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1.  The effects of commensal bacteria on innate immune responses in the female genital tract 
The innate and adaptive immune systems are important mechanisms for resistance to pathogens in the female lower genital tract. Lactobacilli at this site help maintain a healthy vagina by producing several factors including lactic acid. Indeed, bacterial vaginosis, a condition in which the genital microbiota is altered, is strongly associated with increased rates of a number of infections including HIV. However, the precise factors that contribute to increased rates of microbial and viral infections in bacterial vaginosis remain to be elucidated.
We have studied the effects of bacterial microbiota in the lower genital tract on innate immunity and have found that Toll-like receptor ligands and short chain fatty acids, produced by bacterial microbiota, have dramatic effects on immune function. In this review, we will discuss these results, in addition to some recent articles that we believe will enhance our understanding of how microbes might interact with the immune system.
doi:10.1111/j.1600-0897.2010.00943.x
PMCID: PMC3581076  PMID: 21143335
innate immunity; short chain fatty acids; vaginal microbiota
2.  CD8 T Cell Memory Recall Is Enhanced by Novel Direct Interactions with CD4 T Cells Enabled by MHC Class II Transferred from APCs 
PLoS ONE  2013;8(2):e56999.
Protection against many intracellular pathogens is provided by CD8 T cells, which are thought to need CD4 T cell help to develop into effective memory CD8 T cells. Because murine CD8 T cells do not transcribe MHC class II (MHC-II) genes, several models have proposed antigen presenting cells (APCs) as intermediaries required for CD4 T cells to deliver their help to CD8 T cells. Here, we demonstrate the presence of MHC-II molecules on activated murine CD8 T cells in vitro as well as in vivo. These MHC-II molecules are acquired via trogocytosis by CD8 T cells from their activating APCs, particularly CD11c positive dendritic cells (DCs). Transferred MHC-II molecules on activated murine CD8 T cells were functionally competent in stimulating specific indicator CD4 T cells. CD8 T cells that were “helped” in vitro and subsequently allowed to rest in vivo showed enhanced recall responses upon challenge compared to “helpless” CD8 T cells; in contrast, no differences were seen upon immediate challenge. These data indicate that direct CD8∶CD4 T cell interactions may significantly contribute to help for CD8 T cells. Furthermore, this mechanism may enable CD8 T cells to communicate with different subsets of interacting CD4 T cells that could modulate immune responses.
doi:10.1371/journal.pone.0056999
PMCID: PMC3575485  PMID: 23441229
3.  Use of Replication Restricted Recombinant Vesicular Stomatitis Virus Vectors for Detection of Antigen-Specific T Cells 
Journal of Immunological Methods  2011;375(1-2):118-128.
Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.
doi:10.1016/j.jim.2011.09.016
PMCID: PMC3253942  PMID: 22004852
VSV (vesicular stomatitis virus); ELISpot; intracellular cytokine staining (ICS); T cell assays; cytotoxic T cells; helper T cells
4.  Topoisomerase I inhibition in colorectal cancer: biomarkers and therapeutic targets 
British Journal of Cancer  2011;106(1):18-24.
The topoisomerase I (Top 1) poison irinotecan is an important component of the modern treatment of colorectal cancer. By stabilising Top 1-DNA complexes, irinotecan generates Top 1-linked DNA single-strand breaks that can evolve into double-strand breaks and ultimately cause cell death. However, cancer cells may overcome cell killing by releasing the stalled topoisomerase from DNA termini, thereby reducing the efficacy of Top 1 poisons in clinics. Thus, understanding the DNA repair mechanisms involved in the repair of Top 1-mediated DNA damage provides a useful tool to identify potential biomarkers that predict response to this class of chemotherapy. Furthermore, targeting these pathways could enhance the therapeutic benefits of Top 1 poisons. In this review, we describe the cellular mechanisms and consequences of targeting Top 1 activity in cells. We summarise preclinical data and discuss the potential clinical utility of small-molecule inhibitors of the key proteins.
doi:10.1038/bjc.2011.498
PMCID: PMC3251848  PMID: 22108516
irinotecan; colorectal cancer; biomarkers; topoisomerase I; TDP1; PARP
5.  Quantifying excessive mirror overflow in children with attention-deficit/hyperactivity disorder 
Neurology  2011;76(7):622-628.
