Keratin as an effective coating material for in vitro stem cell culture, induced differentiation and wound healing assays.

The utilization of stem cells in tissue engineering holds great promise as efficient tools for tissue regeneration and in treating numerous musculoskeletal diseases. However, several limiting factors, such as precise delivery and control of differentiation of these stem cells as well as mimicking the microenvironment required to modulate stem cell behaviour in-vivo, have given rise to an urgent need for the development of new biomaterials which could be tailored to enhance cell renewal and/or direct cell fates. Keratin-rich biological materials offer several advantages, such as biocompatibility, tailorable mechanical properties, huge bioavailability, non-toxicity, non-immunogenic, and intrinsic tissue repair and/or regeneration capabilities, which makes them highly valued. In the present work, we report the preparation of keratin-based bio-materials from goat hair waste and its effectiveness as a coating material for in vitro culture and induced differentiation of mesenchymal stem cells (MSC's) and primary goat fibroblast cells. Since no known keratinase enzymes are expressed as such in human and/or animal systems, these keratin biomaterials could be used to slow the rate of degradation and deliver keratin-loaded stem cell scaffolds to induce their directed differentiation in vivo. The generated keratin materials have been characterized for surface morphology, protein structures, size and other properties using SDS-PAGE, LC/MS-MS, SEM, FTIR etc. Also, in vitro cell culture assays such as cell adhesion, viability using MTT, live dead assays, differentiation assays and in vitro scratch/wound healing assays were performed. Our results provide important data supporting tissue engineering applications of these keratinous biomaterials by combining the unique biological characteristics of goat hair-derived keratin material with the regenerative power of stem cells and their combinatorial use in applications such as disease treatment and injury repair as well as their use in the preparation of wound healing products, such as dressings and bandages, for management of clinical care in animals.

Scavenger receptor B1 facilitates the endocytosis of Escherichia coli via TLR4 signaling in mammary gland infection.

SCARB1 belongs to class B of Scavenger receptors (SRs) that are known to be involved in binding and endocytosis of various pathogens. SRs have emerging role in regulating innate immunity and host-pathogen interactions by acting in co-ordination with Toll-like receptors.Query Little is known about the function of SCARB1 in milk-derived mammary epithelial cells (MECs). This study reports the role of SCARB1 in infection and its potential association in TLR4 signaling on bacterial challenge in Goat mammary epithelial cells (GMECs). The novelty in the establishment of MEC culture lies in the method that aims to enhance the viability of the cells with intact characteristics upto a higher passage number. We represent MEC culture to be used as a potential infection model for deeper understanding of animal physiology especially around the mammary gland. On E.coli challenge the expression of SCARB1 was significant in induced GMECs at 6 h. Endoribonuclease-esiRNA based silencing of SCARB1 affects the expression of TLR4 and its pathways i.e. MyD88 and TRIF pathways on infection. Knockdown also affected the endocytosis of E.coli in GMECs demonstrating that E.coli uses SCARB1 function to gain entry in cells. Furthermore, we predict 3 unique protein structures of uncharacterized SCARB1 (Capra hircus) protein. Overall, we highlight SCARB1 as a main participant in host defence and its function in antibacterial advances to check mammary gland infections. Video Abstract.

Pathogenesis-Related Gene Expression in Response to Trachyspermum ammi Supplementation Along With Probiotics in Chicken Salmonellosis and Insights in Drug Therapeutics.

