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Bona fide embryonic stem cell (ESC) lines from livestock species have been challenging to derive and maintain, contrasting mouse and human ESCs. However, induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells tender an option, as they display characteristic features of ESC. The comprehension that induced pluripotent stem cells (iPSC) could be created with in no time also holds the potential of allowing pluripotent cells to be derived from animal models vital in biomedical research. Endeavors to produce bona fide pluripotent stem cells (PSC) from livestock have been going on for more than two decades. But, attempts to derive bona fide livestock iPS cells have met with limited success. Recently it’s been reported that small molecules can augment reprogramming efficiency and may be used to substitute few or all transcription factors used for reprogramming. It is assumed that the reprogramming factors are conserved among species, and this small molecule reprogramming approach will probably apply to livestock species as well. So this review will focus mainly on the accomplishments of small molecules on accelerating cell reprogramming and obtaining naive pluripotency, and raise a new insight on, exogenous genes free, livestock naive iPSC generation with a new bullet, small molecule.

Treatments to repair the human heart following regenerative diseases remain a challenge for medical science. Unlike lower vertebrate species the human heart lacks a regenerative pathway meaning that research has to be focused on cell transplantation. Porcines (Sus scrofa) are excellent models for cardiovascular disease and pluripotent stem cells (PSCs) generated from porcines will provide important clinical insights for cardiac cell therapy. This could open a new avenue of research into degenerative conditions as porcine is a more effective human proxy to work with. However, bona fide PSCs are currently available onlyin rodents (mouse, rat) and primates (monkey, human). Attempts to derivepluripotent stem cells (PSCs) from porcine have been going on for more than two decades with slow progress. Despite the fact that the porcine stem cells are under increasing glare of publicity due to milestone achievements in this area of research. Advances in stem cell technology, especially the genetic engineering, innovative cell culturing and induced pluripotency to generate stem cells has dramatically revolutionized the basic and applied investigations and applications of porcine stem cells. This review attempts to summarize the major signaling pathways involved in maintenance of pluripotency and the state of the art conceptual and technical progress for generating bona fide porcine PSCs.

Embryonic pluripotent stem cells (ePSCs) can generate all somatic cell types, as well as functional gametes. In mouse and rat, derivation of ePSCs from the early epiblast is promoted by the double inhibition ("2i") of mitogen-activated protein kinase kinase (MAP2K), antagonizing fibroblast growth factor signaling (FGF), and glycogen synthase kinase 3 (GSK3), stimulating the WNT pathway. However, it has remained unclear whether this culture regime is applicable to nonrodent livestock species. Here we report the generation of bovine ePSCs under minimal conditions. Inner cell masses (ICMs) were immunosurgically isolated from in vitro fertilized bovine blastocysts and cultured feeder-free in 2i medium. Dual kinase inhibition primed bovine ICMs for stem cell derivation and sustained expression of epiblast-specific pluripotency markers SOX2 and NANOG, while repressing the hypoblast marker GATA4. Following mechanical passage, 2i supported limited proliferation for several weeks. Continuously cultured ePSC lines expressed discriminatory markers of naïve pluripotency and primordial germ cells, but not of primed epiblast stem cells. In female ePSCs, most OCT4-positive cells lacked epigenetically silenced X-chromosomes, displaying a diagnostic feature of naïve pluripotency. Bovine ePSCs maintained a normal karyotype and differentiated into derivatives of all three germ layers in suspension culture. This culture system provides a screening platform for factors that maintain long-term proliferation of pluripotent embryonic cattle cells without genetic intervention.

