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The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has previously never been identified with humans, thereby creating devastation in public health. The need for an effective vaccine to curb this pandemic cannot be overemphasized. In view of this, we, therefore, designed a subcomponent antigenic peptide vaccine targeting the N-terminal (NT) and C-terminal (CT) RNA binding domains of nucleocapsid protein that aid in viral replication. Promising antigenic B-cells and T cell epitopes were predicted using computational pipelines. The peptides “RIRGGDGKMKDL” and “AFGRRGPEQTQGNFG” were the B cell linear epitopes with good antigenic index and non-allergenic property. Two CD8+ and Three CD4+ T-cell epitopes were also selected considering their safe immunogenic profiling such as allergenicity, antigen level conservancy, antigenicity, peptide toxicity, and putative restrictions to a number of MHC-I and II alleles. With these selected epitopes, a non-allergenic chimeric peptide vaccine incapable of inducing a Type II hypersensitivity reaction was constructed. The molecular interaction between the toll-like receptor-5 (TLR5) which was triggered by the vaccine was analyzed by molecular docking and scrutinized using dynamics simulation. Finally, in silico cloning was performed to ensure the expression and translation efficiency of the vaccine, utilizing pET-28a vector. This research, therefore, provides a guide for experimental investigation and validation.

Thesearch for a sustainable and environmental friendly energy has resulted to thedevelopment of an alternative renewable energy such as biogas. The objective ofthis work was to identify the presence of methanogenic bacteria in a short-termoperated biogas reactor fed with kitchen waste by PCR-amplification of themethyl coenzyme M reductase (mcrA). Household organic waste wasobtained from Bells University of Technology canteen while pig manure thatserved as the inoculum was obtained from a pig farm. Three replicate sampleswere taken every week and analysed for changes in the physico-chemicalparameters to determine their effects on the rate of biogas production. Resultsobtained showed that the % moisture and carbon contents, before and afterfermentation, were reduced from 88% to 80% and16.7% to 12.5%, while % nitrogen content increased from 1.20% to 1.70%. Themean weekly biogas production ranged from 27.4 ml to 147 ml at 4 weeks and 6weeks, respectively. Methyl coenzyme M reductase(mcrA) gene, which codes for the enzyme thatcatalyse the terminal step in methane production during the anaerobicfermentation of biomass, was amplified both inthe methanogenic bacteria and transformed E.coli with the fragment size of 500 bp.

Twelve medicated soaps: Crusader, Septol, Carat, 14 days, Funbact, Lifebouy, Safeguard, Tetmosol, TCP, Dettol, Delta and Antigal were investigated for their antibacterial activities against Staphylococcus aureus and Escherichia coli. Two cloth washing soaps (Key and Truck) were used as control. Identification of the bacterial species was by standard microbiological techniques which included colonial examination, Gram staining and biochemical testing. Minimum inhibitory and minimum bactericidal activities of the soaps were determined by disc-agar diffusion method. Profloxacin was employed as a positive control antibiotic. Crusader soap had the highest antibacterial activity (25 mm, against Staphylococcus aureus) while Antigal exhibited the least zone of inhibition (9 mm against Staphylococcus aureus). Significant differences (P

Cystic fibrosis (CF) patients and model systems exhibit consistent abnormalities in PUFA metabolism, including increased metabolism of linoleate to arachidonate. Recent studies have connected these abnormalities to increased expression and activity of the Δ6- and Δ5-desaturase enzymes. However, the mechanism connecting these changes to the CF transmembrane conductance regulator (CFTR) mutations responsible for CF is unknown. This study tests the hypothesis that increased activity of AMP-activated protein kinase (AMPK), previously described in CF bronchial epithelial cells, causes these changes in fatty acid metabolism by driving desaturase expression. Using CF bronchial epithelial cell culture models, we confirm elevated activity of AMPK in CF cells and show that it is due to increased phosphorylation of AMPK by Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ). We also show that inhibition of AMPK or CaMKKβ reduces desaturase expression and reverses the metabolic alterations seen in CF cells. These results signify a novel AMPK-dependent mechanism linking the genetic defect in CF to alterations in PUFA metabolism.

