The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Cervical carcinoma is the second most common cause of cancer deaths in women worldwide. Treatments have not changed for decades and survival rates for advanced disease remain low. An exciting new molecular target for the treatment of cervical squamous cell carcinoma (SCC), and possibly for SCCs at other anatomical sites, is the oncostatin M receptor (OSMR). This cell surface cytokine receptor is commonly copy number gained and overexpressed in advanced cervical SCC, changes that are associated with significantly worse clinical outcomes. OSMR overexpression in cervical SCC cells results in enhanced responsiveness to the major ligand oncostatin M (OSM), which induces several pro-malignant effects, including a pro-angiogenic phenotype and increased cell migration and invasiveness. OSMR is a strong candidate for antibody-mediated inhibition, a strategy that has had a major impact on haematological malignancies and various solid tumours such as HER2-positive breast cancers. © 2013 The Authors.  Despite a reduction in incidence and mortality rates since the introduction of population-wide screening programmes in developed countries, cervical carcinoma remains the second most common cause of cancer deaths in women worldwide Most cervical carcinomas are squamous cell carcinomas (SCCs), which arise from precursor squamous intraepithelial lesions (SILs). While infection with HR-HPV is necessary for cervical carcinogenesis, it is not sufficient for progression to malignancy. Low-grade SILs often regress and only a minority of women infected with HPV develop cervical carcinoma, with malignant progression involving a long latent period where additional ‘hits’ are needed in situ hybridization (FISH). One of the most common genomic imbalances in cervical SCC is copy number gain and amplification of chromosome 5p, which occurs in up to half of advanced-stage cervical SCCs in vitro system, which accurately models HPV16-associated cervical squamous carcinogenesis, 5p gain was rapidly selected over ∼15–20 population doublings and was associated with acquisition of the ability to invade collagen in organotypic tissue culture Several groups have performed integrative genomic analysis to identify genetic alterations that contribute to the progression to cervical SCC OSMR) and the microRNA processor Drosha were functionally validated and are likely to contribute to the selection of 5p gain in cervical carcinogenesis p = 0.046) In order to identify driver oncogenes of importance in cervical squamous carcinogenesis, our group first performed aCGH in a set of 46 cervical SCC samples to identify regions of copy number gain that also showed amplification OSMR is a member of the interleukin 6 (IL6) receptor family. It associates with gp130 to form the high affinity receptor for its major ligand, the cytokine oncostatin M (OSM), and is also able to bind IL31. Following OSM binding, OSMR signals mainly through the JAK/STAT pathway, but also activates MAPK/ERK and PI3K/AKT cascades, inducing the transcription of context-dependent target genes n = 321 cases) n = 114 cases) OSM–OSMR signalling has key roles in inflammation, haematopoiesis, and development, and is increasingly being recognized as an important contributor to cancer progression in vivo xenograft models of prostate and breast cancer The role of OSM in cell proliferation seems to be context- and cell type-dependent, with pro-proliferative effects in some cells in vitro approaches including gene depletion and overexpression Our group has studied the biological basis of the association between OSMR overexpression and adverse clinical outcome in cervical SCC. In representative OSMR-overexpressing cervical SCC cell lines, OSM activated (ie phosphorylated) STAT3, p44/42 MAPK, and S6 ribosomal protein, effects that were reduced after OSMR depletion using RNA interference  5. TheseThe principal indirect effect of OSM–OSMR interactions in cervical SCC cells was to promote a pro-angiogenic phenotype. OSM induced a significant increase in VEGFA production in OSMR-overexpressing cells, while conditioned medium from OSM-stimulated cervical SCC cells induced angiogenesis in an endothelial–fibroblast co-culture model system The direct effects of OSMR overexpression on cervical SCC cells were to increase cell migration  and invasiveness (measured using Matrigel Boyden chambers) . While tin vitro and in vivo.We used integrative gene expression profiling to identify potential mediators of the ligand-dependent phenotypic effects of OSMR overexpression in cervical SCC cells. We identified genes that were induced by OSM in cells that overexpressed OSMR but not in cells where OSMR was not overexpressed. The most significant gene ontology category groups for the differentially expressed genes included angiogenesis, cell motility/invasion, and signal transduction, consistent with the phenotype observed in OSMR-overexpressing cells after OSM treatment IL6), which is a well-known STAT3 target and further activates STAT3 HIF2α or EPAS1), which was induced by OSM in normoxic OSMR-overexpressing cells. This gene is an interesting candidate mediator of the pro-angiogenic effects of OSMR-overexpressing cells. A third gene of note was transglutaminase-2 (TGM2), also known as tissue transglutaminase, a multifunctional protein with both enzymatic (crosslinking) and non-enzymatic functions. These functions are closely related to the subcellular location of TGM2 and also depend on the patho-physiological context in vitro study, we showed that TGM2 was an important mediator of the ligand-dependent phenotypic effects of OSMR overexpression in cervical SCC cells (ITGA5) was also one of the 15 genes identified in our comparative microarray analysis of OSMR targets The 15 genes included the pro-inflammatory cytokine interleukin 6  and that the TGM2–integrin-α5β1–fibronectin pathway downstream of OSMR activation is overexpressed in head and neck as well as cervical SCCs Taking advantage of the fact that OSMR is a cell surface receptor and OSM an extracellular ligand, a rational strategy to target OSM–OSMR interactions is antibody-based inhibition. This therapeutic approach has had a major impact on haematological malignancies and solid tumours  via a range of beneficial effects, including growth arrest, induction of apoptosis, inhibition of angiogenesis, etc In vivo studies using appropriate preclinical models are now required to test the benefits of targeting this functionally significant receptor pathway in cervical and other SCCs. In addition to well-studied cell line xenografts


On day 18 or 19, anesthesia was induced 1–3 hours post feeding and a bolus of [methyl-3H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-3H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-3H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis.Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs ( In the cell, creatine (Cr) functions as a high-energy phosphate buffer via its conversion to phosphocreatine and subsequent restoration of ATP by the enzyme creatine kinase. In an omnivorous adult, both diet and endogenous synthesis play an equal role in replenishing the daily Cr loss  of approCreatine has been extensively studied as a nutritional supplement to improve exercise and sports performance –7 and haHowever, the possible metabolic consequences of GAA supplementation must also be examined. Hepatic synthesis of Cr utilizes a significant proportion of methyl groups  which co3H]methionine was obtained from American Radiolabeled Chemicals, Inc. . All other chemicals were of analytical grade and were from Sigma  or Fisher Scientific .L-[Methyl-3H]methionine via one catheter while the other was used to sample blood before and every 7 min after the bolus infusion. Thirty min following the infusion, a laparotomy was performed using cautery and the liver, right kidney, heart, brain and sample of biceps femoris muscle were immediately excised and freeze clamped. Pigs were euthanized via exsanguination while under anesthesia. A labeling period of 30 minutes following the bolus of L-[methyl-3H]methionine was chosen based on a previous study [All animal handling procedures (protocol: 11-55-RB) were performed at Memorial University and approved by the Institutional Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care. Isoflurane anesthesia was used as described below and all efforts were made to minimize suffering. Eighteen pre-pubertal Yucatan miniature pigs (14–16 weeks old) were obtained from Memorial University of Newfoundland breeding colony and weight-matched to control (n = 6), GAA supplemented (n = 6) or Cr supplemented (n = 6) groups. Pigs were group housed in a large room with ample space to run and were supplied with rubber balls for enrichment. Pigs had unlimited access to water and received standard pig grower diet which was fed twice daily. Supplemental GAA (157 mg/kg/day) or Cr monohydrate (200 mg/kg/day) was mixed with an aliquot of grower feed and hand-fed to experimental groups during each feeding for 18–19 days. The supplementation level in this study roughly corresponds to a typical Cr dosing schedule  used by humans to increase muscle Cr levels and achieve positive ergogenic effects ,22. On tus study ; in our Total homocysteine, cysteine and glutathione concentrations were determined by HPLC according to . Plasma Creatine SRA and total Cr concentration were determined using a modified method  by Lamar3H-methyl incorporation into methylated products and liver protein in 30 min was calculated as:Rate of P < 0.05.Data are presented as means ± SD. All normally distributed data were analyzed using one-way ANOVA with Newman-Keuls multiple comparison post-tests when main effects were identified. GAA concentrations were not normally distributed as determined by the D’Agostino Pearson normality test, so non-parametric Kruskal-Wallis analysis was used to determine differences among groups with Dunn’s post-tests when main effects were identified . Differences were considered significant at The percent body weight change from day 0 to day 18–19 of supplementation was 23% ± 6, 22% ± 4 and 28% ± 5 for control, Cr supplemented and GAA supplemented pigs, respectively, with no difference among groups.Compared to control pigs, GAA and Cr supplementation led to ~7.5- and ~4-fold higher hepatic Cr concentrations, respectively, and hepatic Cr was ~2-fold higher with GAA versus Cr