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Identifying which young people living with perinatally acquired HIV (PHIV) are less likely to engage in care is crucial to allow targeted interventions to support them to attend clinic. We adapted an existing Engagement in Care (EIC) algorithm for adults with HIV in England, for use in young people. We applied it to data from young people with PHIV in the Adolescents and Adults Living with Perinatal HIV (AALPHI) cohort. The algorithm predicts the timing of the next scheduled clinic visit, within 1-6 months of current visit, based on routine clinical data. Follow-up was 12-months from AALPHI baseline interview. Each person-month was classified as engaged in care or not. Logistic regression models (allowing for clustered data) were used to explore baseline characteristics associated with being engaged in care, adjusting for a priori variables (time from interview, sex, age, ethnicity, country of birth). Potential characteristics were across 7 domains: sociodemographic; risk behaviour practices; mental health; cognition; clinic setting; HIV management and experience; and HIV clinical markers. Of 316 young people, 187(59%) were female, 271(86%) of black ethnicity and 184(58%) born abroad. At baseline, median [IQR] age was 17[15-18] years, and 202(69%) had viral load ≤50 copies/ml(c/mL). 87% of 3,585 person-months were classified as engaged in care. Characteristics independently associated with poorer odds of being engaged in care were: Asian/mixed/other ethnicity, vs. black ethnicity (OR 0.44, 95% CI 0.25, 0.78, p = 0.02); ever self-harmed, vs. not (OR 0.55, 95% CI 0.32, 0.95, p = 0.03); on antiretroviral therapy (ART) and self-assessed bad/not so good adherence (OR 0.46, 95% CI 0.25, 0.84) or not on ART (OR 0.64, 95% CI 0.64, 1.21) vs. on ART and good/excellent adherence (p = 0.04)); baseline VL>50c/mL, vs VL≤50c/mL (OR 0.47, 95% CI 0.30, 0.75, p = 0.002). These characteristics can help identify individuals requiring enhanced support to maintain service engagement.

Insulin dysregulation in horses is characterised by hyperinsulinaemia and/or tissue insulin resistance and is associated with increased risk of laminitis. There is growing evidence in other species that dopamine attenuates insulin release from the pancreas; however, this has yet to be examined in horses. The present study aimed to identify whether there are cells capable of producing or responding to dopamine within the equine gastrointestinal mucosa and pancreas. Tissue samples were collected from the stomach, small and large intestines, and pancreas of six mature horses following euthanasia. Samples of stomach contents and faeces were also collected. Immunohistochemistry was performed to identify tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine production, and dopamine D2 receptors in tissue sections. Additional immunostaining for glucagon, insulin and chromogranin A was performed to identify α cells, β cells and enteroendocrine cells, respectively. Gastric parietal cells expressed both TH and D2 receptors, indicating that they are capable of both producing and responding to dopamine. Dopamine was quantified in stomach contents and faeces by high-performance liquid chromatography with electrochemical detection, with similar concentrations found at both sites. Dopamine D2 receptors were expressed in duodenal epithelial cells but not more distally. A subset of enteroendocrine cells, located sporadically along the gastrointestinal tract, were found to be immunopositive for the D2 receptor. In pancreatic islets, TH was present in α cells, while D2 receptors were strongly expressed in β cells and variably expressed in α cells. These findings are consistent with studies of other species; however, dynamic studies are required to further elucidate the role of dopamine in the modulation of insulin and glucagon secretion in horses. This descriptive study provides preliminary evidence for a potential role of dopamine to act as a paracrine messenger in the gastrointestinal mucosa and endocrine pancreas of horses.

BackgroundChildren living with perinatally acquired HIV (CLWH) survive into adulthood on antiretroviral therapy (ART). HIV, ART, and malnutrition can all lead to low bone mineral density (BMD). Few studies have described bone health among CLWH in Sub-Saharan Africa. We determined the prevalence and factors associated with low BMD among CLWH switching to second-line ART in the CHAPAS-4 trial (ISRCTN22964075) in Uganda.MethodsBMD was determined using dual-energy X-ray Absorptiometry (DXA). BMD Z-scores were adjusted for age, sex, height and race. Demographic characteristics were summarized using median interquartile range (IQR) for continuous variables and proportions for categorical variables. Logistic regression was used to determine the associations between each variable and low BMD.ResultsA total of 159 children were enrolled (50% male) with median age (IQR) 10 (7-12) years, median duration of first -line ART 5.2(3.3-6.8) years; CD4 count 774 (528-1083) cells/mm3, weight-for-age Z-score -1.36 (-2.19, -0.65) and body mass index Z-score (BMIZ) -1.31 (-2.06, -0.6). Low (Z-score≤ -2) total body less head (TBLH) BMD was observed in 28 (18%) children, 21(13%) had low lumbar spine (LS) BMD, and15 (9%) had both. Low TBLH BMD was associated with increasing age (adjusted odds ratio [aOR] 1.37; 95% CI: 1.13-1.65, p = 0.001), female sex (aOR: 3.8; 95% CL: 1.31-10.81, p = 0.014), low BMI (aOR 0.36:95% CI: 0.21-0.61, pConclusionNearly 20% CLWH failing first-line ART had low BMD which was associated with female sex, older age, first-line ZDV exposure, and low BMI. Prevention, monitoring, and implications following transition to adult care should be prioritized to identify poor bone health in HIV+adolescents entering adulthood.

