Spontaneous Early Withdrawal Behaviors after Chronic 24-hour Free-Choice Access to Ethanol.

AIMS: Abstinence after chronic alcohol consumption leads to withdrawal symptoms, which are exacerbated after repeated cycles of relapse. This study examined withdrawal-like behaviors after chronic ethanol drinking, with or without repeated cycles of deprivation. METHODS: Male alcohol-preferring (P) rats had access to continuous ethanol (CE), chronic ethanol with repeated deprivation (RD), or remained ethanol naïve (EN). The RD group experienced seven cycles of 2 weeks of deprivation and 2 weeks of re-exposure to ethanol after an initial 6 weeks of ethanol access. Withdrawal was measured after an initial 24 h of ethanol re-exposure in the RD group, which coincided with the same day of ethanol access in the CE group. Withdrawal-like behavior was measured by (a) ethanol intake during the initial 24 h of re-exposure, (b) locomotor activity (LMA) in a novel field 9-13 h after removal of ethanol at the beginning of the fifth re-exposure cycle and (c) acoustic startle responding (ASR) 8-15 h after removal of ethanol at the beginning of the sixth re-exposure cycle. RESULTS: The RD rats displayed a 1-h alcohol deprivation effect (ADE) (temporary ethanol increase), relative to CE rats, during the first to fourth and seventh re-exposure cycles. RD and CE rats displayed significant increases in LMA than EN rats. Regarding ASR, RD rats displayed significantly greater ASR relative to EN rats. CONCLUSION: This study confirms that P rats meet the animal model criterion for ethanol-associated dependence, without a reliance on either behavioral (limited fluid access) or pharmacological (seizure threshold manipulation) challenges.

Integrative taxonomy refutes a species hypothesis: The asymmetric hybrid origin of Arsapnia arapahoe (Plecoptera, Capniidae).

Molecular tools are commonly directed at refining taxonomies and the species that constitute their fundamental units. This has been especially insightful for groups for which species hypotheses are ambiguous and have largely been based on morphological differences between certain life stages or sexes, and has added importance when taxa are a focus of conservation efforts. Here, we examine the taxonomic status of Arsapnia arapahoe, a winter stonefly in the family Capniidae that is a species of conservation concern because of its limited abundance and restricted range in northern Colorado, USA. Phylogenetic analyses of sequences of mitochondrial and nuclear genes of this and other capniid stoneflies from this region and elsewhere in western North America indicated extensive haplotype sharing, limited genetic differences, and a lack of reciprocal monophyly between A. arapahoe and the sympatric A. decepta, despite distinctive and consistent morphological differences in the sexual apparatus of males of both species. Analyses of autosomal and sex-linked single nucleotide polymorphisms detected using genotyping by sequencing indicated that all individuals of A. arapahoe consisted of F1 hybrids between female A. decepta and males of another sympatric stonefly, Capnia gracilaria. Rather than constitute a self-sustaining evolutionary lineage, A. arapahoe appears to represent the product of nonintrogressive hybridization in the limited area of syntopy between two widely distributed taxa. This offers a cautionary tale for taxonomists and conservation biologists working on the less-studied components of the global fauna.

Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

BACKGROUND: Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. RESULTS: Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. CONCLUSION: Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

Measuring inhibition of HIV replication by ex vivo CD8⁺ T cells.

HIV replication is unrestrained in vivo in the vast majority of infected subjects, and the ability of some rare individuals to control this virus is poorly understood. Standard immunogenicity assays for detecting HIV-1-specific CD8(+) T-cell responses, such as IFN-γ ELISpot and intracellular cytokine staining, generally fail to correlate with in vivo inhibition of HIV replication. Several viral inhibition assays, which measure the effectiveness of CD8(+) T-cell responses in suppressing HIV replication in vitro, have been described; but most depend on in vitro expansion of CD8(+) T cells, and some show inhibitory activity in HIV-negative individuals. We have optimized an assay to assess the suppressive capability of CD8(+) T cells directly ex vivo, eliminating the potential for altering their function through activation or expansion prior to assay setup, and thereby enhancing the assay's sensitivity by avoiding non-specific inhibition. With this method, the ability of ex vivo CD8(+) T cells to control HIV-1 replication in vitro can be quantified over several orders of magnitude. Specifically, our assay can be used to better define the antiviral function of CD8(+) T cells induced by vaccination, and can provide insight into their ability to control viral replication if HIV infection occurs post-vaccination.

Vaccine-induced gag-specific T cells are associated with reduced viremia after HIV-1 infection.

