representatives Jason Smith (MO) and Dusty Johnson (SD), introduced the Feed America by Incentivizing Rural (FAIR) Meat Packing Act to ensure a level playing field for our nation’s cattlemen and to return to fair prices for both cattlemen and consumers. The FAIR Meat Packing Act creates two tax incentives to facilitate the formation of small- and mid-sized meat processing facilities allowing cattle producers to compete for better prices in the marketplace.
In January, Senator Marshall introduced the Bona Fide Beef Branding Act to help ranchers distinguish their product in the meat case in grocery stores nationwide. This legislation would direct the U.S. Depart of Agriculture (USDA) to eliminate the “Product of the USA” meat label and instead create three new voluntary labels to address consumer confusion.
ICYMI: Senator Marshall is focused on cattle market reform (RFDTV) Sen. Roger Marshall, an outspoken member of the ag committee, shares his top priorities to put producers in the driver’s seat. On the hill, there are several legislative efforts underway to support the livestock industry. Kansas Senator Roger Marshall says that he is working to address transparency in the cattle market. According to Sen. Marshall, “What we have to be careful about is overcorrecting. What I don’t want to do is drive the prices down, and I’m afraid if we overcorrect if we get the government too involved, if we rely upon the USDA to set prices and quotas, it could end up having a negative impact. So, that’s the last thing I want to happen. I think we need to keep emphasizing transparency, and I think we need to keep emphasizing growing the businesses, those small businesses to do more packing plant, and do more meat processing.” Marshall says that he has not seen a “silver bullet” piece of legislation yet… In the meantime, Sen. Marshall says that he is focused on creating more options for producers: “We certainly need more capacity and it seems like online over the next two years, we’re going to increase the capacity, maybe 3-4 percent. So, that’s the good news! We want it to continue to be consolidated with just four factory plants, so that’s the bad news. So, my job is to do everything I can to help lower those barriers to market for some of those small packing plants and make it more feasible for them to do business and get my producers more options.”


5 Simple Techniques For MOT Check
Some repair stores give both of those mechanical and bodywork fix. Automotive maintenance retailers that specialize in bodywork maintenance are often called entire body outlets.
Rotor: The rotor is the main disc portion of one's brake program the pads clamp on to. It truly is linked to the wheel and once the pads strike it, the vehicle slows down. The pads are held on the rotor by calipers.
Check automobile community forums and overview web-sites for suggestions. If you are not familiar with or even a fan of the deep world of automobile boards and enthusiast discussion boards, It can be ok. AutomotiveForums.com is host to forums for specific will make and versions of cars, and It truly is generally frequented by mechanics on their own seeking next opinions or help diagnosing troubles. Talk to all around for garage ideas based on your type of vehicle and your locale.
Brake pads: The brake pads tend to be the element of your brakes that use the frictional power to halt the vehicle. These pads Use a constructed-in warning process that squeals once the brakes are utilized and also the pad is worn down.
The scent of burned toast — a light-weight, sharp odor — usually indicators an electrical limited and burning insulation. For being Protected, consider to not push the auto till the problem is diagnosed.
Certainly, the initial step to doing all your individual get the job done is usually to think you can do it. Like our friends at Jalopnik have reported, "Prevent becoming a coward and fix your own private damn automobile." Then, you should definitely Possess a place to do the get the job done which is wonderful and flat where you can jack the car up, or a place you more info can find the car in the air securely to work on it.
Ever get that sneaking suspicion after you just take your car in for repair service that you just're overpaying…
Check together with your point out Attorney Standard or area client safety company for specifics of your guarantee legal rights.
Should the inspection picks up A serious dilemma relevant to any from the detailed points, your automobile will fail the test. You’ll receive a VT30 certificate outlining The explanations for the failure.
Know a fantastic store? Evaluation it right here to inform others and explain to us! Want to help you folks like you uncover an outstanding shop close to you? Had a nasty practical experience? Allow others know.
Consider a AAA Automobile Treatment Middle or even a AAA permitted car mend shop. When you are a AAA member, look at browsing a store that is possibly operated by or accepted by the Group you pay out membership dues to every year. Furthermore, you'll get a discount over the perform you'll need accomplished.
Your very best defense from fraud and defective maintenance work is to find a highly regarded mechanic or repair service store right before your automobile should be fixed. Check with your friends if they know of a trustworthy mechanic.
When you finally're comfortable correcting small car complications and doing all of your frequent routine maintenance, you happen to be probably intending to need to check out a lot more sophisticated repairs. Except major engine get the job done, you can do a lot of repairs within the ease and comfort of your own private garage or street.
Diagnosing the trouble is the hardest A part of harder repairs, but Vehicle Difficulty make the diagnosing procedure a bit much easier. In case your problem can be a Check Motor light, do not forget that most auto sections retailers will test your automobile for free.


Naresh got Navarasa Raya Award
Yesterday that is on 28th Sept 2015 Monday, at 2 PM Ravindra Bharathi. Organized by Shruthilaya Arts Academy on the eve of Dr. Akkineni Nageswar Rao’s 92nd Birthday celebrations. The Chairman NavaRatna Sri. R.N.Singh Garu presented NavarasaRaya Award to NARESH.