BackgroundThe need to improve support following a diagnosis of dementia is widely recognised, but it is unclear how this can best be achieved within UK health and social care systems. A task-shared and task-shifted approach has been recommended, but there is limited guidance on how to achieve this in practice. As part of a programme of research, we developed an intervention to enhance the role of primary care in post-diagnostic care and support for people living with dementia and carers.MethodsWe used the Theory of Change to develop a complex intervention informed by initial literature reviews and qualitative work. The intervention was developed through an iterative series of workshops, meetings and task groups with a range of stakeholders, including the multidisciplinary project team, people living with dementia and carers, service managers, frontline practitioners, and commissioners.Results142 participants contributed to intervention development through face-to-face or virtual meetings. The intervention comprises three complementary strands of work focusing on: developing systems, delivering tailored care and support, and building capacity and capability. Clinical dementia leads, based in primary care networks, will facilitate the intervention providing tailored expertise and support.ConclusionThe Theory of Change proved useful in providing structure and engaging stakeholders. The process was challenging, took longer and was less participative than intended due to restrictions caused by the COVID-19 pandemic. We will next conduct a feasibility and implementation study to explore whether the intervention can be successfully delivered within primary care. If successful, the intervention offers practical strategies for delivering a task-shared and task-shifted approach to post-diagnostic support that could be adapted for similar health and social care contexts internationally.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.

Natural Killer (NK) cells contribute to HIV control in adults, but HLA-B-mediated T-cell activity has a more substantial impact on disease outcome. However, the HLA-B molecules influencing immune control in adults have less impact on paediatric infection. To investigate the contribution NK cells make to immune control, we studied >300 children living with HIV followed over two decades in South Africa. In children, HLA-B alleles associated with adult protection or disease-susceptibility did not have significant effects, whereas Bw4 (p = 0.003) and low HLA-A expression (p = 0.002) alleles were strongly associated with immunological and viral control. In a comparator adult cohort, Bw4 and HLA-A expression contributions to HIV disease outcome were dwarfed by those of protective and disease-susceptible HLA-B molecules. We next investigated the immunophenotype and effector functions of NK cells in a subset of these children using flow cytometry. Slow progression and better plasma viraemic control were also associated with high frequencies of less terminally differentiated NKG2A+NKp46+CD56dim NK cells strongly responsive to cytokine stimulation and linked with the immunogenetic signature identified. Future studies are indicated to determine whether this signature associated with immune control in early life directly facilitates functional cure in children.

Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.

BackgroundThere has been a shift in focus of international dementia policies from improving diagnostic rates to enhancing the post-diagnostic support provided to people with dementia and their carers. There is, however, little agreement over what constitutes good post-diagnostic support. This study aimed to identify the components of post-diagnostic dementia support.MethodsWe adopted a qualitative design using interviews, focus groups and observation to explore the perspectives of key stakeholders on the content of post-diagnostic dementia support. Purposive sampling was used to identify sites in England and Wales recognised as delivering good practice. Participants included 17 people with dementia, 31 carers, 68 service managers or funders, and 78 frontline staff. Interviews and focus groups were audio recorded and transcribed for analysis. Forty-eight sessions of observation were completed and recorded in fieldnotes. Components were identified through an inductive, thematic approach and cross-checked against national guidelines and existing frameworks; they were subsequently critically reviewed by a range of experts and our mixed stakeholder panel.ResultsTwenty distinct components of post-diagnostic support were identified, related to five themes: timely identification and management of needs; understanding and managing dementia; emotional and psychological wellbeing; practical support; and integrating support. The first and last of these were cross-cutting themes facilitating the delivery of a unique constellation of components of post-diagnostic support to each individual living with dementia or dyad at a particular time.ConclusionsOur work offers an empirically based framework to inform the development and delivery of holistic, integrated and continuous dementia care from diagnosis to end of life. It highlights the relevance of many components to both people living with dementia and their carers. Since the framework was developed in England and Wales, further research is needed to explore the relevance of our components to other sectors, countries and care systems.

