Phase I Randomized Safety Study of Twice Daily Dosing of Acidform Vaginal Gel: Candidate Antimicrobial Contraceptive

Background

Acidform gel, an acid-buffering product that inactivates spermatozoa, may be an effective topical non-hormonal contraceptive. This study was designed to evaluate the safety of vaginal dosing and effects of Acidform on mucosal immune mediators, antimicrobial properties of genital secretions, and vaginal microbiota.

Methods

Thirty-six sexually abstinent U.S. women were randomized to apply Acidform or hydroxyethylcellulose (HEC) placebo gel twice daily for 14 consecutive days. Safety was assessed by symptoms and pelvic examination. The impact of gel on mucosal immunity was assessed by quantifying cytokines, chemokines, antimicrobial proteins and antimicrobial activity of genital secretions collected by cervicovaginal lavage (CVL) at screening, 2 hours after gel application, and on days 7, 14 and 21. Vaginal microbiota was characterized at enrollment and day 14 using species-specific quantitative PCR assays.

Results

The median vaginal and cervical pH was significantly lower 2 hours after application of Acidform and was associated with an increase in the bactericidal activity of CVL against E. coli. However, 65% of women who received Acidform had at least one local adverse event compared with 11% who received placebo (p = 0.002). While there was no increase in inflammatory cytokines or chemokines, CVL concentrations of lactoferrin and interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory protein, were significantly lower following Acidform compared to HEC placebo gel application. There were no significant changes in Lactobacillus crispatus or Lactobacillus jensenii in either group but there was a decrease in Gardnerella vaginalis in the Acidform group (p = 0.08).

Conclusions

Acidform gel may augment mucosal defense as evidenced by an increase in bactericidal activity of genital secretions against E. coli and a decrease in Gardnerella vaginalis colonization. However, Acidform was associated with more irritation than placebo and lower levels of antimicrobial (lactoferrin) and anti-inflammatory (IL-1ra) proteins. These findings indicate the need for additional safety studies of this candidate non-hormonal contraceptive.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00850837","term_id":"NCT00850837"}}NCT00850837     

Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.     

Community composition has greater impact on the functioning of marine phytoplankton communities than ocean acidification

Ecosystem functioning is simultaneously affected by changes in community composition and environmental change such as increasing atmospheric carbon dioxide (CO₂) and subsequent ocean acidification. However, it largely remains uncertain how the effects of these factors compare to each other. Addressing this question, we experimentally tested the hypothesis that initial community composition and elevated CO₂ are equally important to the regulation of phytoplankton biomass. We full‐factorially exposed three compositionally different marine phytoplankton communities to two different CO₂ levels and examined the effects and relative importance (ω²) of the two factors and their interaction on phytoplankton biomass at bloom peak. The results showed that initial community composition had a significantly greater impact than elevated CO₂ on phytoplankton biomass, which varied largely among communities. We suggest that the different initial ratios between cyanobacteria, diatoms, and dinoflagellates might be the key for the varying competitive and thus functional outcome among communities. Furthermore, the results showed that depending on initial community composition elevated CO₂ selected for larger sized diatoms, which led to increased total phytoplankton biomass. This study highlights the relevance of initial community composition, which strongly drives the functional outcome, when assessing impacts of climate change on ecosystem functioning. In particular, the increase in phytoplankton biomass driven by the gain of larger sized diatoms in response to elevated CO₂ potentially has strong implications for nutrient cycling and carbon export in future oceans.     

Sleeping Beauty-mediated down-regulation of huntingtin expression by RNA interference

Huntington disease (HD) is a devastating neurologic disorder that is characterized by abnormal expansion of a CAG nt repeat in the first exon of the huntingtin (htt) gene, producing a mutant protein with an elongated polyglutamine stretch. The presence of this mutant protein is correlated with the characteristic loss of striatal neurons and the clinical manifestation of HD. Currently there is no effective treatment for the associated cell death. The aim of this study was to evaluate an innovative strategy combining RNA interference (RNAi) and gene transfer via the nonviral Sleeping Beauty (SB) transposon system to down-regulate Htt expression. siRNA expression vectors were designed to target exons 1, 4, 6, and 62 of the human htt gene. Real-time RT-PCR and Western blot analysis were used to quantify Htt mRNA and protein levels, respectively, in human cell lines. The results indicated that selected siRNA constructs significantly decreased Htt mRNA and protein levels relative to controls. In addition, SB transposition of the siRNA constructs into the genome reduced long-term protein expression of Htt by approximately 90%. The combination of siRNA, the SB transposon, and an accurate transgenic mouse model may permit evaluation of this approach in preventing the pathogenesis associated with expression of mutant Htt.     

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca²⁺. The elevation of Ca²⁺ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K⁺ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca²⁺ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K⁺ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K⁺ current present in the Slo3 ^−/− sperm arise from CATSPER? Second, can any additional membrane K⁺ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 ^−/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca²⁺ influx in response to alkalization.     

AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain

The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.     

Cyanide-induced transition from endogenous carbohydrate to lipid oxidation as indicated by the carbon-13 content of respiratory CO2

The respiration of thin aerated discs of potato tuber tissue rises sigmoidally through 24 h. Aged disc respiration is ostensibly resistant to concentrations of cyanide which inhibit the respiration of fresh discs. It has been shown that cyanide-resistant respiration does not represent indifference to the inhibitor, but is rather due to the suppression of one respiratory carbon path and the evocation of another. The predominant respiratory carbon path of aged discs in the absence of cyanide comprises glycolysis linked to the tricarboxylic acid cycle. The carbon path mediating the cyanide-induced respiration reflects tricarboxylic acid cycle-independent lipid degradation.

The respiratory substrate at any time was deduced by comparing the ¹³C/¹²C ratio of respired CO₂, collected from discs in the presence or absence of cyanide, with the ¹³C/¹²C ratios characterizing endogenous potential metabolites. The determination of the predominant respiratory substrate in potato discs, which have an endogenous substrate reserve, proved possible because the relative concentrations of the stable carbon isotopes in endogenous compounds such as lipid and starch are widely different.     

Wild‐type p53 enhances endothelial barrier function by mediating RAC1 signalling and RhoA inhibition

Inflammation is the major cause of endothelial barrier hyper‐permeability, associated with acute lung injury and acute respiratory distress syndrome. This study reports that p53 “orchestrates” the defence of vascular endothelium against LPS, by mediating the opposing actions of Rac1 and RhoA in pulmonary tissues. Human lung microvascular endothelial cells treated with HSP90 inhibitors activated both Rac1‐ and P21‐activated kinase, which is an essential element of vascular barrier function. 17AAG increased the phosphorylation of both LIMK and cofilin, in contrast to LPS which counteracted those effects. Mouse lung microvascular endothelial cells exposed to LPS exhibited decreased expression of phospho‐cofilin. 17AAG treatment resulted in reduced levels of active cofilin. Silencing of cofilin pyridoxal phosphate phosphatase (PDXP) blocked the LPS‐induced hyper‐permeability, and P53 inhibition reversed the 17AAG‐induced PDXP down‐regulation. P190RHOGAP suppression enhanced the LPS‐triggered barrier dysfunction in endothelial monolayers. 17AAG treatment resulted in P190RHOGAP induction and blocked the LPS‐induced pMLC2 up‐regulation in wild‐type mice. Pulmonary endothelial cells from “super p53” mice, which carry additional p53‐tg alleles, exhibited a lower response to LPS than the controls. Collectively, our findings help elucidate the mechanisms by which p53 operates to enhance barrier function.