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Neisseria meningitidis historically has been an infrequent and sporadic cause of urethritis and other urogenital infections.

Neisseria gonorrhoeae has developed resistance to all previous antibiotics used for treatment. This highlights a crucial need for novel antimicrobials to treat gonococcal infections. We previously showed that carbamazepine (Cz), one of the most commonly prescribed antiepileptic drugs, can block the interaction between gonococcal pili and the I-domain region of human complement receptor 3 (CR3)-an interaction that is vital for infection of the female cervix. We also show that Cz can completely clear an established N. gonorrhoeae infection of primary human cervical cells. In this study, we quantified Cz in serum, saliva, and vaginal fluid collected from 16 women who were, or were not, regularly taking Cz. We detected Cz in lower reproductive tract mucosal secretions in the test group (women taking Cz) at potentially therapeutic levels using a competitive ELISA. Furthermore, we found that Cz concentrations present in vaginal fluid from women taking this drug were sufficient to result in a greater than 99% reduction (within 24 h) in the number of viable gonococci recovered from

The US Neisseria meningitidis urethritis clade (US_NmUC) harbors gonococcal deoxyribonucleic acid alleles and causes gonorrhea-like urogenital tract disease. A large convenience sample of US_NmUC isolates (N = 122) collected between January 2015 and December 2019 in Columbus, Ohio demonstrated uniform susceptibility to antibiotics recommended for gonorrhea treatment and meningococcal chemoprophylaxis.

Multidrug-resistant (MDR) N. gonorrhoeae is a current public health threat. New therapies are urgently needed. PBT2 is an ionophore that disrupts metal homeostasis. PBT2 administered with zinc is shown to reverse resistance to antibiotics in several bacterial pathogens. Here we show that both N. meningitidis and MDR N. gonorrhoeae are sensitive to killing by PBT2 alone. PBT2 is, thus, a candidate therapeutic for MDR N. gonorrhoeae infections.

Gonorrhea remains a major global public health problem because of the high incidence of infection (estimated 82 million cases in 2020) and the emergence and spread of Neisseria gonorrhoeae strains resistant to previous and current antibiotics used to treat infections. Given the dearth of new antibiotics that are likely to enter clinical practice in the near future, there is concern that cases of untreatable gonorrhea might emerge. In response to this crisis, the World Health Organization (WHO), in partnership with the Global Antibiotic Research and Development Partnership (GARDP), has made the search for and development of new antibiotics against N. gonorrhoeae a priority. Ideally, these antibiotics should also be active against other sexually transmitted organisms, such as Chlamydia trachomatis and/or Mycoplasma genitalium, which are often found with N. gonorrhoeae as co-infections. Corallopyronin A is a potent antimicrobial that exhibits activity against Chlamydia spp. and inhibits transcription by binding to the RpoB switch region. Accordingly, we tested the effectiveness of corallopyronin A against N. gonorrhoeae. We also examined the mutation frequency and modes of potential resistance against corallopyronin A. We report that corallopyronin A has potent antimicrobial action against antibiotic-susceptible and antibiotic-resistant N. gonorrhoeae strains and could eradicate gonococcal infection of cultured, primary human cervical epithelial cells. Critically, we found that spontaneous corallopyronin A-resistant mutants of N. gonorrhoeae are exceedingly rare (≤10sup-10/sup) when selected at 4× the MIC. Our results support pre-clinical studies aimed at developing corallopyronin A for gonorrheal treatment regimens.bIMPORTANCE/bThe high global incidence of gonorrhea, the lack of a protective vaccine, and the emergence of N. gonorrhoeae strains expressing resistance to currently used antibiotics demand that new treatment options be developed. Accordingly, we investigated whether corallopyronin A, an antibiotic which is effective against other pathogens, including C. trachomatis, which together with gonococci frequently cause co-infections in humans, could exert anti-gonococcal actioniin vitro/iandiex vivo/i, and potential resistance emergence. We propose that corallopyronin A be considered a potential future treatment option for gonorrhea because of its potent activity, low resistance development, and recent advances in scalable production.

Despite advances made in our understanding ofNeisseria gonorrhoeaepathogenesis, factors dictating the divergent presentation of gonococcal disease observed between men and women,in vivo, remain unclear. Clinical data indicate that gonococcal pathogenesis of the female genital tract is influenced by steroid hormones. Notwithstanding, there are limited data addressing how steroid hormones modulate gonococcal pathogenesis. Hence, we investigated the effect(s) of physiological concentrations of estrogens and progestogens onN. gonorrhoeaeviability and on complement-mediated infection of primary cervical cells. In contrast to previous studies that showed a bacteriostatic effect of non-physiological concentrations of steroid hormones on gonococci, our data indicate that physiological concentrations of estrogens and progestogens do not inhibit gonococcal growthin vitroor during infection of primary cervical cells. Estradiol promoted complement receptor 3 recruitment to the cervical cell surface and, thus, the ability of gonococci to associate with these cells. Progesterone-predominant assay conditions resulted in decreased expression of Opa proteins by gonococci, increased complement production by cervical cells, and increased iC3b opsonization of gonococci during cervical cell challenge. Collectively, our data support clinical observations and demonstrate that estrogens and progestogens distinctly modulate gonococcal cervical infection.

Neisseria gonorrhoeae is a Gram-negative bacterium that causes the sexually transmitted infection gonorrhea. N. gonorrhoeae has progressively developed resistance to all currently prescribed antibiotics, and no vaccine is available. Here, we report the closed, completed, annotated genome sequences for seven N. gonorrhoeae strains obtained by single-molecule real-time (SMRT) long-read genome sequencing.

Neisseria meningitidis strain C311 has been widely used to study meningococcal pathogenesis in the past 30 years, but its genome is not available. Here, we report that the complete C311 genome is 2,311,508 bp in length, contains a total of 2,274 genes, and has a GC content of 51.25%.

L-lactate is an abundant metabolite in a number of niches in host organisms and represents an important carbon source for bacterial pathogens such as Neisseria gonorrhoeae. In this study, we describe an alternative, iron-sulfur cluster-containing L-lactate dehydrogenase (LutACB), that is distinct from the flavoprotein L-lactate dehydrogenase (LldD). Expression of lutACB was found to be positively regulated by iron, whereas lldD was more highly expressed under conditions of iron-limitation. The functional role of LutACB and LldD was reflected in in vitro studies of growth and in the survival of N gonorrhoeae in primary cervical epithelial cells.