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text:
Manchester United agreed a £30.7million fee over the weekend for Benfica defender Lindelof, who looks set to become Jose Mourinho’s first signing of the summer.
Express Sport understands Lindelof, who still has a medical to undertake, could complete a switch to Old Trafford this week.
Mourinho supposedly doesn’t want to sign anymore centre-backs, should he clinch a deal for the Sweden international.
But there could be departures with the Daily Star suggesting Smalling faces an uncertain future.
The report suggests Mourinho will invite offers for the 27-year-old, who missed six weeks of the season with a broken toe.
Chris Smalling's long-term future at Manchester United is under threat
He knew the picture in terms of liaising with the medical staff
The Daily Star claim West Ham, Everton and Crystal Palace are showing an interest.
Smalling was publicly criticised by Mourinho over his fitness record, but the England international reckons the Portuguese's comment was borne out of ‘frustration’.
He said: “I think that was sort of directed at everybody in many ways … we had a lot of injuries at that time and he was frustrated that quite a lot of players weren’t available.
Victor Lindelof looks set to become Manchester United's first signing of the summer
“He knew the picture in terms of liaising with the medical staff.
“He was just as frustrated as us who want to be on the pitch. That was just a global frustration. (Before that) I had done the injury (broken toe); I played on and there was obviously a break.
“He didn’t know the full extent at that time.”

domain: news


text:
Guess Where I'm Eating?
Welcome to our inaugural post of Guess Where I'm Eating, soon to be a regular feature of this blog where you, the reader, gets to guess where I, the blogger, ate. The reward? A prize to our first correct guesser...
Your only clue (so far)? The above picture. So where am I eating? And also, what am I eating?
Get the Food & Drink Newsletter
Our weekly guide to Orange County dining includes food news and reviews, as well as dining events and interviews with chefs and restaurant owners.

domain: food


text:
Written by Petter Näse
Recorded and produced by Petter Näse at Sthlm Songs studio
Mixed by Markus Sjöberg at Sthlm Songs studio
Mastered by Eric Broyhill at MonsterLab Audio
Vocals, guitars, bass, mellotron: Petter Näse
Drums, glockenspiel and percussion: Fredrik Myhr
© & ℗: Sthlm Songs AB 2019
”Let Me Fall”, the new single by Markus Black Sjoberg, will be out October 12th on all streaming services! Solid songwriting, heartfelt vocals and brilliant production…be sure to check it out!
New single by The Heartbeat Band out now!
Check it out here:
Maritza horn – innan elden brunnit ut
Maritza Horn’s latest album, released on Brus & Knaster, is out now! The album is produced by Maritza Horn and Markus Sjöberg. The album was recorded by Markus Sjöberg at Durango Recording, Sthlm Songs Studio and Stureparken Studio. Markus mixed the record here at Sthlm Songs Studio. Mastering by Classe Persson at CRP. The record consists of Maritza’s interpretations of Ola Magnell’s songs.
Månskensbonden – Vårat ödeland
Månskensbonden:s debut album ”Vårat Ödeland”, released on the artist’s own label Månskensmusik, is out now! The album was recorded by Petter Näse in Sthlm Songs Studio, produced by Petter Näse and Fredrik Myhr, mixed by Markus Sjöberg in Sthlm Songs Studio and mastered by Eric Broyhill at MonsterLab Audio. The backing track for ”Döpt I Kyro Älv” was recorded by Stefan Backas in his studio in Malax, Finland.
Musicians: Markus Bergfors, Petter Näse, Fredrik Myhr, Clas Lassbo, Sofie Björkgren-Näse, Daniel Ventus, Daniel Hjerppe and Eero Paalanen.
New single by our new friends erik & ella
Released May 19th 2017, ”Han Som Har Flest Saker När Han Dör Han Vinner” is the debut single of Sthlm Songs’ artists Erik & Ella. It features the longest title with what may be the sparsest lyrics in quite a while, and yet the song places a firm boot in the male capitalist establishment…while being totally danceable. Dig it!
Music by: Erik Hedrenius & Ella Junghahn
Vocals: Ella Junghahn and Erik Hedrenius
Programming, keyboards: Erik Hedrenius
Drums: Jesper Hättander
Guitars, banjo, mandolin, bass guitar: Petter Näse
Synth bass, piano: Markus Sjöberg
Recorded and produced by Markus Sjöberg and Petter Näse at Sthlm Songs
Mixed by Markus Sjöberg at Sthlm Songs
Mastered by Eric Broyhill at Monsterlab Audio
Additional recording by Erik Hedrenius
Sthlm Songs proudly gives you Felicia’s new single ”Window Nr 9”. About the song and the beautiful animated video she says:
”Window Nr 9 is a story about humanity, sexuality and power. In the song, a woman tells a story about her living as a sex worker. She lives in her own world, a utopia. In her world everyone is equal. She can see that the men that come to her are suffering, in need of help, and she wants to help. Window Nr 9 is a clash between two realities, as in the eyes of the men she is an object giving sexual satisfaction, nothing more. We all see the world through different eyes, and as long as the power resides among those who do not see everyone as equal, there will be oppression.”