In humans injured myocardium cannot avert the onset and progression of ventricular dysfunction because of limited regenerative ability of myocytes. Although limited renaissance of cardiomyocytes has been reported in human infarcted hearts, it is generally accredited that non-functional fibrous tissue replaces the dead myocardium. High cardiovascular morbidity and dearth of donor hearts warrant a constant hunt for radically different approach to treat heart failure. Pluripotent stem (PS) cells possess the ability to produce functional cardiomyocytes for clinical applications and drug development, which may provide the answer to this problem. Although progress has been made in differentiating human PS cells into cardiomyocytes, however, the in vitro differentiation of pluripotent cells into cardiomyocytes involves a poorly defined, inefficient and relatively non-selective process. A thorough understanding of signaling pathways would tender a roadmap for the streamlined development of in vitro cardiac differentiation strategies. The ability to obtain unlimited numbers of human cardiomyocytes would improve development of cell-based therapies for cardiovascular diseases, facilitate the study of cardiovascular biology and improve the early stages of drug discovery. Here in this review, we highlight the interacting endogenous cellular signals and their modulators involved in directing the human PSCs towards cardiac differentiation.

Objective: The effects of Lactobacillus rhamnosus GG (LGG) and Aloe vera (AV) gel on lipid profiles in rats with induced hypercholesterolemia were studied. Methods: Five treatment groups of rats (n ¼ 7) were the fed experimental diets: a normal control diet, a hypercholesterolemic diet (HD), HD þ LGG, HD þ AV gel, and HD þ LGG þ AV gel. Results: Supplementation with LGG decreased serum total cholesterol by 32%; however, in combination with AV, the decrease was 43%. The decreases in triacylglycerol levels in the HD þ LGG, HD þ AV, and HD þ LGG þ AV groups were 41%, 23% and 45%, respectively. High-density lipoprotein increased by 12% in the HD þ LGG þ AV group, whereas very low-density and lowdensity lipoprotein values decreased by 45% and 30%, respectively. The atherogenic index in the HD þ LGG þ AV group decreased to 2.45 from 4.77 in the HD þ LGG group. Furthermore, fecal Lactobacillus species counts increased significantly when LGG was fed in combination with the AV gel. The oral administration of LGG fermented milk alone or in combination with the AV gel increased cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase expression) and absorption (low-density lipoprotein receptor expression), whereas cholesterol 7 a-hydroxylase mRNA expression levels were lower in the HD þ LGG and HD þ LGG þ AV groups compared with the control HD group. Conclusion: The combination of LGG and AV gel may have a therapeutic potential to decrease cholesterol levels and the risk of cardiovascular diseases.

The aim of the present study was to explore the effect of exogenous application of castasterone (CS) on physiologic and biochemical responses in Brassica juncea seedlings under copper (Cu) stress. Seeds were pre-soaked in different concentrations of CS and grown for 7 days under various levels of Cu. The exposure of B. juncea to higher levels of Cu led to decrease of morphologic parameters, with partial recovery of length and fresh weight in the CS pre-treated seedlings. Metal content was high in both roots and shoots under Cu exposure while the CS pre-treatment reduced the metal uptake. Accumulation of hydrogen peroxide (H2O2) and superoxide anion radical (O2-) were chosen as stress biomarker and higher levels of H2O2 (88.89%) and O2- (62.11%) showed the oxidative stress in metal treated B. juncea seedlings, however, CS pre-treatment reduced ROS accumulation in Cu-exposed seedlings. The Cu exposures lead to enhance the plant's enzymatic and non-enzymatic antioxidant system. It was observed that enzymatic activities of ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR), glutathione perxoidase (GPOX) and gultrathione-s-transferase increased while activity of monodehydroascorbate reductase (MDHAR) decreased under Cu stress. The pre-treatment with CS positively affected the activities of enzymes. RT-PCR analysis showed that mRNA transcript levels were correlated with total enzymatic activity of DHAR, GR, GST and GSH. Increase in the gene expression of DHAR (1.85 folds), GR (3.24 folds), GST-1 (2.00 folds) and GSH-S (3.18 folds) was noticed with CS pre-treatment. Overall, the present study shows that Cu exposure induced severe oxidative stress in B. juncea plants and exogenous application of CS improved antioxidative defense system by modulating the ascorbate-glutathione cycle and amino acid metabolism.