Background Neisseria gonorrhoeae is a Gram-negative bacterial pathogen that causes gonorrhoea. No vaccine is available to prevent gonorrhoea and the emergence of MDR N. gonorrhoeae strains represents an immediate public health threat. Objectives To evaluate whether PBT2/zinc may sensitize MDR N. gonorrhoeae to natural cationic antimicrobial peptides. Methods MDR strains that contain differing resistance mechanisms against numerous antibiotics were tested in MIC assays. MIC assays were performed using the broth microdilution method according to CLSI guidelines in a microtitre plate. Serially diluted LL-37 or PG-1 was tested in combination with a sub-inhibitory concentration of PBT2/zinc. Serially diluted tetracycline was also tested with sub-inhibitory concentrations of PBT2/zinc and LL-37. SWATH-MS proteomic analysis of N. gonorrhoeae treated with PBT2/zinc, LL-37 and/or tetracycline was performed to determine the mechanism(s) of N. gonorrhoeae susceptibility to antibiotics and peptides. Results Sub-inhibitory concentrations of LL-37 and PBT2/zinc synergized to render strain WHO-Z susceptible to tetracycline, whereas the killing effect of PG-1 and PBT2/zinc was additive. SWATH-MS proteomic analysis suggested that PBT2/zinc most likely leads to a loss of membrane integrity and increased protein misfolding and, in turn, results in bacterial death. Conclusions Here we show that PBT2, a candidate Alzheimer’s and Huntington’s disease drug, can be repurposed to render MDR N. gonorrhoeae more susceptible to the endogenous antimicrobial peptides LL-37 and PG-1. In the presence of LL-37, PBT2/zinc can synergize with tetracycline to restore tetracycline susceptibility to gonococci resistant to this antibiotic.

Made available in DSpace on 2021-06-25T12:25:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Dairy Research Cluster Initiative II (Dairy Farmers of Canada) Dairy Research Cluster Initiative II (Agriculture and Agri-Food Canada) Dairy Research Cluster Initiative II (Canadian Dairy Network) Dairy Research Cluster Initiative II (Canadian Dairy Commission; Ottawa, Canada) Natural Sciences and Engineering Research Council, Discovery Grant (Ottawa, Canada) USDA-NIFA Hatch/Multistate Project There are multiple factors that contribute to reduced fertility in lactating dairy cows. Recently, a reproductive tract size and position score (SPS) system was developed as a management tool to identify dairy cows with decreased fertility. The aim of this study was to evaluate the association between the SPS on fertility outcomes such as ovulation failure, pregnancy per artificial insemination (P/AI), concentration of pregnancy-associated glycoproteins (PAGs), and pregnancy loss in lactating dairy cows. Primiparous and multiparous lactating Holstein cows (n = 869) were enrolled at two locations. Location 1 (Loc. 1) in Minas Gerais, Brazil (n = 613) and location 2 (Loc. 2) in Agassiz, British Columbia, Canada (n = 256). At the time of AI (d 0), cows were classified as SPS (small [SPS1], medium [SPS2], or large [SPS3] sized reproductive tract) and ovulation failure was determined at 48 h and 7 d post-AI via ultrasonography (Loc. 2 only). Blood samples were collected on d 24 and 31 of gestation for quantification of PAGs and pregnancy diagnosis was performed via ultrasonography at d 31 and 60 post-AI (Loc. 1) and at d 31 +/- 3 and 60 +/- 3 post-AI (Loc. 2). Cows diagnosed pregnant at d 31 post-AI but not pregnant at d 60 were defined to have undergone late embryonic pregnancy loss. Parity was found to impact SPS (P < 0.01), as primiparous cows had a higher frequency of SPS1 and lower frequency of SPS3 when compared with multiparous cows (SPS1: 42.6 vs. 15.0%; SPS3: 7.0 vs. 22.0%, respectively). Cows classified as SPS3 had greater ovulation failure at 48 h (P = 0.04) and 7 d post-AI (P = 0.05). Cows classified as SPS1 had greater P/AI when compared to SPS2 and SPS3 (45.9 +/- 3.3 vs. 37.4 +/- 2.6 and 29.1 +/- 3.5%, respectively; P = 0.004). There was no interaction between parity and SPS on P/AI. Pregnancy loss between 31 and 60 d post-AI was increased in cows classified as SPS3 compared to SPS2 and SPS1 (24.3 +/- 0.05 vs. 11.6 +/- 0.02 and 9.4 +/- 0.02%, respectively; P = 0.04). Cows classified as SPS1 and SPS2 had greater concentrations of PAGs at 31 d post-AI when compared to SPS3 at both Loc.1 (P < 0.01) and Loc. 2 (P < 0.01). There was no interaction between SPS and pregnancy loss on PAGs at 24 and 31 d postAI for either Loc. 1 (P = 0.75 and P = 0.76, respectively) or Loc. 2 (P = 0.61 and P = 0.81, respectively). In conclusion, cows that were classified as SPS3 had greater ovulation failure, reduced P/AI, similar concentrations of PAG on d 24, but decreased on d 31, and a greater incidence of pregnancy loss. Thus, size and position of the reproductive tract is associated with fertility and this scoring system could be used to make reproductive management decisions on dairy operations. (C) 2020 Elsevier Inc. All rights reserved. Univ British Columbia, Fac Land & Food Syst, Appl Anim Biol, Vancouver, BC V6T 1Z4, Canada Sao Paulo State Univ, Dept Anim Prod, BR-18168000 Botucatu, SP, Brazil Univ Tennessee, Dept Anim Sci, Knoxville, TN 37996 USA Texas A&M Univ, Dept Anim Sci, Pregnancy & Dev Programming Area Excellence, College Stn, TX 77843 USA Sao Paulo State Univ, Dept Anim Prod, BR-18168000 Botucatu, SP, Brazil USDA-NIFA Hatch/Multistate Project: W3112-TEX07702