The contribution of host T-cell immunity and HLA class I alleles to the control of human immunodeficiency virus (HIV-1) replication in natural infection is widely recognized. We assessed whether vaccine-induced T-cell immunity, or expression of certain HLA alleles, impacted HIV-1 control after infection in the Step MRKAd5/HIV-1 gag/pol/nef study. Vaccine-induced T cells were associated with reduced plasma viremia, with subjects targeting ≥3 gag peptides presenting with half-log lower mean viral loads than subjects without Gag responses. This effect was stronger in participants infected proximal to vaccination and was independent of our observed association of HLA-B*27, -B*57 and -B*58:01 alleles with lower HIV-1 viremia. These findings support the ability of vaccine-induced T-cell responses to influence postinfection outcome and provide a rationale for the generation of T-cell responses by vaccination to reduce viremia if protection from acquisition is not achieved. Clinical trials identifier: NCT00095576.

Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials.

Various aspects of the human immune system can be analyzed to determine the efficacy of a vaccine. We have developed a B-cell ELISpot to measure HIV-specific antibody-secreting B cells in the peripheral blood as a result of vaccination or natural infection. Our method includes stimulating peripheral blood mononuclear cells with interleukin-2 and a polyclonal activator, R848, to induce memory B cells to differentiate into antibody-secreting cells. Total immunoglobulin-secreting as well as antigen-specific B cells are then quantified. We have tested several HIV Env gp120 and gp140 proteins from different HIV subtypes, as well as a sensitive consensus group M Env gp140. Our findings indicate that the B-cell ELISpot provides a sensitive and specific tool to detect antigen-specific memory B-cell responses, and it is equally suited to detect antibody-secreting plasmablasts present in the circulation shortly after infection or vaccination.

Modeling binge-like ethanol drinking by peri-adolescent and adult P rats.

Alcohol binge-drinking, especially among adolescents and young adults, is a serious public health concern. The present study examined ethanol binge-like drinking by peri-adolescent [postnatal days (PNDs 30-72)] and adult (PNDs 90-132) alcohol-preferring (P) rats with a drinking-in-the-dark-multiple-scheduled-access (DID-MSA) procedure used by our laboratory. Male and female P rats were provided concurrent access to 15% and 30% ethanol for three 1-h sessions across the dark cycle 5 days/week. For the 1st week, adolescent and adult female P rats consumed 3.4 and 1.6g/kg of ethanol, respectively, during the 1st hour of access, whereas for male rats the values were 3.5 and 1.1g/kg of ethanol, respectively. Adult intakes increased to ~2.0 g/kg/h and adolescent intakes decreased to ~2.5 g/kg/h across the 6 weeks of ethanol access. The daily ethanol intake of adult DID-MSA rats approximated or modestly exceeded that seen in continuous access (CA) rats or the selection criterion for P rats (≥5 g/kg/day). However, in general, the daily ethanol intake of DID-MSA peri-adolescent rats significantly exceeded that of their CA counterparts. BELs were assessed at 15-min intervals across the 3rd hour of access during the 4th week. Ethanol intake was 1.7 g/kg vs. 2.7 g/kg and BELs were 57 mg% vs. 100mg% at 15- and 60-min, respectively. Intoxication induced by DID-MSA in female P rats was assessed during the 1st vs. 4th week of ethanol access. Level of impairment did not differ between the 2 weeks (106 vs. 97 s latency to fall, 120 s criterion) and was significant (vs. naïve controls) only during the 4th week. Overall, these findings support the use of the DID-MSA procedure in rats, and underscore the presence of age- and sex-dependent effects mediating ethanol binge-like drinking in P rats.

The GAS41-PP2Cbeta complex dephosphorylates p53 at serine 366 and regulates its stability.

The p53 tumor suppressor is principally regulated by post-translational modifications and proteasome-dependent degradation. Various kinases have been shown to phosphorylate p53, but little is known about the counteracting phosphatases. We demonstrate here that the newly identified complex GAS41-PP2Cß, and not PP2Cß alone, is specifically required for dephosphorylation of serine 366 on p53. Ectopic expression of GAS41 and PP2Cß reduces UV radiation-induced p53 up-regulation, thereby increasing the cell survival upon genotoxic DNA damage. To our knowledge, the GAS41-PP2Cß complex is the first example in which substrate specificity of a PP2C family member is controlled by an associated regulatory subunit. Because GAS41 is frequently amplified in human gliomas, our finding illustrates a novel oncogenic mechanism of GAS41 by p53 dephosphorylation.