The diet of Antarctic fur seals (Arctocephalus gazella) at South Georgia is dominated by Antarctic krill (Euphausia superba). During the breeding season, foraging trips by lactating female fur seals are constrained by their need to return to land to provision their pups. Post-breeding, seals disperse in order to feed and recover condition; estimates indicate c.70% of females remain near to South Georgia, whilst others head west towards the Patagonian Shelf or south to the ice-edge. The krill fishery at South Georgia operates only during the winter, providing the potential for fur seal: fishery interaction during these months. Here we use available winter (May to September) tracking data from Platform Terminal Transmitter (PTT) tags deployed on female fur seals at Bird Island, South Georgia. We develop habitat models describing their distribution during the winters of 1999 and 2003 with the aim of visualising and quantifying the degree of spatial overlap between female fur seals and krill harvesting in South Georgia waters. We show that spatial distribution of fur seals around South Georgia is extensive, and that the krill fishery overlaps with small, highly localised areas of available fur seal habitat. From these findings we discuss the implications for management, and future work.

Most patients with hepatitis C virus (HCV) infection perceive some degree of disease-related stigma. Misunderstandings about diseases may contribute to disease-related stigma. The objective of this study was to evaluate patient-level knowledge about HCV infection transmission and natural history and its association with HCV-related stigma among HCV-infected patients. We conducted a cross-sectional survey study among 265 patients with HCV in Philadelphia using the HCV Stigma Scale and the National Health and Nutrition Examination Survey (NHANES) Hepatitis C Follow-up Survey (2001-2008). The association between HCV knowledge and HCV-related stigma was evaluated via linear regression. Overall knowledge about HCV transmission and natural history was high, with >80% of participants answering ≥9 of 11 items correctly (median number of correct responses, 9 [82%]), HCV-related knowledge was similar between HIV/HCV-coinfected and HCV-monoinfected participants (p = 0.30). A higher level of HCV-related knowledge was associated with greater perceived HCV-related stigma (β, 2.34 ([95% CI, 0.51-4.17]; p = 0.013). Results were similar after adjusting for age, race, ethnicity, HIV status, education level, stage of HCV management, time since diagnosis, and history of injection drug use. In this study, increased HCV-related knowledge was associated with greater perceptions of HCV stigma. Clinicians may consider allotting time to address common misconceptions about HCV when educating patients about HCV infection, which may counterbalance the stigmatizing impact of greater HCV-related knowledge.

BACKGROUND:Stigma around hepatitis C virus (HCV) infection is an important and understudied barrier to HCV prevention, treatment, and elimination. To date, no validated instrument exists to measure patients' experiences of HCV stigma. This study aimed to revise the Berger (2001) HIV stigma scale and evaluate its psychometric properties among patients with HCV infection. METHODS:The Berger HIV stigma scale was revised to ask about HCV and administered to patients with HCV (n = 270) in Philadelphia, Pennsylvania. Scale reliability was evaluated as internal consistency by calculating Cronbach's alpha. Exploratory factor analysis was performed to evaluate construct validity by comparing item clustering to the Berger HIV stigma scale subscales. Item response theory was employed to further evaluate individual items and to calibrate items for simulated computer adaptive testing sessions in order to identify potential shortened instruments. RESULTS:The revised HCV Stigma Scale was found to have good reliability (α = 0.957). After excluding items for low loadings, the exploratory factor analysis indicated good construct validity with 85% of items loading on pre-defined factors. Analyses strongly suggested the predominance of an underlying unidimensional factor solution, which yielded a 33-item scale after items were removed for low loading and differential item functioning. Adaptive simulations indicated that the scale could be substantially shortened without detectable information loss. CONCLUSIONS:The 33-item HCV Stigma Scale showed sufficient reliability and construct validity. We also conducted computer adaptive testing simulations and identified shortened six- and three-item scale alternatives that performed comparably to the original 40-item scale.