Reactive oxygen species (ROS) are generated in various plant organelles under normal conditions and play an important role in different physiological progressions. But under abiotic stress, excessive ROS generation takes place which causes damage to normal functioning of plants. ROS play a dual role as they cause cellular damage and are also involved in abiotic stress signaling. Therefore, it is important to investigate the features of appearance of physiological effects of ROS depending on their cellular localization under the abiotic stress. Plants possess certain antioxidative mechanisms to deal with excess ROS in the cells, which involves enzymatic and nonenzymatic antioxidants. In the review, the mechanisms of ROS formation in different cellular compartments like mitochondria, peroxisomes, chloroplasts, nucleus, vacuole, cell wall, and plasma membranes are considered and summarized.

Dietary interventions for preventing colon cancer have recently attracted increased attention from researchers and clinicians. The probiotics have emerged as potential therapeutic agents but are also regarded as healthy dietary supplements for nutrition and health applications. The probiotic metabolome may interfere with various cellular and molecular processes, including the onset and progression of colon cancer. Probiotic metabolites may lead to the modulation of diverse cellular signal transduction and metabolic pathways. The gut microbial metabolites (organic acids, bacteriocins, peptides, etc.) have been noted to interact with multiple key targets in various metabolic pathways that regulate cellular proliferation, differentiation, apoptosis, inflammation, angiogenesis, and metastasis. Progress in this field suggests that epigenetic alterations will be widely used in the near future to manage colon cancer. The present review provides insights into the molecular basis of the therapeutic applications and the chemopreventive activities of certain probiotic metabolites, with emphasis on the interaction between these metabolites and the molecular signaling cascades that are considered to be epigenetic targets in preventing colon cancer.

Heterogeneous amniotic fluid contains various cell types. The aim of this study was to characterize and differentiate some of the key stemness attributes of the amniotic fluid-derived cells in buffalo (Bubalus bubalis). The amniotic fluid (AF) cells were cultured without feeder cells, in DMEM containing 15% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin/ampicillin, 1% vitamin solution, and 1% l-glutamine in 5% CO(2) in humidified air at 38.5±0.5 °C. After 6 days of culture different types of cells viz., star shaped (62.7%), spherical without nucleus (1.9%), spherical with nucleus (26.4%), pentagonal (0.4%), and free floating/rounded cells (8.3%) were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. Expression of alkaline phosphatase (AP) and Oct-4, Nestin and FGF-5 were observed from the AF cells at different passages. Using species-specific primers, a PCR amplicon of 200, 296 and 210 bp were observed for Oct-4, Nestin and FGF-5, respectively. The cells were found to have a normal karyotype at different passages. These results may contribute towards establishing non-embryonic pluripotent stem cells for various therapeutic and reproductive biotechnological applications in the species.

Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especiallyBifidobacteriumspp. andLactobacillusspp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface, incorporation of cholesterol into the cellular membrane, deconjugation of bile via bile salt hydrolase, coprecipitation of cholesterol with deconjugated bile, binding action of bile by fibre, and production of short-chain fatty acids by oligosaccharides. The present paper reviews the mechanisms of action of anti-cholesterolemic potential of probiotic microorganisms and probiotic food products, with the aim of lowering the risks of cardiovascular and coronary heart diseases.

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B₁ (AFB₁) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B₁ at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB₁ control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P

The present investigation was carried out to evaluate the hepatoprotective effect of probiotic fermented milk (FM) containingLactobacillus rhamnosusGG andLactobacillus caseistrain Shirota, alone as well as in combination with chlorophyllin (CHL) as an antioxidant agent in male Wistar rats administered aflatoxin-B1(AFB1). AFB1was injected intraperitoneally at the rate of 450 μg/kg body weight per animal twice a week for 6 weeks, maintaining an equal time interval between the two consecutive AFB1administrations. A total of 125 male Wistar rats were randomly allocated to five groups, each group having twenty-five animals. Group I was offered FM containingL. rhamnosusGG andL. caseistrain Shirota. Group II was administered AFB1and served as the control group; group III was administered FM-AFB1, in which besides administering AFB1, FM was also offered. Group IV was offered CHL and AFB1, and group V was offered both FM and CHL along with AFB1. The rats were euthanised at the 15th and 25th week of the experiment and examined for the biochemical and hepatopathological profile. A significant reduction in thiobarbituric acid-reactive substances (TBARS) was observed in the FM–CHL–AFB1group compared with the AFB1control group. FM alone or in combination with CHL was found to show a significant (P S-transferase, indicating that probiotic FM alone or in combination with CHL possesses a potent protective effect against AFB1-induced hepatic damage.