Objectives:
Qualitative observations have revealed that children with attention-deficit/hyperactivity disorder (ADHD) show increased overflow movements, a motor sign thought to reflect impaired inhibitory control. The goal of this study was to develop and implement methods for quantifying excessive mirror overflow movements in children with ADHD.
Methods:
Fifty right-handed children aged 8.2–13.3 years, 25 with ADHD (12 girls) and 25 typically developing (TD) control children (10 girls), performed a sequential finger-tapping task, completing both left-handed (LHFS) and right-handed finger sequencing (RHFS). Phasic overflow of the index and ring fingers was assessed in 34 children with video recording, and total overflow in 48 children was measured by calculating the total angular displacement of the index and ring fingers with electrogoniometer recordings.
Results:
Phasic overflow and total overflow across both hands were greater in children with ADHD than in TD children, particularly during LHFS. Separate gender analyses revealed that boys, but not girls, with ADHD showed significantly more total phasic overflow and total overflow than did their gender-matched control children.
Conclusions:
The quantitative overflow measures used in this study support past qualitative findings that motor overflow persists to a greater degree in children with ADHD than in age-matched TD peers. The quantitative findings further suggest that persistence of mirror overflow is more prominent during task execution of the nondominant hand and reveal gender-based differences in developmental neural systems critical to motor control. These quantitative measures will assist future physiologic investigation of the brain basis of motor control in ADHD.
doi:10.1212/WNL.0b013e31820c3052
PMCID: PMC3053337  PMID: 21321336
6.  Motor cortex inhibition 
Neurology  2011;76(7):615-621.
Objective:
Attention-deficit/hyperactivity disorder (ADHD) is a childhood-onset behavioral diagnosis in which children often fail to meet age norms in development of motor control, particularly timed repetitive and sequential movements, motor overflow, and balance. The neural substrate of this motor delay may include mechanisms of synaptic inhibition in or adjacent to the motor cortex. The primary objective of this study was to determine whether transcranial magnetic stimulation (TMS)–evoked measures, particularly short interval cortical inhibition (SICI), in motor cortex correlate with the presence and severity of ADHD in childhood as well as with commonly observed delays in motor control.
Methods:
In this case-control study, behavioral ratings, motor skills, and motor cortex physiology were evaluated in 49 children with ADHD (mean age 10.6 years, 30 boys) and 49 typically developing children (mean age 10.5 years, 30 boys), all right-handed, aged 8–12 years. Motor skills were evaluated with the Physical and Neurological Examination for Subtle Signs (PANESS) and the Motor Assessment Battery for Children version 2. SICI and other physiologic measures were obtained using TMS in the left motor cortex.
Results:
In children with ADHD, mean SICI was reduced by 40% (p < 0.0001) and less SICI correlated with higher ADHD severity (r = −0.52; p = 0.002). Mean PANESS motor development scores were 59% worse in children with ADHD (p < 0.0001). Worse PANESS scores correlated modestly with less SICI (r = −.30; p = 0.01).
Conclusion:
Reduced TMS-evoked SICI correlates with ADHD diagnosis and symptom severity and also reflects motor skill development in children.
doi:10.1212/WNL.0b013e31820c2ebd
PMCID: PMC3053341  PMID: 21321335
7.  A prospective study of chemotherapy-induced febrile neutropenia in the South West London Cancer Network. Interpretation of study results in light of NCAG/NCEPOD findings 
British Journal of Cancer  2010;104(3):407-412.
Background:
Chemotherapy-induced febrile neutropenia is a medical emergency complicating the treatment of many cancer patients. It is associated with considerable morbidity and mortality, as well as impacting on healthcare resources.
