Utilization of phosphorus from dicalcium phosphate and defluorinated phosphate supplements by rumen bacteria in vitro and by lambs fed a semi-purified ration

The primary functions of phosphorus in animal metabolism, structure and reproduction are as follows: (1) it is concerned in the maintenance of a proper reaction in the blood; (2) with calcium it is of great importance in the formation of bone and teeth; (3) it is necessary for the maintenance of a normal calcium concentration in the blood; (4) it plays an important role in carbohydrate metabolism and the transfer of energy in cellular activity. The requirements of phosphorus for cattle and sheep have long been estimated based upon the needs of the animal body. Bone development, blood phosphorus levels and animal performance have been the principal indicators of phosphorus adequacy in much of the work done for establishing the phosphorus level in the diet that is needed for optimum animal production. More recently rumen bacteria are also being employed for this purpose. Cellulose is the main constituent of the crude fiber in roughages. Herbivores in general and ruminants in particular digest and use large amounts of cellulose with the help of the rumen bacteria. A number of workers have studied the factors affecting rumen microorganisms and cellulose digestion in vivo and in vitro. Vigorous rumen microbial activity which is essential for proper and efficient feed utilization by cattle and sheep depends upon the presence and availability of various nutrients in the rations fed. Experiments have shown that rumen bacteria require a source of nitrogen, readily available energy, minerals, fatty acids and other factors. It is now known that phosphorus is one of the essential minerals required by these bacteria. The amount of phosphorus required for optimum bacterial activity in the rumen may be an important criterion for fixing the need of the animal, for this mineral. The use of phosphatic supplements which are available today in rather large numbers for livestock feeding assumes importance in this context. The chemical content of phosphorus in them may often be misleading when an attempt is made to compute the feed requirements of farm animals. Not all the phosphorus in these phosphatic compounds may be available to the animal. Therefore several phosphatic compounds presently available as feed supplements for livestock were obtained to evaluate their availability. The artificial rumen technique was employed for the first part of the experiment for its obvious advantages. The assay is based on the fact that phosphorus depleted rumen microorganisms will rapidly digest cellulose only when supplied with an adequate amount of available phosphorus. The following study was undertaken therefore: (1) To determine the effect of phosphorus from each of three commercial supplements of dicalcium phosphate and defluorinated phosphate upon cellulose digestion by rumen microorganisms in vitro. (2) To determine the effect of one good and one poor commercial defluorinated phosphate supplement as revealed by item (1) above, on digestibility of a semi-purified phosphorus-poor basal ration by sheep. (3) To compare the relative availability of the two samples of phosphorus used in (2) above to sheep

Mycobacterial transcriptional signals: requirements for recognition by RNA polymerase and optimal transcriptional activity

Majority of the promoter elements of mycobacteria do not function well in other eubacterial systems and analysis of their sequences has established the presence of only single conserved sequence located at the −10 position. Additional sequences for the appropriate functioning of these promoters have been proposed but not characterized, probably due to the absence of sufficient number of strong mycobacterial promoters. In the current study, we have isolated functional promoter-like sequences of mycobacteria from the pool of random DNA sequences. Based on the promoter activity in Mycobacterium smegmatis and score assigned by neural network promoter prediction program, we selected one of these promoter sequences, namely A(37) for characterization in order to understand the structure of housekeeping promoters of mycobacteria. A(37)–RNAP complexes were subjected to DNase I footprinting and subsequent mutagenesis. Our results demonstrate that in addition to −10 sequences, DNA sequence at −35 site can also influence the activity of mycobacterial promoters by modulating the promoter recognition by RNA polymerase and subsequent formation of open complex. We also provide evidence that despite exhibiting similarities in −10 and −35 sequences, promoter regions of mycobacteria and Escherichia coli differ from each other due to differences in their requirement of spacer sequences between the two positions

Global assessment of dengue Virus-Specific CD4+ T cell responses in Dengue-Endemic areas

Background: Dengue is a major public health problem worldwide. Assessment of adaptive immunity is important to understanding immunopathology and to define correlates of protection against dengue virus (DENV). To enable global assessment of CD4+ T cell responses, we mapped HLA-DRB1-restricted DENV-specific CD4+ T cell epitopes in individuals previously exposed to DENV in the general population of the dengue-endemic region of Managua, Nicaragua. Methods: HLA class II epitopes in the population of Managua were identified by an in vitro IFNγ ELISPOT assay. CD4+ T cells purified by magnetic bead negative selection were stimulated with HLA-matched epitope pools in the presence of autologous antigen-presenting cells, followed by pool deconvolution to identify specific epitopes. The epitopes identified in this study were combined with those previously identified in the DENV endemic region of Sri Lanka, to generate a “megapool” (MP) consisting of 180 peptides specifically designed to achieve balanced HLA and DENV serotype coverage. The DENV CD4MP180 was validated by intracellular cytokine staining assays. Results: We detected responses directed against a total of 431 epitopes, representing all 4 DENV serotypes, restricted by 15 different HLA-DRB1 alleles. The responses were associated with a similar pattern of protein immunodominance, overall higher magnitude of responses, as compared to what was observed previously in the Sri Lanka region. Based on these epitope mapping studies, we designed a DENV CD4 MP180 with higher and more consistent coverage, which allowed the detection of CD4+ T cell DENV responses ex vivo in various cohorts of DENV exposed donors worldwide, including donors from Nicaragua, Brazil, Singapore, Sri Lanka, and U.S. domestic flavivirus-naïve subjects immunized with Tetravalent Dengue Live-Attenuated Vaccine (TV005). This broad reactivity reflects that the 21 HLA-DRB1 alleles analyzed in this and previous studies account for more than 80% of alleles present with a phenotypic frequency ≥5% worldwide, corresponding to 92% phenotypic coverage of the general population (i.e., 92% of individuals express at least one of these alleles). Conclusion: The DENV CD4 MP180 can be utilized to measure ex vivo responses to DENV irrespective of geographical location