supplementation . Muscle 3H-methyl incorporation into Cr was ~4-fold and ~3-fold higher than control and Cr supplemented pigs, respectively (3H-methyl incorporation into PC and ~50% lower 3H-methyl incorporation into hepatic protein, with no difference between Cr supplemented and control pigs. There were no differences in 3H-methyl incorporation into DNA or in the SRA of SAM among groups (In GAA supplemented pigs, the rate of ectively . Compareg groups .Supplementation with GAA resulted in a ~50% lower hepatic SAM concentration compared to both control and Cr supplemented groups while there were no differences in hepatic SAH or SAM/SAH ratio among groups . There wWe supplemented young pigs with equimolar amounts of either GAA or Cr for 18–19 days and found that while both GAA and Cr supplementation led to higher Cr concentrations in the liver and kidney compared to control pigs, GAA supplementation was more effective than Cr at increasing hepatic Cr concentrations. Furthermore, only GAA supplemented pigs had a higher concentration of muscle Cr compared to control pigs, suggesting supplemental GAA could be a potential approach to increase body Cr stores.Because of the positive ergogenic and therapeutic effects of Cr, the use of dietary Cr supplementation to increase muscle concentrations has been a growing area of interest. Although experiments in both humans and animals have demonstrated that dietary Cr supplementation can be an effective means to increase muscle Cr , some stAlthough in this study supplemental Cr had no significant effect on muscle concentrations of the metabolite, it is interesting that GAA supplementation both increased Cr in the muscle and was more effective than Cr supplementation at increasing concentrations in the liver. Because the liver is the primary site for Cr synthesis, the higher level of hepatic Cr following GAA supplementation is a result of endogenous synthesis in the liver. However, the mechanism leading to a higher level of muscle Cr could be a result of transport of hepatic Cr, endogenous synthesis in the muscle or a combination of both. Higher concentrations of extracellular Cr have been shown to downregulate Cr transport into rat muscle, with 50% inhibition occuring at physiological Cr concentrations . So it im of the transporter for Cr is similar to plasma Cr concentrations. In contrast, under normal conditions, plasma GAA concentration is more than 90% lower than the Km for gamma-aminobutyric acid transporter-2, a proposed transporter for hepatic GAA [m for its transporter, suggesting higher transport of GAA into muscle following supplementation. Although the liver has the highest level of GAMT activity, detectable levels have been found in the muscle of young pigs [Although less is known about GAA transport, experiments using isolated rat hepatocytes demonstrate a higher GAA uptake activity compared to Cr uptake . In ratsatic GAA ; howeverung pigs  so this We previously demonstrated in an acute model of GAA supplementation that Cr synthesis is not feedback regulated at GAMT , and othInterestingly, unlike rats, Cr supplementation did not lead to lower plasma homocysteine levels . The kidAlthough an omnivorous adult human typically synthesizes half of their Cr requirement , it is nThe effectiveness of GAA at increasing both hepatic and muscle Cr concentrations suggests GAA may be more favorable than Cr at increasing Cr stores. However, the higher methyl group utilization for Cr synthesis following supplementation with GAA can lead to a limited methyl group supply for other transmethylation reactions. Although a 6 week GAA supplementation trial in humans concluded that GAA is safe with a low incidence of biochemical abnormalities and an acceptable side-effects profile , the pot


This perspective considers measurements of mitochondrial function and Ca2+ handling made in both motor neuron somata and motor nerve terminals of SOD1-G93A mice at different disease stages. These complementary studies are integrated into a model of how mitochondrial dysfunction disrupts handling of stimulation-induced Ca2+ loads in presymptomatic and end-stages of this disease. Also considered are possible mechanisms underlying the findings that some treatments that preserve motor neuron somata fail to postpone degeneration of motor axons and terminals.Abundant evidence indicates that mitochondrial dysfunction and Ca Whole cell recordings using fluorescent Ca2+ indicators demonstrate that mitochondria normally buffer about 50% of large Ca2+ loads in hypoglossal motor neurons  muscles, because these motor neurons tend to have low levels of cytoplasmic Ca2+ buffers  , which favors Ca2+ entry  accelerates, restoring most of ΔΨm  , a high-conductance channel in the inner mitochondrial membrane, likely formed by dimers of ATP synthase  spinal cord or brainstem, only a fraction of which originate from motor neurons. However, Fuchs et al.  measuredm depolarization. Damiano et al.  to wild-type motor neurons found similar elevations of cytosolic [Ca2+] at moderate stimulation frequencies (40 Hz), but greater elevations at maximal stimulation frequencies  has no significant effect on stimulation-induced elevations of cytosolic [Ca2+]  and thus may be slower than the rapid passive uptake of Ca2+ into mitochondria that becomes possible following opening of the MCU.When mitochondrial Cas et al.  report i2+ (Giorgio et al., 2+] required for opening. CyPD knockout had many beneficial effects within the spinal cord, including retention of normal mitochondrial morphology, improved mitochondrial ATP synthesis, increased mitochondrial Ca2+ buffering capacity, reduced glial activation, reduced aggregation of misfolded SOD1, and improved motor neuron survival. These beneficial effects suggest an important role for mPTP opening in multiple aspects of motor neuron pathology. Nonetheless, the CyPD knockout had no beneficial effect on muscle denervation, motor axon degeneration or disease progression. This result reinforces evidence from multiple studies indicating that this disease includes a dying-back pathology (Fischer et al., In an effort to understand how mitochondrial dysfunction contributes to pathology in mutant SOD1 mice Parone et al.  complete2+, large elevations of matrix [Ca2+] can still open the mPTP in the absence of CyPD, and stimulation evokes larger-than-normal elevations of matrix [Ca2+] in motor nerve terminals of symptomatic SOD1-G93A mice (Vila et al., m depolarization and oxidative stress (Bernardi and Petronilli, 2+ influx, since motor terminals have a much higher surface-to-volume ratio and a higher density of depolarization-activated Ca2+ channels than the soma.CyPD knockout may not be sufficient to prevent mPTP opening in motor nerve terminals. Although CyPD sensitizes the mPTP to Ca2+ overload. CyPD knockout increased mitochondrial Ca2+ buffering capacity, but this single aspect of mitochondrial Ca2+ handling gives no information concerning the speed of Ca2+ uptake, a critical determinant of mitochondrial efficiency as buffers for stimulation-induced elevations of cytosolic [Ca2+]. Mutant SOD1-induced reductions in the activity of respiratory chain complexes (reviewed in Cozzolino and Carri, 2+ uptake due to reduced ability to maintain ΔΨm and reduced dynamic buffering of matrix Ca2+, and will also reduce the supply of ATP needed for Ca2+ extrusion across the plasma membrane. Thus, mutant SOD1-induced mitochondrial dysfunction might be expected to result in greater and longer-lasting stimulation-induced elevations of cytosolic [Ca2+] in motor nerve terminals, with or without mPTP opening.Inhibition of mPTP opening may not protect motor terminals from degeneration caused by chronic Ca2+-handling loads imposed on each remaining mitochondrion would be greater, increasing the likelihood of chronic Ca2+ overload and mPTP opening, as mentioned above. Consistent with this idea, although CyPD knockout preserved mitochondria in motor neuron somata, CyPD knockout did not prevent mitochondrial swelling in motor axons (motor terminal mitochondria were not examined, Parone et al., Defective fusion and axonal transport of mitochondria in mutant SOD1 mice (Magrané et al., Some of the mechanisms involved in death may be different for motor axons/ nerve terminals compared to motor neuron soma.irreversible mPTP opening is a trigger for cell death, the reversible opening of the mPTP that appears to occur in repetitively stimulated motor terminals of symptomatic SOD1-G93A mice (Nguyen et al., 2+, then close the mPTP and re-establish Ψm (reviewed by Bernardi and von Stockum, 2+ loads only episodically, as might be expected from the discharge pattern of motor neurons that innervate FF muscle fibers.While CyPD knockout may have preserved motor neuron somata by inhibiting a death mechanism specific to the central nervous system. For example, Re et al.  found thCyPD knockout may fail to prevent disease-related changes in skeletal muscles, which dominate the environment experienced by motor nerve terminals. Skeletal muscles in mutant SOD1 mice evidence signs of hypermetabolism and oxidative stress even at asymptomatic stages of disease (reviewed in Cozzolino and Carri, Maintenance of motor nerve terminals may require some activity of SOD1 in addition to its dismutase role. Degeneration of motor nerve terminals is one of the earliest events in all fALS-causing SOD1 mutations studied to date (Parone et al., Multiple possible mechanisms might explain why degeneration of motor axons/motor terminals was not postponed by CyPD knockout. Some examples:These considerations suggest that the search for mechanisms underlying ALS pathologies and effective ALS therapeutics needs to include a focus on motor axons, motor nerve terminals and/or skeletal muscle.