Animator: Beatrice Lorén
Background artist: Erik J. Olofsson
Music & Lyrics by Felicia
Produced by Markus Sjöberg and Petter Näse.
Recorded by Markus Sjöberg and Petter Näse.
Released by Sthlm Songs AB.
Mixed by Markus Sjöberg.
Mastered by Eric Broyhill.
Piano: Jonas Westberg.
Drums, harmonium, omnichord: Markus Sjöberg.
Guitars, bass: Petter Näse.
℗ Sthlm Songs AB 2016
© Sthlm Songs AB 2016
Sthlm Songs proudly presents a new single from Markus Black Sjoberg. ”The Lucky Ones” is a bittersweet story about when life surprises you. Written sincerely from one person to another. Dreamy, acoustic pop from deep within. Intimate yet grand. Available on all streaming services August 26th 2016.
Written by Markus Black Sjoberg and Leif Larson. Produced, and mixed by Markus Black Sjoberg. Mastered by Eric Broyhill at MonsterLab Audio.
Cello: Anna Dager. Drums and percussion: Jesper Hättander. Electric guitars: Petter Näse. All other instruments: Markus Black Sjoberg.
Pills – released may 27th
”Pills” is the first single from the follow-up to The Heartbeat Band’s debut album Sailor Heart. In “Pills”, the band’s songwriter Marcus Granfors deals with the fact that, in today’s society, we feel worse day by day while we all really strive to feel loved and valuable. Burned-out and lonely people choose the easy way out either to make it through the day or to end it, while sincere relationships might be the only thing that really helps. Only true love love makes us do the good and bad things we never thought we could.
Written by Marcus Granfors
Produced and mixed by Markus Sjöberg at Sthlm Songs.
Recorded by Pelle Henricsson at Tonteknik Recording.
Additional recording by Markus Sjöberg.
Mastered by Eric Broyhill at MonsterLab Audio.
Artwork by Ulrika Öhman.
Malin’s take on Bon Iver’s Skinny Love has now been played 1,236,842 times on Spotify. One point two million and counting…Nothing short of amazing!!!

domain: artistic


text:
MicroRNA‑103a‑3p potentiates chemoresistance to cisplatin in non‑small cell lung carcinoma by targeting neurofibromatosis 1
Affiliations: Department of Second Pulmonary Medicine, The Third Hospital Affiliated of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China, Department of Video Center, The Third Hospital Affiliated of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China, Department of Lymphoma and Breast Cancer Internal Medicine, The Third Hospital Affiliated of Xinjiang Medical University, Urumqi, Xinjiang 830011, P.R. China
- Published online on: January 3, 2020 https://doi.org/10.3892/etm.2020.8418
- Pages: 1797-1805
Copyright: © Zhu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
This article is mentioned in:
Non-small cell lung cancer (NSCLC) accounts for ~85% of all primary lung cancer cases worldwide in 2018 (1). Chemoresistance is responsible for the high prevalence and mortality of NSCLC, as this phenomenon enhances NSCLC progression and makes NSCLC difficult to treat (2). The underlying molecular mechanisms of chemoresistance are not yet understood (3). The aim of the present study was to elucidate these mechanisms in order to identify a method to decrease chemotherapy resistance in NSCLC.
MicroRNAs (miRNAs or miRs) are an important component of epigenetic mechanisms that decrease gene expression at the post-transcriptional level by binding to 3′-untranslated regions (UTRs) of target mRNAs (4). miRNA is an important regulator in a number of biological processes, such as cell proliferation, invasion, metastasis and apoptosis (5,6). Abnormal expression of miRNAs has been demonstrated to be associated with tumor chemotherapy resistance, including resistance to cisplatin (7). However, how miRNAs regulate cisplatin resistance is not yet clear. Previous studies have demonstrated the suppressive role of miR-103a-3p in chemotherapy-resistance in numerous different types of cancer such as bladder carcinoma, malignant mesothelioma and glioma (8–11). However, whether miR-103a-3p regulates cisplatin resistance in NSCLC remains unknown.
Neurofibromin 1 (NF1) plays a role as a key negative regulator of the Ras signaling pathway, negatively regulating mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) signaling (12). Recent studies revealed that primary and acquired chemotherapy-resistance of lung adenocarcinomas in patients was significantly correlated with lower NF1expression (12,13). However, the underlying molecular mechanism of how NF1 downregulates cisplatin resistance in NSCLC remains unknown.
In the present study, it was demonstrated that expression levels of miR-103a-3p were significantly increased in patients with cisplatin-resistant NSCLC and can induce cisplatin resistance in NSCLC cells by directly targeting NF1 to activate ERK signaling. Furthermore, the results revealed that overexpression of miR-103a-3p can overcome cisplatin resistance of NSCLC cells.