Novel therapies that avert the problem of Neisseria gonorrhoeae with acquired antibiotic resistance are urgently needed. Gonococcal infection of the human cervix is initiated by an interaction between a galactose modification made to its surface appendages, pili, and the I-domain region of (host) complement receptor 3 (CR3). By targeting this crucial gonococcal–I-domain interaction, it may be possible to prevent cervical infection in females. To this end, we identified the I-domain galactose-binding epitope of CR3 and characterized its galactose lectin activity. Moreover, we identified two drugs, carbamazepine and methyldopa, as effective host-targeted therapies for gonorrhea treatment. At doses below those currently used for their respective existing indications, both carbamazepine and methyldopa were more effective than ceftriaxone in curing cervical infection ex vivo. This host-targeted approach would not be subject to N. gonorrhoeae drug resistance mechanisms. Thus, our data suggest a long-term solution to the growing problem of multidrug-resistant N. gonorrhoeae infections., In the absence of a vaccine, multidrug-resistant Neisseria gonorrhoeae has emerged as a major human health threat, and new approaches to treat gonorrhea are urgently needed. N. gonorrhoeae pili are posttranslationally modified by a glycan that terminates in a galactose. The terminal galactose is critical for initial contact with the human cervical mucosa via an interaction with the I-domain of complement receptor 3 (CR3). We have now identified the I-domain galactose-binding epitope and characterized its galactose-specific lectin activity. Using surface plasmon resonance and cellular infection assays, we found that a peptide mimic of this galactose-binding region competitively inhibited the N. gonorrhoeae-CR3 interaction. A compound library was screened for potential drugs that could similarly prohibit the N. gonorrhoeae-CR3 interaction and be repurposed as novel host-targeted therapeutics for multidrug-resistant gonococcal infections in women. Two drugs, methyldopa and carbamazepine, prevented and cured cervical cell infection by multidrug-resistant gonococci by blocking the gonococcal-CR3 I-domain interaction.

Neisseria gonorrhoeae is one of the most common causes of sexually transmitted infections, with an estimated more than 100 million cases of gonorrhea each year worldwide. N. gonorrhoeae has gained recent increasing attention because of the alarming rise in incidence and the widespread emergence of multidrug-resistant gonococcal strains. Vaccine development is one area of renewed interest. Herein, we review the recent advances in this area.Vaccine development for N. gonorrhoeae has been problematic, but recent progress in the field has provided new hope that a gonococcal vaccine may be feasible. Several new vaccine antigens have been characterized in various models of infection. Furthermore, the first potential vaccine-induced protection against gonorrhea in humans has been reported, with decreased rates of gonorrhea described among individuals vaccinated with the Neisseria meningitidis serogroup B vaccine, MeNZB.As antibiotic resistance continues to increase, vaccine development for N. gonorrhoeae becomes more urgent. The MeNZB vaccine is shown to have efficacy, albeit relatively low, against N. gonorrhoeae. This finding has the potential to reinvigorate research in the field of gonococcal vaccine development and will guide future studies of the antigens and mechanism(s) required for protection against gonococcal infection.

Background Complement receptor 3 (CR3) is a leukocytic, pattern recognition receptor that plays a pivotal role in innate immunity. CR3 is also uniquely expressed by human cervical epithelial cells where it is the key receptor mediating Neisseria gonorrhoeae (Ng) cervicitis. Binding of the Ng surface appendage, pilus, to the I-domain region of CR3 (CR3ID) is critical to cervical cell adherence and modulates the host response to infection. Thus, the pilus-CR3ID interaction may pose a novel target for critically-needed, new strategies to treat or prevent Ng disease in women. Methods To identify potential inhibitors of the Ng-CR3 interaction, recombinant human I-domain or purified CR3 were immobilized on biosensor chips. Interactions between these immobilized proteins and a library of 3141 drugs were investigated by surface plasmon resonance (SPR). Drugs that bound the CR3ID were further examined by competitive SPR studies and in Ng, primary human cervical epithelial (Pex) cell infection assays. Results Using SPR, we identified fourteen drugs that bound to the CR3ID with disassociation constants in the nanomolar range. Competitive SPR analysis demonstrated that six of these fourteen drugs blocked the pilus-CR3ID interaction. Moreover, these drugs also blocked Ng adherence to Pex cells as well as to Chinese hamster ovary cells expressing CR3 (CHO-CR3). One drug, carbamazepine, was chosen for further analysis using a panel of low-passage and multidrug-resistant Ng strains. Carbamazepine blocked adherence to Pex and CHO-CR3 cells for all strains tested and also could cure Pex cells that had an established Ng infection. In this regard, a single dose of carbamazepine was effective in curing (≥99% Ng killing by 24h post-treatment) Pex cells infected with multidrug-resistant Ng strains, including the ceftriaxone-resistant strains WHO-X (H041) and WHO-Y (F89). Conclusion Our data identify safe, repurposed, drugs that may have efficacy in preventing and treating Ng cervical infection in women. Disclosure No significant relationships.

Made available in DSpace on 2018-11-26T17:42:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-15 Cattle producers are limited to day 28-30 of gestation as the earliest time point for accurate pregnancy diagnosis due to the effectiveness of ultrasound and chemical based methods, including commercially available pregnancy associated glycoproteins (PAG) tests. The objective of the current studies were to determine if early gestation circulating PAG concentrations at day 24 could be used to diagnose pregnancy in dairy cattle undergoing embryo transfer. In vitro produced embryos were transferred into Holstein x Gir cows and heifers on day 7 following ovulation. Study 1 utilized only cows (n = 101) determined to be pregnant on day 24 of gestation by progesterone concentration, as well as CL and PAG presence. In study 2, animals were not predetermined to be pregnant and both heifers (n = 111) and cows (n = 242) were used. In both studies, blood was collected at day 24 for PAG analysis as well as day 31. Final pregnancy confirmation occurred on day 60 via transrectal ultrasonography. Serum PAG concentrations were quantified using an in house PAG ELISA. Following timed embryo transfer (TET) in study 1, of the 101 cows diagnosed as pregnant on day 24, 80 cows were identified as still pregnant on day 31 of gestation (77%). Study 2 had a pregnancy rate at day 31 of 33.7% of total embryos transferred. Mean circulating PAG concentration at day 24 differed (P < 0.001) between animals diagnosed pregnant and non-pregnant at day 31 in both studies (study 1, 2.964 +/- 0.262 ng/mL vs 0.946 +/- 0.168 ng/mL and study 2, 1.962 +/- 0.261 ng/mL vs 0.731 +/- 0.109 ng/mL). Concentration of PAG between pregnant and nonpregnant cows in study 1 and 2 was significant, however, pregnant heifers in study 2 (1.562 +/- 0.266 ng/mL) had concentration of PAGs that only had a tendency to differ compared to nonpregnant heifers (non-pregnant, 0.799 +/- 0.290 ng/mL; P = 0.0669). Only animals that were pregnant at day 31 were analyzed in late embryo mortality analysis (heifers, n = 54; cows, n = 159), defined as pregnancy loss between day 31 and 60. Between day 31 and 60, 39 (12 in study 1 and 28 in study 2) animals experienced late embryo mortality. Circulating concentrations of PAG were not significantly different (P > 0.05), in either study, at day 24 of gestation in animals that maintained pregnancy until day 60 compared to animals that lost pregnancy between day 31 and 60 (late embryo mortality, LEM). In summary, early gestation circulating PAG concentration may have application in diagnosing pregnancy at day 24 of gestation and more work is needed to determine the potential of early gestation PAGs in predicting embryo loss in dairy. (C) 2017 Elsevier Inc. All rights reserved. Univ Tennessee, Dept Anim Sci, 2506 River Dr, Knoxville, TN 37901 USA Sao Paulo State Univ, Dept Anim Prod, Botucatu, SP, Brazil Sao Paulo State Univ, Dept Anim Prod, Botucatu, SP, Brazil

