Strengthening Warfighter Resiliency Using Broad-Spectrum or Host-Directed Therapies within the Rapid Acquisition and Investigation of Drugs for Repurposing Program.

To maintain cadence with looming threats in a prolonged field-care environment, the broader medical countermeasure (MCM) enterprise must adopt new strategies for chemical, biological, radiological, and nuclear (CBRN)-addressing drug development. The Countering Emerging Threats - Rapid Acquisition and Investigation of Drugs for Repurposing (CET RAIDR) program within the Joint Program Manager for CBRN Medical is designed to rapidly tackle known, unknown, and emerging threats by utilizing late-stage or licensed therapeutics. The focus of the CET RAIDR effort is to bridge treatment gaps between threat identification and the implementation of licensed targeted MCMs, thereby strengthening warfighter resiliency.

Approach to Strain Selection and the Propagation of Viral Stocks for Venezuelan Equine Encephalitis Virus Vaccine Efficacy Testing under the Animal Rule.

Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical.

Comparison of Aerosol- and Percutaneous-acquired Venezuelan Equine Encephalitis in Humans and Nonhuman Primates for Suitability in Predicting Clinical Efficacy under the Animal Rule.

Licensure of medical countermeasure vaccines to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires the use of the Animal Rule to assess vaccine efficacy, because human studies are not feasible or ethical. We therefore performed a retrospective study of VEE cases that occurred in at-risk laboratory workers and support personnel during the United States Biowarfare Program (1943-1969) to better define percutaneous- and aerosol-acquired VEE in humans and to compare these results with those described for the NHP model (in which high-dose aerosol VEEV challenge led to more severe encephalitis than parenteral challenge). Record review and analysis of 17 aerosol- and 23 percutaneous-acquired human cases of VEE included incubation period, symptoms, physical examination findings, and markers of infection.

Prevention and removal of lipid deposits by lens care solutions and rubbing.

Purpose: Despite the prevalence of silicone hydrogel (SiHy) contact lenses, there are relatively few studies that evaluate the efficacy of multipurpose lens care solutions (MPSs) in reducing lipid deposition on these lenses and the effect of rubbing on the removal. Therefore, we used an in vitro soaking and rubbing model to compare the effectiveness of borate buffered saline (BBS) and two commercial MPSs, PureMoist and Biotrue, in preventing sorption of representative polar and nonpolar lipids.

Methods: Radiolabeled cholesterol (CH) and dipalmitoylphosphatidylcholine (DPPC) were sorbed on two SiHy lenses (senofilcon A and balafilcon A) from an artificial tear fluid.

The role of multi-purpose solutions in prevention and removal of lipid depositions on contact lenses.

The sorption and desorption of radiolabeled dipalmitoylphosphatidylcholine (DPPC) and cholesterol (CH) were measured on 5 types of commercial contact lenses. The lenses were soaked in vitro in an artificial tear fluid for 16h. The effects of borate buffered saline and two commercial multi-purpose lens-care solutions (MPSs) on reducing the lipid (DPPC and CH) sorption and increasing the lipid removal were examined.

Adenoviruses isolated from wild gorillas are closely related to human species C viruses.

We have isolated and cultured three distinct adenoviruses from wild gorillas. Phylogenetic analysis grouped the viruses with human adenovirus species C based on DNA polymerase, hexon, and E4ORF6 genes. The three wild gorilla adenoviruses clustered with the other species C captive gorilla adenoviruses, forming a branch separate from human and chimpanzee/bonobo adenoviruses.

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions.

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion.

Probing for pH-regulated proteins in Sinorhizobium medicae using proteomic analysis.

To elucidate the mechanisms of pH response in an acid-tolerant Sinorhizobium medicae strain we have identified acid-activated gene transcription and now complement this approach by using a proteomic analysis to identify the changes that occur following exposure to acidity. Protein profiles of persistently or transiently acid-stressed S. medicae cells were compared to those grown in pH neutral, buffered media.

Probing for pH-regulated genes in Sinorhizobium medicae using transcriptional analysis.

The low pH sensitivity of Sinorhizobium species is one of the major causes of reduced productivity of Medicago species (such as lucerne) sown in acidic soils. To investigate the pH response of an acid-tolerant Sinorhizobium medicae strain, a pool of random promoter fusions to gusA was created using minitransposon insertional mutagenesis. Acid-activated expression was identified in 11 mutants; rhizobial DNA flanking insertions in 10 mutants could be cloned and the DNA sequences obtained were used to interrogate the genome database of Sinorhizobium meliloti strain 1021.

Sinorhizobium medicae genes whose regulation involves the ActS and/or ActR signal transduction proteins.

ActS-ActR proteins belong to a highly conserved family of two-component signal transduction systems involved in global regulation in the alpha-proteobacteria; they were first identified in Sinorhizobium medicae (previously Sinorhizobium meliloti) as essential for acid-tolerance. This paper reports on the identification of genes regulated by ActS and/or ActR in S. medicae.

ActP controls copper homeostasis in Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti preventing low pH-induced copper toxicity.

Two 'calcium-irreparable' acid-sensitive mutants were identified after mutagenizing Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti with Tn5. Each mutant contains a single copy of the transposon which, inserted within the actP gene, prevents expression of a P-type ATPase that belongs to the CPx heavy metal-transporting subfamily.

Survival and exopolysaccharide production in Sinorhizobium meliloti WSM419 are affected by calcium and low pH.

Cells of Sinorhizobium meliloti WSM419 showed an adaptive acid-tolerance response when grown at pH 5.8 instead of pH 7.0.

Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes.

The transcriptional regulator gene phrR in Sinorhizobium meliloti WSM419 is regulated by low pH and other stresses.

The phrR gene in Sinorhizobium meliloti (previously known as Rhizobium meliloti) WSM419, directly downstream from actA, is induced by low pH or certain stresses (e.g. high concentrations of Zn2+, Cu2+, H2O2 or ethanol), but not in stationary phase or by other stresses (e.

Rhizobium leguminosarum bv. viciae produces a novel cyclic trihydroxamate siderophore, vicibactin.

Trihydroxamate siderophores were isolated from iron-deficient cultures of three strains of biovar , two from Japan (WSM709, WSM710) and one from the Mediterranean (WU235), and from a Tn5-induced mutant of WSM710 (MNF7101). The first three all produced the same compound (vicibactin), which was uncharged and could be purified by solvent extraction into benzyl alcohol. The gallium and ferric complexes of vicibactin were extractable into benzyl alcohol at pH 5.

Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR.

A mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38.

An essential role for actA in acid tolerance of Rhizobium meliloti.

The actA gene, which is disrupted by Tn5 in the acid-sensitive mutant of Rhizobium meliloti TG2-6, was cloned and sequenced. It encodes a protein of 541 amino acids with a calculated molecular mass of 57,963 Da and an estimated pl of 9.0.

Maintenance of Intracellular pH and Acid Tolerance in Rhizobium meliloti.

The development and function of the Rhizobium meliloti-Medicago sp. symbiosis are sensitive to soil acidity. Physiological criteria that can be measured in culture which serve to predict acid tolerance in soil would be valuable.