Materials and methods
Human samples and cell lines
A total of 20 patients (age, 38–69 years; male 12, female 8) with primary NSCLC who underwent surgical resections from January 2016 to December 2017 at The Affiliated Tumor Hospital of Xinjiang Medical University were included in the present study, and their adjacent normal lung tissues (5 cm away from the tumor tissue) were obtained. The aforementioned 20 patients' serum was also included in the present study. All samples were properly preserved for future use. The tissues were stored in liquid nitrogen. Venous blood samples of all participants were collected in EDTAK2 anticoagulation tubes from an antecubital vein, which were stored in −80°C fridge. The present study was approved by the Research Ethics Board of the Xinjiang Medical University. All patients who provided tissues and serum provided written informed consent and all of them agreed to the use of their samples in scientific research.
Human lung adenocarcinoma A549 and PC-9 cells, which were purchased from the American Type Culture Collection, were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) with 10 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO2. The cisplatin-resistant A549/PC-9 cell lines were established in the center laboratory of The Affiliated Tumor Hospital of Xinjiang Medical University and were preserved in 1 µmol/l cisplatin (cat. no. BP809; Sigma-Aldrich; Merck KGaA).
miR-103a-3p inhibitors (100 nM), miR-103a-3p mimics (50 nM) and negative control (NC), ERK siRNAs, NF1 siRNAs and their negative controls were synthesized by RiboBio. The final concentration of siRNAs was 50 nM and the sequences are presented in Table I. Transfection was performed using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 10 µl lentivirus (1×108 TU/ml; Shanghai Genechem Co., Ltd.) carries a puromycin resistance gene, which can be used to reject untransfected cells following transfection with lentivirus. The pre-experimental results revealed that when 2 mg/ml puromycin was added to the culture medium then cultured at 37°C for 48-h, A549 cell viability was 0% without any transfection. Total RNA and protein were prepared 72-h after transfection and were used for reverse transcription-quantitative PCR (RT-qPCR) or western blot analysis.
Cell viability assay
A549 and PC9 cells were seeded into 96-well plates (5×103 cells/well) either directly or 24 h after transfection and allowed to attach overnight. Freshly prepared cisplatin was then added at 5, 10, 20, 50 and 100 µM at 37°C. After 24-h, cell viability was assessed using a Cell Counting Kit-8 (cat. no. HY-K0301; MedChemExpress) according to the manufacturer's protocol.
RNA preparation and RT-qPCR
Total RNA was isolated from NSCLC tissues or cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA (All-in-One™ miRNA First-Strand cDNA Synthesis Kit for miRNA; cat. no. AMRT-0020 and All-in-One™ First-Strand cDNA Synthesis Kit for mRNA; cat. no. AORT-0020; GeneCopoeia, Inc.) under the following protocol: 37°C for 60 min then 85°C for 5 min. miRs from serum were isolated using the miRNeasy Serum/Plasma kit (Qiagen) according to the manufacturer's protocol.
Mature miR-103a-3p and the RNU6 (GeneCopoeia, Inc.) endogenous control were analyzed according to the manufacturer's protocol. The expression levels of miR-103a-3p were quantified in relation to the expression of RNU6 using the 2−ΔΔCq method (14). Thermocycling condition were as follows; 95°C for 10 sec; 58°C for 20 sec; 72°C for 10 sec and 40 cycles. For the analysis of NF1 expression, RT and qPCR were performed using a High-Capacity cDNA Reverse Transcription kit and QuantiTect SYBR Green PCR kit (Thermo Fisher Scientific, Inc.), respectively. The thermocycling conditions were as follows: 95°C for 10 sec; 60°C for 20 sec; 72°C for 10 sec and 40 cycles. The expression levels of NF1 were quantified in relation to the expression levels of GAPDH using the 2−ΔΔCq method (14). The primer sequences were described in Table II. All primers were obtained from Guangzhou RiboBio Co., Ltd.
Protein extraction and western blotting
Total protein was collected using radioimmunoprecipitation assay buffer (cat. no. R0278; Sigma Aldrich; Merck KGaA) containing protease inhibitors (Merck KGaA). The supernatant protein concentration was measured using a bicinchoninic acid kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.). A total of 30 µg of protein per lane was separated via 10% SDS-PAGE electrophoresis and transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). After the membrane was transferred, PVDF was blocked with 5% skim milk powder at room temperature for 1 h. Membranes were then incubated with primary antibodies against ERK (1:1,000; cat. no. 9102), p-ERK (Thr202/Tyr204) (1:2,000; cat. no. 4370), NF1 (1:100; cat. no. 14623), (all from Cell Signaling Technology, Inc.) and GAPDH (1:10,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.) overnight at 4°C, followed by incubation with secondary antibodies conjugated to horseradish peroxidase for 2 h at room temperature (goat anti rabbit; Thermo Fisher Scientific, Inc.; 1:10,000 and goat anti mouse; Thermo Fisher Scientific, Inc; 1:10,000). Protein bands were developed using Enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc.), with images taken by imager (ChemiDoc™ Touch Imaging System; Bio-Rad Laboratories, Inc.). Density analysis was performed using Quantity One (Bio-Rad Laboratories, Inc.; Software version 4.6.2).