IntroductionThere are few studies describing colonisation with extended spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) and carbapenem-resistant Enterobacterales (CRE) among children in sub-Saharan Africa. Colonisation often precedes infection and multi-drug-resistant Enterobacterales are important causes of invasive infection.MethodsIn this prospective cross-sectional study, conducted between April and June 2017, 200 children in a tertiary academic hospital were screened by rectal swab for EBSL-PE and CRE. The resistance-conferring genes were identified using polymerase chain reaction technology. Risk factors for colonisation were also evaluated.ResultsOverall, 48% (96/200) of the children were colonised with at least one ESBL-PE, 8.3% (8/96) of these with 2 ESBL-PE, and one other child was colonised with a CRE (0.5% (1/200)). Common colonising ESBL-PE were Klebsiella pneumoniae (62.5%, 65/104) and Escherichia coli (34.6%, 36/104). The most frequent ESBL-conferring gene was blaCTX-M in 95% (76/80) of the isolates. No resistance- conferring gene was identified in the CRE isolate (Enterobacter cloacae). Most of the Klebsiella pneumoniae isolates were susceptible to piperacillin/tazobactam (86.2%) and amikacin (63.9%). Similarly, 94.4% and 97.2% of the Escherichia coli isolates were susceptible to piperacillin/tazobactam and amikacin, respectively. Hospitalisation for more than 7 days before study enrolment was associated with ESBL-PE colonisation.ConclusionApproximately half of the hospitalised children in this study were colonised with ESBL-PE. This highlights the need for improved infection prevention and control practices to limit the dissemination of these microorganisms.

INTRODUCTION:There are few studies describing Escherichia coli (E. coli) bloodstream infection (BSI) among children in Africa, yet E.coli is increasing in importance as a cause of antibiotic resistant infection in paediatric settings. METHODS:In this retrospective, descriptive study aspects of E. coli BSI epidemiology are described over a 10-year period including incidence risk, risk factors for extended-spectrum β-lactamase (ESBL)-producing E. coli BSI, antibiotic susceptibility of the bacterial isolates and outcome including risk factors for severe disease. RESULTS:There were 583 new E. coli BSI episodes among 217,483 admissions, an overall incidence risk of 2.7 events/1,000 hospital admissions. Of 455 of these E. coli BSI episodes that were analysed, 136 (29.9%) were caused by ESBL-producing isolates. Risk factors for ESBL-producing E. coli BSI included hospitalization in the 28-day period preceding E. coli BSI episodes, having an underlying chronic illness other than HIV infection at the time of the E. coli BSI and having a temperature of 38° Celsius or higher at the time of the E. coli BSI. None of the E. coli isolates were resistant to carbapenems or colistin. The mortality rate was 5.9% and admission to the intensive care unit was required in 12.3% of BSI episodes. Predictors of severe disease included age less than 1 month, hospitalization in the 28-day period preceding E. coli BSI and BSI without a definable focus. CONCLUSIONS:These findings extend our understanding of E. coli BSI in a sub-Saharan African setting, provide useful information that can guide empiric treatment choices for community- and hospital-acquired BSI and help inform prevention strategies.

In reverse genetic experiments we have isolated recombinant mumps viruses (rMuV) that carry large numbers of mutations clustered in small parts of their genome, which are not caused by biased hyper-mutation. In two separate experiments we obtained such recombinant viruses: one virus had 11 mutations in the V/P region of the genome; the other, which also contained an extra transcription unit encoding green fluorescent protein (EGFP), had 32 mutations in the N gene. These specific sets of mutations have not been observed in naturally occurring MuV isolates. Unusually, the vast majority of the mutations (48/51) were synonymous. On passage in Vero cells and human B-LCL cells, a B lymphocyte-like cell line, these mutations appear stable as no reversion occurred to the original consensus sequence, although mutations in other parts of the genome occurred and changed in frequency during passage. Defective interfering RNAs accumulate in passage in Vero cells but not in B-LCL cells. Interestingly, in all passaged samples the level of variation in the EGFP gene is the same as in the viral genes, though it is unlikely that this gene is under any functionality constraint. What mechanism gave rise to these viruses with clustered mutations and their stability remains an open question, which is likely of interest to a wider field than mumps reverse genetics.