XYZ, a marginal carbonate field in Western Offshore India was undertaken for development recently. During its exploration phase, around two decade ago, thick oil layer was discovered with high sour gas content (around 14000 ppm). The reservoir was delineated in small oil pool, where six wells were completed for production, including one water injector for pressure maintenance to support oil recovery. Production behavior of most of the wells had a unique response of rapid decline in short period of time with similar drastic loss in THP without any change in water cut. Production response of all these wells led the suspicion of near well bore skin development as the primary reason behind sharp decline in production. Acid treatment as a quick fix solution in carbonates was ruled out because of possible communication with bottom aquifer; coil tubing had multiple operational challenges in such a high H2S environment. Hence, to overcome the challenges and perform root cause analysis, it was planned to carry out the best possible set of data acquisition with rigless operations and plan ahead for productivity improvement and design future intervention as well. Production Logging, being an ideal diagnostic tool, was chosen to study the production profile and understand the reservoir and well behavior. Post analysis, a relatively new concept of selective stimulation with post perforation controlled dynamic underbalance technique was considered to be the best fit solution to address the skin and production decline problem in the field. The technique proved to be very beneficial and provided substantial increase in the wells production. The skin debris obtained in the process were analyzed to identify the cause of skin development and production plunge. Based on the results, further interventions were carried out in the well, which again proved to be very efficacious and increased the field production significantly. The paper has a detailed discussion on the root cause analysis and arriving to the appropriate solution for this kind of wells. It emphasizes on the importance of production logging prior to operation for better and successful planning and execution of the well intervention jobs leading to production gain. It also highlights the operational challenges in such hostile high H2S environment and the importance of planning and preparation to ensure safety of one and all.

Genetically unmodified cardiomyocytes mandated for cardiac regenerative therapy is conceivable by “foot-print free” reprogramming of somatic cells to induced pluripotent stem cells (iPSC). In this study, we report generation of foot-print free hiPSC through messenger RNA (mRNA) based reprograming. Subsequently, we characterize cardiomyocytes derived from these hiPSC using molecular and electrophysiological methods to characterize their applicability for regenerative medicine. Our results demonstrate that mRNA-iPSCs differentiate ontogenetically into cardiomyocytes with increased expression of early commitment markers of mesoderm, cardiac mesoderm, followed by cardiac specific transcriptional and sarcomeric structural and ion channel genes. Furthermore, these cardiomyocytes stained positively for sarcomeric and ion channel proteins. Based on multi-electrode array (MEA) recordings, these mRNA-hiPSC derived cardiomyocytes responded predictably to various pharmacologically active drugs that target adrenergic, sodium, calcium and potassium channels. The cardiomyocytes responded chronotropically to isoproterenol in a dose dependent manner, inotropic activity of nifidipine decreased spontaneous contractions. Moreover, Sotalol and E-4031 prolonged QT intervals, while TTX reduced sodium influx. Our results for the first time show a systemic evaluation based on molecular, structural and functional properties of cardiomyocytes differentiated from mRNA-iPSC. These results, coupled with feasibility of generating patient-specific iPSCs hold great promise for the development of large-scale generation of clinical grade cardiomyocytes for cardiac regenerative medicine.

For decades, the hallmark of medical treatment for cancer has been intravenous cytotoxic chemotherapy, where these drugs target rapidly dividing cells, including cancer cells and certain normal tissues. As a result, many patients experience the classic toxicities of alopecia, gastrointestinal symptoms, and myelosuppression. In the past decade, however, a dramatic shift has been witnesses in the cancer therapy. Although traditional cytotoxic chemotherapy still remains the treatment of choice for many malignancies, targeted therapies are now a component of treatment for many types of cancer, including breast, colorectal, lung, and pancreatic cancers, as well as lymphoma, leukemia, and multiple myeloma.