Immunohistochemistry (IHC) in NSCLC xenograft specimens
Specimens were formalin (10%) fixed for 24 h in 4°C and paraffin-embedded tumor tissues (3 µM) were examined to ensure a tumor content of >75% by a pathologist. IHC was performed using Antigen Retrieval Dako Target Retrieval solution (pH 6.0) and Histostain-Plus 3rd Gen IHC Detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) on FFPE slides according to the manufacturer's protocols. Xylene was used for dewaxing and then samples were blocked with 100% goat serum (cat. no. E510009-0100) Sangon Biotech Co., Ltd.) for 30 min at 37°C. The sections were stained with human rabbit Ki-67 antibody (1:10; cat. no. TA801577 OriGene Technologies, Inc.) overnight at 4°C. The secondary antibody working solution kit containing DAB (Maxim Biotech, Inc.) was added to the tissue and incubated for 30 min in 37°C and the slides were reviewed using a light microscope (magnification, ×100).
Target gene prediction
National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) and TargetScan databases (http://www.targetscan.org/vert_72; http://starbase.sysu.edu.cn/) were downloaded and analyzed to comprehensively screen miR-103a-3p target genes.
Luciferase reporter assay
QuickMutation kit (cat. no. D0206; Beyotime Institute of Biotechnology) was used to process the wild type plasmid (Promega Corporatino) to obtain a mutant plasmid. The NF1 promoter region was cloned using the following primer sequence: 5′-TTAGGTTTAAAATTGGTTAAATTAATGGTG-3′ was inserted into a luciferase reporter plasmid (pRL-TK; Promega Corporation). A wild-type 3′-UTR and a mutant 3′-UTR of NF1 that contained the predicted miR-103a-3p target sequence were amplified using PCR as described previously. Either the wild-type 3′-UTR or the mutant 3′-UTR of NF1 was incorporated into a luciferase miRNA expression reporter vector (pMIR-REPORT) at MluI and HindIII sites. miR-103a-3p and the miRNA expression reporter vector with wild-type or mutant NF1 3′-UTR and the pRL-TK were transiently co-transfected into the cells using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The luciferase activity was measured following incubation for 24 h at 37°C with Dual luciferase reporter gene system according to the manufacturer's protocol (Promega Cor). The luciferase activity was normalized to the activity of Renilla luciferase. The experiment was performed in triplicate.
Animal experiments were performed on female BALB/C nude mice, (6 weeks of age; average weight 18 g). The mice were kept in specific pathogen-free conditions, with a 12-h light/dark cycle and had free access to food and water, The room temperature was 26–28°C, and the relative temperature was maintained at 40–60%.
A549/cisplatin cells were transfected with control lentivirus or miR-103a-3p inhibitors expression lentivirus as previously described. After drug (puromycin, 2 mg/ml) screening for transfection, 1×107 cells in 100 µl of phosphate-buffered saline were subcutaneously injected into left side of each mouse. When the tumors reached ~100 mm3, mice were treated with or without cisplatin (3 mg/kg body weight; 6 mice per group) by intraperitoneal injection every 3 days. After 4 weeks of treatment, the mice, average weight 20 g, were sacrificed by cervical dislocation (maximum tumor volume was 1,300 mm3), and the tumor weight was measured. The methods of the animal models used in the present study were approved by the Research Ethics Board of The Affiliated Tumor Hospital of Xinjiang Medical University.
All data are presented as the mean ± standard deviation. One-way analysis of variance followed by Tukey's post hoc test was used to evaluate the comparisons of multiple groups the SAS statistical software package (version 6.12; SAS Institute, Inc.). All experiments were performed in triplicate at minimum. P<0.05 was considered to indicate a statistically significant difference.
Cisplatin resistance is closely associated with miR-103a-3p overexpression in NSCLC cells
The miR-103a-3p expression levels in 20 human NSCLC samples (10 cisplatin-resistant samples and 10 cisplatin-sensitive samples) from different patients were analyzed in the present study, in order to investigate the association between miR-103a-3p levels and cisplatin resistance. It was revealed that miR-103a-3p was significantly increased in the samples from patients with cisplatin-resistant NSCLC in both serum (Fig. 1A) and solid tumor (Fig. 1B). A549/cisplatin had increased remarkably compared to parental cell A549 (Fig. 1C) in vitro, and PC-9/cisplatin demonstrated also changed (Fig. 1D). These results demonstrated that miR-103a-3p exhibits high expression levels in NSCLC cells and could affect the development of cisplatin resistance.
miR-103a-3p expression levels are increased in patients with cisplatin-resistant NSCLC. The level of miR-103a-3p was significantly increased in (A) the serum and (B) the tumors of patients with NSCLC that acquired resistance to cisplatin treatment compared with cisplatin-sensitive patients. (C) The level of miR-103a-3p was significantly increased in the cisplatin resistant cell line, A549/cisplatin, compared with the cisplatin-sensitive cell line, A549. (D) The level of miR-103a-3p was significantly increased in cisplatin resistant cell line, PC-9/cisplatin, compared with the cisplatin-sensitive cell line, PC-9. The levels of miR-103a-3p were assessed using reverse transcription-quantitative PCR. *P<0.05 and **P<0.01 with comparisons indicated by lines. miR, microRNA; NSCLC, non-small cell lung carcinoma; cDDP, cisplatin.