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and β-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic β-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis  and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and β-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel. Aptenodytes forsteri) doubles its body weight in fat to survive for up to 4 months on the Antarctic ice during brooding [Body weight is tightly regulated in most species. For example, Keesey et al have shown that rats return to their baseline weight after either force feeding or force fasting . Migratibrooding . HibernaMarmota flaviventris) and 13-lined ground squirrel (Ictidomys tridecemlineatus) also become insulin resistant [Ground squirrels and other sciurid rodent hibernators actively accumulate fat in the fall, and then live off their fat stores throughout the many months of winter hibernation ,6. In thesistant ,8. Throuesistant ,10. Duriesistant  Animals esistant .The overall response to temperature changes in a circannual hibernator involve local adaptations from multiple central and peripheral organs including the skeletal muscle, adiposeOne of the most important enzymes involved in controlling fat oxidation is adenosine monophosphate-activated protein kinase (AMPK). In response to an increased ratio of AMP to total ATP, AMPK is activated by phosphorylation . TherefoWe therefore tested the hypothesis that hepatic AMPD2 and AMPK have opposing patterns of activity during periods of fat accumulation (summer) and fat oxidation (winter hibernation) in 13-lined ground squirrels traversing their yearly cycle of weight gain and loss.All Animal experiments were performed according to protocols approved by the University of Colorado Animal Care and Use Committee.ad libitum until late September or early October when they were transferred to a hibernaculum in which temperatures were maintained at 4°C in darkness; food and water were removed after the animals entered torpor until they began to emerge from hibernation in spring. To identify the various stages of hibernation based on body temperature (Tb), each ground squirrel was surgically implanted intraabdominally with a radiotelemeter and a data logger  [Ground squirrels were obtained from the University of Wisconsin 13-lined ground squirrel captive breeding program at Oshkosh. Ground squirrels were housed at the University of Colorado and maintained under 14 h light-10 h dark conditions with cat chow Systems) .en bloc and samples were used for immunohistochemistry and oil red O staining (from samples embedded in OCT and frozen) or snap frozen for protein and fat determination.Ground squirrels (n = 3 to 6 for each of the 7 timepoints) were anesthetized with isoflurane, euthanized by cardiac exsanguination, and the liver tissue snap frozen with liquid nitrogen. Livers were excised Summer active (SA), obtained in July and early August; Fall transition (FT), obtained in September and October; Interbout Arousal (IBA), approximately 3 h after reaching Tb of 35–37°C following a period of torpor; Entrance (Ent), Tb decreasing to between 27 and 23°C as animals enter a new bout of torpor; Early Torpor (ET), Tb of 4°C for less than 10% of previous torpor bout length; Late Torpor (LT), Tb 4°C for 80–95% of the time of the previous torpor bout; Arousing (Ar), Tb between 7–12°C during spontaneous arousal from torpor; Spring Active (Sp), after emergence from hibernation, the ground squirrel was homeothermic for 11–20 days and had resumed eating, but the hibernaculum was still dark and cold (4°C).The timepoints were as follows: Protein lysates were prepared from livers employing MAP Kinase lysis buffer as previously described . Six ani2O). After incubation for 30 min at 25°C, the absorbance of the samples was measured at 625 nm with a spectrophotometer. To determine the absolute specific activity of ammonia production (micromoles ammonia/min), a calibration curve was determined in the range of 5μM to 1 mM of ammonia.AMPD2 activity was determined by estimating the production of ammonia by a modification of the method described by Chaney and Marbach from celμm) were dipped in formalin, washed with running tap water, rinsed with 60% isopropanol, stained for lipids with Oil Red O and counterstained with hematoxylin. Oil red O was then extracted from the slides with isopropanol containing 4% Nonidet P-40, transferred to plastic cuvettes, and optical density (OD) was then measured at a wavelength of 520 nm.Liver tissue was embedded in Optimal Cutting Temperature gel  and frozen in liquid nitrogen. Air-dried cryostat tissue sections (8 2O2) and carbon dioxide by uricase. The H2O2 then, is determined by its reaction with the probe to generate color at approximately 571 nm. Values obtained were normalized per mg of soluble protein in the lysates. Hepatic β-hydroxybutyrate, inosine and phosphate levels in MAPK lysates were determined by enzymatic kits .For triglyceride (TG) determination in liver, fat was solubilized by homogenization in 1 ml solution containing 5% nonidet P40 (NP-40) in water, slowly samples were exposed to 80–100°C in a water bath for 5 minutes until the NP-40 became cloudy, then cooled down to room temperature. Samples were then centrifuged for 2 min to remove any insoluble material. Triglyceride determination with the VetAce autoanalyzer consisted in their initial breakdown into fatty acids and glycerol. Glycerol oxidation generates a product that reacts with the probe to produce color at 570 nm. Similarly, uric acid determination is based in the conversion of uric acid to allantoin, hydrogen peroxide , uric acid and allantoin were identified and quantified in ground squirrel plasma samples by Metabolon by mass spectrometry as described previously .All data are presented as the mean ± standard deviation (SD). Data graphics and statistical analysis were performed using Instat (version 3.0) and Prism 5 . Data were analyzed using ANOVA followed by the Tukey-Kramer multiple comparison test with α = 0.05. In all cases experiments were performed 3 times with independent replicates. Total data points  are identified in Figure legends.We first evaluated the ground squirrel livers for fat by the presence of oil red-O staining and triglyceride content Fig –1C. ThesWe also examined the abundance of both total and activated AMP kinase. The active AMPK, phosphorylated at threonine 172 (P-AMPK), is important in oxidation of fatty acids. ECH1 (enoyl CoA hydratase-1) and CPT1A -rate-limiting enzymes in β-fatty acid oxidation- and β-hydroxybutyrate (a ketone generated during fat oxidation) levels were also assessed for a more complete picture of fatty acid oxidation activity. As shown in Fig 10 effects (a measure of how changes in temperature affects enzyme activity) and indicating that the enzymatic activity of AMPD2 is immeasurably low while animals are in torpor (ET and LT). Hibernating animals warm briefly to 37°C during each interbout arousal (IBA). The ratio of AMPD2 activity to P-AMPK (reflecting AMPK activity) when the animal’s body temperature is at 37°C  is lowest during IBA (ratio 15.71), compared to summer active , fall transition (FT ratio 71.42), and spring (Sp ratio 61.11). Thus, the relative activity of AMPD2 to AMPK is higher during the non-hibernating months, declines during the period of hibernation, with marked reduction  at the physiologically relevant temperature of torpor  in the liver. AMPD2 protein abundance tended to be highest in summer (SA) and during the fall transition (FT), with lower levels during the hibernation period, but the differences were not significant . AMPD2 epor 4°C, .Inosine and uric acid are products from AMP metabolism through AMPD2. In this regard, in states where AMPD2 is active, levels of uric acid and inosine significantly raise. Consistently, hepatic uric acid, inosine and IMP levels were highest in the summer and fall transition and decreased during the hibernation season Fig  and 5D. We also measured plasma levels of fat oxidation markers  during torpor-arousal cycles. Uric acid levels were low in IBA and Entering, but accumulated during torpor and early arousing periods. The decreased serum uric acid during IBA was followed by decreased allantoin in Entering. Allantoin began to reaccumulate by ET and continued to accumulate through LT and early arousing.Circannual hibernators accumulate fat during the summer and live off their fat stores throughout winter ,6. WhileA summary schematic is shown in During summer the animal must store fat in preparation for the winter season. We provide evidence for increased fat synthesis with minimal evidence for fat oxidation in the liver in SA animals, in association with the development of moderate hepatic steatosis. During the summer months and fall transition there remains high AMPD2 activity with low levels of activated AMPK in the liver, consistent with a pattern of fat synthesis and storage.With hibernation a more complex pattern was observed, especially as it relates to AMPK activity and fatty acid oxidation. AMP kinase is well known to be activated during starvation where it has a role in oxidizing fat, and generating ATP ; as suchIt should be mentioned that there have been two prior studies that examined hepatic AMPK activation in tissues from hibernating ground squirrels ,41. One Besides stimulating AMPK, AMP can also act as a substrate for AMPD2, which depletes AMP by converting it to inosine monophosphate (IMP) and eventually to inosine, hypoxanthine, xanthine, uric acid and, in most mammals including ground squirrels, allantoin. As such, AMPD2 may act to block the activation of AMP kinase by reducing the available pool of AMP . In addiTo evaluate this possibility, we evaluated AMPD2 activity and hepatic uric acid levels at various time points throughout the year. During the summer AMPD2 protein levels were high, associated with high AMPD2 activity and high intrahepatic uric acid, inosine and IMP levels, and this was paralleled by increased abundance of enzymes involved in fat synthesis and elevated hepatic triglyceride levels. During this period P-AMPK was low and there was minimal evidence for fatty acid oxidation.A more complex pattern was observed during torpor-arousal cycles in hibernation. P-AMPK, increased markedly during interbout arousal and then fell continuously throughout the remainder of the torpor-arousal cycle, consistent with slow degradation in torpor coupled with the inability to initiate protein synthesis until body temperature increases during arousal . On the Previous studies have reported how metabolic rate is modified during hibernation and torpor in mammals,19,20. MWe are unaware of any previous studies that have examined AMPD2 activity in the liver during hibernation. Nevertheless, it is known that both inosine  and uricWe also examined plasma uric acid and allantoin, the latter being a metabolite of uric acid. Previous studies have found a fall in serum uric acid in the hedgehog when they enter into torpor , and likIn summary, the hibernating 13-lined ground squirrel actively increases its fat stores in the liver during the summer, and this is associated with relatively high AMPD2 activity, high intrahepatic fat and uric acid stores, and low P-AMPK activity. During hibernation fat synthesis is turned off while fat oxidation is increased and P-AMPK is activated, especially during the IBA. Meanwhile AMPD2 activity remains the same or falls slightly and intrahepatic uric acid levels fall. During torpor both P-AMPK and AMPD2 decline, but some evidence for fat oxidation is still present, likely due to persistent effects of P-AMPK on ECH1 and CPT1A. In the spring the liver fat is nearly absent, and fat oxidation is again turned off and fat synthesis stimulated. During this time intrahepatic uric acid levels are still low. However, while we could not demonstrate this, others have reported that serum uric acid levels increase with emergence from hibernation in spring ,49,50 co