Contains fulltext : 169834.pdf (Publisher’s version ) (Open Access) Contains fulltext : 169834pos.pdf (Author’s version postprint ) (Open Access) BACKGROUND: Neuroimaging studies have shown structural alterations in several brain regions in children and adults with attention deficit hyperactivity disorder (ADHD). Through the formation of the international ENIGMA ADHD Working Group, we aimed to address weaknesses of previous imaging studies and meta-analyses, namely inadequate sample size and methodological heterogeneity. We aimed to investigate whether there are structural differences in children and adults with ADHD compared with those without this diagnosis. METHODS: In this cross-sectional mega-analysis, we used the data from the international ENIGMA Working Group collaboration, which in the present analysis was frozen at Feb 8, 2015. Individual sites analysed structural T1-weighted MRI brain scans with harmonised protocols of individuals with ADHD compared with those who do not have this diagnosis. Our primary outcome was to assess case-control differences in subcortical structures and intracranial volume through pooling of all individual data from all cohorts in this collaboration. For this analysis, p values were significant at the false discovery rate corrected threshold of p=0.0156. FINDINGS: Our sample comprised 1713 participants with ADHD and 1529 controls from 23 sites with a median age of 14 years (range 4-63 years). The volumes of the accumbens (Cohen's d=-0.15), amygdala (d=-0.19), caudate (d=-0.11), hippocampus (d=-0.11), putamen (d=-0.14), and intracranial volume (d=-0.10) were smaller in individuals with ADHD compared with controls in the mega-analysis. There was no difference in volume size in the pallidum (p=0.95) and thalamus (p=0.39) between people with ADHD and controls. Exploratory lifespan modelling suggested a delay of maturation and a delay of degeneration, as effect sizes were highest in most subgroups of children (21 years): in the accumbens (Cohen's d=-0.19 vs -0.10), amygdala (d=-0.18 vs -0.14), caudate (d=-0.13 vs -0.07), hippocampus (d=-0.12 vs -0.06), putamen (d=-0.18 vs -0.08), and intracranial volume (d=-0.14 vs 0.01). There was no difference between children and adults for the pallidum (p=0.79) or thalamus (p=0.89). Case-control differences in adults were non-significant (all p>0.03). Psychostimulant medication use (all p>0.15) or symptom scores (all p>0.02) did not influence results, nor did the presence of comorbid psychiatric disorders (all p>0.5). INTERPRETATION: With the largest dataset to date, we add new knowledge about bilateral amygdala, accumbens, and hippocampus reductions in ADHD. We extend the brain maturation delay theory for ADHD to include subcortical structures and refute medication effects on brain volume suggested by earlier meta-analyses. Lifespan analyses suggest that, in the absence of well powered longitudinal studies, the ENIGMA cross-sectional sample across six decades of ages provides a means to generate hypotheses about lifespan trajectories in brain phenotypes. FUNDING: National Institutes of Health. 10 p.

The prevalence of reproductive losses costing 1 billion dollars annually to the beef and dairy industries has led researchers to evaluate strategies to improve reproductive efficiency. The microbiome of the reproductive tract has been a recent focus in humans due to its effects on health and fertility; however, little research has evaluated the uterine and vaginal microbiomes of cattle. The present study evaluated the reproductive tract bacterial communities of postpartum beef cows undergoing estrus synchronization and the differences between cows who became pregnant and those who failed to conceive. Forty postpartum Angus cows were subjected to a 7 Day Co-Synch protocol with a pre-synchronization step 21 days prior (day -21) to artificial insemination (AI). Uterine and vaginal flushes were collected on day -21, -9, and -2 of the protocol for pH and bacterial DNA extraction to sequence the V1-V3 hypervariable regions of the 16S rRNA gene. Transrectal ultrasound was performed 30 days after AI for pregnancy diagnosis. Ten pregnant and non-pregnant animals were selected and used for analysis. Decreased numbers of bacterial species present in the uterus of cows were identified, independent of pregnancy status, leading up to AI with an average of 1180 ± 97 species on day -21 to 427 ± 99 on day -2 (P < 0.0001). Principal coordinate analyses depicted significant clustering of bacterial communities by day in the uterus (P = 0.001) and vagina (P = 0.001) as well as pregnancy status in the uterus at day -2 (P = 0.005). Additionally, at day -2, multiple significantly different uterine bacterial genera were present at >1% in non-pregnant cows while present at

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria . Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria , including the human pathogen Neisseria gonorrhoeae . Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit ( ccoP ), nitrite reductase ( aniA ), and nitric oxide reductase ( norB ). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus , showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

The objective was to examine growth of the ovulatory follicle after FSH (Folltropin-V; Bioniche Animal Health, Belleville, Ontario, Canada) was given at the onset of induced luteolysis during a synchronization of ovulation protocol. Using GnRH or hCG for inducing ovulation enabled assessing ovulatory follicle responsiveness to an endogenous versus exogenous surge of LH activity. At 8 to 10 days after estrus (synchronized estrus = Day 0), lactating dairy cows received an Eazi-Breed CIDR (Pfizer Animal Health) plus 100 μg GnRH. After 7 days, controlled internal drug release devices (CIDRs) were removed, cows were given 500 μg cloprostenol, and then randomly allocated to receive 80 mg Folltropin-V (FSH; N = 19) or 4 mL sterile saline (SAL; N = 16). After 49 hours, FSH and SAL cows were randomly allocated to receive 100 μg GnRH or 3000 IU hCG. Five cows ovulated 30 to 42 hours (38.4 ± 1.2 hours) after FSH treatment. In the remaining FSH (N = 14) or SAL (N = 16) cows, ovulatory follicle size was similar at CIDR removal (14.5 ± 0.6 and 14.7 ± 0.6 mm, respectively; P = 0.85) and when GnRH/hCG was given (16.6 ± 0.6 and 17.7 ± 0.6 mm, respectively; P = 0.23). Estradiol-17β concentrations were lower in FSH cows at 36 and 49 hours after CIDR removal (FSH by time interaction, P < 0.005). After GnRH or hCG treatment, four FSH cows failed to ovulate. In cows exhibiting ovulation, the last recorded size of the ovulatory follicle was not influenced by FSH (18.1 ± 0.9 and 17.5 ± 0.6 mm for FSH and SAL, respectively; P = 0.59) or hormonal induction approach (18.4 ± 0.9 and 17.2 ± 0.7 mm for GnRH and hCG, respectively; P = 0.29). The interval from onset of luteolysis to ovulation and pharmaceutical induction to ovulation was shorter in FSH cows given GnRH (FSH by pharmaceutical inducer [GnRH vs. hCG] interaction; P = 0.01). Cows receiving GnRH had an LH surge; hCG-treated cows did not. Maximum LH concentrations were greater (P < 0.04) in SAL versus FSH cows after GnRH treatment (10.9 ± 1.2 vs. 6.7 ± 1.4 ng/mL, respectively). In three FSH cows failing to ovulate after GnRH treatment, the maximum LH concentration was