In order to investigate this hypothesis, miR-103a-3p overexpressed A549 cells were treated with cisplatin and cell viability assays were performed. The results revealed that the miR-103a-3p expression levels increased following treatment with miR-103a-3p mimics or decreased following treatment with inhibitors (Fig. 2A). High expression levels of miR-103a-3p caused A549 cells to exhibit significantly greater levels of resistance to cisplatin treatment compared to the control and miR-103a-3p inhibitors group (Fig. 2B). miR-103a-3p mimics reversed the inhibitory effect of cisplatin on A549 cells (Fig. 2C). Overall, these results demonstrate that high expression levels of miR-103a-3p significantly contribute to the development of cisplatin resistance in NSCLC cells.
Overexpression of miR-103a-3p results in cisplatin resistance in NSCLC cells. (A) Transfection of miR-103a-3p mimics and inhibitors significantly changed the miR-103a-3p level in A549 cells. (B) Cell viability assays revealed that overexpression of miR-103a-3p can protect A549 cells from cisplatin-induced cell death. (C) Cell viability assays revealed that overexpression of miR-103a-3p abolished cisplatin-induced inhibition of A549 cell proliferation. *P<0.05 and **P<0.01 with comparisons indicated by lines. miR, microRNA; NSCLC, non-small cell lung cancer; NC, negative control; cDDP, cisplatin.
miR-103a-3p activates the ERK signaling pathway, leading to cisplatin resistance in NSCLC
A number of studies have demonstrated that the ERK signaling pathway contributes to the development of cisplatin resistance in cancer (11–13). The present study therefore investigated whether miR-103a-3p was able to affect ERK signaling in NSCLC. p-ERK was significantly increased when miR-103a-3p was overexpressed compare to the NC and inhibitors group, whereas phosphorylation of ERK was decreased by the miR-103a-3p inhibitor compared to the NC and mimics group (Fig. 3A). These results indicate that miR-103a-3p serves an important role in the ERK signaling pathway leading to cisplatin resistance of NSCLC cells. ERK was knocked-down with siRNA to observe ERK expression (Fig. 3B). In addition, the cell viability assay data revealed that miR-103A-3p did not induce cisplatin resistance following ERK silencing in NSCLC cells (Fig. 3C). Taken together, these data demonstrate that miR-103a-3p induces cisplatin resistance in NSCLC cells by activating the ERK signaling pathway.
Overexpression of miR-103a-3p induces cisplatin resistance in NSCLC cells by activating ERK signaling. (A) miR-103a-3p positively regulates the phosphorylation of ERK in A549 cells. PC-9 cells were transfected with miR-103a-3p mimic or miR-103a-3p inhibitor. (B) ERK expression was significantly inhibited by ERK siRNA treatment in A549 cells. Cells were transfected with ERK siRNA. (C) Silencing of ERK abolished miR-103a-3p-induced cisplatin resistance in A549 cells. A549 cells were transfected with miR-103a-3p mimics and/or siRNA of ERK. After 24 h transfection, cells were treated with indicated concentration of cisplatin for 48 h. *P<0.05 and **P<0.01 with comparisons indicated by lines. miR, microRNA; NSCLC, non-small cell lung cancer; ERK, extracellular signal-regulated protein kinase; siRNA, small interfering RNA; NC, negative control; cDDP, cisplatin.
miR-103a-3p induces ERK signaling in NSCLC cells by targeting NF1 expression
In order to investigate the role of miR-103a-3p in the regulation of ERK signaling, the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) and TargetScan databases (http://www.targetscan.org/vert_72; http://starbase.sysu.edu.cn/) to comprehensively to screen miR-103a-3p target genes and identified NF1 as a tentative target of miR-103a-3 A different 3′-UTR of the NF1 gene was constructed in the present study, which contained three sites that interacted with miR-103a-3p (Fig. 4A). In order to confirm whether miR-103a-3p decreases NF1 expression in A549 cells, NF1 expression was measured following overexpression or inhibition of miR-103a-3p in A549 cells. At the protein (Fig. 4B) or mRNA (Fig. 4C) levels, miR-103a-3p negatively regulated NF1 expression in NSCLC cells. In addition, the present study assessed the binding of miR-103a-3p to NF1 3′-UTR by synthesizing each site of the NF1 3′-UTR that could interact with miR-103a-3p into the firefly luciferase reporter plasmid. These sites were then transfected into A549 cells with miR-103a-3p mimics or control oligonucleotides. The luciferase assay results revealed that the signal was significantly decreased following transfection with the third site of the NF1 3′-UTR (Fig. 4D). These results indicate that miR-103a-3p directly interacts with the third site of the NF1 3′-UTR. A three-nucleotide mutation site of miR-103a-3p was inserted into the 3′-UTR to confirm whether the expression of NF1-luciferase is dependent on the 3′-UTR sequence (third binding site) complementary to the miR-103a-3p seed sequence, as presented in Fig. 4A. The data from the present study indicate that the 3′-UTR mutation had no effect on miR-103a-3p overexpression or luciferase activity, but wild-type 3′-UTR significantly repressed the luciferase activity following miR-103a-3p overexpression in A549 cells. Inhibition of miR-103a-3p significantly enhanced the luciferase activity associated with the wild-type 3′-UTR (Fig. 4E). Furthermore, it was demonstrated that miR-103a-3p regulates ERK signaling by targeting NF1.