Results indicated several positively selected sites located in the region comprising the two heptad repeats (HR1 and HR2) and their linker. Two sites (R652 and V1060) were positively selected in the betaCoVs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. During the most recent evolution of MERS-CoV, adaptive mutations in the HR1 (Q/R/H1020) arose in camels or in a previous host and spread to humans. We determined that different residues at position 1020 establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the HRs. A similar effect on stability was observed for a nearby mutation (T1015N) that increases MERS-CoV infection efficiency in vitro. Data herein indicate that the heptad repeat region was a major target of adaptive evolution in MERS-CoV-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function.Middle East respiratory syndrome coronavirus (MERS-CoV) originated in bats and spread to humans via zoonotic transmission from camels. We analyzed the evolution of the  The spike protein assembles as a trimer on the viral surface and belongs to the class I fusion protein familyHost proteases cleave the CoV spike protein into two functionally distinct domains: the N-terminal region  contains the receptor binding domain (RBD), whereas the C-terminal portion (S2 subunit) includes the fusion peptide, two heptad repeats (HR1 and HR2), and the transmembrane (TM) domain1313Because of its central role in membrane fusion, a number of antiviral peptides that interfere with the 6HB formation have been developed as potential therapeutic compounds against HIV17Although the RBD of spike proteins is generally considered the major determinant of host range, several reports have suggested that variation in the C-terminal portion of spike proteins, particularly in the HR1 and HR2, determine host range expansion78Motivated by the notion that evolutionary analyses can provide information on the molecular events that underlie host shifts and, more generally, host-pathogen interactionsS genes of viruses isolated from humans/camels (MERS-CoV), bats, and hedgehogs  from unreliably aligned codons and we screened them for evidence of additional recombination events using GARD (Genetic Algorithm Recombination Detection)codeml branch-site testThe saturation of substitution rates represents a major problem in the detection of positive selection among distantly related sequences. Computation of the nonsynonymous (dN) and synonymous (dS) substitution rates over whole phylogenies allows breaking of long branches, resulting in improved rate estimation. Thus, evidence of episodic positive selection was searched for by using the spike gene as a result of in vitro adaptation of zoonotic strains to primate cells26One positively selected site was found in the S1 region, 7 sites in the S2 subunit , Table 1No positively selected site was found to be located in the RBD . Neverthspike gene was therefore analyzed using the codeml site models, which test the hypothesis that a subset of codons evolve with dN/dS > 1. No evidence of positive selection was found, even using the relatively non-conservative model M7/model M8 comparison We next wished to determine whether positive selection occurred at the spike sequences of MERS-CoV isolated from camels or humans  showed better fit to the data than the null models (NSsite models M1a and M7), strongly supporting the action of positive selection  A and 2A The structure of the 6HB of MERS-CoV has been solved13in vitroTo gain further insight into the effect of adaptive evolution at position 1020, we performed a stability computational analysis after in silico mutagenesis. Q1020 was replaced with all other possible aminoacids: even if changes of different magnitude in ΔG were obtained using three different methods3233We analyzed the evolution of the S protein in betaCoVs, in bat Ty-BatCoV HKU4 and Pi-BatCoV HKU5 viral populations, as well as in MERS-CoV isolates from the current outbreak. Different strategies were applied, as appropriate depending on divergence and recombination.Results indicated that several adaptive changes are located in the S2 region, with fewer in the S1 domain and none of these within the RBD. It should be noted, though, that in all analyses we applied quite conservative approaches and we intersected two different methods to declare a site as positively selected. Whereas this approach was meant to limit the false positive rate it may have yielded some false negative results. In particular, the branch-site test we used to analyze the S1 and S2 regions of betaCoVs is robust to saturation issues and has a minimal false positive rate, but lacks power20coronavirus genus. The Beaudette strain of IBV has been adapted to embryonated chicken eggs; following passages in culture, the strain was further adapted to infect Vero cells (from African Green monkey) and primary chicken kidney cellscoronavirus genus, but that use a class I fusion protein. For instance, one single mutation in the HR1 of a simian-human immunodeficiency virus (SHIV) strain (KB9) increases by two- to three-fold infection efficiency in cells expressing the marmoset cellular receptorsAnalysis of both betaCoVs and MERS-CoV strains revealed evidence of positive selection in the S2 region. Most positively selected sites were found to be located either in the heptad repeats or in the intervening linker. Among these sites, position 1060 is particularly interesting, as it almost corresponds to substitutions that modify the host range and/or cell tropism in MHV and IBV26in vitro4041The molecular mechanisms underlying the altered phenotype of HR1 and HR2 mutants remain to be determined in all these instances, although changes in conformational structure have been suggested as a possible explanation. Unfortunately, no coronavirus S protein fusion intermediate or pre-fusion state has been solved to date, hampering investigation of molecular interactions. We therefore analyzed the effect of variation at the 1020 position of MERS-CoV on the stability of the 6HB in the post-fusion conformationCoronavirus spike proteins are highly exposed on the virus surface and represent major targets for antibody responsein vitroFinally, it is worth noting that HRs have been studied in different viruses because synthetic peptides interfering with 6HB formation are promising antiviral molecules151617Virus sequences were retrieved from the NCBI database and a list of accession numbers is provided as Errors in the inferred multiple sequence alignment (MSA), which may be common when highly divergent sequences are analyzed, can inflate estimates of positive selection. We therefore used PRANKS gene of clade c betaCoVs we applied the branch-site test from the PAML suite2 distribution with one degree of freedomTo detect positive selection at the Positively selected sites were identified through the BEB analysis , which calculates the posterior probability that each site belongs to the site class of positive selection on the foreground branch(es). Sites were validated using MEME (with the default cutoff of 0.1), which allows the distribution of dN/dS  to vary from site to site and from branch to branch at a site, therefore allowing the detection of episodic positive selectionThe site models implemented in PAML were applied -independently- for the analysis of HKU4 and MERS-CoV sequences, which display very limited divergence and do not suffer from saturation problems. To detect selection, site models that allow  or disallow  a class of sites to evolve with ω > 1 were fitted to the dataTo assure consistency, all models were run using the F3 × 4 and the F61 codon frequency models.MSAs were screened for the presence of recombination using GARD. Recombination breakpoints were considered significant if the HK (Kishino-Hasegawa) p value was <0.01.Simultaneous inference of selection and recombination for analysis of positive selection was performed using omegaMapThe REL (random effects likelihood) analysis models variation in both dN and dS across sites according to a predefined distribution with different rate classes; positively selected sites are identified through an empirical Bayes methodhttp://www.datamonkey.org).For GARD, MEME, and REL the nucleotide substitution models were chosen using a Genetic Algorithm implemented in the dataMonkey suiteThe crystal structure of MERS-CoV HR1 and HR2 region was obtained from PDB (PDB ID: 4MOD).Histidine or arginine residues were introduced at positions 1020 and suitable rotamers were sampled through the rapid torsion scan utility in Maestro . Intraprotein interactions were calculated with PIC Because programs that calculate stability changes achieve only moderate accuracyΔΔG = ΔGmutant − ΔGwild-type. In FoldX and I-Mutant ΔΔG values > 0 kcal/mol indicate mutations that decrease protein stability, whereas in PoPMuSiC ΔΔG values > 0 kcal/mol are mark of mutations increasing protein stability. Therefore, PoPMuSiC ΔΔG values were multiplied by −1 to obtain homogeneous results.In FoldX and I-Mutant the ΔΔG values are calculated as follows: In the analysis carried out with FoldX 3D, the three-dimensional structure of the protein was repaired using the <RepairPDB> command. Mutations were introduced using the <BuildModel> command with <numberOfRuns> set to 5 and <VdWdesign> set to 0. Temperature (298K), ionic strength (0.05 M) and pH (7) were set to default values and the force-field was used to predict the water molecules on the protein surface.How to cite this article: Forni, D. et al. The heptad repeat region is a major selection target in MERS-CoV and related coronaviruses. Sci. Rep.5, 14480; doi: 10.1038/srep14480 (2015).