Salmonella enterica serovar typhimurium (S. typhimurium) is the leading cause of foodborne illness. Since Salmonella continues to have a detrimental effect on public health, there is an ongoing need to develop more advanced methods for combating Salmonellosis in foods before they reach consumers. In addition, the quest for alternative natural products has recently intensified due to increasingly stringent regulations regarding the use of antibiotics as growth promoters and consumer demand for antibiotic-free poultry products. This study evaluated the effect of Ajwain extract (AJE) on immune response and antioxidant status in broiler chicks challenged with Salmonella typhimurium. The chicks were infected with S. typhimurium and were divided into the different groups, except for the control group (CON). The challenged chicks received different treatments with 3 × 109 colony-forming unit (CFU) AciproTM-WS probiotic (PRO), 200 mg/kg Ajwain extract (AJE), 200 mg/100 kg of enrofloxacin (ENR), and a combination of 3 × 109 CFU AciproTM-WS probiotic and 200 mg/kg Ajwain extract (COM). Five days posttreatment, the tissue samples (liver and spleen) were analyzed. The results showed that basal diet supplemented with Ajwain extract (AJE) and a combination of probiotic and Ajwain extract (COM) significantly (P < 0.0.5) reduced the cytokine expression in broiler chicks challenged with S. typhimurium. Our findings suggest that AJE can clear the bacterial infection, improve antioxidant status, and suppress the inflammation response. Additionally, AJE supplementation significantly mitigated the S. typhimurium-induced increase in the interleukin-6 (IL-6) (liver and spleen), interleukin-8 (IL-8) (liver and spleen), interleukin-17A (IL-17A) (liver and spleen), and inducible nitric oxide (iNOS) (spleen and liver) levels (P < 0.05). We conclude that Ajwain is an efficient feed additive with antioxidant and anti-inflammatory properties. The interaction networks developed in this study provide a novel lead that could be targeted for anti-inflammatory and antioxidant properties.

Actin Modulation Regulates the Alpha-1-Syntrophin/p66Shc Mediated Redox Signaling Contributing to the RhoA GTPase Protein Activation in Breast Cancer Cells.

SNTA1 signaling axis plays an essential role in cytoskeletal organization and is also implicated in breast cancers. In this study, we aimed to investigate the involvement of actin cytoskeleton in the propagation of SNTA1/p66shc mediated pro-metastatic cascade in breast cancer cells.The effect of actin filament depolymerization on SNTA1-p66Shc interaction and the trimeric complex formation was analyzed using co-immunoprecipitation assays. Immunofluorescence and RhoA activation assays were used to show the involvement of SNTA1-p66Shc interaction in RhoA activation and F-actin organization. Cellular proliferation and ROS levels were assessed using MTT assay and Amplex red catalase assay. The migratory potential was evaluated using transwell migration assay and wound healing assay.We found that cytochalasin D mediated actin depolymerization significantly declines endogenous interaction between SNTA1 and p66Shc protein in MDA-MB-231 cells. Results indicate that SNTA1 and p66Shc interact with RhoA protein under physiological conditions. The ROS generation and RhoA activation were substantially enhanced in cells overexpressing SNTA1 and p66Shc, promoting proliferation and migration in these cells. In addition, we found that loss of SNTA1-p66Shc interaction impaired actin organization, proliferation, and migration in breast cancer cells. Our results demonstrate a novel reciprocal regulatory mechanism between actin modulation and SNTA1/p66Shc/RhoA signaling cascade in human metastatic breast cancer cells.

Genome wide expression analysis of circular RNAs in mammary epithelial cells of cattle revealed difference in milk synthesis.

Milk is an excellent source of nutrients for humans. Therefore, in order to enhance the quality and production of milk in cattle, it is interesting to examine the underlying mechanisms. A number of new investigations and research have found that, circRNA; a specific class of non-coding RNAs, is linked with the development of mammary gland and lactation. In the present study, genome wide identification and expression of the circRNAs in mammary epithelial cells of two distinct cattle breeds viz Jersey and Kashmiri at peak lactation was conducted. We reported 1554 and 1286 circRNA in Jersey and Kashmiri cattle, respectively, with 21 circRNAs being differentially expressed in the two breeds. The developmental genes of the established differentially expressed circRNAs were found to be largely enriched in antioxidant activity, progesterone, estradiol, lipid, growth hormone, and drug response. Certain pathways like MAPK, IP3K and immune response pathways were found significantly enriched in KEGG analysis. These results add to our understanding of the controlling mechanisms connected with the lactation process, as well as the function of circRNAs in bovine milk synthesis. Additionally, the comparative analysis of differentially expressed circRNAs showed significant conservation across different species.

Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stem Cells from Sheep: Culture Characteristics.

The current study demonstrates the culture characteristics of adipose tissue and bone marrow-derived mesenchymal stem cells (MSC). The study evaluates the effect of ambient temperature, physiological status of the donor and the tissue source on sheep (Ovis aries) mesenchymal stem cells. The tissue samples were harvested from full term pregnant female sheep (n = 9) and male sheep (n = 10). Adipose tissue was harvested from n = 9 sheep and bone marrow from n = 10 sheep. The samples (adipose tissue, n = 2; bone marrow, n = 3) transported at cold ambient temperature (<10 °C) failed to yield MSCs while those (n = 14) at higher (>20 °C) ambient temperature successfully yielded MSCs. Bone marrow mononuclear cell (MNC) fraction was higher than the adipose tissue-derived stromal vascular fraction (SVF), but the percent adherent cells (PAC) was higher in the later cell fraction. Adipose tissue-derived MSCs from the full term female sheep had a significantly (p < 0.05) higher proliferation potential as compared to those of the male sheep-derived MSCs. Female sheep MSCs also had rapid differentiation potential. The cryopreserved MSCs had morphological features comparable to that of the fresh cells. In conclusion, the tissue type and physiological status of donor animal may affect MSCs' characteristics and should be taken into consideration while applying in clinical settings.

Jasplakinolide Attenuates Cell Migration by Impeding Alpha-1-syntrophin Protein Phosphorylation in Breast Cancer Cells.

BACKGROUND: Alpha-1-syntrophin (SNTA1) is emerging as a novel modulator of the actin cytoskeleton. SNTA1 binds to F-actin and regulates intracellular localization and activity of various actin organizing signaling molecules. Aberration in syntrophin signaling has been closely linked with deregulated growth connected to tumor development/metastasis and its abnormal over expression has been observed in breast cancer. In the present work the effect of jasplakinolide, an actin-binding cyclodepsipeptide, on the SNTA1 protein activity and SNTA1 mediated downstream cellular events was studied in MDA-MB-231 breast cancer cell line. METHODS: SNTA1 protein levels and phosphorylation status were determined in MDA-MB-231 cells post jasplakinolide exposure using western blotting and immunoprecipitation techniques respectively. MDA-MB-231 cells were transfected with WT SNTA1 and DM SNTA1 (Y215/229 phospho mutant) and simultaneously treated with jasplakinolide. The effect of jasplakinolide and SNTA1 protein on cell migration was determined using the boyden chamber assay. RESULTS: Jasplakinolide treatment decreases proliferation of MDA-MB-231 cells in both dose and time dependent manner. Results suggest that subtoxic doses of jasplakinolide induce morphological changes in MDA-MB-231 cells from flat spindle shape adherent cells to round weakly adherent forms. Mechanistically, jasplakinolide treatment was found to decrease SNTA1 protein levels and its tyrosine phosphorylation status. Moreover, migratory potential of jasplakinolide treated cells was significantly inhibited in comparison to control cells. CONCLUSION: Our results demonstrate that jasplakinolide inhibits cell migration by impairing SNTA1 functioning in breast cancer cells.

Mesenchymal Stem Cell-Mediated Immuno-Modulatory and Anti- Inflammatory Mechanisms in Immune and Allergic Disorders.

BACKGROUND: Mesenchymal Stem Cells (MSCs) are present in almost all the tissues of the body and act as the backbone of the internal tissue homeostasis. Among their various characteristic features, immuno-modulatory and/ anti-inflammatory properties play an important role in therapeutics. OBJECTIVE: The current topic focuses on the characterization and immuno-modulatory and/ antiinflammatory properties of MSCs. To present and discuss the current status of MSCs immunomodulatory properties. METHODS: Available literature on MSCs properties and patents have been detailed, critically interpreted, and discussed based upon available literature. The main focus has been on their characteristic immunomodulatory and anti-inflammatory properties though some of the basic characterization markers have also been detailed. The databases searched for the literature include PubMed, Med Line, PubMed Central, Science Direct and a few other scientific databases. RESULTS: MSCs are present in a very limited concentration in the tissues, and as such their culture expansion becomes imperative. MSCs immuno-modulatory and anti-inflammatory roles are achieved through direct cell-cell contact and / by the release of certain factors. Such properties are controlled by micro-environment upon which currently very limited control can be exerted. Besides, further insights in the xeno-protein free culture media as against the fetal bovine serum is required. CONCLUSION: MSCs have been well-isolated, cultured and characterized from numerous tissues of the body. The majority of the studies have shown MSCs as immuno-compromised with immunomodulatory and / or anti-inflammatory properties except some of the latest studies that have failed to achieve the desired results and thus, demand further research. Further research is required in the area to translate the results into clinical application.