Inflammation is viewed as one of the major causes for the development of different diseases like cancer, cardiovascular disease, diabetes, obesity, osteoporosis, rheumatoid arthritis, inflammatory bowel disease, asthma, and CNS related diseases such as depression and parkinson's disease; and this fervent phenomenon provides space for understanding different inflammatory markers. Increasing evidences have elucidated the outcome of inflammatory pathways dysregulation resulting in many symptoms of chronic diseases. The detection of transcription factors such as nuclear factor kappa-B (NF-κB), STAT and their gene products such as COX-2, cytokines, chemokines and chemokine receptors has laid molecular foundation for the important role of inflammation in chronic diseases in which the NF-κB is reported as a major mediator which makes a possible way for the development of new therapeutic approaches using synthetic and natural compounds that might eventually decrease the prevalence of these diseases. Even if many inflammatory markers like TNF-α, IL-1, IL-6, IL-8 and C-reactive protein (CRP) are reported to be the major key factors with proved role in several inflammatory diseases, IL-1 and TNF-α are the important cytokines that can induce the expression of NF-κB which is the potential target in these inflammatory diseases. This review aims to explore and summarize that how some drugs and natural compounds show their modulatory activity in inflammatory pathways and chronic inflammatory markers in these inflammatory diseases.

Human pluripotent stem cells (hPSCs) offer unique opportunities to discover and develop a new generation of drugs. Their ability to differentiate into virtually any cell type renders them a cost-effective, renewable source of tissue-specific cell types capable of predicting human responses towards novel chemical entities. Using these improved in vitro models based on physiologically relevant human cell types could result in identifying highly precise and safe compounds, thereby reducing drug attrition rates. Moreover, ability to develop humanised disease models for patient-stratified drug screening makes hPSCs an impeccable tool in translational medicine. In this mini-review we focus on the positives and negatives of utilising hPSC-derived cell types as drug discovery platforms with special emphasis on cardio-, hepato- and embryotoxicity.

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B₁ (AFB₁) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B₁ at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB₁ control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B₁ control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB₁ control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB₁-induced molecular alterations in hepatic cells during carcinogenesis.

Authentic induced pluripotent stem cells (iPSCs), capable of giving rise to all cell types of an adult animal, are currently only available in mouse. Here, we report the first generation of bovine iPSC-like cells following transfection with a novel virus-free poly-promoter vector. This vector contains the bovine cDNAs for OCT4, SOX2, KLF4 and c-MYC, each controlled by its own independent promoter. Bovine fibroblasts were cultured without feeders in a chemically defined medium containing leukaemia inhibitory factor (LIF) and inhibitors of MEK1/2 and glycogen synthase kinase-3 signaling (‘2i’). Non-invasive real-time kinetic profiling revealed a different response of bovine vs human and mouse cells to culture in 2i/LIF. In bovine, 2i was necessary and sufficient to induce the appearance of tightly packed alkaline phosphatase-positive iPSC-like colonies. These colonies formed in the absence of DNA synthesis and did not expand after passaging. Following transfection, non-proliferative primary colonies expressed discriminatory markers of pluripotency, including endogenous iPSC factors, CDH1, DPPA3, NANOG, SOCS3, ZFP42, telomerase activity, Tra-1-60/81 and SSEA-3/4, but not SSEA-1. This indicates that they had initiated a self-sustaining pluripotency programme. Bovine iPSC-like cells maintained a normal karyotype and differentiated into derivatives of all three germ layers in vitro and in teratomas. Our study demonstrates that conversion into induced pluripotency can occur in quiescent cells, following a previously undescribed route of direct cell reprogramming. This identifies a major species-specific barrier for generating iPSCs and provides a chemically defined screening platform for factors that induce proliferation and maintain pluripotency of embryo-derived pluripotent stem cells in livestock.