Feeding conditions can influence dopamine neurotransmission and impact behavioral and neurochemical effects of drugs acting on dopamine systems. This study examined whether eating high fat chow alters the locomotor effects of cocaine and dopamine transporter activity in adolescent  and adult  male Sprague-Dawley rats.Dose-response curves for cocaine-induced locomotor activity were generated in rats with free access to either standard or high fat chow or restricted access to high fat chow (body weight matched to rats eating standard chow).Compared with eating standard chow, eating high fat chow increased the sensitivity of adolescent, but not adult, rats to the acute effects of cocaine. When tested once per week, sensitization to the locomotor effects of cocaine was enhanced in adolescent rats eating high fat chow compared with adolescent rats eating standard chow. Sensitization to cocaine was not different among feeding conditions in adults. When adolescent rats that previously ate high fat chow ate standard chow, sensitivity to cocaine returned to normal. As measured by chronoamperometry, dopamine clearance rate in striatum was decreased in both adolescent and adult rats eating high fat chow compared with age-matched rats eating standard chow.These results suggest that high fat diet-induced reductions in dopamine clearance rate do not always correspond to increased sensitivity to the locomotor effects of cocaine, suggesting that mechanisms other than dopamine transporter might play a role. Moreover, in adolescent but not adult rats, eating high fat chow increases sensitivity to cocaine and enhances the sensitization that develops to cocaine. Drug use during adolescence often predicts an increased likelihood of continued use into adulthood . For exaCocaine increases extracellular dopamine (DA) by blocking DA uptake via the high-affinity DA transporter (DAT) . ConvergThe aim of this study was to determine the effects of eating high fat chow on the locomotor-stimulating effects of cocaine and on DAT activity in striatum in adolescent and adult male rats. In experiment 1, rats ate either standard or high fat chow for either 1 or 4 weeks and then were tested once with cocaine. It was hypothesized that eating high fat chow would increase sensitivity of rats to the acute effects of cocaine based on the evidence that rats eating high fat chow have increased sensitivity to drugs acting directly at D2 and D3 receptors , 2010, aoC, 50 ±10% relative humidity) under a 12-h-light/-dark cycle with water available continuously. Rats had either free or restricted access to chow (see below), except during experimental sessions when no food was available . Animals were maintained and experiments were conducted in accordance with the Institutional Animal Care and Use Committee, the University of Texas Health Science Center at San Antonio, and with the Guide for Care and Use of Laboratory Animals .A total of 152 male Sprague-Dawley rats  were used in the study. Seventy-eight adolescent rats at postnatal day (PND) 19 to 21 weighed 80 to 85g upon arrival, and 76 adult rats at PND 68 to 72 weighed 250 to 300g upon arrival; all rats were housed individually in an environmentally controlled room (24±1Rats were fed either standard or high fat chow beginning at either PND 25/26 (for adolescents) or PND 75/76 (for adults) and according to the conditions described below also see . The nutIn experiment 1, a single test with cocaine was conducted after adolescent and adult rats (n = 6/feeding condition) had free access to either standard or high fat chow or restricted access (adjusted daily to match body weights of rats with free access to standard chow) to high fat chow for either 1 or 4 weeks. Next, all rats in experiment 1 had free access to standard chow for 4 weeks followed by a second test with cocaine. In experiment 2 , tests wFour Plexiglass chambers, measuring 26×61×23cm high  and located within ventilated sound-attenuating cubicles , were used to assess locomotor activity . Two of At the beginning of experiment 1, adolescent (n=36) and adult (n=36) rats with free access to standard chow were acclimated to the locomotor chamber for 120-minute periods on each of 2 days . After the 2-day acclimation period, all adolescent and adult rats were tested again with saline then assigned to their respective feeding condition at PND 25 or 75, respectively. After either 1 (PND 32 or 82) or 4 (PND 53 or 103) weeks on these feeding conditions, adolescent and adult rats (n= 6/feeding condition) were tested once with cumulative doses of cocaine . Two days after the test with cocaine, all rats were again tested with saline to determine whether acute testing with cocaine or eating high fat chow changed basal locomotion . Next, the 36 rats (18 adolescents and 18 adults) in experiment 1 that were tested with cocaine after 4 weeks on a particular diet were given free access to standard chow for an additional 4 weeks prior to redetermination of a second cocaine dose-response curve . Initially, adolescent (n=18) and adult (n=18) rats in experiment 2 had free access to standard chow and were acclimated to the locomotor chamber as described above for experiment 1. After the acclimation period, rats were tested with cumulative doses of cocaine  at PND 25 or 75. Tests with cocaine were identical to experiment 1 except that smaller doses were studied and rats received only 5 injections in a session. After an initial cocaine test, adolescent and adult rats were assigned to their respective feeding condition  and thereafter were tested with cocaine once per week for 4 weeks. Two days after the last cocaine test, all rats were tested again with saline to determine whether repeated testing with cocaine or eating high fat chow changed basal locomotion .Chronoamperometry was performed in adolescent (n=7–8/feeding condition) and adult (n=6–8/feeding condition) rats that had free access to either standard or high fat chow or restricted access to high fat chow for 1 week immediately prior to testing . Adolesc80, the time (s) for the signal to decline by 80% of the maximal amplitude. The effects of feeding condition on T80 and signal amplitude for 80 pmol DA in adolescent rats and 100 pmol DA in adult rats were analyzed using a 1-way ANOVA (feeding condition). The mean area under the curve  was also calculated for each cocaine dose-response curve and for chronoamperometry functions. A 1-way, repeated-measures ANOVA was used to determine whether the AUC in adolescent or adult rats with free or restricted access to high fat chow was significantly different from the AUC in adolescent or adult rats with free access to standard chow (GraphPad Prism), respectively; posthoc multiple comparisons were made with the Dunnett’s test. For all tests, significance was set at P<.05.For locomotor activity experiments, data for 5-minute periods beginning 10 minutes after injections are expressed as mean (±SEM) activity counts for each group and plotted as a function of dose. A 2-way (dose and feeding condition), repeated-measures ANOVA with posthoc Bonferroni’s test was used to determine whether cocaine-induced locomotor activity in adolescent or adult rats with free or restricted access to high fat chow was significantly different from activity in adolescent or adult rats with free access to standard chow . Chronoamperometry data were analyzed using the following 3 signal parameters: (1) the maximal amplitude (in μM) of the signal; (2) clearance rate (in nM/s), defined as the slope of the decay curve from 20% to 60% of maximal signal amplitude ; and (3) TCocaine was provided by the Research Technology Branch, National Institute on Drug Abuse  and dissolved in sterile 0.9% saline. DA hydrochloride, DOPAC, ascorbic acid, Nafion (5%), chloralose, and urethane were purchased from Sigma-Aldrich . Chloralose, urethane, DA, DOPAC, and ascorbic acid were dissolved in 0.1M phosphate buffered saline. Drug and saline were administered i.p. in a volume of 1mL/kg.Cocaine increased locomotor activity in a dose-related manner in adolescent and adult rats, with maximum increases occurring at cumulative doses of 32 and 56mg/kg, respectively  . After eCocaine increased locomotor activity in adolescent (PND 25) rats eating standard chow . The effData from cocaine dose-response curves in the left panels (A-C) of Cocaine increased locomotor activity in adult (PND 75) rats eating standard chow . The eff80 values in rats eating high fat chow compared with rats eating standard chow (The rate of DA clearance in the striatum was decreased in adolescent (PND 32 or 33) rats eating high fat chow (free or restricted access) compared with adolescent rats eating standard chow . The AUCard chow . Furtherard chow . Lastly,The first major finding of the current study is that eating high fat chow, in adolescent but not adult male rats, increases sensitivity to the locomotor stimulating effects of acutely administered cocaine and enhances the development of sensitization to repeatedly administered cocaine. These findings in male rats are in contrast to the effects reported for the same dietary conditions in female rats, whereby eating high fat chow in both adolescent and adult female rats increased sensitivity to acutely administered cocaine and facilitated the development of sensitization to repeatedly administered cocaine . Thus, aThe underlying mechanism(s) whereby eating high fat chow might alter sensitivity to the locomotor effects of cocaine and DAT activity remains unclear. However, eating high fat chow can modify circulating concentrations of insulin, and insulin directly alters the expression and activity of DAT in a manner such that low concentrations of insulin decrease  and highCompared with DAT and DA clearance, much less is known about the effects of reduced insulin signaling and eating high fat chow on DA receptor expression and function as well as extracellular DA concentrations see . In addiThat eating high fat chow did not increase sensitivity to cocaine in adult rats (as it did in adolescent rats) might be due to age-related differences in DA systems and/or the manner in which high fat chow impacts these systems. In rats, DA receptor density, particularly D1 and D2 receptors, increases between PND 25 and 40 (adolescent) then decreases between PND 40 and 120 (late adolescent to adult) . WhetherOne important aspect of the present study is that in adolescent rats, diet-induced changes in sensitivity to the acute effects of cocaine were not accompanied by differences in body weight. Thus, it is the type of chow  that adolescent rats eat, and not changes in body weight, which most likely contributes to their increased sensitivity to cocaine. Moreover, and as shown in other studies, not all effects of a drug change in a similar manner across feeding conditions, suggesting that changes in the locomotor effects of cocaine were not due to pharmacokinetic factors , 2011. IRepeated administration of cocaine can induce a progressive and enduring enhancement of locomotor stimulating effects . Although there are a number of reports showing cocaine-induced sensitization in adult rats , sensitiWhen adolescent rats that ate high fat chow were returned to eating standard chow, sensitivity to the locomotor stimulating effects of cocaine returned to control values . It is possible that longer access to high fat chow  would have more enduring effects on sensitivity to cocaine even when rats are returned to standard chow. Several studies have shown that the younger rats are when they are exposed to some drugs, the greater their sensitivity is to the same drugs in adulthood . This fiIn summary, the current study demonstrates that eating high fat chow during adolescence significantly enhances sensitivity to acutely administered cocaine as well as to repeatedly administered cocaine . This finding is sex-dependent, with both adolescent males (present study) and females  showing None.