It is currently believed that type I and III interferons (IFNs) have redundant functions. However, the preferential distribution of type III IFN receptor on epithelial cells suggests functional differences at epithelial surfaces. Here, using human intestinal epithelial cells we could show that although both type I and type III IFNs confer an antiviral state to the cells, they do so with distinct kinetics. Type I IFN signaling is characterized by an acute strong induction of interferon stimulated genes (ISGs) and confers fast antiviral protection. On the contrary, the slow acting type III IFN mediated antiviral protection is characterized by a weaker induction of ISGs in a delayed manner compared to type I IFN. Moreover, while transcript profiling revealed that both IFNs induced a similar set of ISGs, their temporal expression strictly depended on the IFNs, thereby leading to unique antiviral environments. Using a combination of data-driven mathematical modeling and experimental validation, we addressed the molecular reason for this differential kinetic of ISG expression. We could demonstrate that these kinetic differences are intrinsic to each signaling pathway and not due to different expression levels of the corresponding IFN receptors. We report that type III IFN is specifically tailored to act in specific cell types not only due to the restriction of its receptor but also by providing target cells with a distinct antiviral environment compared to type I IFN. We propose that this specific environment is key at surfaces that are often challenged with the extracellular environment.

As antimicrobial signalling molecules, type III or lambda interferons (IFNλs) are critical for defence against infection by diverse pathogens, including bacteria, fungi and viruses. Counter-intuitively, expression of one member of the family, IFNλ4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural frameshift mutation that abrogates IFNλ4 production improves HCV clearance. To further understand how genetic variation between and within species affects IFNλ4 function, we screened a panel of all known extant coding variants of human IFNλ4 for their antiviral potential and identify three that substantially affect activity: P70S, L79F and K154E. The most notable variant was K154E, which was found in African Congo rainforest 'Pygmy' hunter-gatherers. K154E greatly enhanced in vitro activity in a range of antiviral (HCV, Zika virus, influenza virus and encephalomyocarditis virus) and gene expression assays. Remarkably, E154 is the ancestral residue in mammalian IFNλ4s and is extremely well conserved, yet K154 has been fixed throughout evolution of the hominid genus Homo, including Neanderthals. Compared to chimpanzee IFNλ4, the human orthologue had reduced activity due to amino acid K154. Comparison of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFNλ4 correlates with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data show that the human-specific K154 negatively affects IFNλ4 activity through a novel means by reducing its secretion and potency. We thus demonstrate that attenuated activity of IFNλ4 is conserved among humans and postulate that differences in IFNλ4 activity between species contribute to distinct host-specific responses to-and outcomes of-infection, such as HCV infection. The driver of reduced IFNλ4 antiviral activity in humans remains unknown but likely arose between 6 million and 360,000 years ago in Africa.

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

Mechanistic differences in the development and function of adaptive, high-affinity antibody-producing B-2 cells and innate-like, "natural" antibody-producing B-1a cells remain poorly understood. Here we show that the multi-functional dynein light chain (DYNLL1/LC8) plays important roles in the establishment of B-1a cells in the peritoneal cavity and in the ongoing development of B-2 lymphoid cells in the bone marrow of mice. Epistasis analyses indicate that Dynll1 regulates B-1a and early B-2 cell development in a single, linear pathway with its direct transcriptional activator ASCIZ (ATMIN/ZNF822), and that the two genes also have complementary functions during late B-2 cell development. The B-2 cell defects caused by loss of DYNLL1 were associated with lower levels of the anti-apoptotic protein BCL-2, and could be supressed by deletion of pro-apoptotic BIM which is negatively regulated by both DYNLL1 and BCL-2. Defects in B cell development caused by loss of DYNLL1 could also be partially suppressed by a pre-arranged SWHEL Igm-B cell receptor transgene. In contrast to the rescue of B-2 cell numbers, the B-1a cell deficiency in Dynll1-deleted mice could not be suppressed by the loss of Bim, and was further compounded by the SWHEL transgene. Conversely, oncogenic MYC expression, which is synthetic lethal with Dynll1 deletion in B-2 cells, did not further reduce B-1a cell numbers in Dynll1-defcient mice. Finally, we found that the ASCIZ-DYNLL1 axis was also required for the early-juvenile development of aggressive MYC-driven and p53-deficient B cell lymphomas. These results identify ASCIZ and DYNLL1 as the core of a transcriptional circuit that differentially regulates the development of the B-1a and B-2 B lymphoid cell lineages and plays a critical role in lymphomagenesis.