Methods:
A prospective study of all cases of chemotherapy-induced febrile neutropenia in the South West London Cancer Network was conducted over a 4-month period. Factors including demographics, treatment history, management of febrile neutropenia and outcome were recorded.
Results and conclusi:
Our results reflect those of the recent National Chemotherapy Advisory Group (NCEPOD, 2008)/National Confidential Enquiry into Patient Outcomes and Death reports (NCAG, 2009) and highlight the need for network-wide clinical care pathways to improve outcomes in this area.
doi:10.1038/sj.bjc.6606059
PMCID: PMC3049562  PMID: 21179036
neutropenic sepsis; chemotherapy; infection; febrile neutropenia
8.  Motor Tics, Stereotypies, and Self-Flagellation in Primrose Syndrome 
Neurology  2010;75(3):284-286.
doi:10.1212/WNL.0b013e3181e8e754
PMCID: PMC3463006  PMID: 20644156
9.  Engineering a novel self-powering electrochemical biosensor 
Systems and Synthetic Biology  2010;4(3):203-214.
This paper records the efforts of a multi-disciplinary team of undergraduate students from Glasgow University to collectively design and carry out a 10 week project in Synthetic Biology as part of the international Genetic Engineered Machine competition (iGEM). The aim of the project was to design and build a self-powering electrochemical biosensor called ‘ElectrEcoBlu’. The novelty of this engineered machine lies in coupling a biosensor with a microbial fuel cell to transduce a pollution input into an easily measurable electrical output signal. The device consists of two components; the sensor element which is modular, allowing for customisation to detect a range of input signals as required, and the universal reporter element which is responsible for generating an electrical signal as an output. The genetic components produce pyocyanin, a competitive electron mediator for microbial fuel cells, thus enabling the generation of an electrical current in the presence of target chemical pollutants. The pollutants tested in our implementation were toluene and salicylate. ElectrEcoBlu is expected to drive forward the development of a new generation of biosensors. Our approach exploited a range of state-of-the-art modelling techniques in a unified framework of qualitative, stochastic and continuous approaches to support the design and guide the construction of this novel biological machine. This work shows that integrating engineering techniques with scientific methodologies can provide new insights into genetic regulation and can be considered as a reference framework for the development of biochemical systems in synthetic biology.
doi:10.1007/s11693-010-9063-2
PMCID: PMC2955201  PMID: 21189841
Biosensor; Synthetic biology; Genetic engineering; Computational modelling
10.  Engineering a novel self-powering electrochemical biosensor 
Systems and Synthetic Biology  2010;4(3):203-214.
This paper records the efforts of a multi-disciplinary team of undergraduate students from Glasgow University to collectively design and carry out a 10 week project in Synthetic Biology as part of the international Genetic Engineered Machine competition (iGEM). The aim of the project was to design and build a self-powering electrochemical biosensor called ‘ElectrEcoBlu’. The novelty of this engineered machine lies in coupling a biosensor with a microbial fuel cell to transduce a pollution input into an easily measurable electrical output signal. The device consists of two components; the sensor element which is modular, allowing for customisation to detect a range of input signals as required, and the universal reporter element which is responsible for generating an electrical signal as an output. The genetic components produce pyocyanin, a competitive electron mediator for microbial fuel cells, thus enabling the generation of an electrical current in the presence of target chemical pollutants. The pollutants tested in our implementation were toluene and salicylate. ElectrEcoBlu is expected to drive forward the development of a new generation of biosensors. Our approach exploited a range of state-of-the-art modelling techniques in a unified framework of qualitative, stochastic and continuous approaches to support the design and guide the construction of this novel biological machine. This work shows that integrating engineering techniques with scientific methodologies can provide new insights into genetic regulation and can be considered as a reference framework for the development of biochemical systems in synthetic biology.
doi:10.1007/s11693-010-9063-2
PMCID: PMC2955201  PMID: 21189841
Biosensor; Synthetic biology; Genetic engineering; Computational modelling
11.  Evidence-based pragmatic guidelines for the follow-up of testicular cancer: optimising the detection of relapse 
British Journal of Cancer  2008;98(12):1894-1902.