We developed a reproductive tract size and position score (SPS) system as a reproductive management tool to identify lactating dairy cows with decreased fertility. This system, relying solely on transrectal palpation, considers the size (cervical and uterine) and position of the reproductive tract relative to the pelvis. Cows undergoing pre-breeding exams were identified as having reproductive tracts that were small (SPS1), medium (SPS2), or large (SPS3). Cows designated SPS1 had small and compact uterine horns that rested within the pelvic cavity; SPS2 cows had reproductive tracts that were intermediate in cervical and uterine horn diameter, with longer uterine horns resting partially outside the pelvic cavity; and SPS3 cows had reproductive tracts that were larger and rested mostly outside the pelvic cavity. Cows that were SPS1 had a higher rate of pregnancy per artificial insemination (43.3 ± 3.7%) than cows that were SPS2 (36.9 ± 3.6%) or SPS3 (27.7 ± 4.3%). The percentage of cows with an SPS2 score differed in pregnancies per artificial insemination compared with SPS3 cows. The average days in milk was similar for SPS1, SPS2, and SPS3 cows (104.3 ± 3.5, 98.4 ± 3.4, and 94.7 ± 7.7, respectively). Ultrasound measurements of the uterine horn and cervical diameter, and length measurements of the uterine horns, cervix, and vagina confirmed differences among the SPS groups derived by transrectal palpation. The ease with which transrectal palpation can be used to determine the size and position of the reproductive tract attests to the relevance and usefulness of this scoring system to identify less fertile lactating dairy cows. The ability to do so with ease provides an opportunity to make economically relevant management decisions and maximize reproductive efficiency in a given herd.

Gonorrhea is a major, global public health problem for which there is no vaccine. The continuing emergence of antibiotic-resistant strains raises concerns that untreatable Neisseria gonorrhoeae may become widespread in the near future. Consequently, there is an urgent need for increased efforts towards the development of new anti-gonococcal therapeutics and vaccines, as well as suitable models for potential pre-clinical vaccine trials. Several current issues regarding gonorrhea are discussed herein, including the global burden of disease, the emergence of antibiotic-resistance, the status of vaccine development and, in particular, a focus on the model systems available to evaluate drug and vaccine candidates. Finally, alternative approaches to evaluate vaccine candidates are presented. Such approaches may provide valuable insights into the protective mechanisms, and correlates of protection, required to prevent gonococcal transmission, local infection and disease sequelae.

Consequences of heat stress exposure during the first 12 h of meiotic maturation differed depending on how and when bovine oocytes were activated. If heat-stressed oocytes underwent IVF at ∼24 h, blastocyst development was less than for respective controls and similar to that obtained for nonheat-stressed oocytes undergoing IVF at 30 h (i.e. slightly aged). In contrast, if heat-stressed oocytes underwent chemical activation with ionomycin/6-dimethylaminopurine at 24 h, blastocyst development was not only higher than respective controls, but also equivalent to development obtained after activation of nonheat-stressed oocytes at 30 h. Developmental differences in chemically activated vs IVF-derived embryos were not related to fertilization failure or gross alterations in cytoskeletal components. Rather, ionomycin-induced calcium release and MAP kinase activity were less in heat-stressed oocytes. While underlying mechanisms are multifactorial, ability to obtain equivalent or higher development after parthenogenetic activation demonstrates that oocytes experiencing heat stress during the first 12 h of meiotic maturation have the necessary components to develop to the blastocyst stage, but fail to do so after fertilization.

Expression of type IV pili by Neisseria gonorrhoeae plays a critical role in mediating adherence to human epithelial cells. Gonococcal pilin is modified with an O-linked glycan, which may be present as a di- or monosaccharide because of phase variation of select pilin glycosylation genes. It is accepted that bacterial proteins may be glycosylated; less clear is how the protein glycan may mediate virulence. Using primary, human, cervical epithelial (i.e. pex) cells, we now provide evidence to indicate that the pilin glycan mediates productive cervical infection. In this regard, pilin glycan-deficient mutant gonococci exhibited an early hyper-adhesive phenotype but were attenuated in their ability to invade pex cells. Our data further indicate that the pilin glycan was required for gonococci to bind to the I-domain region of complement receptor 3, which is naturally expressed by pex cells. Comparative, quantitative, infection assays revealed that mutant gonococci lacking the pilin glycan did not bind to the I-domain when it is in a closed, low-affinity conformation and cannot induce an active conformation to complement receptor 3 during pex cell challenge. To our knowledge, these are the first data to directly demonstrate how a protein-associated bacterial glycan may contribute to pathogenesis.

Neisseria gonorrhoeae is an obligate human pathogen that causes gonorrhea. We have shown previously that complement receptor 3 and Akt kinase play important roles in mediating cervical infection. At present, there are limited data to indicate how hormonally induced changes to the mucosal epithelia of the female genital tract mediate the course of gonococcal disease. Hence, I have expanded upon previous work to investigate the interaction of gonococci with primary human cervical epithelial (pex) cells under the variable estrogen and progesterone concentrations likely to be encountered in vivo throughout the female menstrual cycle. My data indicated that the ability of gonococci to survive and to replicate within pex cells was increased under progesterone-predominant conditions. Using bacterial survival, immunological, and kinase assays, I show that progesterone functioned in an additive manner with gonococcal phospholipase D to augment Akt kinase activity. This, in turn, resulted in a parallel increase in nitric oxide synthase expression. Nitric oxide production by pex cells was dependent upon Akt activity and was increased under progesterone-predominant conditions. Whereas both inducible and endothelial nitric oxide synthase contributed to nitric oxide production, only inducible nitric oxide synthase activity promoted gonococcal survival within pex cells. Collectively, these data provide the first clues as to how steroid hormones potentially modulate the course of gonococcal disease in women. In addition, these data demonstrate that host-derived nitric oxide likely is not protective against gonococci, in vivo ; rather, nitric oxide may be required to sustain cervical bacterial disease.