INTRODUCTION:Accurate etiological diagnosis of meningitis is important, but difficult in resource-limited settings due to prior administration of antibiotics and lack of viral diagnostics. We aimed to develop and validate 2 real-time multiplex PCR (RT-PCR) assays for the detection of common causes of community-acquired bacterial and viral meningitis in South African children. METHODS:We developed 2 multiplex RT- PCRs for detection of S. pneumoniae, N. meningitidis, H. influenzae, enteroviruses, mumps virus and herpes simplex virus. We tested residual CSF samples from children presenting to a local paediatric hospital over a one-year period, whose CSF showed an abnormal cell count. Results were compared with routine diagnostic tests and the final discharge diagnosis. We calculated accuracy of the bacterial RT-PCR assay compared to CSF culture and using World Health Organisation definitions of laboratory-confirmed bacterial meningitis. RESULTS:From 292 samples, bacterial DNA was detected in 12 (4.1%) and viral nucleic acids in 94 (32%). Compared to CSF culture, the sensitivity and specificity of the bacterial RT-PCR was 100% and 97.2% with complete agreement in organism identification. None of the cases positive by viral RT-PCR had a bacterial cause confirmed on CSF culture. Only 9/90 (10%) of patients diagnosed clinically as bacterial meningitis or partially treated bacterial meningitis tested positive with the bacterial RT-PCR. DISCUSSION:In this population the use of 2 multiplex RT-PCRs targeting 6 common pathogens gave promising results. If introduced into routine diagnostic testing, these multiplex RT-PCR assays would supplement other diagnostic tests, and have the potential to limit unnecessary antibiotic therapy and hospitalisation.

Evidence consistently shows that people with advanced dementia experience suboptimal end of life care compared to those with cancer; with increased hospitalisation, inadequate pain control and fewer palliative care interventions. Understanding the views of those service managers and frontline staff who organise and provide care is crucial in order to develop better end of life care for people with dementia.Qualitative interviews and focus groups were conducted from 2013 to 2015 with 33 service managers and 54 staff involved in frontline care, including doctors, nurses, nursing and care home managers, service development leads, senior managers/directors, care assistants and senior care assistants/team leads. All were audio recorded and transcribed verbatim. Participants represented a diverse range of service types and occupation. Transcripts were subject to coding and thematic analysis in data meetings. Analysis of the data led to the development of seven key themes: Recognising end of life (EOL) and tools to support end of life care (EOLC), Communicating with families about EOL, Collaborative working, Continuity of care, Ensuring comfort at EOL, Supporting families, Developing and supporting staff. Each is discussed in detail and comprise individual and collective views on approaches to good end of life care for people with dementia.The significant challenges of providing good end of life care for people with dementia requires that different forms of expertise should be recognised and used; including the skills and knowledge of care assistants. Successfully engaging with people with dementia and family members and helping them to recognise the dying trajectory requires a supportive integration of emotional and technical expertise. The study strengthens the existing evidence base in this area and will be used with a related set of studies (on the views of other stakeholders and observations and interviews conducted in four services) to develop an evidence-based intervention.

Reflecting a global trend, freshwater wetlands in Madagascar have received little conservation or research attention. Madagascar is a global conservation priority due to its high level of species endemism but most work has focused on protecting forests. For the first time, we investigated the state of wetlands across the country to determine the effects of human disturbance. We conducted a rapid survey of 37 wetlands, using waterbirds and benthic invertebrates as ecological indicators. We recorded nine variables relating to human disturbance, revealing widespread wetland destruction. Principal Components Analysis reduced the nine variables to a single Principal Component (PC) that explained 50% of the dataset variance, demonstrating that different forms of human disturbance are ubiquitous and inseparable. The disturbance PC provides an index of how pristine a lake is and in Generalized Linear Models (GLMs) was significantly inversely related to the number of waterbird species present and the density of Chironomidae. The disturbance PC was estimated for every wetland in a GIS-derived dataset of wetland locations in Madagascar, giving a country-wide frequency distribution of disturbance. To validate the estimated PC values, we used the GLMs to predict the number of endemic bird species at an independent sample of 22 lakes. The predicted values correlated with the observed number of species, demonstrating that our procedure can identify lakes with high biodiversity value. The disturbance PC provides a convenient method for ranking sites, and a country-wide ranking demonstrates that the only near-pristine lakes in Madagascar are small sites that have been preserved by remoteness from human activity and not conservation management. The strategy of conserving high biodiversity remnants is insufficient because existing remnants suffer some degree of degradation and only support small populations of threatened species. Large-scale restoration of degraded wetlands is required for the long-term conservation of Madagascar's freshwater biodiversity.