Snake venoms contain many proteinaceous toxins that can cause severe pathology and mortality in snakebite victims. Interestingly, mRNA encoding such toxins can be recovered directly from venom, although yields are low and quality is unknown. It also remains unclear whether such RNA contains information about toxin isoforms and whether it is representative of mRNA recovered from conventional sources, such as the venom gland. Answering these questions will address the feasibility of using venom-derived RNA for future research relevant to biomedical and antivenom applications.Venom was extracted from several species of snake, including both members of the Viperidae and Elapidae, and either lyophilized or immediately added to TRIzol reagent. TRIzol-treated venom was incubated at a range of temperatures (4–37°C) for a range of durations (0–48 hours), followed by subsequent RNA isolation and assessments of RNA quantity and quality. Subsequently, full-length toxin transcripts were targeted for PCR amplification and Sanger sequencing. TRIzol-treated venom yielded total RNA of greater quantity and quality than lyophilized venom, and with quality comparable to venom gland-derived RNA. Full-length sequences from multiple Viperidae and Elapidae toxin families were successfully PCR amplified from TRIzol-treated venom RNA. We demonstrated that venom can be stored in TRIzol for 48 hours at 4–19°C, and 8 hours at 37°C, at minimal cost to RNA quality, and found that venom RNA encoded multiple toxin isoforms that seemed homologous (98–99% identity) to those found in the venom gland.The non-invasive experimental modifications we propose will facilitate the future investigation of venom composition by using venom as an alternative source to venom gland tissue for RNA-based studies, thus obviating the undesirable need to sacrifice snakes for such research purposes. In addition, they expand research horizons to rare, endangered or protected snake species and provide more flexibility to performing fieldwork on venomous snakes in tropical conditions. Venomous snakes use venom proteins to capture their prey. When a human is bitten, these proteins cause severe pathology and often lead to death, disability or other serious complications. Understanding the composition of snake venom is essential not only for developing more effective snakebite treatments but also for acquiring a better understanding of the biology that underpins this extraordinary group of animals. Currently, to determine the genes expressing these venom proteins requires sacrificing the snake and isolation of mRNA from the venom gland. This is undesirable for ethical reasons and limits the scope of this type of research. Encouragingly, we and others have recently demonstrated that mRNA encoding venom proteins can be obtained from lyophilized snake venom. In this study we show that venom RNA in these samples is in fact considerably degraded. We report a protocol to improve the yield of undegraded RNA (immediate addition of TRIzol reagent to the venom). We also explored the limitations of this new protocol, in terms of time and temperature applicability. We then showed that considerable isoform diversity can be captured from snake venom RNA, and show that the isoforms identified are identical to those found in the venom gland of sacrificed snakes. Our findings provide protocols enabling researchers working in a range of fields to considerably expand the scope of their research because venom composition data can now be reliably acquired from venom which in turn can be obtained from live snakes without harming the animals. Venomous snakes are highly proficient predators that occupy almost all natural habitats: from desert to tropical jungle and all terrestrial environments within this range, as well as marine and freshwater bodies ,2. The eEach venomous snake species/genus has evolved a distinct repertoire of venom proteins to achieve their predatory objective . Thus, tShaw’s group in Belfast were the first to document that mRNA encoding toxins can be recovered from the venom or skin secretions of several species ,25, and All snakes were maintained in individual cages within the temperature, humidity and light-controlled environment of the herpetarium of the Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine. The facility and its protocols for the expert husbandry of the snakes are regularly inspected and approved by the UK Home Office and the LSTM Animal Welfare and Ethical Review Board.Bitis arietans), (ii) a single, captive bred, adult specimen of the intermediate shield-nose snake (Aspidelaps scutatus intermedius), (iii) six Angolan coral snakes (Aspidelaps lubricus cowlesi), (iv) four cape coral snakes (Aspidelaps lubricus lubricus) and (v) a single monocled cobra (Naja kaouthia). The number of snakes used in this study was the maximum possible given the limited availability of these animals in our facility. We also used data from a separate study wherein venom glands were dissected from terminally-anaesthetised specimens of B. arietans, and immediately snap frozen in liquid nitrogen.Venom was extracted and pooled from (i) ten, wild-caught, Nigerian puff adders  after 8, 24 or 48 hours. A sample of venom was also conventionally lyophilized and stored at 4°C until required. Each aliquot equated to 500 μL of liquid venom . All other venom samples had TRIzol added at a ratio of 5:1 and were processed using the TRIzol Plus RNA Purification System. All samples were DNase treated on column during the TRIzol Plus purification system protocol  and total RNA was eluted in 30 μL nuclease free water (QIAGEN).To assess the potential benefit of mRNA-stabilising reagents, we added TRIzol reagent (Life Technologies) to the venoms at a ratio of 5:1 as soon after venom extraction as safety permitted. RNA was isolated from the venom using the TRIzol Plus RNA Purification System following the manufacturer’s instructions (unless stated otherwise). To assess the effect of storage time and temperature on venom RNA stability, a sample of the Bitis arietans venom gland tissue (272 mg) was immediately homogenised under liquid nitrogen using a pestle and mortar and then a TissueRuptor (QIAGEN). Samples were DNase treated  and total RNA was extracted and eluted in 30 μL nuclease free water (QIAGEN) using the TRIzol Plus RNA Purification System protocol. Venom gland transcriptomes were sequenced, assembled and annotated as previously described .LYO) or TRIzol treated (VTRZ) venom samples (including after variant storage temperature and times), was compared using a Bioanalyzer . The Bioanalyzer uses the entire electrophoretic trace (electropherogram) of the RNA sample including the presence/absence of degradation products in a sample concentration-independent manner. We added 1 μL of each total RNA sample into Agilent RNA 6000 Nano/Pico Chips (following the manufacturer’s instructions) and then inserted them into the Agilent 5000 Bioanalyzer for analysis. The quality of the two ribosomal RNA peaks are used as a reliable indicator of the integrity of the other RNA molecules. Calculations are based upon quantitating the amount of fluorescent dye intercalated within the RNA, thereby efficiently discounting contaminants such as phenol (a common carry over from RNA isolation preparations) that can vitiate the accuracy of standard UV-spectrometry. Agilent’s RNA 2100 Expert software was used to calculate total RNA concentration and assign a RNA integrity number (RIN), which range from 1–10; with 10 being completely undegraded, and 1 being completely degraded RNA [The quality and quantity of RNA isolated from conventionally lyophilized  primers and processed it into single-stranded cDNA using the Superscript III first-strand synthesis system (Invitrogen). Each single-stranded cDNA sample was treated with RNase H to eliminate contaminating RNA. This process provided a stable and reproducible copy of each venom/venom gland RNA preparation.B. arietans serine proteases (SP), snake venom metalloproteinases (SVMPs), L-amino acid oxidases (LAO), C-type lectins (CTL), vascular endothelial growth factors (VEGF) and aminopeptidases (AMP) were designed (B. arietans transcriptome data [Primers complimentary to genes encoding designed  and syntome data .2 (PLA2), CTLs, SVMPs and Kunitz-type serine protease inhibitors (KSPI) (Aspidelaps spp. (the objective of the experiment). We therefore decided to pool the venom RNA of N. kaouthia (that has known venom gland gene isoform diversity) with that from A. s. intermedius, A. l. cowlesi, and A. l. lubricus, thereby ensuring the presence of multiple gene targets for PCR amplification. Furthermore, since there are no published genes encoding Aspidelaps 3FTXs, PLA2s, CTLs, SVMPs or KSPIs, our primers were designed upon conserved sequences flanking the open reading frames of published sequences from several species of the genus Naja (cobras), Walterinnesia aegyptia (desert black cobra) and Ophiophagus hannah (king cobra). These snakes are the closest phylogenetic relatives of the genus Aspidelaps [To assess whether mRNA recovered from venom includes representation of transcripts encoding multiple diverse isoforms, we generated primers complementary to the three-finger neurotoxin (3FTX)  gene fams (KSPI) . Howeverpidelaps . To maxi2O (QIAGEN) to a final volume of 50 μL. PCR experiments were performed using a Techne TC-512 thermal cycler. Touchdown PCR conditions were as follows: Initial denaturation at 94°C for 3 min. Denaturing at 94°C, 30 sec; annealing temperature gradient 66–50°C, 1 min (-1.6°C/cycle); extension at 72°C, 1 min; 10 cycles. Denaturing at 94°C, 30 sec; annealing at 50°C, 1 min; extension at 72°C, 1 min; 25 cycles. Final extension at 72°C for 10 min. Amplicons were visualised on a 1% agarose in TBE gel, and analysed using the Bio-Rad Gel Doc System and Image Lab Software (Bio-Rad).One μL of venom cDNA was added to 5 μL 10 x PCR buffer, 5 μL 2 mM dNTP mixture (Fisher Scientific), 2.5 μL 10 μM forward primer, 2.5 μL 10 μM reverse primer, 1 μL DreamTaq polymerase (Life Technologies) and 33 μL nuclease-free HBitis arietans amplicons, generated from highly stringent, gene-specific primers for SVMP, LAO, SP, VEGF and AMP, were submitted for Sanger sequencing . The amplicons from the pooled elapid venoms were generated