Testicular germ cell tumours (TGCTs) are the most common cause of cancer in men between the ages of 15 and 40 years, and, overall, the majority of patients should expect to be cured. The European Germ Cell Cancer Consensus Group has provided clear guidelines for the primary treatment of both seminoma and nonseminomatous germ cell tumours. There is, however, no international consensus on how best to follow patients after their initial management. This must promptly and reliably identify relapses without causing further harm. The standardising of follow-up would result in optimising risk-benefit ratios for individual patients, while ensuring economic use of resources. We have identified the seven common scenarios in managing seminomas and nonseminomas of the various stages and discuss the pertinent issues around relapse and follow-up. We review the available literature and present our comprehensive TGCT follow-up guidelines. Our protocols provide a pragmatic, easily accessible user-friendly basis for other centres to use or to adapt to suit their needs. Furthermore, this should enable future trials to address specific issues around follow-up giving meaningful and useful results.
doi:10.1038/sj.bjc.6604280
PMCID: PMC2441965  PMID: 18542063
12.  Phenotypic Resistance of Staphylococcus aureus, Selected Enterobacteriaceae, and Pseudomonas aeruginosa after Single and Multiple In Vitro Exposures to Ciprofloxacin, Levofloxacin, and Trovafloxacin 
The phenotypic resistance of selected organisms to ciprofloxacin, levofloxacin, and trovafloxacin was defined as a MIC of ≥4 μg/ml. The dynamics of resistance were studied after single and sequential drug exposures: clinical isolates of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MSSA and MRSA), Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa were utilized. After a single 48-h exposure of a large inoculum to four times the initial MIC for the organism, the frequency of selection of resistant mutants of MSSA was greater for trovafloxacin than levofloxacin (P = 0.008); for E. cloacae, the frequency was highest for ciprofloxacin and lowest for levofloxacin and trovafloxacin; for S. marcescens, the frequency was highest for trovafloxacin and lowest for ciprofloxacin (P = 0.003). The results of serial passage experiments were analyzed both by the Kaplan-Meier product-limited method as well as by analysis of variance of mean inhibitory values. By both methods, MSSA and MRSA expressed mutants resistant to ciprofloxacin after fewer passages than were required for either levofloxacin or trovafloxacin. For the aerobic gram-negative bacilli, two general patterns emerged. Mutants resistant to trovafloxacin appeared sooner and reached higher mean MICs than did mutants resistant to levofloxacin or ciprofloxacin. Mutants resistant to ciprofloxacin appeared later and reached mean MICs lower than the MICs of the other two drugs studied. Even though individual strain variation occurred, the mean MICs were reproduced when the serial passage experiment was repeated using an identical panel of E. coli isolates. In summary, the dynamic selection of fluoroquinolone-resistant bacteria can be demonstrated in experiments that employ serial passage of bacteria in vitro.
doi:10.1128/AAC.45.3.883-892.2001
PMCID: PMC90388  PMID: 11181375
13.  Identification of a novel Sry-related gene and its germ cell-specific expression. 
Nucleic Acids Research  1999;27(12):2503-2510.
Sox family proteins are characterized by a unique DNA-binding domain, a HMG box which shows at least 50% sequence similarity with mouse Sry, the sex-determining factor. At present almost 30 Sox genes have been identified. Members of this family have been shown to be conserved during evolution and to play key roles during animal development. Some are involved in human diseases, including sex reversal. Here we report the isolation of a novel member of the Sox gene family, Sox30, which may constitute a distinct subgroup of this family. Using a bacterially expressed DNA-binding domain of Sox30, we show that it is able to specifically recognize the ACAAT motif. Furthermore, Sox30 is capable of activating transcription from a synthetic promoter containing the ACAAT motif. The specific expression of Sox30 in normal testes, but not in maturing germ cell-deficient testes, suggests the involvement of Sox30 in differentiation of male germ cells. Mapping analyses revealed that the Sox30 gene is located on human chromosome 5 (5q33) and on mouse chromosome 11.