IntroductionLocalised infections, and burn wound sepsis are key concerns in the treatment of burns patients, and prevention of colonisation largely relies on biocides. Acetic acid has been shown to have good antibacterial activity against various planktonic organisms, however data is limited on efficacy, and few studies have been performed on biofilms.ObjectivesWe sought to investigate the antibacterial activity of acetic acid against important burn wound colonising organisms growing planktonically and as biofilms.MethodsLaboratory experiments were performed to test the ability of acetic acid to inhibit growth of pathogens, inhibit the formation of biofilms, and eradicate pre-formed biofilms.ResultsTwenty-nine isolates of common wound-infecting pathogens were tested. Acetic acid was antibacterial against planktonic growth, with an minimum inhibitory concentration of 0.16-0.31% for all isolates, and was also able to prevent formation of biofilms (at 0.31%). Eradication of mature biofilms was observed for all isolates after three hours of exposure.ConclusionsThis study provides evidence that acetic acid can inhibit growth of key burn wound pathogens when used at very dilute concentrations. Owing to current concerns of the reducing efficacy of systemic antibiotics, this novel biocide application offers great promise as a cheap and effective measure to treat infections in burns patients.

INTRODUCTION:We aimed to obtain an in-depth understanding on recent antimicrobial resistance trends and molecular epidemiology trends of S. aureus bacteraemia (SAB). METHODS:Thirteen academic centres in South Africa were included from June 2010 until July 2012. S. aureus susceptibility testing was performed on the MicroScan Walkaway. Real-time PCR using the LightCycler 480 II was done for mecA and nuc. SCCmec and spa-typing were finalized with conventional PCR. We selected one isolate per common spa type per province for multilocus sequence typing (MLST). RESULTS:S. aureus from 2709 patients were included, and 1231 (46%) were resistant to methicillin, with a significant decline over the three-year period (p-value = 0.003). Geographical distribution of MRSA was significantly higher in Gauteng compared to the other provinces (P

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.

Live bacteria and archaea have been isolated from several rock salt deposits of up to hundreds of millions of years of age from all around the world. A key factor affecting their longevity is the ability to keep their genomic DNA intact, for which efficient repair mechanisms are needed. Polyploid microbes are known to have an increased resistance towards mutations and DNA damage, and it has been suggested that microbes from deeply buried rock salt would carry several copies of their genomes. Here, cultivable halophilic microbes were isolated from a surface sterilized middle-late Eocene (38-41 million years ago) rock salt sample, drilled from the depth of 800 m at Yunying salt mine, China. Eight unique isolates were obtained, which represented two haloarchaeal genera, Halobacterium and Halolamina. We used real-time PCR to show that our isolates are polyploid, with genome copy numbers of 11-14 genomes per cell in exponential growth phase. The ploidy level was slightly downregulated in stationary growth phase, but the cells still had an average genome copy number of 6-8. The polyploidy of halophilic archaea living in ancient rock salt might be a factor explaining how these organisms are able to overcome the challenge of prolonged survival during their entombment.

Noise often has detrimental effects on performance. However, because of the phenomenon of stochastic resonance (SR), auditory white noise (WN) can alter the "signal to noise" ratio and improve performance. The Moderate Brain Arousal (MBA) model postulates different levels of internal "neural noise" in individuals with different attentional capacities. This in turn determines the particular WN level most beneficial in each individual case-with one level of WN facilitating poor attenders but hindering super-attentive children. The objective of the present study is to find out if added WN affects cognitive performance differently in children that differ in attention ability.Participants were teacher-rated super- (N = 25); normal- (N = 29) and sub-attentive (N = 36) children (aged 8 to 10 years). Two non-executive function (EF) tasks (a verbal episodic recall task and a delayed verbal recognition task) and two EF tasks (a visuo-spatial working memory test and a Go-NoGo task) were performed under three WN levels. The non-WN condition was only used to control for potential differences in background noise in the group testing situations.There were different effects of WN on performance in the three groups-adding moderate WN worsened the performance of super-attentive children for both task types and improved EF performance in sub-attentive children. The normal-attentive children's performance was unaffected by WN exposure. The shift from moderate to high levels of WN had little further effect on performance in any group.The predicted differential effect of WN on performance was confirmed. However, the failure to find evidence for an inverted U function challenges current theories. Alternative explanations are discussed. We propose that WN therapy should be further investigated as a possible non-pharmacological treatment for inattention.

Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.