We previously identified the G6PC2 locus as a strong determinant of fasting plasma glucose (FPG) and showed that a common G6PC2 intronic single nucleotide polymorphism (SNP) (rs560887) and two common G6PC2 promoter SNPs (rs573225 and rs13431652) are highly associated with FPG. However, these promoter SNPs have complex effects on G6PC2 fusion gene expression, and our data suggested that only rs13431652 is a potentially causative SNP. Here we examine the effect of rs560887 on G6PC2 pre-mRNA splicing and the contribution of an additional common G6PC2 promoter SNP, rs2232316, to the association signal.Minigene analyses were used to characterise the effect of rs560887 on G6PC2 pre-mRNA splicing. Fusion gene and gel retardation analyses characterised the effect of rs2232316 on G6PC2 promoter activity and transcription factor binding. The genetic association of rs2232316 with FPG variation was assessed using regression adjusted for age, sex and BMI in 4,220 Europeans with normal FPG.The rs560887-G allele was shown to enhance G6PC2 pre-mRNA splicing, whereas the rs2232316-A allele enhanced G6PC2 transcription by promoting Foxa2 binding. Genetic analyses provide evidence for association of the rs2232316-A allele with increased FPG (β = 0.04 mmol/l; p = 4.3 × 10(-3)) as part of the same signal as rs560887, rs573225 and rs13431652.As with rs13431652, the in situ functional data with rs560887 and rs2232316 are in accord with the putative function of G6PC2 in pancreatic islets, and suggest that all three are potentially causative SNPs that contribute to the association between G6PC2 and FPG.

Patients with cystic fibrosis, caused by mutations in CFTR, exhibit specific and consistent alterations in the levels of particular unsaturated fatty acids compared with healthy controls. Evidence suggests that these changes may play a role in the pathogenesis of this disease. Among these abnormalities are increases in the levels of n-7 and n-9 fatty acids, particularly palmitoleate (16:1n-7), oleate (18:1n-9), and eicosatrienoate or mead acid (20:3n-9). The underlying mechanisms of these particular changes are unknown, but similar changes in the n-3 and n-6 fatty acid families have been correlated with increased expression of fatty acid metabolic enzymes. This study demonstrated that cystic fibrosis cells in culture exhibit increased metabolism along the metabolic pathways leading to 16:1n-7, 18:1n-9, and 20:3n-9 compared with wild-type cells. Furthermore, these changes are accompanied by increased expression of the enzymes that produce these fatty acids, namely Δ5, Δ6, and Δ9 desaturases and elongases 5 and 6. Taken together, these findings suggest that fatty acid abnormalities of the n-7 and n-9 series in cystic fibrosis are as a result, at least in part, of increased expression and activity of these metabolic enzymes in CFTR-mutated cells.

Summary Background Factor XIa is traditionally assigned a role in FIX activation during coagulation. However, recent evidence suggests this protease may have additional plasma substrates. Objective To determine whether FXIa promotes thrombin generation and coagulation in plasma in the absence of FIX, and to determine whether FXI-deficiency produces an antithrombotic effect in mice independently of FIX. Methods FXIa, FXIa variants and anti-FXIa antibodies were tested for their effects on plasma coagulation and thrombin generation in the absence of FIX, and for their effects on the activation of purified coagulation factors. Mice with combined FIX and FXI deficiency were compared with mice lacking either FIX or FXI in an arterial thrombosis model. Results In FIX-deficient plasma, FXIa induced thrombin generation, and anti-FXIa antibodies prolonged clotting times. This process involved FXIa-mediated conversion of FX and FV to their active forms. Activation of FV by FXIa required the A3 domain on the FXIa heavy chain, whereas activation of FX did not. FX activation by FXIa, unlike FIX activation, was not a calcium-dependent process. Mice lacking both FIX and FXI were more resistant to ferric chloride-induced carotid artery occlusion than FXI-deficient or FIX-deficient mice. Conclusion In addition to its predominant role as an activator of FIX, FXIa may contribute to coagulation by activating FX and FV. As the latter reactions do not require calcium, they may make important contributions to in vitro clotting triggered by contact activation. The reactions may be relevant to FXIa's roles in hemostasis and in promoting thrombosis.

The detection of Entamoeba histolytica in stool and tissue samples or in culture has been hampered by several constraints that are related to traditional microscopic examination of fresh samples and those stained with other chemical reagents. In this study, a direct immunoperoxidase (DIP) technique was used for the identification of E. histolytica in stool samples and compared to direct microscopy and permanent staining. Immunopurified antigens of axenic E. histolytica were used to produce rabbit hyper-immune sera. Immunoglobulin G (IgG) was purified from hyper-immune sera and coupled to peroxidase using a two-step procedure. The IgG-peroxidase conjugate was then evaluated by detection of E. histolytica in 128 stool samples and 20 controls from different hospitals in Cameroon, using a direct immunoperoxidase method. The trophozoites and cysts of E. histolytica were strongly stained; both intact and damaged cysts or vegetative forms could be detected in all the positive samples. The immunoperoxidase method was more specific than the iron haematoxylin with no significant difference in sensitivity between the two methods. Results from this study demonstrated that the DIP staining is a reliable, specific and rapid method for the diagnosis of E. histolytica infections. Keywords : amoebiasis, diagnosis, direct immuno-peroxidase, Entamoeba histolytica, Entamoeba dispar , endemic areas, parasites Journal of Tropical Microbiology and Biotechnology Vol. 2(1) 2006: 10-18

Abstract 2244 In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV. We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not. In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX. Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures: Tucker: Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.