A two-tube combined TaqMan/SYBR Green assay to identify mycobacteria and detect single global lineage-defining polymorphisms in Mycobacterium tuberculosis.

We have developed a novel real-time PCR assay to identify and perform preliminary genotyping of mycobacteria in a manner tailored to our local service. Within a single thermocycler run, mycobacterial 16S rDNA and the Mycobacterium tuberculosis global lineage-defining RD750 polymorphism are targeted in separate reaction tubes, each of which includes both TaqMan and SYBR Green chemistries. The results of this 16S-RD assay differentiate M. tuberculosis complex (MTBC) from nontuberculous mycobacteria (NTM) and recognize whether or not MTBC isolates belong to the East African-Indian lineage, the single most frequently isolated global MTBC lineage in our service. If required, NTM amplicons may be sequenced to provide more specific identities. We report the technical performance of this assay on 88 mycobacteria-positive cultures and discuss its use in the initial management of mycobacterial infections. The 16S-RD assay correctly identified all 70 MTBC-positive cultures and 17 NTM-positive cultures while contemporaneously recognizing 26 MTBC isolates as within and 44 outside the East African-Indian lineage. In artificial samples, the combined assay also showed limited potential to detect mixed mycobacterial infections (MTBC/NTM) and tuberculosis infections involving more than one global MTBC lineage. The approach we have established can be readily tailored to targets of particular value for any mycobacterial diagnostic service, thereby optimizing the value of the results for local clinical and public health management of mycobacterial infections.

Somatoform dissociation in depersonalization disorder.

Along with psychoform dissociation, somatoform dissociation has been put forth as a core aspect of dissociative states, possibly as reliable as psychoform dissociation in the screening for dissociative disorders. The goal of this study was to investigate the prominence and correlates of somatoform dissociation in one of the major Diagnostic and Statistical Manual of Mental Disorders (4th ed., text rev.) dissociative disorders, depersonalization disorder (DPD). A total of 54 adults with DPD and 47 healthy control participants free of lifetime Axis I and II disorders were administered the 20-item Somatoform Dissociation Questionnaire (SDQ) as well as the Dissociative Experiences Scale, the Cambridge Depersonalization Scale, and the Childhood Trauma Questionnaire-Short Form. Somatoform dissociation scores were statistically significantly, but clinically only modestly, elevated in the DPD as compared to the healthy control group. SDQ items significantly elevated in the DPD group were mostly perceptual in nature. Depersonalization scores were significantly correlated with somatoform dissociation in the DPD group, whereas absorption and amnesia scores were not. With respect to childhood interpersonal trauma, although emotional abuse was significantly associated with depersonalization severity, none of the 5 categories of trauma were significantly associated with somatoform dissociation in the DPD group. In conclusion, somatoform dissociation is modest in DPD, and the SDQ is a weak instrument for the screening of dissociation in this disorder, detecting only one third of the sample when using the traditional SDQ cutoff score of 30.

Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.

BACKGROUND: Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis. METHODS AND FINDINGS: Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body-positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body-positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R(2) = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population. CONCLUSION: As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

In situ Raman spectroscopy as a probe for the effect of power on microwave-promoted Suzuki coupling reactions.

We report the use of in situ Raman spectroscopy as a probe for the effect of power on microwave-promoted Suzuki coupling reactions. We find that increased initial microwave power leads to greater acceleration of the reaction but that the product yield obtained is essentially independent of initial microwave power. The application of simultaneous cooling lengthens the reaction time but does not alter the relative rates of the Suzuki coupling and deboronation processes. Performing the reaction at an initial microwave power of 5 W leads to an improvement in product yield.

Pili mediate specific adhesion of Streptococcus pyogenes to human tonsil and skin.

Very little is known about the biological functions of pili that have recently been found to be expressed by important Gram-positive pathogens such as Corynebacterium diphtheriae, Streptococcus agalacticae, S. pneumoniae and S. pyogenes. Using various ex vivo tissue and cellular models, here we show that pili mediate adhesion of serotype M1 S. pyogenes strain SF370 to both human tonsil epithelium and primary human keratinocytes, which represent the two main sites of infection by this human-specific pathogen. Mutants lacking minor pilus subunits retained the ability to express cell-surface pili, but these were functionally defective. In contrast to above, pili were not required for S. pyogenes adhesion to either immortalized HEp-2 or A549 cells, highlighting an important limitation of these extensively used adhesion/invasion models. Adhering bacteria were internalized very effectively by both HEp-2 and A549 cells, but not by tonsil epithelium or primary keratinocytes. While pili acted as the primary adhesin, the surface M1 protein clearly enhanced adhesion to tonsil, but surprisingly, had the opposite effect on adhesion to keratinocytes. These studies provide clear evidence that S. pyogenes pili display an adhesive specificity for clinically relevant human tissues and are likely to play a critical role in the initial stages of infection.