PMCID: PMC148454  PMID: 10359848
14.  The genes encoding the peripheral cannabinoid receptor and alpha-L-fucosidase are located near a newly identified common virus integration site, Evi11. 
Journal of Virology  1997;71(9):6796-6804.
A new common region of virus integration, Evi11, has been identified in two retrovirally induced murine myeloid leukemia cell lines, NFS107 and NFS78. By interspecific backcross analysis, it was shown that Evi11 is located at the distal end of mouse chromosome 4, in a region that shows homology with human 1p36. The genes encoding the peripheral cannabinoid receptor (Cnr2) and alpha-L-fucosidase (Fuca1) were identified near the integration site by using a novel exon trapping system. Cnr2 is suggested to be the target gene for viral interference in Evi11, since proviruses are integrated in the first intron of Cnr2 and retroviral integrations alter mRNA expression of Cnr2 in NFS107 and NFS78. In addition, proviral integrations were demonstrated within the 3' untranslated region of Cnr2 in five independent newly derived CasBrM-MuLV (mouse murine leukemia virus) tumors, CSL13, CSL14, CSL16, CSL27, and CSL97. The Cnr2 gene encodes a seven-transmembrane G-protein-coupled receptor which is normally expressed in hematopoietic tissues. Our data suggest that the peripheral cannabinoid receptor gene might be involved in leukemogenesis as a result of aberrant expression of Cnr2 due to retroviral integration in Evi11.
PMCID: PMC191960  PMID: 9261404
15.  The replication origin decision point is a mitogen-independent, 2-aminopurine-sensitive, G1-phase event that precedes restriction point control. 
Molecular and Cellular Biology  1997;17(8):4312-4321.
At a distinct point during G1 phase (the origin decision point [ODP]), Chinese hamster ovary (CHO) cell nuclei experience a transition (origin choice) that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. We have investigated the relationship between the ODP and progression of CHO cells through G1 phase. Selection of the DHFR origin at the ODP was rapidly inhibited by treatment of early G1-phase cells with the protein kinase inhibitor 2-aminopurine (2-AP). Inhibition of the ODP required administration of 2-AP at least 3 h prior to phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and the restriction point (R point). Cells deprived of either serum or isoleucine from metaphase throughout early G1 phase acquired the capacity to replicate in Xenopus egg extract (replication licensing) and subsequently passed through the ODP on the same schedule as cells cultured in complete growth medium. After growth arrest at the R point with hypophosphorylated Rb protein, serum- or isoleucine-deprived cells experienced a gradual loss of replication licensing. However, recognition of the DHFR origin by Xenopus egg cytosol remained stable in growth-arrested cells until the point at which all nuclei had lost the capacity to initiate replication. These results provide evidence that the ODP requires a mitogen-independent protein kinase that is activated after replication licensing and prior to R-point control.
PMCID: PMC232284  PMID: 9234688
16.  FlyBase: a Drosophila database. The FlyBase consortium. 
Nucleic Acids Research  1997;25(1):63-66.
FlyBase is a database of genetic and molecular data concerning Drosophila. FlyBase is maintained as a relational database (in Sybase) and is made available as html documents and flat files. The scope of FlyBase includes: genes, alleles (and phenotypes), aberrations, transposons, pointers to sequence data, clones, stock lists, Drosophila workers and bibliographic references. The Encyclopedia of Drosophila is a joint effort between FlyBase and the Berkeley Drosophila Genome Project which integrates FlyBase data with those from the BDGP.
PMCID: PMC146418  PMID: 9045212
17.  Net effect of inoculum size on antimicrobial action of ampicillin-sulbactam: studies using an in vitro dynamic model. 
To examine the predictable effect of inoculum size on the kinetics of the antimicrobial action of ampicillin-sulbactam, five TEM-1 beta-lactamase-producing Escherichia coli strains were studied in an in vitro dynamic model at two different initial inocula (N0S). All bacteria were exposed to ampicillin-sulbactam in a simulated system reflecting the pharmacokinetic profiles in human tissue after the administration of a single intravenous dose of ampicillin (2 g) plus sulbactam (1 g). Each strain was studied at low (4.0 to 5.2 log CFU/ml) and high (5.0 to 7.1 log CFU/ml) N0S. Despite pronounced differences in susceptibilities, the patterns of the killing curves observed with a given strain at different N0S were similar. As expected, viable bacterial counts increased with inoculum size. Striking visual contrasts in the respective curves for each organism were reflected by the area under the bacterial count-time curve (AUBC) but not by the difference between the N0 and the lowest bacterial counts (Nmin) at the nadir of the killing curve: the N0-associated changes in the AUBC on average were 75%, versus 2.5% for log N0--logNmin. To examine qualitative differences in antimicrobial effects at different N0S (i.e., the net effect of the inoculum), the difference in the high and low N0S was subtracted from each point on the killing curve obtained at the higher N0 for each strain. These adjusted curves were virtually superimposable on the observed killing curves obtained at the lower N0. Moreover, by using adjusted data, the AUBC values were similar at the two inocula, although slight (average, 11%) but systematic increases in the AUBC occurred at high N0S. Thus, there was only a weak net effect of inoculum size on the antibacterial effect of ampicillin-sulbactam. Due to similar slopes of the AUBC-log N0 plots, the antibacterial action at different N0S may be easily predicted by an approximate equation; the predicted AUBCs were unbiased and well correlated with the observed AUBCs (r = 0.997). Compiled data obtained with normalized AUBCs for different strains at different N0S yielded a positive correlation (r = 0.963) between the N0-normalized AUBC and the MIC of ampicillin-sulbactam. The adjustment and normalization procedure described might be a useful tool for revealing the net effect of the inoculum and to predict the inoculum effect if there are no qualitative differences in antimicrobial action at different inocula.
PMCID: PMC163651  PMID: 8980746
18.  Cloning and characterization of mouse CCAAT binding factor. 
Nucleic Acids Research  1996;24(6):1091-1098.
Isolation of cDNA clones for the mouse CCAAT binding factor (mCBF) has revealed the expression of two distinct forms of mCBF that are generated by alternative splicing of a single primary transcript from a gene that maps to chromosome 17. The mCBF1 mRNA encodes a protein of 997 amino acids, whereas the mCBF2 protein is predicted to be only 461 amino acids in length; mCBF1 and human CBF (hCBF) share>80% amino acid sequence identity. Analysis of adult mouse tissue RNAs has revealed that the mCBF1 and mCBF2 mRNAs are ubiquitously expressed, but that mCBF1 mRNA is 5- to 10-fold more abundant than mCBF2 mRNA. Similarly, mCBF mRNA was detected through-out the placenta and in all tissues of the developing embryo from day 8 to day 18 of gestation. Overexpression of the two forms of mCBF in mammalian cells has demonstrated that the mCBF1 and mCBF2 proteins localize to different cellular compartments, with mCBF1 found predominantly in the nucleus and mCBF2 restricted to the cytoplasm. Co-expression of these two forms influences their localization, however, indicating that CBF activity can be regulated by the relative amounts of the two forms expressed in a cell.
PMCID: PMC145757  PMID: 8604343
19.  Predictors of effect of ampicillin-sulbactam against TEM-1 beta-lactamase-producing Escherichia coli in an in vitro dynamic model: enzyme activity versus MIC. 
The clinical outcome in patients treated with ampicillin-sulbactam may not always be predictable by disc susceptibility testing or with the MIC as determined with a constant level (4 micrograms/ml) of the beta-lactamase inhibitor (MIC1). The enzyme activities (EA) and the MICs estimated at a constant ratio of ampicillin to sulbactam of 2:1 (MIC2) for 15 TEM-1 beta-lactamase-producing strains of Escherichia coli were examined as alternatives to MIC1 as predictors of the antibacterial effects of this combined drug as studied in an in vitro model which simulates ampicillin-sulbactam pharmacokinetic profiles observed in human peripheral tissues. Integral parameters describing the area under the bacterial count-time curve (AUBC), the area between the normal growth curve, and the killing curve of bacteria exposed to antibiotic (ABBC), and the second parameter expressed as a percentage of its maximal hypothetical value (ABBC/ABBCmax) were calculated. All three parameters correlated well with EA (AUBC, r = 0.93; ABBC, r = -0.88; ABBC/ABBCmax, r = -0.91) and with MIC2 (r = 0.94, -0.94, and -0.95, respectively) but not with MIC1. Both EA and MIC2 can be considered reliable predictors of the antibacterial effect of ampicillin-sulbactam in an in vitro model. These correlations suggest that in vitro kinetic-dynamic models might be useful to reexamine established susceptibility breakpoints obtained with data based on the MIC1 (MICs obtained with constant levels of beta-lactamase inhibitors). These data also suggest that quantitative determinations of bacterial beta-lactamase production and MICs based on the component concentration ratio observed in vivo might be useful predictors of the effect of ampicillin-sulbactam and other beta-lactam-inhibitor combinations.
PMCID: PMC163189  PMID: 8851602
20.  Staphylococcus aureus septic arthritis in patients on hemodialysis treatment. 
Western Journal of Medicine  1995;163(2):128-132.
We retrospectively reviewed hospital discharge diagnoses of septic arthritis over an 11-year period (1982 through 1992) at 3 medical centers; 11 episodes of septic arthritis were identified in patients on hemodialysis treatment. Of the 11 episodes, 9 were caused by Staphylococcus aureus; in 8 of 9, the blood cultures were positive for the organism and the infection was monoarticular. Concurrent infection of the dialysis access site occurred in 4 cases. Two patients died (22%). We postulate that repeated skin trauma and contact with health care personnel and facilities result in a high rate of nasal carriage of S aureus and, hence, an increased risk of bacteremia with its attendant complications such as septic arthritis. The use of mupirocin nasal ointment is reported to eradicate or suppress carriage in a high percentage of patients; some studies report that long-term suppressive therapy reduces the frequency of S aureus bacteremia.
PMCID: PMC1303006  PMID: 7571559
21.  Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure. 
Molecular and Cellular Biology  1995;15(6):2942-2954.
Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.
PMCID: PMC230525  PMID: 7760792
22.  Characteristics of African-American college students with HIV/AIDS. 
This article examines the risky sexual behaviors, condom and drug usage, sexually transmitted diseases (STDs), and attitudes of African-American college students with the human immunodeficiency virus (HIV), which is the precursor of the acquired immunodeficiency syndrome (AIDS). A total of 408 (199 males, 209 females) African-American college students, representing 75% of the students enrolled in a southern university, were surveyed. The results revealed that 3.18% of the students reported having HIV/AIDS. The students with HIV/AIDS exhibited significant deficits in AIDS knowledge, particularly information concerning the transmission of HIV/AIDS. While subjects with HIV/AIDS did not differ from subjects without HIV/AIDS with regard to their perceived risk of being exposed to AIDS or their attitudes about using condoms, a significantly larger percentage of subjects with HIV/AIDS reported that they "always" used condoms with their partner. Nevertheless, subjects with HIV/AIDS were more likely to engage in anal intercourse, experience sex with prostitutes, and use drugs. Sexually transmitted diseases were more prevalent among subjects with HIV/AIDS, and syphilis was found to be the best predictor of HIV/AIDS.
PMCID: PMC2607687  PMID: 7861472
23.  Pharmacodynamics of piperacillin alone and in combination with tazobactam against piperacillin-resistant and -susceptible organisms in an in vitro model of infection. 
Antimicrobial Agents and Chemotherapy  1994;38(10):2351-2356.