NF1 is a target gene of miR-103a-3. (A) Sequence alignment of miR-103a-3p with the 3′-UTR of NF1 gene. The red labeled nucleotides represent point mutations in the 3′-UTR sequence. miR-103a-3p negatively regulates NF1 expression at both the (B) protein and (C) mRNA levels. A549 cells were transfected with miR-103a-3p mimic or inhibitor. After 72 h of transfection, cells were subjected to reverse transcription-quantitative PCR and western blot analysis. (D) 3′-UTR luciferase reporter assay for NF1. Each miR-103a-3p binding site of NF1 3′-UTR was cloned into luciferase reporter constructs and transfected into A549 cells with miR-103a-3p mimic or control oligonucleotides. The luciferase assay results revealed that the signal was significantly decreased following transfection with the third site of the NF1 3′-UTR. (E) The third NF1 3′-UTR binding site inhibits the expression of firefly luciferin. The indicated luciferin plasmid was transfected with miR-103a-3p mimic or control oligonucleotides. *P<0.05 and **P<0.01 with comparisons indicated by lines. NF1, neurofibromin 1; miR, microRNA; 3′-UTR, 3′-untranslated region; NC, negative control; mut, mutant.
Overexpression of NF1 reversed miR-103a-3p-induced upregulation of phosphor-ERK in A549 cells (Fig. 5A). In contrast, silencing NF1 reverses the inhibition of miR-103a-3p on ERK phosphorylation (Fig. 5B). Consistent with these results, the cell viability assay indicated that miR-103a-3p overexpression and cisplatin-induced resistance were overcome by NF1 overexpression in A549 cells (Fig. 5C). In summary, these data indicate that miR-103a-3p regulates cisplatin resistance of NSCLC cells via NF1, which activates ERK signaling.
miR-103a-3p activates ERK signaling by inhibiting NF1 expression in NSCLC cells. (A) Overexpression of NF1 inhibits miR-103a-3p-induced ERK phosphorylation. A549 cells were transfected with miR-103a-3p mimic or/and NF1 expression plasmid 72 h. (B) Silencing of NF1 suppressed miR-103a-3p inhibitor-induced inhibition of ERK phosphorylation. A549 cells were transfected with miR-103a-3p inhibitor and/or NF1 siRNA 72 h. (C) Overexpression of NF1 inhibits miR-103a-3p induced cisplatin resistance in A549 cells. Cells were transfected with the indicated nucleotides and/or plasmid, then treated with indicated concentration of cisplatin for 72 h. *P<0.05 and **P<0.01 with comparisons indicated by lines. NF1, neurofibromin 1; miR, microRNA; NC, negative control; ERK, extracellular signal-regulated protein kinase; siRNA, small interfering RNA; p, phosphorylated.
Inhibition of miR-103a-3p can reverse cisplatin resistance of NSCLC cells in vivo
The results of the present study indicate that overexpression of miR-103a-3p induces cisplatin resistance, but whether miR-103a-3p interference can overcome cisplatin resistance in NSCLC cells remains unclear. To demonstrate this hypothesis, cell viability changes in two different cisplatin-resistant A549 cell clones treated with miR-103a-3p inhibitor and cisplatin were then examined. The results revealed that the combination of miR-103a-3p inhibitor and cisplatin was more effective than cisplatin only treatment (Fig. 6A). Furthermore, a xenograft model generated by the lentiviral transfected cisplatin-resistant cell line A549/cisplatin was used to observe whether this theory is correct in vivo. When the average volume of the xenograft tumor reached 100 mm3, the mice received 4 weeks of cisplatin treatment. The results revealed that in the A549/cisplatin xenograft model, the combination of miR-103a-3p inhibitor and cisplatin was superior to cisplatin or miR-103a-3p inhibitor alone in inhibition of A549/cisplatin xenograft growth (Fig. 6B). The combination of the two can significantly slowed tumor growth in volume and in weight (Fig. 6C and D). The expression levels of Ki-67 were markedly lower in the miR-103a-3p inhibitor combined with cisplatin treatment group compared to the control (Fig. 6E).
Inhibition of miR-103a-3p enhances the sensitivity of cisplatin-resistant NSCLC cells to cisplatin treatment in vivo. (A) A combination of miR-103a-3p inhibitor and cisplatin treatment synergistically inhibited A549/cisplatin cell viability. A549/cisplatin cells were transfected with negative control oligonucleotides or miR-103a-3p inhibitor for 24 h. Cells were then reseeded in a 96-well plate. After 12 h of seeding, cells were treated with or without 10 µg/ml cisplatin for 48 h. (B) Xenograft tumor morphology. (C) A combination of miR-103a-3p inhibition and cisplatin treatment synergistically suppressed tumor growth in A549/cisplatin xenograft models. (D) Tumor weight. At the end of experiments, tumors were collected, and the weight was measured. miR-103a-3p inhibitors combine cisplatin treatment synergistically suppressed tumor weight compare to other groups. (E) Immunohistochemical analysis of Ki-67 derived from xenograft model tumors (magnification, ×100). miR-103a-3p inhibitors combine cisplatin treatment could suppress the expression of Ki-67. *P<0.05 and **P<0.01 with comparisons indicated by lines. miR, microRNA; NSCLC, non-small cell lung cancer.
Over the past 10 years, NSCLC has become a prevalent malignancy worldwide, but cisplatin-based chemotherapy resistance is one obstacle standing in the way of treatment (15). A number of studies have demonstrated that dysregulation of specific miRs results in the development of chemoresistance in a number of different types of cancer (16,17). In the present study, NF1 was identified as a target of miR-103a-3. It was revealed that miR-103a-3p overexpression could decrease NF1, improve cell viability and desensitize NSCLC to cisplatin both in vivo and in vitro. Chen et al (12) reported that miR-641 can contribute to erlotinib resistance in NSCLC cells by targeting NF1. miR and NF1 play an important role in NSCLC treatment resistance. Furthermore, the present study demonstrates the association between miR-103a-3p and the development of cisplatin chemoresistance in NSCLC.
There are numerous reasons underlying drug resistance, which include factors such as increases in drug efflux, alterations in drug targets, DNA repair, cell cycle regulation and evasion of apoptosis (12,18). It has previously been demonstrated that selective regulation of miR activity can improve responsiveness to chemotherapy (18) miR-103a-3p expression has been demonstrated in several different cancer cell lines such as bladder carcinoma cell and glioma cell line (8–10), and miR-103a-3p has been indicated to be important in proliferation and metastasis (8,10). In the present study, it was revealed that miR-103a-3p was significantly increased in patients with NSCLC who acquired resistance to cisplatin treatment, as well as increased cisplatin resistance in NSCLC cell lines. It was also demonstrated that overexpression of miR-103a-3p can decrease NF1 levels, desensitize A549/cisplatin cells to cisplatin, and promote tumor growth in a nude mice model. In addition, it was revealed that miR-103a-3p is partially complementary to the 3′-UTR of the NF1 mRNAs using bioinformatics (TargetScan) and that miR-103a-3p can affect luciferase activity due to canonical binding to the NF1 3′-UTR. Thus, the present study clearly established an inverse association between miR-103a-3p and NF1. Furthermore, overexpression of NF1 can reverse high ERK phosphorylation levels, which had been induced by overexpression of miR-103a-3. On the other hand, low phosphorylation levels, which had been caused by inhibition of miR-103a-3p, were increased via inhibition of NF1.
miR-103a-3p levels are highly expressed in breast cancer, pancreatic cancer and ovarian cancer, and are closely associated with tumor invasion and metastasis (19–22). There are some inconsistent results, however; Fasihi et al (23) assessed whether hsa-miR-103a-3p plays an important role in colorectal carcinoma via regulation of the Wnt signaling pathway. They hypothesized that miR-103a-3p overexpression resulted in cell cycle progression and decreased apoptotic rate in SW480 cells. A single miR may disrupt multiple pathways involved in regulating cancer cell survival or drug response. Thus, the effect of miR-103a-3p was determined by the function of target genes in the present study. However, the molecular mechanism underlying the differing functions of miR-103a-3p in different cancers remains unclear; in addition, whether miR-103a-3p affect the therapeutic effect of tyrosine kinase inhibitors treatment in NSCLC requires further examination.
In summary, the present study combined clinical and experimental studies to establish a role for miR-103a-3p in regulating cisplatin chemoresistance in NSCLC. Upregulated expression of miR-103a-3p dramatically enhances the sensitivity of NSCLC cells to cisplatin chemotherapy. These results are also helpful for developing potential therapeutics for the treatment of NSCLC chemoresistance.
We thank all patients who participated in this study. We thank Professor Yunquan Guo (Department of Pathology, Affiliated Tumor Hospital, Xinjiang Medical University) for his support in pathology.
The study was supported by the foundation of Xinjiang Uygur Autonomous Region Natural Science (grant. no. 2018D01C277).
Availability of data and materials
The datasets used and/or analyzed during the currenty study are available from the corresponding author on reasonable request.
SY conceived and designed this study. HZ was responsible for doing the main experimental. HZ and JY were jointly involving in extracting data and writing the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Research Ethics Board of The Affiliated Tumor Hospital of Xinjiang Medical University (Urumqi, China). All patients who provided tissues and serum provided written informed consent and all of them agreed to the use of their samples in scientific research. The methods of the animal models used in the present study were approved by the Research Ethics Board of The Xinjiang Medical University.
Patient consent for publication
The authors declare they have no competing interests.
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