with less species- and gene-specific primers and therefore were likely to contain divergent sequences. Since we sought to capture this diversity, we purified amplicons from each PCR reaction from the agarose gel using the Promega Wizard Kit (Promega), cloned these into pCR2.1-TOPO vectors (Life Technologies), transformed chemically competent E. coli  to amplify the recombinant plasmids, and submitted the purified plasmids  for sequencing as above. This was also completed for the B. arietans venom CTL amplicons.Sequencing files were trimmed of vector sequence and base calls with <30 PHRED scores using SeqTrace , translaLYO) because most venom collections, including the samples used in all previous publications on venom mRNA, are/will be prepared in this manner (extracted venom is frozen for 3+ hours and then lyophilized into a dehydrated powder). The RIN number of VLYO was 4.6  had an RIN number of 7.7  was substantially greater than that from the same amount of VLYO sample (5 ng). Nevertheless, these quantities of venom RNA are 1000-fold less than that obtained from venom gland tissue (35 μg from 272 mg of tissue). Current Next Generation Sequencing (NGS) technologies routinely require greater than 1 μg total RNA as a starting point. Thus, although the total RNA obtained from VTRZ is of sufficient quality, there is insufficient quantity of total RNA for standard (non-PCR amplified) transcriptome NGS.Consistent with the RNA quality analysis, the total yield of RNA  obtainedTRZ, but they provide no indication as to the quality/protein-encoding validity of the underlying mRNA. To investigate this, we designed primers to PCR-amplify full-length transcripts encoding B. arietans venom proteins from the VLYO and VTRZ samples. We selected toxin sequence templates that we had verified as differing with respect to transcript size and proportional representation [The Bioanalyzer results suggest that total RNA is largely undegraded in Ventation , i.e. SVB. arietans VLYO and VTRZ cDNA  and for different durations  than those used above (4°C and 0 hours) to determine if the TRIzol-treatment protocol was suited to recovering RNA from venoms collected under field conditions.Having shown the benefit of TRIzol-treating venom to acquire undegraded full-length toxin transcripts, we next explored the effects of storing VTRZ after storage at all time and temperature conditions tested show distinct ribosomal peaks (LYO (red) samples. The quantity of total RNA recovered from VTRZ samples decreased with extended storage periods and for all storage temperatures  different sequencing techniques, (ii) from venom glands and (iii) from Viperidae venoms, we compared PCR-derived B. arietans CTL genes obtained from VTRZ RNA to those obtained from venom gland samples (a) after PCR amplification, (b) as Expressed Sequence Tags (EST) from a cDNA library, and (c) as NGS sequences. As anticipated, the VTRZ CTL sequences included analogues (98–99% nucleotide sequence identity) of the isoforms recovered from venom gland and by whatever sequence protocol used  was in fact extensively degraded. We therefore modified the experimental approach, by omitting the lyophilization step and instead treating venom with TRIzol immediately after extraction (VTRZ). We show here that the complete coding regions of the transcripts can be PCR amplified from both VLYO and VTRZ samples  at minimal cost to RNA degradation Figs . FinallyLYO, but as the incubation time increased the yield of total RNA decreased, although the quality remained stable. This is important, particularly for the collection of venom during fieldwork, where access to adequate storage conditions or laboratory access for sample processing are often very limited. Our study has identified the value of TRIzol processing of snake venom. This therefore suggests that other similar products may also have comparable scientific benefits, and will require the similar protocol-standardising analysis as performed here with TRIzol.That RNA quality remains high, irrespective of an increase in temperature or time, likely explains why the majority of transcripts were successfully PCR amplified. These results suggest that the incubation of venom in TRIzol helps to reduce degradation of RNA, including mRNA, but that it comes at a duration-to-yield cost. For example, incubation of venom in TRIzol at 19°C for all durations resulted in a greater yield of high quality RNA than was possible from VTRZ over VLYO .Considering the fact that snake venom contains a diverse array of destructive nucleases and phosphodiesterases  and is aLYO Figs  and 2 suB. arietans venom and venom gland . The pooling of venom sourced from many snakes of the same species would reduce this limitation, but would of course impart its own set of limitations on subsequent analysis, particularly given reports of intra-specific variation in venom composition –15. RNA S1 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.


Clostridium difficile infection (CDI), with increases in incidence and severity of disease in many countries worldwide. The incidence of CDI has also increased in surgical patients. Optimization of management of C difficile, has therefore become increasingly urgent. An international multidisciplinary panel of experts prepared evidenced-based World Society of Emergency Surgery (WSES) guidelines for management of CDI in surgical patients.In the last two decades there have been dramatic changes in the epidemiology of  Clostridium difficile infection (CDI) in many countries worldwide  of 51) than with vancomycin . Fidaxomicin may be useful for treating patients who are considered at high risk for recurrence (elderly patients with multiple comorbidities who are receiving concomitant antibiotics). However, it is important to note that there are no data available on the efficacy of Fidaxomicin in severe life-threatening disease.Fidaxomicin was non-inferior to vancomycin for initial cure of CDI in two prospective trials , 165. InThe use of other antibiotics such as tigecycline , 175 fusPatients with fulminant colitis (FC) who progress to systemic toxicity require surgical intervention.3), hemodynamic instability, use of anti-peristaltic medications, and a clinical triad of increasing abdominal pain, distention and diarrhea.To determine clinical predictors for the development of fulminant colitis in patients with CDI a 10-year retrospective review of FC patients who underwent colectomy was performed and compared with randomly selected age- and sex-matched non-fulminant CDI patients at a single institution study by Girotra in 2012 . Predict18) Patients with severe CDI who progress to systemic toxicity should undergo early surgical consultation and evaluated for potential surgical intervention (Recommendation 1 C).Patients with severe CDI who progress to systemic toxicity are likely to have serious comorbidities. Delaying surgery in this group leads to increased likelihood of adverse outcomes , althougThere are no reliable clinical and/or laboratory findings that can predict those patients who will respond to medical therapy and those who will subsequently need surgery .C. difficile colitis were published in a recent systematic review by Stewart et al. [C. difficile colitis.Data comparing mortality rates between surgical and medical treatment for fulminant t et al. . Five huPatients presenting with organ failure  also need prompt intervention.The timing of surgical intervention is the key for survival of patients with FC –185.Seder et al.  describe9/L or equal to or less than 2,000 x 109/L was assigned 1 point, cardiorespiratory failure was assigned 7 points, and diffuse abdominal tenderness on physical examination was assigned 6 points. A value of 6 points was determined to be the threshold for reliably dividing low-risk (<6) from high-risk (≥6) patients. Only patients with cardiorespiratory failure or diffuse abdominal tenderness were high risk.Recently a risk scoring system (RSS) for daily clinical practice was designed by van der Wilden et al. . Age greFerrada et al.  reviewedMany factors have been described as predictors of mortality in patients who undergo emergency intervention.C difficile colitis. In 199 patients (4.1 %) with fulminant C difficile colitis the in-hospital mortality rate was 34.7 %. Independent predictors of mortality included age 70 years or older, severe leukocytosis or leukopenia  or bandemia , and cardiorespiratory failure (intubation or vasopressors). Survival rates were higher in patients who were cared for by surgical vs nonsurgical departments.Sailhamer et al.  reviewedC difficile colitis in the USA [3/mm3), coagulopathy , and renal insufficiency (blood urea nitrogen > 40 mg/dL) were also associated with a higher mortality.The ACS-NSQIP database from 2005 to 2010 was used by Lee et al. to study emergency open colectomies performed for  the USA . The oveC. difficile colitis was published by Banghu et al. [C. difficile colitis. It concluded that the strongest predictors for postoperative death were those relating to preoperative physiological status: preoperative intubation, acute renal failure, multiple organ failure and shock requiring vasopressors.A systematic review and meta-analysis of outcomes following emergency surgery for u et al. . Thirty-19) Resection of the entire colon should be considered to treat patients with fulminant colitis (FC) (Recommendation 1 B).20) Diverting loop ileostomy with colonic lavage may be a useful alternative to resection of entire colon (Recommendation 2 C).21) Patients with FC should be treated with high dose oral or by enema vancomycin  in combination with intravenous metronidazole . (Recommendation 1 C).C. difficile colitis.In the Bhangu et al. meta-analysis  the most9/L or lactate between 2.2 and 4.9 mmol/L.To evaluate the role of emergency colectomy in patients with FC, and to identify subgroups of patients that may benefit Lemontagne et al.  publisheDiverting loop ileostomy with antegrade colonic lavage may be a colon preserving alternative to total colectomy , 194. To22) Supportive measures, including intravenous fluid resuscitation and electrolyte replacement, should be provided to all patients with severeC. difficileinfection (Recommendation 1 C).Diarrhea results in significant volume depletion and electrolyte