Cancer is a serious global public health problem. Cancer incidence and mortality have been steadily rising throughout the past century in most places of the world. There are several epidemiological evidences that support a protective role of probiotics against cancer. Lactic acid bacteria and their probioactive cellular substances exert many beneficial effects in the gastrointestinal tract, and also release various enzymes into the intestinal lumen and exert potential synergistic (LAB) effects on digestion and alleviate symptoms of intestinal malabsorption. Consumption of fermented dairy products with LAB may elicit anti-tumor effects. These effects are attributed to the inhibition of mutagenic activity, the decrease in several enzymes implicated in the generation of carcinogens, mutagens, or tumor-promoting agents, suppression of tumors, and epidemiology correlating dietary regimes and cancer. Specific cellular components in lactic acid bacteria seem to induce strong adjuvant effects including modulation of cell-mediated immune responses, activation of the reticulo-endothelial system, augmentation of cytokine pathways, and regulation of interleukins and tumor necrosis factors. Studies on the effect of probiotic consumption on cancer appear promising, since recent in vitro and in vivo studies have indicated that probiotic bacteria might reduce the risk, incidence and number of tumors of the colon, liver and bladder. The protective effect against cancer development may be ascribed to binding of mutagens by intestinal bacteria, may suppress the growth of bacteria that convert procarcinogens into carcinogens, thereby reducing the amount of carcinogens in the intestine, reduction of the enzymes beta-glucuronidase and beta-glucosidase and deconjugation of bile acids, or merely by enhancing the immune system of the host. There are isolated reports citing that administration of LAB results in increased activity of anti-oxidative enzymes or by modulating circulatory oxidative stress that protects cells against carcinogen-induced damage. These include glutathione-S-transferase, glutathione, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase. However, there is no direct experimental evidence for cancer suppression in human subjects as a result of the consumption of probiotic cultures in fermented or unfermented dairy products, but there is a wealth of indirect evidence based largely on laboratory studies.

The swamp buffalo holds tremendous potential in the livestock sector in Asian and Mediterranean countries. Current needs are the faster multiplication of superior genotypes and the conservation of endangered buffalo breeds. Recent advances in assisted reproductive technologies, including in vitro embryo production methodologies, offer enormous opportunities to not only improve productivity, but also to use buffaloes to produce novel products for applications to human health and nutrition. The use of molecular genomics will undoubtedly advance these technologies for their large-scale application and resolve the key problems currently associated with advanced reproductive techniques, such as animal cloning, stem cell technology and transgenesis. Preliminary success in the application of modern reproductive technologies warrants further research at the cellular and molecular levels before their commercial exploitation in buffalo breeding programmes.

Microbial metagenomics—the applications of the genomics suit of technologies to nonculturable microorganisms, is coming of age. These approaches can be used for the screening and identification of nonculturable gastrointestinal (GI) microflora for assessing and exploiting them in nutrition and the health of the host. Advances in technologies designed to access this wealth of genetic information through environmental nucleic acids extraction and analysis have provided the means of overcoming the limitations of conventional culture-dependent microbial genetic exploitation. The molecular techniques and bioinformatics tools will result in reliable insights into the animals’ GI microbial structure and activity of the livestock gut microbes in relation to functional interactions, temporal and spatial relationships among different microbial consortia and dietary ingredients. Further developments and applications of these methods promise to provide the opportunity to link distribution and identity of various GI microbes in their natural habitats, and explore their use for promoting livestock health and industrial development.