Using in situ Raman monitoring as a tool for rapid optimisation and scale-up of microwave-promoted organic synthesis: esterification as an example.

Microwave-promoted esterification reactions have been monitored using in situ Raman spectroscopy. Having optimised a reaction on a 23 mmol scale, it was transferred to a larger reaction vessel and scaled up to 0.26 mol, again with Raman monitoring. With conditions in hand, an automated stop-flow apparatus was used to prepare 5.7 moles of product.

Development of a new, combined rapid method using phage and PCR for detection and identification of viable Mycobacterium paratuberculosis bacteria within 48 hours.

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.

A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.

Real-time monitoring of microwave-promoted Suzuki coupling reactions using in situ Raman spectroscopy.

The progress of microwave-promoted Suzuki reactions has been monitored using an in situ Raman spectroscopy apparatus assembled from commercially available components. It was possible to see if any reaction occurred and, if so, when it reached completion. In addition, the monitoring technique has given us an insight into the reaction, confirming that, when run in aqueous media, the coupling is in competition with the rapid deboronation of the boronic acid.

Use of genome level-informed PCR as a new investigational approach for analysis of outbreak-associated Mycobacterium tuberculosis isolates.

Mycobacterium tuberculosis strain CH, the index isolate linked to a major tuberculosis outbreak associated with high levels of transmissibility and virulence, was characterized by microarray analysis by use of a PCR product array representative of the genome of M. tuberculosis strain H37Rv. Seven potential genomic deletions were identified in CH, five of which were confirmed by PCR analysis across the predicted deletion points. The panel of five PCRs required to individually interrogate these loci was collectively referred to as the genome level-informed PCR (GLIP) assay. GLIP analysis was performed with CH, 12 other epidemiologically linked isolates, and 43 recent, non-outbreak-associated isolates derived from patients within the local area. All 13 outbreak-linked isolates showed a profile corresponding to the presence of all five deletions. These 13 isolates were also found to share common variable-number tandem repeat and mycobacterial interspersed repetitive unit profiles. None of the 43 non-outbreak-associated isolates exhibited the five-deletion profile. Although three individual deletions were present in upwards of 44% of the non-outbreak-associated isolates, no single-deletion isolates were detected. Interestingly, none of these deletions had been previously recognized, and sequence analysis of the immediate flanking regions in CH failed to identify a likely mechanism of deletion for four of the five loci. The GLIP assay also proved valuable in ongoing surveillance of the outbreak, rapidly identifying a further two outbreak-associated cases months after the initial cluster and, importantly, dismissing a further 12 epidemiologically suspect cases, which allowed the optimum deployment of public health resources.

Use of information technology in general practice.

The internet and NHS Net are used increasingly in UK general practice. A questionnaire survey conducted in southern England examined these applications. 77 (55%) of 141 practices responded. Of these, 71 were connected to one or other service and 27 offered a practice website. Only a small minority used a website for direct patient booking or access to pathology results. Moreover, among those with a practice website, none paid the necessary attention to data security. The survey revealed some fundamental misunderstandings that may partly account for the slow uptake of these technologies in British general practice.

Active but nonculturable cells of Salmonella enterica serovar Typhimurium do not infect or colonize mice.

The possibility that nonculturable cells of a normally culturable bacterial pathogen may constitute a source or reservoir for infective disease was investigated. In multiple experiments and with careful attention to the statistical limitations of the assays used, Salmonella enterica serovar Typhimurium cells rendered nonculturable by carbon and nitrogen stress in the presence of chloramphenicol were administered orally and intraperitoneally to over 300 female BALB/c mice. Neither infection nor colonization was detected in these studies, even when active but nonculturable (ABNC) cells, as defined by the Kogure cell elongation assay, were present in the inoculum. Doses of ABNC cells exceeding the oral and intraperitoneal LD(50) values by 3.5 and 2 orders of magnitude, respectively, were administered. It was concluded that ABNC cells of the salmonella strains used could not be considered potentially infective and that their detection in samples from material being evaluated as a potential source or reservoir of infection by the Kogure test does not specifically represent an infective hazard.