MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than four MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.     

Structure of the gene of tum- transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. We found that the gene conferring expression of tum- antigen P91A contains 12 exons, encoding a 60 kd protein lacking a typical N-terminal signal sequence. The sequence shows no significant similarity with sequences in current data bases. A mutation that causes expression of the antigen is located in exon 4; it is the only apparent difference between the normal and the antigenic alleles. A short synthetic peptide corresponding to a region of exon 4 located around this mutation makes P815 cells sensitive to lysis by anti-P91A cytolytic T cells. The mutation creates a strong aggretope enabling the peptide to bind the H-2 Ld molecule. Several secondary tumor cell variants that no longer express tum- antigen P91A were found to carry deletions in the gene.     

Creatine–facilitated protection of stress caused by disrupted circadian rhythm

Disturbances in natural Circadian rhythm are well-known stress factors, affecting a range of metabolic pathways in the living body including the brain. Hence, discovery of natural compounds that could help to prevent and cure of adverse changes is very important. One of the recently discussed substances is creatine, that is believed to have anti-stressor properties. Recent paper describes the impact of intraperitoneally injected creatine (140 mg/kg) into rats with a disturbed natural circadian rhythm for an extended period of time (30 days). Markedly, creatine-treated animals show positive changes in open-field behavioral parameters, and an increase in certain antioxidant enzymes’ (SOD, catalase) activity in the hippocampus, whereas the concentration of nitric oxide, H₂O₂, and Ca²⁺ are approximated to the control value. Similar findings were also observed in case of Na⁺/K⁺- and Ca²⁺-ATPases. To sum up, the recent findings allow the conclusion that oxidative stress induced by long-term disturbances in natural circadian rhythm is accompanied and likely provoked by an increase in Ca²⁺-cytotoxicity, which is supposedly normalized by the creatine’s indirect action on the NMDA receptor. Therefore, impact on energy mediating pathways has a positive effect on stabilization of antioxidant and various metabolic systems and protecting hippocampal cells from stress.     

Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol

Biofuels provide a potential route to avoiding the global political instability and environmental issues that arise from reliance on petroleum. Currently, most biofuel is in the form of ethanol generated from starch or sugar, but this can meet only a limited fraction of global fuel requirements. Conversion of cellulosic biomass, which is both abundant and renewable, is a promising alternative. However, the cellulases and pretreatment processes involved are very expensive. Genetically engineering plants to produce cellulases and hemicellulases, and to reduce the need for pretreatment processes through lignin modification, are promising paths to solving this problem, together with other strategies, such as increasing plant polysaccharide content and overall biomass.     

Streamlined production of genetically modified T cells with activation,transduction and expansion in closed-system G-Rex bioreactors

BackgroundGas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes.ResultsThe simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically.DiscussionThis study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

Influence of exogenous corticosterone on testicular function and mating behavior of Nigerian indigenous cocks

In bridging the knowledge gap on stress physiology of Nigerian indigenous chickens, this study investigated the effect of exogenous corticosterone (eCORT) as stress inducing agent on the testicular function and mating behavior of Nigerian indigenous cocks. Twenty-four (24) cocks and one hundred and forty four (144) hens (mating ratio of 1 cock: 6 hens) were grouped into four and assigned to each of the four eCORT treatments (0, 2, 4 and 6 mgeCORT/KgBW) daily for 14 days. Semen samples were collected on days 0, 7 and 14 and analyzed for semen volume (SV), progressive sperm motility (PSM), membrane integrity (MI) and sperm abnormality (SA). Mating behaviors were monitored on days 3, 5 and 8. Blood samples, for hormonal (Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH) Testosterone (TEST) and stress analysis (heterophil/lymphocyte ratio, H/L) were collected from brachial vein on days 7 and 14. On day 15, cocks were euthanized and testes harvested for histomorphometry. Data were analyzed using multivariate analysis, one–way ANOVA and Kruskal-Wallis tests all in SPSS 23. Administration of 4 mgeCORT/KgBW declined (P<0.05) PSM while 4 mgeCORT/KgBW and 6 mgeCORT/KgBW cocks had reduced (P<0.05) SV and MI with increased SA. Compared to baseline values, progressive sperm motility of cocks administered 6 mgeCORT for 7 and 14 days decreased (P<0.05) by 57.5% and 52.4%, respectively. Exogenous CORT had no significant (P>0.05) influence on the mating behaviors, H/L ratio, FSH and TEST. However, 2 mgeCORT/KgBW enhanced LH levels. Administration of eCORT did not affect the testicular epithelial height and seminiferous tubular diameter. In conclusion, optimal stress induced by eCORT impaired semen quality but with less impact on reproductive hormones, H/L and mating behaviors of intensively raised Nigerian indigenous cocks.     

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

Background

Recurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.

Methodology/Principal Findings

Analysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.

Conclusions

Impaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.     

Single chain Fab (scFab) fragment

Background  

The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display.