IGF-1 stabilizes goat sperm mitochondrial transmembrane potential and reduces dna fragmentation.

BACKGROUND: Antioxidant present in sperm cells protects them from oxidative damage. However, sperm are more susceptible to peroxidative damages due to the loss of these enzymes during cryopreservation and their survival and fertility may be compromised. Insulin like growth factor-1 (IGF-1) has an antioxidant effect and could maintain sperm motility. OBJECTIVE: To improve seminal parameters, mitochondrial membrane potential (MMP), oxidative status and DNA integrity of buck semen after freeze-thawing by fortification of goat semen diluent with various concentrations of IGF-1. MATERIALS AND METHODS: Fifty ejaculates were collected and were extended with tris- citric acid- fructose diluent with 10% egg yolk and 6% glycerol with sperm concentrations of 1×108 mL-1. Post-cryopreserved sperm were assessed for motility and a range of other functional parameters. RESULTS: In post-thaw semen sperm motility, live sperm count, acrosome integrity, hypo-osmotic swelling positive spermatozoa, malondialdehyde (MDA), protein carbonyl content (PCC), TUNEL positive sperm differed significantly (P<0.05) with the various concentrations of IGF-1 used. Sperm functional parameters post-thawing were significantly (P<0.05) better in 250 ng/mL IGF-1. IGF-1 protects against lipid peroxidation by lowering MDA and PCC production, thus reducing the harmful effect of reactive oxygen species. The kidding percentage using the artificial insemination technique was significantly higher ( i.e., 40%) in the group supplemented with 250 ng/mL of IGF-1 than in the non-supplemented group (i.e., 30%). CONCLUSION: IGF-1 may be used to improve post-thaw semen quality and fertility as measured by actual kidding rate. Doi.org/10.54680/fr23610110312.

Diagnosis of urogenital tuberculosis by multiplex-nested PCR targeting mpt64 (Rv1980c) and IS6110: comparison with multiplex PCR and GeneXpert® MTB/RIF.

A multiplex-nested PCR (M-nested PCR) targeting mpt64 (Rv1980c) + IS6110 was designed to detect Mycobacterium tuberculosis (Mtb) DNA within urine (n = 35), endometrial biopsies (n = 22) and menstrual blood (n = 3) of male/female UGTB patients, and results were compared with M-PCR using the same targets. Detection limit of the purified Mtb DNA was found to be 1 fg by M-nested PCR, which was 106 -fold lower than M-PCR. Moreover, sensitivities of 100% and 81·8% were obtained in confirmed (n = 5) and clinically suspected UGTB (n = 55) cases, respectively, by M-nested PCR, with a specificity of 97·1% (n = 70). Sensitivities attained by M-nested PCR were significantly higher (p < 0·05) than M-PCR in both clinically suspected and total UGTB (n = 60) cases. To confirm the true PCR-negative results, an internal amplification control, that is, human ß-globin gene (hbb) was incorporated in the M-nested PCR/M-PCR assays, wherein all the clinical specimens (positive/negative for mpt64/IS6110) were found to be positive for hbb. Some UGTB specimens (n = 35) were also subjected to GeneXpert® MTB/RIF assay that revealed a significantly lower (p < 0·001) sensitivity (17·1 vs 88·6%) than M-nested PCR, although high specificity (100%) was attained with GeneXpert. After validating the results in a higher number of UGTB specimens, our M-nested PCR may be translated into an attractive diagnostic kit.

Parthenogenetic activation of buffalo (Bubalus bubalis) oocytes: comparison of different activation reagents and different media on their developmental competence and quantitative expression of developmentally regulated genes.

This study was carried out to compare the efficacy of different methods to activate buffalo A + B and C + D quality oocytes parthenogenetically and to study the in vitro developmental competence of oocytes and expression of some important genes at the different developmental stages of parthenotes. The percentage of A + B oocytes (62.16 ± 5.06%, range 53.8-71.3%) was significantly higher (P < 0.001) compared with that of C + D oocytes (37.8 ± 5.00%, range 28.6-46.1%) retrieved from slaughterhouse buffalo ovaries. Among all combinations, ethanol activation followed by culture in research vitro cleave medium gave the highest cleavage and blastocyst yields for both A + B and C + D grade oocytes. Total cell numbers, inner cell mass/trophectoderm ratio and apoptotic index of A + B group blastocysts were significantly different (P < 0.05) from their C + D counterpart. To determine the status of expression patterns of developmentally regulated genes, the expression of cumulus-oocyte complexes, fertilization, developmental competence and apoptotic-related genes were also studied in parthenogenetically produced buffalo embryos at different stages, and indicated that the differential expression patterns of the above genes had a role in early embryonic development.