The objective of this experiment was to evaluate the impact of trace mineral supplementation on reproductive performance in lactating beef cows. Thirty days before AI (Day –30), 68 postpartum cows were stratified by weight, body condition score, and parity before being equally and randomly assigned to either a treatment or a control group. Each group received a weekly mineral supplement allotment of 1.2 kg/week per cow-calf pair for 15 weeks. Cows in the treatment group received a mineral supplement that contained zinc, copper, and manganese amino acid complexes as well as cobalt glucoheptonate (Availa® Plus; Zinpro Corp., Eden Prairie, MN, USA), whereas cows assigned to the control group received a mineral supplement that contained the same concentrations of these trace minerals from inorganic sources. All cows were submitted to a 7-day CO-Synch + CIDR protocol on Day –10 and bred using fixed time AI on Day 0. Pregnancy diagnosis was performed via transrectal ultrasound on Day 28. Cows diagnosed as nonpregnant were then removed from the experiment. Pregnant cows were divided into 10 groups of 3 or 4 cows per group: 5 treatment groups (20 total cows) and 5 control groups (18 total cows). On Day 52 and 67, all pregnant cows were subjected to an ovum pick-up (OPU). Collected oocytes were evaluated before performing in vitro fertilization. Number of oocytes cleaving (dividing into more than one cell) and developing to blastocyst stage was recorded. Analysis of variance was conducted using PROC GLIMMIX (SAS 9.4; SAS Institute Inc., Cary, NC, USA) to determine differences between treatment and control, and group was the experimental unit. Mineral consumption did not differ (P = 0.48) between treatments (average of 1.16 ± 0.065 v. 1.09 ± 0.065 kg/week per cow-calf pair, for treatment and control, respectively). Pregnancy per AI was numerically higher in mineral-supplemented cows versus controls (64.7% v. 52.9%, respectively; P = 0.33). Consumption of the complexed trace mineral supplement increased (P = 0.06) total oocyte yield (22.6 ± 1.44 mean ± SEM v. 16.4 ± 2.04 oocytes/group for treatment and control, respectively). Moreover, groups receiving the complexed trace minerals had more (P = 0.05) culturable oocytes (grades A through C) compared with control groups (15.9 ± 1.6 mean ± SEM v. 11.8 ± 1.01 oocytes/group, respectively). In addition, production of transferable embryos (grades 1 through 3) was greater (P = 0.04) for groups receiving complexed trace minerals than inorganic trace minerals (4.2 ± 0.64 mean ± SEM v. 2.6 ± 0.65 embryos/group, respectively). In summary, supplementing with zinc, manganese, and copper amino acid complexes plus cobalt glucoheptonate improved oocyte production and embryo development in pregnant beef cows when compared with cows supplemented with inorganic forms of these trace minerals.

Thioredoxin-like proteins of the TlpA/ResE/CcmG subfamily are known to face the periplasm in gram-negative bacteria. Using the tlpA gene of Bradyrhizobium japonicum as a query, we identified a locus (NGO1923) in Neisseria gonorrhoeae that encodes a thioredoxin-like protein (NG_TlpA). Bioinformatics analysis indicated that the predicted NG_TlpA protein contained a cleavable signal peptide at the N terminus, and secondary structure analysis identified a thioredoxin fold with a helical insertion (∼25 residues), similar to that found in B. japonicum TlpA but absent in cytoplasmic thioredoxins. Biochemical characterization of a recombinant form of NG_TlpA revealed a standard redox potential (E 0 ′) of −206 mV. This property and the observation that the oxidized form of the protein exhibited greater thermal stability than the reduced species indicated that NG_TlpA is a reducing thioredoxin and not an oxidizing thiol-disulfide oxidoreductase like DsbA. The thioredoxin activity of NG_TlpA was confirmed in an insulin disulfide reduction assay. A tlpA mutant of N. gonorrhoeae strain 1291 was found to be highly sensitive to oxidative killing by paraquat and hydrogen peroxide, indicating an antioxidant role for the NG_TlpA in this bacterium. The tlpA mutant also exhibited reduced intracellular survival in human primary cervical epithelial cells.

estD encodes a carboxylic ester hydrolase and is part of the NmlR regulon in Neisseria gonorrhoeae. An estD mutant was found to be susceptible to nitrite and to S-nitrosoglutathione. This mutant was also unable to infect and survive within human cervical epithelial cells, and it showed reduced ability to form a biofilm on these cells. We conclude that esterase D is an integral part of the nitrosative stress defense system of N. gonorrhoeae and that it has potential importance in pathogenesis.

Because multiple ovulation embryo transfer procedures are occasionally performed in cows experiencing heat stress, the goal of this study was to assess the developmental competence of otherwise morphologically normal embryos from heat-stressed ova. To this end, the ability of compact morulae from heat-stressed and non-heat-stressed bovine ova to undergo blastocyst development after culture at 38.5 or 41.0 degrees C was examined. It was hypothesized that heat-induced perturbations in the ooplasm carry over to increase the susceptibility of the preattachment embryo to heat stress. Initially, ova were matured at 38.5 or 41.0 degrees C. The consequences of heat stress did not include altered cleavage, but did reduce the proportion of 8- to 16-cell-stage embryos (55.3 vs. 50.6%; SEM +/- 1.9). Although proportionately fewer, compact morulae from heat-stressed ova were equivalent in quality to those from non-heat-stressed ova (2.1 and 2.1; SEM = 0.04). Culture of compact morulae from non-heat-stressed ova at 41.0 degrees C did not affect blastocyst development (71.9 and 71.5%; SEM = 3.0). Furthermore, the development of compact morulae from heat-stressed ova was similar to that of non-heat-stressed ova after culture at 38.5 degrees C (68.2 vs. 71.9 and 71.5%; SEM = 3.0). However, blastocyst development was reduced when compact morulae from heat-stressed ova were cultured at 41.0 degrees C (62.3 vs. 71.9, 71.5 and 68.2; SEM = 3.1). In summary, reduced compaction rates of heat-stressed ova explained in part why fewer develop to the blastocyst stage after fertilization. The thermolability of the few embryos that develop from otherwise developmentally challenged ova emphasizes the importance of minimizing exposure to stressor(s) during oocyte maturation.