Comparative milk proteome analysis of Kashmiri and Jersey cattle identifies differential expression of key proteins involved in immune system regulation and milk quality.

BACKGROUND: Exploration of the bioactive components of bovine milk has gained global interest due to their potential applications in human nutrition and health promotion. Despite advances in proteomics profiling, limited studies have been carried out to fully characterize the bovine milk proteome. This study explored the milk proteome of Jersey and Kashmiri cattle at day 90 of lactation using high-resolution mass spectrometry based quantitative proteomics nano-scale LC-MS/Q-TOF technique. Data are available via ProteomeXchange with identifier PXD017412. RESULTS: Proteins from whey were fractionated by precipitation into high and low abundant proteins. A total of 81 high-abundant and 99 low-abundant proteins were significantly differentially expressed between Kashmiri and Jersey cattle, clearly differentiating the two breeds at the proteome level. Among the top differentiating proteins, the Kashmiri cattle milk proteome was characterised by increased concentrations of immune-related proteins (apelin, acid glycoprotein, CD14 antigen), neonatal developmental protein (probetacellulin), xenobiotic metabolising enzyme (flavin monooxygenase 3 (FMO3), GLYCAM1 and HSP90AA1 (chaperone) while the Jersey milk proteome presented higher concentrations of enzyme modulators (SERPINA1, RAC1, serine peptidase inhibitor) and hydrolases (LTF, LPL, CYM, PNLIPRP2). Pathway analysis in Kashmiri cattle revealed enrichment of key pathways involved in the regulation of mammary gland development like Wnt signalling pathway, EGF receptor signalling pathway and FGF signalling pathway while a pathway (T-cell activation pathway) associated with immune system regulation was significantly enriched in Jersey cattle. Most importantly, the high-abundant FMO3 enzyme with an observed 17-fold higher expression in Kashmiri cattle milk seems to be a characteristic feature of the breed. The presence of this (FMO3) bioactive peptide/enzyme in Kashmiri cattle could be economically advantageous for milk products from Kashmiri cattle. CONCLUSION: In conclusion, this is the first study to provide insights not only into the milk proteome differences between Kashmiri and Jersey cattle but also provides potential directions for application of specific milk proteins from Kashmiri cattle in special milk preparations like infant formula.

Comparative transcriptome analysis of mammary epithelial cells at different stages of lactation reveals wide differences in gene expression and pathways regulating milk synthesis between Jersey and Kashmiri cattle.