Effects of treatment with a microRNA mimic or inhibitor on the developmental competence, quality, epigenetic status and gene expression of buffalo (Bubalus bubalis) somatic cell nuclear transfer embryos.

Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.

Utility of 18 F-FDG PET/CT in pre-surgical risk stratification of patients with breast cancer.

OBJECTIVE: To determine the correlation between fluorine-18-fluorodeoxyglucose (18F-FDG) uptake values and clinicopathological prognostic markers using preoperative 18F-FDG positron emission tomography/computed tomography (PET/CT) in primary breast cancer (BC). SUBJECTS AND METHODS: One hundred and twelve patients with primary BC were studied prospectively. Pretreatment 18F-FDG PET/CT was performed. Maximum standardized uptake values (SUVmax) were compared with various clinicopathological variables. RESULTS: In a univariate analysis, SUVmax correlated well with the following prognostic variables: T stage, absence of progesterone receptor (PR), absence of estrogen receptor (ER), triple negative lesions (ER/PR and Her 2 negative) and high histologic grade. Metastatic lesions and ductal lesions had higher SUVmax than lobular carcinoma. No significant correlation was found between SUVmax,and human epidermal growth factor receptor 2 (Her-2) statusor perineural and lymphovascular invasion. Multivariate analyses showed that breast density, tumor size and PR negativity were significantly correlated with SUVmax (P=0.046 and 0.009, respectively). CONCLUSION: The pre-treatment tumor SUVmax could be utilized as an independent imaging biomarker of the tumor aggressiveness and poor prognosis. Risk stratification based on this index could play a pivotal role in alteration of treatment planning, such as neoadjuvant chemotherapy (precision oncology).

m-carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos.

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50 µM) for 10 hr from the start of reconstruction till activation. At 10 µM, but not at other concentrations examined, CBHA increased (p < .05) the blastocyst rate (63.77 ± 3.97% vs 48.63 ± 3.55%) and reduced (p < .05) the apoptotic index of the cloned blastocysts (8.91 ± 1.94 vs 4.36 ± 1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78 ± 0.74). CBHA treatment, at all the concentrations examined, increased (p < .05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10 µM) decreased (p < .05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p < .05) than in IVF blastocysts. CBHA (10 µM) treatment increased (p < .05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p < .05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10 µM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.

Treatment of buffalo (Bubalus bubalis) donor cells with trichostatin A and 5-aza-2'-deoxycytidine alters their growth characteristics, gene expression and epigenetic status and improves the in vitro developmental competence, quality and epigenetic status of cloned embryos.

We examined the effects of treating buffalo skin fibroblast donor cells with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a DNA methyltransferase (DNMT) inhibitor, on the cells and embryos produced by hand-made cloning. Treatment of donor cells with TSA or 5azadC resulted in altered expression levels of the HDAC1, DNMT1, DNMT3a, P53, CASPASE3 and CASPASE9 genes and global levels of acetylation of lysine at position 9 or 14 in histone 3 (H3K9/14ac), acetylation of lysine at position 5 in histone 4 (H4K5ac), acetylation of lysine at position 18 in histone 3 (H3K18ac) and tri-methylation of lysine at position 27 in histone 3 (H3K27me3). Moreover, global levels of DNA methylation and activity of DNMT1 and HDAC1 were decreased, while global acetylation of H3 and H3K9 was significantly increased in comparison to untreated cells. Simultaneous treatment of donor cells with TSA (50nM) and 5azadC (7.5nM) resulted in higher in vitro development to the blastocyst stage, reduction of the apoptotic index and the global level of H3K27 me3 and altered expression levels of HDAC1, P53, CASPASE3, CASPASE9 and DNMT3a in cloned blastocysts. Transfer of cloned embryos produced with donor cells treated with TSA led to the birth of a calf that survived for 21 days. These results show that treatment of buffalo donor cells with TSA and 5azadC improved developmental competence and quality of cloned embryos and altered their epigenetic status and gene expression, and that these beneficial effects were mediated by a reduction in DNA and histone methylation and an increase in histone acetylation in donor cells.

Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation.

In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

Genome-wide association study of bipolar disorder accounting for effect of body mass index identifies a new risk allele in TCF7L2.

Bipolar disorder (BD) is associated with higher body mass index (BMI) and increased metabolic comorbidity. Considering the associated phenotypic traits in genetic studies of complex diseases, either by adjusting for covariates or by investigating interactions between genetic variants and covariates, may help to uncover the missing heritability. However, obesity-related traits have not been incorporated in prior genome-wide analyses of BD as covariates or potential interacting factors. To investigate the genetic factors underlying BD while considering BMI, we conducted genome-wide analyses using data from the Genetic Association Information Network BD study. We analyzed 729,454 genotyped single-nucleotide polymorphism (SNP) markers on 388 European-American BD cases and 1020 healthy controls with available data for maximum BMI. We performed genome-wide association analyses of the genetic effects while accounting for the effect of maximum BMI, and also evaluated SNP-BMI interactions. A joint test of main and interaction effects demonstrated significant evidence of association at the genome-wide level with rs12772424 in an intron of TCF7L2 (P=2.85E-8). This SNP exhibited interaction effects, indicating that the bipolar susceptibility risk of this SNP is dependent on BMI. TCF7L2 codes for the transcription factor TCF/LF, part of the Wnt canonical pathway, and is one of the strongest genetic risk variants for type 2 diabetes (T2D). This is consistent with BD pathophysiology, as the Wnt pathway has crucial implications in neurodevelopment, neurogenesis and neuroplasticity, and is involved in the mechanisms of action of BD and depression treatments. We hypothesize that genetic risk for BD is BMI dependent, possibly related to common genetic risk with T2D.

Comparative Expression Analysis of Gametogenesis-Associated Genes in Foetal and Adult Bubaline (Bubalus bubalis) Ovaries and Testes.

This study was conducted to identify and analyse the expression of gametogenesis-associated genes and proteins in foetal and adult buffalo gonads of both the sexes. Relative quantification of the genes was determined by qPCR and Western blotting. Immunohistochemistry was also performed for various gametogenesis-associated proteins in foetal and adult gonads of both the sexes. We observed significantly (p < 0.05) increased expression of primordial germ cell-specific, meiotic as well as genes associated with oocyte maturation and development in foetal ovaries as compared to the adult ones. However, significantly (p < 0.05) increased expression of proteins associated with oocyte maturation like GDF9 and ZP4 was found in adult ovaries, indicating temporal regulation of mRNA translation during oogenesis. Meiotic genes showed significantly (p < 0.05) increased expression in adult testes as compared to foetal testes and ovaries, indicating onset of meiosis at a later stage in spermatogenesis. In general, the expression of primordial germ cell-associated as well as meiotic genes was higher in adult testes, indicating the increased biological activity in the organ. Immunohistochemistry revealed localized expression of gametogenesis-associated proteins in ovarian follicles and seminiferous tubules of testes, while the surrounding somatic tissues were devoid of these proteins. The study gives an understanding of the sequential and temporal events of gene expression as well as mRNA translation during male and female gametogenesis. It could also be concluded that follicles and seminiferous tubules are the functional units of the female and male gonads, respectively, and their function could be enhanced by appropriate chemical and genetic intervention of the somatic tissue immediately surrounding them. This assumes importance in the context that buffalo attains sexual maturity at an older age of 2-3 years and have smaller ovaries with lesser number of primordial follicles in comparison with cattle, which is suggested to be the main reason of their poor breeding performance.

Early cleavage of handmade cloned buffalo (Bubalus bubalis) embryos is an indicator of their developmental competence and quality.