In Neisseria gonorrhoeae, the MerR family transcription factor NmlR activates 3 operons in response to disulfide stress. In the present study, we show that trxB, a monocistronic operon under the control of NmlR, encodes a functional thioredoxin reductase. It is shown that neisserial TrxB has biochemical properties similar to those of its homologue from Escherichia coli. Analysis of a trxB mutant of N. gonorrhoeae showed that it was more sensitive to disulfide stress and to stress induced by organic hydroperoxides, superoxide, and nitric oxide than wild-type gonococcus. TrxB was found to be essential for the microaerobic induction of aniA and norB, the genes encoding nitrite reductase and nitric oxide reductase, respectively. The importance of TrxB during natural infection was demonstrated by the fact that the survival of gonococci within human cervical epithelial cells, as well as biofilm formation on these cells, was greatly reduced for a trxB mutant compared with a wild-type strain.

The objectives were to examine the development of embryos derived from control (38.5 degrees C) or heat-stressed ova [41.0 degrees C during the first 12 h of in vitro maturation (hIVM)] when in vitro fertilization (IVF) was performed at 16, 18, 20, 24, or 30 hIVM. Effects of heat stress in compromising ovum development depended on when IVF was performed (in vitro maturation temperature x IVF time interaction). When IVF was performed at 24 or 30 hIVM, fewer heat-stressed ova developed to the blastocyst stage compared with the respective controls. In contrast, when IVF was performed at 16, 18, or 20 hIVM, more heat-stressed ova developed to the blastocyst stage compared with the respective controls. Performing IVF earlier than usual was beneficial, because the ability of heat-stressed ova to develop to the blastocyst stage was improved when IVF was performed at 18 or 20 vs. 24 hIVM. Blastocyst stage and quality were equivalent to non-heat-stressed controls regardless of IVF time. Control ova undergoing IVF at 20, 24, 30, or 32 hIVM and heat-stressed ova undergoing IVF at 16, 18, 20, or 24 hIVM were compared for blastocyst development by multisource regression. Although linear and quadratic slopes were similar, heat stress reduced the peak and shifted the developmental response of ova by 7.3 h. In other words, obtaining optimal blastocyst development from heat-stressed ova would depend on performing IVF at 19.5 hIVM compared with 26.7 hIVM for non-heat-stressed controls. Heat-induced reductions in peak blastocyst development significantly reduced the window of time available to perform IVF and obtain > or = 20% blastocyst development. In summary, results support an effect of heat stress to hasten developmentally important events during oocyte maturation. The inability of earlier IVF to fully restore the development of heat-stressed ova to that of non-heat-stressed controls highlights the importance of further study.

Today, infections attributed to Neisseria gonorrhoeae continue to be a national and international problem, despite the fact that antibiotic therapy is available. This chapter attempts to contrast the different mechanisms of pathogenesis used by N. gonorrhoeae to infect, to invade, and to colonize the mucosal epithelia of the urethra and the uterine cervix, the initial sites of infection in males and females, respectively. Included among the better-studied outer membrane gonococcal constituents contributing to its virulence are pili, porin, the opacity-associated (Opa) outer membrane proteins, and lipooligosaccharide (LOS). Work by many researchers has revealed additional factors that may contribute to N. gonorrhoeae virulence; however, the roles of these factors in human disease are less well defined than those described for pili, porin, Opa proteins, and LOS. Before the 1970s, the understanding of gonococcal pathogenesis in the lower female genital tract was based on light microscopy studies performed by Harkness in the 1940s. The male urethra and the female uterine cervix, the primary sites of the majority of gonococcal infections, are distinctly different epithelial cell surfaces, which result from different embryological origins. While the importance of virulence factors such as pili, porin, Opa, and LOS in gonococcal infection both in vitro and in vivo is well established, the contribution of other virulence factors is undetermined or requires further elucidation.

In previous studies it has been established that resistance to superoxide by Neisseria gonorrhoeae is dependent on the accumulation of Mn(II) ions involving the ABC transporter, MntABC. A mutant strain lacking the periplasmic binding protein component (MntC) of this transport system is hypersensitive to killing by superoxide anion. In this study the mntC mutant was found to be more sensitive to H2O2 killing than the wild-type. Analysis of regulation of MntC expression revealed that it was de-repressed under low Mn(II) conditions. The N. gonorrhoeae mntABC locus lacks the mntR repressor typically found associated with this locus in other organisms. A search for a candidate regulator of mntABC expression revealed a homologue of PerR, a Mn-dependent peroxide-responsive regulator found in Gram-positive organisms. A perR mutant expressed more MntC protein than wild-type, and expression was independent of Mn(II), consistent with a role for PerR as a repressor of mntABC expression. The PerR regulon of N. gonorrhoeae was defined by microarray analysis and includes ribosomal proteins, TonB-dependent receptors and an alcohol dehydrogenase. Both the mntC and perR mutants had reduced intracellular survival in a human cervical epithelial cell model.

The objective of this study was to evaluate nuclear (progression to metaphase II) and cytoplasmic (translocation of cortical granules to the oolemma) maturation in control (38.5 degrees C) and heat-stressed (41.0 degrees C) oocytes. Hoechst staining indicated that a similar proportion of control and heat-stressed oocytes progressed to meta-phase II. More heat-stressed oocytes had type III cortical granule distribution suggesting that heat stress accelerated cytoplasmic maturation. The kinetics of nuclear maturation was examined in a second experiment in which a higher proportion of heat-stressed oocytes progressed to metaphase I by 8 h and arrested at meta-phase II at 16 and 18 h after placement into maturation medium. However, differences related to maturation temperature were no longer apparent by 21 h. Heat-induced alterations in kinetics of nuclear and cytoplasmic maturation prompted a third experiment to evaluate if earlier insemination of heat-stressed oocytes ameliorates heat-induced reductions in development. A significant temperature x insemination time interaction was noted when evaluating blastocyst development. Blastocyst development was reduced when heat-stressed oocytes were inseminated with sperm 24 h after placement into maturation medium compared with controls. In contrast, blastocyst development was similar to controls when heat-stressed oocytes were inseminated at 19 h. Based on this interaction, earlier insemination in vitro prevented heat-induced reductions in oocyte development. Collectively, these studies suggest a cumulative effect of heat stress to hasten in vitro maturation in bovine oocytes.