Jersey and Kashmiri cattle are important dairy breeds that contribute significantly to the total milk production of the Indian northern state of Jammu and Kashmir. The Kashmiri cattle germplasm has been extensively diluted through crossbreeding with Jersey cattle with the goal of enhancing its milk production ability. However, crossbred animals are prone to diseases resulting to unsustainable milk production. This study aimed to provide a comprehensive transcriptome profile of mammary gland epithelial cells at different stages of lactation and to find key differences in genes and pathways regulating milk traits between Jersey and Kashmiri cattle. Mammary epithelial cells (MEC) isolated from milk obtained from six lactating cows (three Jersey and three Kashmiri cattle) on day 15 (D15), D90 and D250 in milk, representing early, mid and late lactation, respectively were used. RNA isolated from MEC was subjected to next-generation RNA sequencing and bioinformatics processing. Casein and whey protein genes were found to be highly expressed throughout the lactation stages in both breeds. Largest differences in differentially expressed genes (DEG) were between D15 vs D90 (1,805 genes) in Kashmiri cattle and, D15 vs D250 (3,392 genes) in Jersey cattle. A total of 1,103, 1,356 and 1,397 genes were differentially expressed between Kashmiri and Jersey cattle on D15, D90 and D250, respectively. Antioxidant genes like RPLPO and RPS28 were highly expressed in Kashmiri cattle. Differentially expressed genes in both Kashmiri and Jersey were enriched for multicellular organismal process, receptor activity, catalytic activity, signal transducer activity, macromolecular complex and developmental process gene ontology terms. Whereas, biological regulation, endopeptidase activity and response to stimulus were enriched in Kashmiri cattle and, reproduction and immune system process were enriched in Jersey cattle. Most of the pathways responsible for regulation of milk production like JAK-STAT, p38 MAPK pathway, PI3 kinase pathway were enriched by DEG in Jersey cattle only. Although Kashmiri has poor milk production efficiency, the present study suggests possible physicochemical and antioxidant properties of Kashmiri cattle milk that needs to be further explored.

Mesenchymal stem cell: Basic research and potential applications in cattle and buffalo.

Characteristic features like self-renewal, multilineage differentiation potential, and immune-modulatory/anti-inflammatory properties, besides the ability to mobilize and home distant tissues make stem cells (SCs) a lifeline for an individual. Stem cells (SCs) if could be harvested and expanded without any abnormal change may be utilized as an all-in-one solution to numerous clinical ailments. However, slender understanding of their basic physiological properties, including expression potential, behavioral alternations during culture, and the effect of niche/microenvironment has currently restricted the clinical application of SCs. Among various types of SCs, mesenchymal stem cells (MSCs) are extensively studied due to their easy availability, straightforward harvesting, and culturing procedures, besides, their less likelihood to produce teratogens. Large ruminant MSCs have been harvested from various adult tissues and fetal membranes and are well characterized under in vitro conditions but unlike human or other domestic animals in vivo studies on cattle/buffalo MSCs have mostly been aimed at improving the animals' production potential. In this document, we focused on the status and potential application of MSCs in cattle and buffalo.

ABC of multifaceted dystrophin glycoprotein complex (DGC).

Dystrophin protein in association with several other cellular proteins and glycoproteins leads to the formation of a large multifaceted protein complex at the cell membrane referred to as dystrophin glycoprotein complex (DGC), that serves distinct functions in cell signaling and maintaining the membrane stability as well as integrity. In accordance with this, several findings suggest exquisite role of DGC in signaling pathways associated with cell development and/or maintenance of homeostasis. In the present review, we summarize the established facts about the various components of this complex with emphasis on recent insights into specific contribution of the DGC in cell signaling at the membrane. We have also discussed the recent advances made in exploring the molecular associations of DGC components within the cells and the functional implications of these interactions. Our review would help to comprehend the composition, role, and functioning of DGC and may lead to a deeper understanding of its role in several human diseases.

TM-Aligner: Multiple sequence alignment tool for transmembrane proteins with reduced time and improved accuracy.

Membrane proteins plays significant role in living cells. Transmembrane proteins are estimated to constitute approximately 30% of proteins at genomic scale. It has been a difficult task to develop specific alignment tools for transmembrane proteins due to limited number of experimentally validated protein structures. Alignment tools based on homology modeling provide fairly good result by recapitulating 70-80% residues in reference alignment provided all input sequences should have known template structures. However, homology modeling tools took substantial amount of time, thus aligning large numbers of sequences becomes computationally demanding. Here we present TM-Aligner, a new tool for transmembrane protein sequence alignment. TM-Aligner is based on Wu-Manber and dynamic string matching algorithm which has significantly improved its accuracy and speed of multiple sequence alignment. We compared TM-Aligner with prevailing other popular tools and performed benchmarking using three separate reference sets, BaliBASE3.0 reference set7 of alpha-helical transmembrane proteins, structure based alignment of transmembrane proteins from Pfam database and structure alignment from GPCRDB. Benchmarking against reference datasets indicated that TM-Aligner is more advanced method having least turnaround time with significant improvements over the most accurate methods such as PROMALS, MAFFT, TM-Coffee, Kalign, ClustalW, Muscle and PRALINE. TM-Aligner is freely available through http://lms.snu.edu.in/TM-Aligner/ .

Molecular characterization of RNA binding motif protein 3 (RBM3) gene from Pashmina goat.

Pashmina goat inhabits the high altitude cold arid desert of Ladakh, India. This goat is known for its finest and costliest under fiber. Though the under fiber may be a part of its complex thermoregulation mechanism, the genetics of its adaptability under cold conditions is not known. As an attempt to understand its adaptive genetics, and the role of RNA-binding proteins at the cellular response, this study was conducted to characterize the RBM3 gene in Pashmina goat and its expression during hypothermia. The ORF of Pashmina RBM3 gene was 273 bp. Phylogenetic analysis revealed that Pashmina RBM3 is closely related to Bos taurus RBM3. Pashmina RBM3 was characterized by comparative modeling studies. The final 3-D model contained two α-helices and four ß-sheets. qRT-PCR data showed that Pashmina RBM3 gene expression was significantly higher (P < 0.05) at moderate (30 °C) hypothermic stress conditions as compared with deep (15 °C) hypothermia.

Liver transplantation with a strongly positive crossmatch: case study and literature review.

A positive crossmatch has been associated with increased risk in liver transplantation. To study the clinical significance of preformed donor-specific human leukocyte antigen antibodies (DSAs) in liver transplantation, we reviewed patients who underwent liver transplantation with a strongly positive flow cytometry crossmatch. DSAs were evaluated with a Luminex solid phase assay. The complement-fixing ability of DSAs was tested with a complement component 1q (C1q) assay. Using an assay correlation between complement-dependent cytotoxicity crossmatch, flow cytometry crossmatch, and DSA results, we reviewed the effects of DSAs on the outcomes of our patients as well as reported cases in the literature. Five of 69 liver recipients had a strongly positive crossmatch: 4 had a positive T cell crossmatch [median channel shift (MCS) = 383.5 ± 38.9], and 5 had a positive B cell crossmatch (MCS = 408.8 ± 52.3). The DSAs were class I only in 1 patient, class I and II in 3 patients, and class II only in 1 patient. Cholestasis, acute rejection, or both were observed in 3 of the 4 patients with a positive T cell crossmatch with an MCS approximately greater than 300. The C1q assay was positive for 3 patients. Two had either persistent cholestasis or early acute rejection. One patient who was treated with preemptive intravenous immunoglobulin had an unremarkable outcome despite a positive C1q result. One of the 2 patients with a negative C1q assay experienced persistent cholestasis and early and recurrent acute rejection; the other had an unremarkable outcome. None of the patients died or lost a graft within the first year of transplantation. Our study suggests that human leukocyte antigen antibody screening, flow cytometry crossmatch MCS levels, DSA mean fluorescent intensity levels, and C1q assays may be useful in assessing the risk of antibody-mediated rejection and timely interventions in liver transplantation.

Equivalency of buffalo (Bubalus bubalis) embryonic stem cells derived from fertilized, parthenogenetic, and hand-made cloned embryos.

This study was aimed at establishing buffalo embryonic stem cells (ESCs) from in vitro fertilized (IVF), parthenogenetic, and hand-made cloned (HMC) embryos and to check their equivalency in terms of stem cell marker expression, longevity, proliferation, and differentiation pattern. ESCs derived from all three sources were found by immunofluorescence to express the pluripotency markers SSEA-4, TRA-1-60, TRA-1-81, OCT4, and SOX2 and were able to form embryoid bodies containing cells expressing genes specific to endoderm (AFP, HNF4, and GATA4), mesoderm (MSX1, BMP4, and ASA), and ectoderm (cytokeratin 8 and NF68). Reverse transcriptase PCR (RT-PCR) showed cells from all sources to be positive for pluripotency markers OCT4, SOX2, NANOG, STAT3, REX1, FOXD3, NUCLEOSTEMIN, and TELOMERASE. Pluripotency markers OCT4, SOX2, NANOG, and c-MYC were also analyzed by real-time PCR. No significant differences were observed among ESCs from all three sources for all these genes except NANOG, whose expression was higher (p<0.05) in HMC-derived ESCs (6.897±2.3) compared to that in parthenogenesis- and IVF-derived cells (1.603±0.315 and 1±0, respectively). Pluripotent, stable buffalo ESC lines derived from IVF, parthenogenesis, and HMC embryos may be genetically manipulated to provide a powerful tool for studies involving embryonic development, genomic imprinting, gene targeting, cloning, chimera formation, and transgenic animal production.

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.

Delayed trunk muscle reflex responses increase the risk of low back injuries.

STUDY DESIGN: Prospective observational study with a 2- to 3-year follow-up. OBJECTIVES: To determine whether delayed muscle reflex response to sudden trunk loading is a result of or a risk factor for sustaining a low back injury (LBI). SUMMARY OF BACKGROUND DATA: Differences in motor control have been identified in individuals with chronic low back pain and in athletes with a history of LBI when compared with controls. However, it is not known whether these changes are a risk for or a result of LBI. METHODS: Muscle reflex latencies in response to a quick force release in trunk flexion, extension, and lateral bending were measured in 303 college athletes. Information was also obtained regarding their personal data, athletic experience, and history of LBI. The data were entered into a binary logistic regression model to identify the predictors of future LBI. RESULTS.: A total of 292 athletes were used for the final analysis (148 females and 144 males). During the follow-up period, 31 (11%) athletes sustained an LBI. The regression model, consisting of history of LBI, body weight, and the latency of muscles shutting off during flexion and lateral bending load releases, predicted correctly 74% of LBI outcomes. The odds of sustaining LBI increased 2.8-fold when a history of LBI was present and increased by 3% with each millisecond of abdominal muscle shut-off latency. On average, this latency was 14 milliseconds longer for athletes who sustained LBI in comparison to athletes who did not sustain LBI (77 [36] vs. 63 [31]). There were no significant changes in any of the muscle response latencies on retest following the injury. CONCLUSIONS: The delayed muscle reflex response significantly increases the odds of sustaining an LBI. These delayed latencies appear to be a preexisting risk factor and not the effect of an LBI.

Neuromuscular function in athletes following recovery from a recent acute low back injury.

STUDY DESIGN: Observational case control design. OBJECTIVES: To examine muscle response to sudden trunk loading in athletes with and without a recent history of acute low back injury (LBI). BACKGROUND: Impaired neuromuscular function is associated with chronic low back pain. This study examined whether such impairment persists after recovery from an acute LBI. METHODS AND MEASURES: Seventeen athletes who had a recent history of acute LBI and 17 matched healthy controls were tested. At the time of testing (mean = 56 days postinjury, range = 7-120 days postinjury), all athletes were symptom free and had returned to regular competition. Subjects performed isometric exertions in trunk flexion, extension, and left and right lateral bending against a trunk restraining cable. Upon reaching the target isometric force, the cable was released to impose sudden loading on the lumbar spine. Surface EMG signals from 12 major trunk muscles were recorded. The shut-off and switch-on latencies and number of muscles responding to sudden loading were compared between the 2 groups. RESULTS: In all 4 testing directions, the athletes with a recent history of acute LBI shut off significantly fewer muscles and did so with delayed latency. On average, the injured subjects shut off 4.0 out of 6.0 (SD = 1.3) muscles compared to 4.6 out of 6.0 (SD = 1.3) muscles in the control group. The average muscle shut-off latency was 71 (SD = 31) milliseconds for the injured and 50 (SD = 21) milliseconds for the control subjects. No differences were found in number or latency of muscles switching on. CONCLUSIONS: These objective measures of neuromuscular function indicated an altered muscle response pattern to sudden trunk loading in athletes following their clinical recovery from a recent acute LBI.