Following IVF, embryos which cleave early have been shown to have higher developmental competence and quality than those that cleave relatively later across many species. We investigated the effect of time of cleavage on the developmental competence, quality, epigenetic status and gene expression in buffalo embryos produced by handmade cloning (HMC). Following classification of embryos as early cleaving (EC) or late cleaving (LC) based on whether they had cleaved or not at 24 h post in vitro culture, 54% (164/303) were found to be EC and the rest to be LC. The blastocyst rate (58.1 ± 3.4 vs 36.9 ± 1.6%, p < 0.01) and the total cell number (285.5 ± 41.9 vs 141.4 ± 36.1, p < 0.05) were higher, whereas the apoptotic index (3.6 ± 0.6 vs 12.2 ± 1.7, p < 0.01) and the global level of H3K9ac and H3K27me3 were lower (p < 0.05) in the blastocysts produced from EC than in those produced from LC embryos. The relative transcript level of CASPASE3, CASPASE7, DNMT1, DNMT3a and CDX2 was higher (p < 0.05) and that of SOX2 was lower (p < 0.05) in blastocysts produced from LC than in those produced from EC embryos, whereas the expression level of CASPASE6, P53, P21, HDAC1, OCT4 and NANOG was not significantly different between the two groups. These results show that (i) following HMC, blastocysts produced from embryos that cleave early differ from those produced from late cleaving embryos in terms of epigenetic status and expression level of many important apoptosis-, pluripotency-, trophectoderm- and epigenetics-related genes, and (ii) EC embryos are superior to LC embryos in view of their higher developmental competence and quality.

Spontaneous abortion is associated with elevated systemic C5a and reduced mRNA of complement inhibitory proteins in placenta.

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto-maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion.

Developmental competence and expression pattern of bubaline (Bubalus bubalis) oocytes subjected to elevated temperatures during meiotic maturation in vitro.

OBJECTIVE: To determine the direct effect of physiologically relevant high temperatures (40.5 and 41.5 °C) for two time periods (12 and 24 h) on bubaline oocytes during in vitro maturation. METHOD: The control group oocytes were cultured at 38.5 °C for 24 h. The treatment 1 (T1) and 3 (T3) group oocytes were cultured at 40.5 and 41.5 °C respectively, for the first 12 h and at 38.5 °C for rest of the 12 h. However, treatment 2 (T2) and 4 (T4) group oocytes were cultured at 40.5 and 41.5 °C for complete 24 h. RESULTS: Development of oocytes to blastocyst was severely compromised (p < 0.001) when matured at 40.5 and 41.5 °C for both exposure periods (12 h and 24 h). It was found that the cleavage rates, blastocyst yield and mean cell number decreased remarkably (p < 0.001) in the treatment groups compared to control. The relative mRNA expression of heat shock protein (Hsp 70.1, 70.2, 70.8, 60, 10 and HSF1), pro-apoptotic (caspases-3, -7, -8, Bid and Bax) and oxidative stress (iNOS) related genes was significantly higher (p < 0.05) in all the treatment groups compared to control. However, mRNA abundance of anti-apoptotic (Bcl-2, Mcl-1, Bcl-xl), glucose transport (Glut1, Glut3 and IGF1R), developmental competence (ZAR1 and BMP15) and oxidative stress (MnSOD) related genes was significantly decreased (p < 0.05) in the treatment groups compared to control. CONCLUSION: The present study clearly establishes that physiologically relevant elevated temperatures during in vitro meiotic maturation reduce developmental competence of bubaline oocytes.

Production of wild buffalo (Bubalus arnee) embryos by interspecies somatic cell nuclear transfer using domestic buffalo (Bubalus bubalis) oocytes.

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin-18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open-pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re-expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified-warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.

Death of a child due to venom toxicity following multiple honey bee stings: A case report.

Most bee stings are not life-threatening. Bee venom often causes local, mild allergic reactions in people, but even a single bee sting may induce a fatal anaphylactic reaction. Usually, anaphylactic reaction is the cause of death, but, when a child suffers multiple stings (more than 30), direct toxicity of venom can also be fatal. A three-year-old male child was brought to the hospital with pain, swelling and redness at the sting sites. He had more than 35 stings at various sites over his face, on his tongue and over his body. He died 10 hours after the incidence of the honey bee stings and was maintaining oxygen saturation until the terminal stage of his life. At autopsy, the honey bee sting sites showed redness, swelling and a small effusion of blood surrounding the stinger tracks. On the tongue two stingers were found in situ. Facial puffiness and eyelid swelling, along with congested organs, were also found, but features suggestive of anaphylactic death like airway oedema, mucous plug or cyanosis were absent. Hospital treatment records show that blood pressure remained low with tachycardia despite treatment. Having regard for all the evidence it was concluded that death was due to multiple honey bee stings that caused direct venom toxicity.

Suicide by a construction nail fired from a pistol.

Penetrating head injury to accomplish suicide by a non-ammunition-related projectile discharged from a nail-gun is a very rare entity. The authors describe even much rarer, and the first reported case of a suicide penetrating head injury by a construction nail discharged from a blank cartridge of a pistol. The absence of beveling and muzzle impression, the non-ejection of the discharged cartridge, and the exit of just the tip of the nail from the other side of wound were the atypical features in this firearm fatality sustained at a contact-range. The entry wound prototypes like abrasion and grease collar, and blackening were absent. An improvisation to insert a construction nail into the chamber of firearm, for utilization as a projectile was another unique highlight here. The deceased was a construction builder. Being debt-ridden, he probably could not manage to purchase even one live cartridge for his licensee pistol to bring suicidal ideation to culmination.

Oral Cavity Cancers: Ethnic Differences in Radiotherapy Outcomes in a Majority South Asian Leicester Community.

AIMS: Squamous cell carcinoma oral cavity cancers (SCCOCCs) have a higher reported incidence in South Asian countries. We sought to compare presenting stage and outcome by ethnicity in patients with SCCOCC treated with radical radiotherapy in a single centre in the UK. MATERIALS AND METHODS: All patients with SCCOCC treated with radical radiotherapy at an oncology department in Leicester (UK) between 2011 and 2017 were identified. Baseline demographic, clinical data and 2-year treatment outcomes were reported. RESULTS: Of the 109 patients included, 40 were South Asian and 59 were non-South Asian. South Asians had significantly poorer 2-year disease-free survival compared with non-South Asians (54.6% versus 73%, P = 0.01). CONCLUSION: Our analysis suggests that South Asians with SCCOCC have poorer outcomes despite a younger age and similar disease characteristics. Environmental, social factors and differing biology of disease may be responsible and further research is required to inform targeted interventions.

Effect of TGF-ß1 superfamily members on survival of buffalo (Bubalus bubalis) embryonic stem-like cells.

This study examined the effects of supplementation of ES-like cell culture medium with bone morphogenetic protein (BMP)-4 (0, 10, 20 or 100 ng/ml) or Noggin (250, 500 or 750 ng/ml) or TGF-ß1 (0, 0.1, 1 or 10 ng/ml) or SB431542 (0, 10, 25 or 50 µm), an inhibitor of TGF-ß1 signalling, on survival, colony area and expression level of pluripotency genes in buffalo ES-like cells at passage 40-80, under different culture conditions. BMP-4 supplementation significantly reduced (p < 0.05) colony survival rate, percentage increase in colony area and relative mRNA abundance of OCT4, whereas that of NANOG and SOX-2 was increased significantly (p < 0.05). Noggin supplementation did not affect the colony survival rate and percentage increase in colony area in the presence of FGF-2 and LIF. In the presence of FGF-2 alone, it significantly reduced (p < 0.05) the relative mRNA abundance of OCT4 and SOX-2 and increased (p < 0.05) that of NANOG. Supplementation with TGF-ß1 at 1.0 ng/ml but not at other concentrations increased colony survival rate but had no effect on percentage increase in colony area at any concentration. Supplementation with SB-431542 decreased (p < 0.05) colony survival rate at 50 µm but not at other concentrations. The percentage increase in colony area was lower (p < 0.05) with 10 µm SB-431542 than that in the controls, whereas at higher concentrations of 25 or 50 µm, SB-431542 decreased (p < 0.05) the colony size instead of increasing it. In conclusion, these results suggest that BMP-4 induces differentiation in buffalo ES-like cells, whereas TGF-ß/activin/nodal pathway may not be playing a crucial role in maintaining pluripotency in these cells.

Buffalo (Bubalus bubalis) ES cell-like cells are capable of in vitro skeletal myogenic differentiation.

When buffalo embryonic stem (ES) cell-like cells that expressed surface markers SSEA-4, TRA-1-60, TRA-1-81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX-1 and NUCLEOSTEMIN as confirmed by RT-PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three-dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF-68 and NESTIN (ectodermal lineage), BMP-4 and α-skeletal actin (mesodermal lineage), and α-fetoprotein, GATA-4 and HNF-4 (endodermal lineage). When these EBs were cultured on gelatin-coated dishes, they spontaneously differentiated to several cell types such as epithelial- and neuron-like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10(-8) M or 10(-7) M retinoic acid for 25 days, ES cells could be directed to form muscle cell-like cells, the identity of which was confirmed by expression of α-actinin by immunofluorescence and of MYF-5, MYOD and MYOGENIN genes by RT-PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell-like cells to undergo directed differentiation to cells of skeletal myogenic lineage.