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.

The results of proper orthogonal decomposition analyses on CO 2 number density and vorticity magnitude data from reacting rectangular jet simulations are presented. The resulting proper orthogonal decomposition eigenfunctions are used to develop physical insight of the vortex formations and dynamics of these jets and their related mixing and spreading characteristics. It is seen that different vortex structures are captured in the eigenfunctions and that CO 2 and vorticity eigenfunctions are very similar indicating that vortex-driven mixing dominates in these jets. The eigenvalue spectra associated with these eigenfunctions are used to evaluate the information content of the eigenfunctions and the potential for reduced-order models. Using subsets of eigenfunctions with high information content, CO 2 and vorticity magnitude distributions can be represented with relatively few eigenfunctions. However, as the flows develop downstream, more eigenfunctions are needed to represent them to the same level of accuracy. The potential for reduced-order modeling of each field is approximately the same for the jets of aspect ratios 1, 2, and 3; however, there is stronger potential for reduced-order modeling of the CO 2 field than of the vorticity field

This paper describes the economically optimal adoption and operation of distributed energy resources (DER) by a hypothetical California microgrid (μGrid) consisting of a group of commercial buildings over an historical test year, 1999. The optimization is conducted using a customer adoption model developed at Berkeley Lab and implemented in the General Algebraic Modeling System. A μGrid is a semiautonomous grouping of electricity and heat loads interconnected with the existing utility grid (macrogrid) but able to island from it. The μGrid minimizes the cost of meeting its energy requirements (consisting of both electricity and heat loads) by optimizing the installation and operation of DER technologies while purchasing residual energy from the local combined natural gas and electricity utility. The available DER technologies are small-scale generators (

SUMMARYThe molecular mechanisms used by the gonococcus to initiate infection exhibit gender specificity. The clinical presentations of disease are also strikingly different upon comparison of gonococcal urethritis to gonococcal cervicitis. An intimate association occurs between the gonococcus and the urethral epithelium and is mediated by the asialoglycoprotein receptor. Gonococcal interaction with the urethral epithelia cell triggers cytokine release, which promotes neutrophil influx and an inflammatory response. Similarly, gonococcal infection of the upper female genital tract also results in inflammation. Gonococci invade the nonciliated epithelia, and the ciliated cells are subjected to the cytotoxic effects of tumor necrosis factor alpha induced by gonococcal peptidoglycan and lipooligosaccharide. In contrast, gonococcal infection of the lower female genital tract is typically asymptomatic. This is in part the result of the ability of the gonococcus to subvert the alternative pathway of complement present in the lower female genital tract. Gonococcal engagement of complement receptor 3 on the cervical epithelia results in membrane ruffling and does not promote inflammation. A model of gonococcal pathogenesis is presented in the context of the male and female human urogenital tracts.

The effect of drop size on the degradation rate of VX, O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate, in fresh concrete has been examined using (31)P NMR. Drops of neat VX, ranging in size from 4 microL to 0.2 microL, applied to small concrete coupons (8 mm x 15 mm) were observed to degrade at different rates, with the 1 microL and smaller drops reacting in less than 4 days, and the larger droplets reacting in less than 11 days. Additionally, 4 microL VX predissolved in hexane to evenly spread it over the concrete coupon likewise reacted faster, degrading in less than 5 days. The fresh concrete, less than 2 months old, exhibited significantly faster VX degradation for all drop sizes than that observed for "aged" concrete in a previous study where VX persisted for months. The enhanced reactivity of the "fresh" concrete for VX was maintained for at least a 1-year period. The pH of water containing crushed "fresh" and "aged" concrete was 10.0 and 9.0, respectively. The higher pH of the "fresh" concrete is one reason for its enhanced reactivity toward VX. An additional contribution to the enhanced reactivity of the "fresh" concrete is suggested by the increased mobility of its sorbed VX as evidenced by its significantly narrower peak in (31)P NMR spectra.

In 1997, Wilmut et al. announced the birth of Dolly, the first ever clone of an adult animal. To date, adult sheep, goats, cattle, mice, pigs, cats and rabbits have been cloned using somatic cell nuclear transfer. The ultimate challenge of cloning procedures is to reprogram the somatic cell nucleus for development of the early embryo. The cell type of choice for reprogramming the somatic nucleus is an enucleated oocyte. Given that somatic cells are easily obtained from adult animals, cultured in the laboratory and then genetically modified, cloning procedures are ideal for introducing specific genetic modifications in farm animals. Genetic modification of farm animals provides a means of studying genes involved in a variety of biological systems and disease processes. Moreover, genetically modified farm animals have created a new form of 'pharming' whereby farm animals serve as bioreactors for production of pharmaceuticals or organ donors. A major limitation of cloning procedures is the extreme inefficiency for producing live offspring. Dolly was the only live offspring produced after 277 attempts. Similar inefficiencies for cloning adult animals of other species have been described by others. Many factors related to cloning procedures and culture environment contribute to the death of clones, both in the embryonic and fetal periods as well as during neonatal life. Extreme inefficiencies of this magnitude, along with the fact that death of the surrogate may occur, continue to raise great concerns with cloning humans.

The use of primary, human, ecto- and endocervical epithelial cell cultures has increased our understanding of the pathogenesis of gonococcal infection in women. Primary cervical epithelial cells express complement (C') receptor type 3 (CR3) and C' proteins required for alternative pathway (AP) activity. Gonococcus -induced membrane ruffling and cellular invasion of primary cervical epithelia is mediated by CR3 and requires co-operative CR3 binding by gonococcus-bound iC3b, porin and pilus. We have extended these studies to identify the site of C3 deposition upon gonococci within the cervical microenvironment. By immunoprecipitation and ELISA we demonstrate that covalent and non-covalent associations occurred between gonococcal LOS and C' protein C3. Sialylation or LOS truncation did not alter the gonococcus-CR3 interaction. By Western blot analysis we observed comparable C3 opsonization patterns among a panel of LOS truncation mutants, sialylated wild-type gonococci, or wild-type bacteria that were not sialylated. Quantitative association/invasion assays performed in the presence or absence of LOS competimers support C3b deposition on the lipid A core structure. Our findings demonstrate a role for lipid A as a C3 acceptor site and suggest that multiple factors govern C3b deposition and its subsequent conversion to iC3b on the surface of the gonococcus within the cervical microenvironment.

Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium.