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We examined the effects of treatment with pulsed electromagnetic fields (PEMFs) on cumulus cells and buffalo somatic cell nuclear transfer (SCNT) embryos. PEMF treatment (30 μT for 3 hours) of cumulus cells increased (p < 0.05) the relative cell viability and cell proliferation and the expression level of OCT4, NANOG, SOX2, P53, CCNB1, and GPX, but decreased (p < 0.05) that of DNMT1, DNMT3a, GSK3b, and BAX, whereas the expression level of DNMT3b, GLUT1, BCL2, CASPASE3, SOD1, and CATALASE was not affected. PEMF treatment of SCNT embryos at the beginning of in vitro culture increased (p < 0.05) the blastocyst rate (51.4% ± 1.36% vs. 42.8% ± 1.29%) and decreased (p < 0.01) the apoptotic index to the level in in vitro fertilization blastocysts, but did not significantly alter the total cell number and the inner cell mass:trophectoderm cell number ratio of blastocysts compared to the controls. PEMF treatment increased the expression level of NANOG, SOX2, CDX2, GLUT1, P53, and BCL2 and decreased that of BAX, CASPASE3, GSK3b, and HSP70, but not OCT4, DNMT1, DNMT3a, DNMT3b, HDAC1, and CCNB1 in blastocysts. It increased (p < 0.001) the global level of H3K27me3 but not H3K18ac. These results suggest that PEMF treatment of SCNT embryos improves their developmental competence, reduces the level of apoptosis, and alters the expression level of several important genes related to pluripotency, apoptosis, metabolism, and stress.

Skeletal muscle wasting causes both morbidity and mortality of cancer patients. Accumulating evidence suggests that the markers of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways are increased in skeletal muscle under multiple catabolic conditions, including cancer. However, the signaling mechanisms and the role of individual arms of the UPR in the regulation of skeletal muscle mass remain largely unknown. In the present study, we demonstrated that gene expression of Toll-like receptors (TLRs) and myeloid differentiation primary response gene 88 (MyD88) was increased in skeletal muscle in a Lewis lung carcinoma (LLC) model of cancer cachexia. Targeted ablation of MyD88 inhibits the loss of skeletal muscle mass and strength in LLC tumor-bearing mice. Inhibition of MyD88 attenuates the LLC-induced activation of the UPR in skeletal muscle of mice. Moreover, muscle-specific deletion of X-box binding protein 1 (XBP1), a major downstream target of IRE1α arm of the UPR, ameliorates muscle wasting in LLC tumor-bearing mice. Our results also demonstrate that overexpression of an active form of XBP1 caused atrophy in cultured myotubes. In contrast, knockdown of XBP1 inhibits myotube atrophy in response to LLC or C26 adenocarcinoma cell conditioned medium. Collectively, our results demonstrate that TLR/MyD88-mediated activation of XBP1 causes skeletal muscle wasting in LLC tumor-bearing mice.

Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. These factors are either secretory, membrane associated or cytosolic in nature. The cytosolic factors facilitate the bacterium to undergo quick adaptive—metabolic, physiological and morphological shifts. The membrane associated virulence factors aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors act synergistically to kill the host cells. In this review, we revisit the role of some of the secreted virulence factors of two human pathogens: Mycobacterium tuberculosis—an intracellular pathogen and Bacillus anthracis—an extracellular pathogen. The advances in research on the role of secretory factors of these pathogens during infection are discussed.

Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events.

The objective of this study was to see the effect of purified heparin binding oviduct specific proteins (OSP) as media supplement on in vitro embryo developmental competence in cattle. The oviduct specific proteins were isolated from abattoir cattle oviducts and precipitated, dialyzed and at the end purified by high performance liquid chromatography system. The SDS-PAGE profile of eluted heparin binding protein (HBP) fraction showed bands between approximately 66-approximately 97 kDa, while heparin unbinding protein (HUBP) fraction showed two bands at approximately 66 kDa and in total protein (TP) bands were approximately 60-approximately 95 kDa. Collected all three OSP fractions were used as a media supplement in three different concentrations (0, 5 and 20 microg/mL) for in vitro maturation of immature oocytes, in vitro fertilization and culture of presumptive embryos at 38.5 degrees C in 5% CO2 incubator with maximum humidity. The highest cleavage rate (73.40 +/- 2.36%) was observed at 5 microg/mL concentration level and lowest cleavage rate (27.63 +/- 1.89%) was obtained in 20 microg/mL total protein (TP) fraction. The highest blastocyst formation (26.47 +/- 1.47%) also occurred in 5 microg/mL concentration of total protein (TP) fraction and the lowest blastocyst rate (3.60 +/- 1.80%) was achieved at 20 microg/mL HBP fraction. The highest cleavage rate in the control group was 60.45 +/- 2.66% in TP fraction and blastocyst formation was 11.66 +/- 2.54% in HUBP fraction which was not significantly differ from HBP fraction. These results indicate that at 5 microg/mL of total OSP fraction (TP) and HBP used as media supplement increased the cleavage rate significantly as compared to HUBP fraction, and total OSP fraction (TP) increased blastocyst formation significantly (P0.05) as compared to HBPHUBP fraction.

Phagocytosis is a complex process that eliminates microbes and is performed by specialised cells such as macrophages. Toll-like receptor 4 (TLR4) is expressed on the surface of macrophages and recognizes Gram-negative bacteria. Moreover, TLR4 has been suggested to play a role in the phagocytosis of Gram-negative bacteria, but the mechanisms remain unclear. Here we have used primary human macrophages and engineered THP-1 monocytes to show that the TLR4 sorting adapter, TRAM, is instrumental for phagocytosis of Escherichia coli as well as Staphylococcus aureus. We find that TRAM forms a complex with Rab11 family interacting protein 2 (FIP2) that is recruited to the phagocytic cups of E. coli. This promotes activation of the actin-regulatory GTPases Rac1 and Cdc42. Our results show that FIP2 guided TRAM recruitment orchestrates actin remodelling and IRF3 activation, two events that are both required for phagocytosis of Gram-negative bacteria., Author summary The Gram-negative bacteria E. coli is the most common cause of severe human pathological conditions like sepsis. Sepsis is a clinical syndrome defined by pathological changes due to systemic inflammation, resulting in paralysis of adaptive T-cell immunity with IFN-β as a critical factor. TLR4 is a key sensing receptor of lipopolysaccharide on Gram-negative bacteria. Inflammatory signalling by TLR4 is initiated by the use of alternative pair of TIR-adapters, MAL-MyD88 or TRAM-TRIF. MAL-MyD88 signaling occurs mainly from the plasma membrane giving pro-inflammatory cytokines like TNF, while TRAM-TRIF signaling occurs from vacuoles like endosomes and phagosomes to give type I interferons like IFN-β. It has previously been shown that TLR4 can control phagocytosis and phagosomal maturation through MAL-MyD88 in mice, however, these data have been disputed and published before the role of TRAM was defined in the induction of IFN-β. A role for TRAM or TRIF in phagocytosis has not previously been reported. Here we describe a novel mechanism where TRAM and its binding partner Rab11-FIP2 control phagocytosis of E. coli and regulate IRF3 dependent production of IFN-β. The significance of these results is that we define Rab11-FIP2 as a potential target for modulation of TLR4-dependent signalling in different pathological states.