The pharmacodynamics of dosage regimens of piperacillin alone or in combination with tazobactam against piperacillin-resistant or -susceptible bacteria were studied in an in vitro model of infection. Experiments were conducted by using a fixed daily exposure of 12 g of piperacillin, given as 3 g alone or in combination with tazobactam at 0.375 g every 6 h, or the same total dose of the combination given as 4 g of piperacillin plus 0.5 g of tazobactam every 8 h. The addition of tazobactam to piperacillin, irrespective of the dosing interval, did not alter the killing of piperacillin-susceptible organisms (Escherichia coli J53 and Pseudomonas aeruginosa ATCC 27853). In contrast, experiments with an isogenic TEM-3-containing transconjugant of E. coli J53 (E. coli J53.2-TEM-3) that was resistant to piperacillin (MIC, 128 micrograms/ml) showed that the addition of tazobactam resulted in bacterial killing similar to that observed with the wild-type strain. Although tazobactam concentrations fell to less than 4 mg/liter (the concentration associated with a reduction in the piperacillin MIC from 128 to 2 mg/liter) 2 to 3 h after a dose, a similar degree of bacterial killing was observed when the same total 24-h dose of piperacillin-tazobactam was fractionated into dosing intervals of every 6 or 8 h. Investigations with Staphylococcus aureus 7176 (piperacillin MIC, 128 micrograms/ml) showed that the addition of tazobactam, again irrespective of dosing interval, also resulted in net bacterial killing which was not seen with piperacillin alone. These data support the use of extended dosing intervals (every 8 h) of piperacillin-tazobactam in the treatment of infections caused by piperacillin-resistant bacteria.
PMCID: PMC284743  PMID: 7840569
24.  What is the significance of black-white differences in risky sexual behavior? 
A sample of African-American and white young adults were classified as having multiple sex partners or one sexual partner. Subjects with multiple sexual partners were more likely to use drugs and practice risky sexual behaviors such as having anal intercourse, having sexual experiences with a prostitute, and having a history of gonorrhea (P < .001) and genital warts (P < .01). Additional analyses were conducted to determine African-American versus white differences in risky sexual behaviors. Results indicated that whites in the multiple partners and single partner groups were more likely to engage in anal and oral sex, while African Americans were more likely to have sex with prostitutes. Attitudes about the use of condoms differed significantly by multiple partner status (P < .004) and gender (P < .007), but not ethnicity. However, angry reactions about the use of condoms occurred more with African Americans (P < .003) and males (P < .05) than with whites or females. While whites reported a greater use of drugs and a significantly higher level of knowledge about HIV/AIDS, African Americans reported a significantly greater perception of risk for being exposed to human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) (P < .01) and significantly more gonorrhea (P < .10), syphilis (P < .05), and HIV/AIDS (P < .05). No whites in our sample were treated for syphilis nor had they tested positive for HIV/AIDS. On the other hand, 4.5% of the total sample of African Americans reported testing positive for HIV/AIDS. Finally, the results from discriminant analysis indicate that a large number of variables significantly discriminate between subjects who engage in risky sexual behaviors and those who do not. Although there is some similarity in the variables for African Americans and whites, there was tremendous variability between the ethnic groups in the factors that predict risky behaviors. These findings are discussed with reference to the need to develop HIV/AIDS prevention programs for African Americans that are based on data derived from African-American populations rather than from black versus white comparison studies.
PMCID: PMC2607704  PMID: 7807559
25.  Pig kidney (LLC-PK1) cell membrane fluidity during exposure to gentamicin or tobramycin. 
The surface membrane properties of LLC-PK1 cells grown with and without various amounts of gentamicin or tobramycin for various lengths of time were determined by measuring the diffusion coefficient of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L- alpha-phosphatidylethanolamine (NBD-PE) and the percentage of NBD-PE free to diffuse after photobleaching. One hour of exposure to tobramycin decreased the percentage that was free to diffuse. After 1 day or longer of exposure to either aminoglycoside the percentage that was free to diffuse returned to preexposure levels and the diffusion coefficient decreased.
PMCID: PMC284703  PMID: 7811038

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