Isolation and culture of embryo-derived cell lines have been reported in many mammals, however, there is not even a single report toward initiation of such work in buffalo (Bubalus bubalis). Therefore the present study was carried out to isolate the inner cell mass from in vitro-produced buffalo blastocysts and to grow this inner cell mass for formation of embryoid bodies in a gelatin-coated dish and on an homologous fetal fibroblast feeder layer. Immature buffalo oocytes were isolated from the slaughterhouse ovaries. In vitro production of blastocysts was carried as reported by Chauhan et al. (1999 J. Dairy Science 82, 918-926). A total of 26 buffalo blastocysts were produced in vitro. These blastocysts were transferred into a 100-mL drop of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS) for further culturing. Thirteen blastocysts hatched on the next day of culture. The hatched mass was separated, suspended in Dulbecco's phosphate buffer saline (DPBS, without Ca++ and Mg++) containing 5% FBS + 0.25% trypsin and examined under the zoom stereomicroscope until disappearance of the trophectoderm cells. The remaining cells of seven blastocysts were cultured in DMEM supplemented with 20% FBS on 0.2% gelatin coated culture dish (group 1), and cells of six blastocysts in DMEM medium supplemented with 20% FBS were cultured on a mitomycin-c-treated (10 �L/mL) feeder layer (group 2), for 14 days. The isolated cells attached to the bottom of the dish in both the groups, spreading was noticed on Day 5 of the culture in group 1 and on Day 3 of culture in group 2. The attached cells were trypsinized using DPBS with 0.25% trypsin, isolated, and subcultured further. Attachment and spreading was noticed only in group 2 subcultured cells. The cellular integrity was homogeneous and the plasma membrane was clearly visible in group 2, but not in group 1. Less than 10% of the attached cells formed embryoid bodies in group 1, where as more than 30% attached cells in group 2 formed embryoid bodies; the latter expressed alkaline phosphatase activity and were blue after staining. These results indicate that the culturing of the inner cell mass on an homologous fetal fibroblast feeder layer is a better choice for production of embryonic stem cells in buffaloes.

A study was carried out to investigate whether the structure of IUD-exposed human endometrium changes, which might possibly account for the noted heavy uterine bleeding among IUD users. 17 women using a Lippes loop, 18 using a copper IUD, and 10 controls who had never used an IUD were included in the study. Duration of IUD use varied from 5 months to 13 years. The endometrial glands of IUD users had some shrunken and degenerated cells. In women who had experienced excessive menstrual bleeding, there were numerous pyknotic cells and a tremendous increase in microfilaments. These microfilaments might possibly have moved the cell fluid towards the pyknotic cells. These morphological changes in themselves do not explain the outflow of erythrocytes. Some inflammatory mechanism which alters the permeability of capillaries is, perhaps, involved.

A total of 75 preterm infants with gestation less than 32 wk received total parenteral nutrition (TPN) using Vamin and Aminosyn as protein base lasting more than 20 days. They were monitored for signs of liver dysfunction, cholestatic jaundice, and TPN-induced metabolic bone disease (osteopenia of prematurity). It was observed that severity of TPN-induced cholestasis depends on the duration of TPN and quantity of protein infused. When used as a protein base, Vamin seemed to be superior to Aminosyn. TPN-induced metabolic bone disease was strongly correlated to the duration of TPN. We suggest close monitoring of critically ill preterm infants on TPN for quantity of protein infusate, liver dysfunction, cholestatic jaundice, and TPN-induced metabolic bone disease. Intravenous protein intake should be limited to less than 2.5 g/kg/day in preterm infants with gestation less than 32 wk. (Journal of Parenteral and Enteral Nutrition9:439-442, 1985)

Biopsies of endometrium from women using intrauterine contraceptive devices (IUDs) were examined by electron microscopy to elucidate the possible cause of IUD-associated uterine hemorrhage. The findings reveal the presence of degenerated, shrunken cells in the endometrial glands of IUD users. These cells, termed pyknotic or apoptotic, are more prevalent in tissue from women with complaints of excessive bleeding than in cases without such complaints. The pyknotic cells often show discontinuities of the plasma membrane at their luminal pole, from which cell contents stream into the lumen. Sometimes erythrocytes are also observed in the lumen. A second alteration in the ultrastructure of the endometrium is an abnormal increase in microfilaments in the epithelial cells. These two changes, a large number of pyknotic cells on the one hand and increase in microfilaments on the other, are correlated with excessive bleeding but they do not explain in simple terms the outflow of erythrocytes. Probably some inflammatory mechanism is involved. The increased number of microfilaments might be helping the movement of fluid from healthy to pyknotic cells.

Degenerating changes after long durations of denervation have been studied in frog motor end-plate. During the 2nd month of denervation, the Schwann cell undergoes fissuration and the post-synaptic membrane displays the presence of furrows. The extent of the furrows in the post-synaptic membrane and fissuration of Schwann cell increases with increasing periods of denervation. The formation of furrows seem to be correlated with fissure formation in the Schwann cell cytoplasm. During the 4th month of denervation, at places, sequestration of postsynaptic fold from parent muscle occurs. The furrows in the postsynaptic membrane are eventually responsible for the sequestration of folds. In spite of the remarkable morphological changes at certain end-plates after long periods of denervation, the percentage of end-plates displaying spontaneous activity remains the same during this period (3-4 months) as after short periods of denervation (I2 months). This permits us to conclude that only at certain end-plate regions is the Schwann cell capable of producing acetylcholine responsible for miniature endplate potentials (min. e.p.p.s.) and this capacity is maintained until very long periods.

It is known that, in a denervated striated muscle, the satellite cells multiply by mitotic division. A liaison between these satellite cells and the Schwann cell in front of the post-synaptic membrane in denervated frog muscle has been observed. It is probable that such cell connections help in the subsistence of the Schwann cell in a denervated muscle.

The mechanism of re-innervation depends on the period of denervation. After 2 1/2 months, when post-synaptic folds are intact but for the furrows, the reinner vation takes place at the former synaptic sites. The Schwann cell which is present in contact with the post-synaptic membrane till that time is withdrawn at the moment of reinnervation and the terminal bouton establishes contact with it. After 4 months of denervation, the post-synaptic sites are not always intact; at places there is sequestration and hence flattening of the folds. Thus, the reinnervation takes place, firstly, at old synaptic gutters where the synaptic sites are intact and secondly, by the formation of new neuromuscular junctions. The latter is marked by the specialisation of the parts of muscular membrane before the nerve is in contact with it. The process of reinnervation is gradual after short durations and is rapid after long durations. Similarly, the reappearance of the electric activity is gradual after short and abrupt after long durations. The miniature end-plate potentials (min. e.p.p.s.) in recently innervated muscle in both cases are characterised by their slow time course but normal in their frequency and distribution of the amplitudes. In case of some fibres, the min. e.p.p.s. had Gaussian as well as skewed amplitude distribution. This could be due to the unequal receptivity of the ACh or to the liberation of cholinesterases in variable amounts. The newly formed end-plates are capable of functionning before acquiring their normal morphological characters.

Structural and functional behaviour of motor end-plates after transection of the motor nerve has been studied in two species of frog: Rana esculenta and Rana temporaria. The physiological results show that in both species there is a transient cessation of spontaneous activity followed by a resumption of miniature end-plate potentials (min. e.p.p.s.) after denervation. The characteristics of these potentials (frequency, distribution of amplitudes, time-course) are similar in the two species. However, some differences have been observed: Firstly, the period of silence lasts for 2--4 days in the case of Rana temporaria whereas it is prolonged to about 15 days in Rana esculenta. Secondly, the resumption of min. e.p.p.s. is gradual and after the 10th day of denervation remains constant in Rana temporaria. It is inconstant independent of the period of denervation in Rana esculenta. The morphological results show that the Schwann cell is constantly in contact with the post-synaptic membrane after about 6 days of denervation in both species. It is suggested that either the Schwann cell is capable of functioning for a limited period of time in Rana esculenta or is activated to produce min. e.p.p.s. only in certain cases.

A review of the ultrastructure of normal human endometrium at different phases of the cycle is presented. The structural changes are correlated to the functional implications in the light of the recent knowledge on the functioning of cell organelles. Relatively undifferentiated cells of the beginning of the menstrual phase become functionally matured, proliferate and later degenerate. The appearance of the nucleolar channel system during the secretory phase coincides with the rise of progesterone level and facilitates the nucleocytoplasmic exchange. The secretion of the endometrial glands consists of glycogen, glycoproteins, lipids and mucopolysaccharides. Large autophagic vacuoles (giant lysosomes) digest various cell components at the end of the cycle. All the lamina functionalis of the endometrium is not shed, a part of it is regressed by focal death (autophagic vacuoles) and by total death (apoptosis) of individual cells.

The frog skeletal muscle resists atrophy for a long time after denervation. If, however, the entry of the nerve is prevented for more than 3.5 months, small muscle portions, sometimes containing nucleus, are sequestered from the parent muscle fibre. The basal lamina does not dissociate from these detached muscle portions.