abnormalities, and fluid and electrolyte imbalance should be promptly corrected , 120.23) Early detection of shock and aggressive management of underlying organ dysfunction are essential for optimum outcomes in patients with fulminant colitis (Recommendation 1 C).Early detection and prompt aggressive treatment of the underlying organ dysfunction is an essential component of improving outcome of critical ill patients .Severe CDI may present with a fulminant course and may be associated with great morbidity and high mortality. Physiologic support including close invasive monitoring in an intensive care unit setting and aggressive resuscitation are often necessary in fulminant colitis.C difficile infection (RCDI) occurs in approximately 20 % of patients and is challenging to treat [Recurrence is diagnosed when CDI recurs <8 weeks after the onset of a previous episode, provided the symptoms from the previous episode resolved after completion of initial treatment and other causes have been excluded. Symptomatic recurrent to treat . Patient24) Agents that may be used to treat the first recurrence of CDI include metronidazole, for non-severe RCDI, and vancomycin for severe RCDI. (Recommendation 1 B).25) Fidaxomicin may be used as an alternative agent (Recommendation 1 B).A systematic review on the treatment of RCDI was recently published . Metroni26) In subsequent recurrence of CDI (2nd or later) oral vancomycin or fidaxomicin is recommended (Recommendation 1 B).Vancomycin and fidaxomicin are equally effective in resolving CDI symptoms but fidaxomicin has been shown to be associated with a lower likelihood of CDI recurrence after a first recurrence , 197. Ho27) Probiotics may be considered as an adjunctive treatment to antibiotics for immunocompetent patients with RCDI (Recommendation 2 B).Saccharomyces boulardii CNCM I-745 in recurrent CDI. The first demonstrated a lower relapse rate compared with a placebo control group (35 vs 65 % in the placebo group) [S. boulardii (1 g/d) with high dose vancomycin (2 g/d) was more effective than high dose vancomycin and placebo (17 vs 50 % recurrence rate) [L. rhamnosus GG or L. plantarum 299v) were stopped prematurely due to enrollment problems [Little evidence exists to support the use of probiotics in the first episode of CDI . Two rano group)  and the ce rate) . Other sproblems . Probiotproblems .Lactobacillus acidophilus CL1285 and Lactobacillus casei LBC80R and a combined analysis of those studies with four studies that used Saccharomyces boulardii, showed lower CDI rates in recipients of probiotics compared with recipients of placebo  Intestinal or faecal microbiota transplantation (IMT or FMT) may be an effective option for the treatment of RCDI (Recommendation 1 B).Intestinal or faecal microbiota transplantation (IMT or FMT) has been considered as an alternative therapy to treat RCDI –208. It C. difficile strains to grow and produce CDI. By reintroducing normal flora via donor faeces, the imbalance may be corrected, and normal bowel function re-established [The rationale of FMT is that disruption of the normal balance of colonic flora allows ablished .FMT has not been widely adopted as a therapeutic tool probably due to concerns regarding safety and acceptability .A systematic literature review of IMT treatment for RCDI and pseudomembranous colitis was published in 2011 by Gough et al. . In 317 Recently a systematic review was published by Cammarota et al. . Twenty In a recently published randomized clinical trial by van Nood et al.  patientsFMT may be administered via enemas or as a slurry given via a nasogastric tube. In the fall of 2014, Youngster et al.  reported29) FMT may be effective in immunocompromised patients and patients who have had solid organ transplants (Recommendation 2 B).Patients who are immunocompromised are at increased risk of CDI. During the last two years the first data on FMT in immunocompromised patients began to appear in the medical literature .A multicenter retrospective series on the use of FMT in immunocompromised (IC) patients with CDI that was recurrent, refractory, or severe was published in 2014 . ReasonsThis series demonstrated the effective use of FMT for CDI in IC patients with few serious adverse events or related adverse events.30) IVIG should only be used as adjunct therapy in patients with multiple recurrent or fulminant CDI until results from large, randomized controlled trials are available (Recommendation 2 C).C. difficile colonization is a major determinant of magnitude and duration of clinical manifestations. Passive immunization with IVIG has been reported to be successful in several small series. A review by Abourgergi [IVIG treatment has been proposed based on the evidence that the level of immune response to ourgergi  of fifte31) Infusion with monoclonal antibodies may be of use to prevent recurrences of CDI, particularly in patients with CDI due to the 027 epidemic strain (Recommendation 2 C).P = 0.06); among patients with more than one previous episode of CDI, recurrence rates were 7 and 38 %, respectively (P = 0.006). The authors concluded that the addition of monoclonal antibodies against C. difficile toxins to antibiotic agents significantly reduced the recurrence of C. difficile infection. The findings of this study require confirmation before firm recommendations can be made.In a phase II clinical trial , the use32) Tube feeding patients should be clinically assessed due to their risk for developing CDI (Recommendation 2 C).C difficile associated diarrhea (20 versus 8 % p = 0.03). One of the reasons may be prolonged use of elemental diets. It is known that critically ill patients tolerate feeding well if the feed is given in elemental form and delivered beyond the stomach into the jejunum because it is totally absorbed within the upper small intestine [C. difficile colonization and subsequent infection. In feeding tube patients the conversion of elemental diet feeding to a diet containing adequate indigestible carbohydrate after the first week of critical illness may, in theory, be useful.It is widely accepted that enteral nutrition (EN) maintains gut mucosal integrity which leads to decreased intestinal permeability, decreased infections, and an improved immunological status. EN during episodes of diarrhea may be well tolerated and may improve enterocyte healing and maintenance of enzyme activity , 215. ENntestine . Elementntestine . The resRecently, Puri et al.  reportedC. difficile toxin A though additional studies are needed to demonstrate its clinical benefit before recommendations can be made [Studies have also investigated the possible value of exogenous Phosphatidylcholene (PC) administration for reinforcement of the mucus layer , 223. Mu be made , 223.33) The use of anti-peristaltic agents for the treatment of CDI should be discouraged. If anti-peristaltic, if used in isolation agents, are used to control persistent symptoms in patients with CDI they must always be accompanied by medical therapy (Recommendation 2 C).A review of the literature regarding anti-motility treatment of CDI found 55 patients with CDI who were exposed to anti-motility agents .Nine patients (16 %) died, and 27 patients (49 %) had unknown outcomes. Seventeen patients (31 %) with CDI developed colonic dilation; 5 of these patients with severe CDI died. However, all patients who experienced complications or died were given anti-motility agents alone initially, without an appropriate antibiotic and 23 patients who received metronidazole or vancomycin co-administered with the anti-motility agent experienced no complications. Further study of the role of anti-motility agents in providing symptomatic relief and reducing environmental contamination with infectious stool may be warranted though, until there is clear evidence of benefit, their use in patients with CDI should be avoided .34) Proper antimicrobial stewardship in selecting an appropriate antibiotic and optimizing its dose and duration to cure an infection may prevent the emergence ofC. difficile(Recommendation 1 B).Despite vigorous infection control measures until recently, CDI was causing an increasing problem in healthcare facilities worldwide. As CDI is thought to follow disruption to the normal bacterial flora of the colon occurring as a consequence of antibiotic use , it is l35) Patients with suspected or proven CDI should be placed in contact (enteric) precautions (Recommendation 1 B).Prompt identification of patients with symptomatic CDI is essential so that appropriate isolation precautions can be put into effect.This is particularly important in reducing environmental contamination as spores can survive for months in the environment , despiteContact (enteric) precautions patients with CDI should be maintained until the resolution of diarrhea, which is demonstrated by passage of formed stool for at least 48 h. Patients with known or suspected CDI should ideally be placed in a private room , 230 witThis is supported by a retrospective cohort of 2859 patients by Chang et al. . Patient36) Hand hygiene with soap and water is a cornerstone of the prevention ofC. difficile. Hand hygiene, contact precautions and good cleaning and disinfection of the environment and patient care equipment, should be used by all health-care workers contacting any patient with known or suspected CDI (Recommendation 1 B).C. difficile. Alcohol-based hand sanitizers are highly effective against non–spore-forming organisms, but they may not kill C. difficile spores or remove C. difficile from the hands [Hand hygiene with soap and water and the use of contact precautions along with good cleaning and disinfection of the environment and patient equipment, should be used by all health-care workers contacting any patient with known or suspected CDI. Hand hygiene is a cornerstone of prevention of nosocomial infections, including he hands , 234.The most effective way to remove them from hands is through hand washing with soap and water.C. difficile transmission is ongoing [For environmental cleaning, hypochlorite disinfection such as sodium hypochlorite solutions are suggested for regular use in patient areas where  ongoing .C. difficile [Though disposable glove use during care of a patient with CDI may be effective in preventing the transmission of ifficile , these m