Human innate immune cytokine response to dengue virus

Fluorescence minus four controls for flow cytometry experiments of innate immune cytokine production in response to dengue virus. Pseudo color flow cytometry plots for a representative patient comparing the fluorescence minus four (FMF; top row) control with completely stained sample (bottom) for secretion of (A) TNF-α, (B) IP-10, (C) IL-10, (D) IL-6 and (E) IFN-γ from the indicated cell subsets. Abbreviations: CM – CD14+CD16- classical monocytes; IM – CD14+CD16+ intermediate monocytes; NCM – CD14-CD16+ non-classical monocytes; G – Granulocytes; NKT – CD56+CD3+ natural killer T cells; NK++ - CD56+CD16+ natural killer cells; NK+- - CD56+CD16- natural killer cells; B – CD19+ cells

JEV Human T cell

JEV exposed human volunteers comprising healthy contacts and recovered JEV patients were recruited for this study. Individuals who received the live attenuated JE vaccine SA14-14-2 were also included. Blood drawn was stimulated with capsid, envelope and NS3 proteins of JEV, PS cells uninfected and infected with JEV along with cells alone controls. The FACS antibody panel consisting of anti-CD3-APC-H7 (clone SK7), anti-CD8-PerCP (clone SK1), anti-IFN--PECy7 (clone B27), anti-IL-2-FITC (MQ1-17H12), anti-TNFα-APC (6401-1111) and anti-MIP-1β-PE (11A3), from BD Pharmingen, San Diego, CA

Characterization of RNA synthesis, replication mechanism, and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus

In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15-18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. Among divalent cations, $Mg^{2+}$ was essential and exhibited cooperative binding for its two replicase-binding sites. $Mn^{2+}$, despite sixfold higher affinity for the replicase, elicited only 70% of the maximum $Mg^{2+}$-dependent activity, and deficit of either cation led to synthesis of incomplete RNA products. We also determined as a first instance for a flavivirus RC, kinetic parameters using cytoplasmic "virus-induced heavy membranes" after depleting endogenous nucleotides. Exhaustive trypsin treatment, which degraded the bulk of NS3 and NS5, had no effect on replicase activity, suggesting that the active flaviviral RC resides behind a membrane barrier and recruits minuscule proportions of the replicase proteins

Availability of a second upstream AUG can completely overcome inhibition of protein synthesis initiation engendered by mRNA secondary structure encompassing the start codon

Secondary structure analysis of the mRNA from a nonproductive construct carrying the nonstructural gene 3 (NS3) of Japanese Encephalitis Virus revealed the presence of a potential 28 nucleotide long stem and loop beginning with the guanine of the initiation codon AUG that had a calculated stabilization energy of -13 kcal/mol (Delta G(f)(0)). Provision of an additional AUG along with three codons upstream resulted in complete relief of inhibition. N-terminal amino acid sequence of the recombinant protein was consistent with initiation of protein synthesis having occurred from the upstream AUG. Similar levels of NS3 specific RNA in E. coli cells carrying the expressing and nonexpressing constructs and restoration of recombinant protein expression following deletion of segments beginning with the stem and loop confirmed the role of this structure in blocking expression at the level of translation initiation. Our approach exploits the ability of a ribosome in motion to open up downstream secondary structural elements of considerable stability and represents a novel and widely applicable strategy to overcome a block in translation initiation caused by mRNA secondary structure around the translation start site

JEV Human T cell

JEV exposed human volunteers comprising healthy contacts and recovered JEV patients were recruited for this study. Individuals who received the live attenuated JE vaccine SA14-14-2 were also included. Blood drawn was stimulated with capsid, envelope and NS3 proteins of JEV, PS cells uninfected and infected with JEV along with cells alone controls. The FACS antibody panel consisting of anti-CD3-APC-H7 (clone SK7), anti-CD8-PerCP (clone SK1), anti-IFN--PECy7 (clone B27), anti-IL-2-FITC (MQ1-17H12), anti-TNFα-APC (6401-1111) and anti-MIP-1β-PE (11A3), from BD Pharmingen, San Diego, CA