BACKGROUND: Antibiotic consumption is a major driver of bacterial resistance. To address the increasing burden of multi-drug resistant bacterial infections, antibiotic stewardship programmes are promoted worldwide to rationalize antibiotic prescribing and conserve remaining antibiotics. Few studies have been reported from developing countries and none from Africa that report on an intervention based approach with outcomes that include morbidity and mortality. METHODS: An antibiotic prescription chart and weekly antibiotic stewardship ward round was introduced into two medical wards of an academic teaching hospital in South Africa between January-December 2012. Electronic pharmacy records were used to collect the volume and cost of antibiotics used, the patient database was analysed to determine inpatient mortality and 30-day re-admission rates, and laboratory records to determine use of infection-related tests. Outcomes were compared to a control period, January-December 2011. RESULTS: During the intervention period, 475.8 defined daily doses were prescribed per 1000 inpatient days compared to 592.0 defined daily doses/1000 inpatient days during the control period. This represents a 19.6% decrease in volume with a cost reduction of 35% of the pharmacy's antibiotic budget. There was a concomitant increase in laboratory tests driven by requests for procalcitonin. There was no difference in inpatient mortality or 30-day readmission rate during the control and intervention periods. CONCLUSIONS: Introduction of antibiotic stewardship ward rounds and a dedicated prescription chart in a developing country setting can achieve reduction in antibiotic consumption without harm to patients. Increased laboratory costs should be anticipated when introducing an antibiotic stewardship program.

OBJECTIVE: To describe an outbreak of multi-resistant Pseudomonas aeruginosa bloodstream infections (MRPA-BSI) that occurred in the haematology ward of a tertiary academic hospital in Cape Town, South Africa, and determine risk factors for acquisition of MRPA-BSI. METHODS: The outbreak investigation included a search for additional cases, review of patient records, environmental and staff screening, molecular typing using pulsed-field gel electrophoresis (PFGE) and Multi-locus sequencing (MLST) and a retrospective case-control study. RESULTS: Ten MRPA-BSI cases occurred in the haematology ward between January 2010 and January 2011. The case fatality rate was 80%. Staff screening specimens were negative for MRPA and an environmental source was not identified. PFGE showed that 9/10 isolates were related. MLST showed that 3 of these 9 isolates belonged to Sequence type (ST) 233 while the unrelated isolate belonged to ST260. CONCLUSION: We have described an outbreak of MRPA-BSI occurring over an extended period of time among neutropenic haematology patients. Molecular typing confirms that the outbreak was predominantly due to a single strain. The source of the outbreak was not identified, but the outbreak appears to have been controlled following intensive infection control measures.

Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria) co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four strains of F. columnare were exposed to three isolates of lytic phage and the development of phage resistance and changes in colony morphology were monitored. Using zebrafish (Danio rerio) as a model system, the ancestral rhizoid morphotypes were associated with a 25-100% mortality rate, whereas phage-resistant rough morphotypes that lost their virulence and gliding motility (which are key characteristics of the ancestral types), did not affect zebrafish survival. Both morphotypes maintained their colony morphologies over ten serial passages in liquid culture, except for the low-virulence strain, Os06, which changed morphology with each passage. To our knowledge, this is the first report of the effects of phage-host interactions in a commercially important fish pathogen where phage resistance directly correlates with a decline in bacterial virulence. These results suggest that phage can cause phenotypic changes in F. columnare outside the fish host, and antagonistic interactions between bacterial pathogens and their parasitic phage can favor low bacterial virulence under natural conditions. Furthermore, these results suggest that phage-based therapies can provide a disease management strategy for columnaris disease in aquaculture.

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.

The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.

The coincidental evolution hypothesis predicts that traits connected to bacterial pathogenicity could be indirectly selected outside the host as a correlated response to abiotic environmental conditions or different biotic species interactions. To investigate this, an opportunistic bacterial pathogen, Serratia marcescens, was cultured in the absence and presence of the lytic bacteriophage PPV (Podoviridae) at 25°C and 37°C for four weeks (N = 5). At the end, we measured changes in bacterial phage-resistance and potential virulence traits, and determined the pathogenicity of all bacterial selection lines in the Parasemia plantaginis insect model in vivo. Selection at 37°C increased bacterial motility and pathogenicity but only in the absence of phages. Exposure to phages increased the phage-resistance of bacteria, and this was costly in terms of decreased maximum population size in the absence of phages. However, this small-magnitude growth cost was not greater with bacteria that had evolved in high temperature regime, and no trade-off was found between phage-resistance and growth rate. As a result, phages constrained the evolution of a temperature-mediated increase in bacterial pathogenicity presumably by preferably infecting the highly motile and virulent bacteria. In more general perspective, our results suggest that the traits connected to bacterial pathogenicity could be indirectly selected as a correlated response by abiotic and biotic factors in environmental reservoirs.

Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.

The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.

The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.