Eliminating a Set of Four Penicillin Binding Proteins Triggers the Rcs Phosphorelay and Cpx Stress Responses in Escherichia coli

Penicillin binding proteins (PBPs) are responsible for synthesizing and modifying the bacterial cell wall, and in Escherichia coli the loss of several nonessential low-molecular-weight PBPs gives rise to abnormalities in cell shape and division. To determine whether these proteins help connect the flagellar basal body to the peptidoglycan wall, we surveyed a set of PBP mutants and found that motility in an agar migration assay was compromised by the simultaneous absence of four enzymes: PBP4, PBP5, PBP7, and AmpH. A wild-type copy of any one of these restored migration, and complementation depended on the integrity of the PBP active-site serine. However, the migration defect was caused by the absence of flagella instead of improper flagellar assembly. Migration was restored if the flhDC genes were overexpressed or if the rcsB or cpxR genes were deleted. Thus, migration was inhibited because the Rcs and Cpx stress response systems were induced in the absence of these four specific PBPs. Furthermore, in this situation Rcs induction depended on the presence of CpxR. The results imply that small changes in peptidoglycan structure are sufficient to activate these stress responses, suggesting that a specific cell wall fragment may be the signal being sensed. The fact that four PBPs must be inactivated may explain why large perturbations to the envelope are required to induce stress responses.     

Biofabrication: an overview of the approaches used for printing of living cells

The development of cell printing is vital for establishing biofabrication approaches as clinically relevant tools. Achieving this requires bio-inks which must not only be easily printable, but also allow controllable and reproducible printing of cells. This review outlines the general principles and current progress and compares the advantages and challenges for the most widely used biofabrication techniques for printing cells: extrusion, laser, microvalve, inkjet and tissue fragment printing. It is expected that significant advances in cell printing will result from synergistic combinations of these techniques and lead to optimised resolution, throughput and the overall complexity of printed constructs.     

Nitrogen and oxygen isotopes in nitrate in the groundwater and surface water discharge from two rural catchments: implications for nitrogen loading to coastal waters

In Prince Edward Island, Canada, widespread intensive potato production has contributed to elevated nitrate concentrations in groundwater and streams, and eutrophic or anoxic conditions occur regularly in several estuarine systems. In this research, the stable isotopes of nitrogen and oxygen in nitrate in intertidal groundwater discharge and stream water were used, in conjunction with water quality and quantity data and land use information, to better understand the characteristics of nitrate delivered to two small estuaries with contrasting land use in their contributory catchments. Most of the water samples collected during the two-year study had isotopic signatures that fell in the range expected for nitrate derived from ammonium-based fertilizers (26.5 % of the samples) or in the overlapping range formed between ammonium-based fertilizers and nitrate derived from soil (64 % of the samples). Overall, isotopic signatures spanned over relatively narrow ranges, and correlations with other water quality parameters, or catchment characteristics, were weak. Nitrate in groundwater discharge and surface water in the Trout River catchment exhibited significantly different isotopic signatures only for the nitrogen isotope, while in the McIntyre Creek catchment groundwater discharge and surface water had similar isotopic signatures. When the isotopic results for the waters from the two catchments were compared, the surface waters were found to be similar, while the isotopic signatures of nitrate in groundwater were distinct only for the nitrogen isotope. Denitrification in the two study catchments was not evident based on the isotopic results for nitrate; however, in the case of the Trout River catchment, where a small freshwater pond exists, an average nitrate load reduction of 14 % was inferred based on a comparison of nitrate loads entering and leaving the pond. Overall, it appears that natural attenuation processes, occurring either in the streams or groundwater flow systems, do not significantly reduce nitrate loading to these estuaries.     

Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

AKAP9 Is Essential for Spermatogenesis and Sertoli Cell Maturation in Mice

Mammalian male fertility relies on complex inter- and intracellular signaling during spermatogenesis. Here we describe three alleles of the widely expressed A-kinase anchoring protein 9 (Akap9) gene, all of which cause gametogenic failure and infertility in the absence of marked somatic phenotypes. Akap9 disruption does not affect spindle nucleation or progression of prophase I of meiosis but does inhibit maturation of Sertoli cells, which continue to express the immaturity markers anti-Mullerian hormone and thyroid hormone receptor alpha in adults and fail to express the maturation marker p27^Kip1. Furthermore, gap and tight junctions essential for blood–testis barrier (BTB) organization are disrupted. Connexin43 (Cx43) and zona occludens-1 are improperly localized in Akap9 mutant testes, and Cx43 fails to compartmentalize germ cells near the BTB. These results identify and support a novel reproductive tissue-specific role for Akap9 in the coordinated regulation of Sertoli cells in the testis.     

Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes

The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2. Antibodies against Anopheles gambiae serpin-2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.     

Primary cilia and forebrain development

With a microtubule-based axoneme supporting its plasma membrane-ensheathed projection from the basal body of almost all cell types in the human body, and present in only one copy per cell, the primary cilium can be considered an organelle sui generis. Although it was first observed and recorded in histological studies from the late 19th century, the tiny structure was essentially forgotten for many decades. In the past ten years, however, scientists have turned their eyes once again upon primary cilia and realized that they are very important for the development of almost all organs in the mammalian body, especially those dependent upon the signaling from members Hedgehog family, such as Indian and Sonic hedgehog. In this review, we outline the roles that primary cilia play in forebrain development, not just in the crucial transduction of Sonic hedgehog signaling, but also new results showing that cilia are important for cell cycle progression in proliferating neural precursors. We will focus upon cerebral cortex development but will also discuss the importance of cilia for the embryonic hippocampus, olfactory bulb, and diencephalon.     

Integrating HIV Care into Primary Care Services: Quantifying Progress of an Intervention in South Africa

Background

Integration of human immunodeficiency virus (HIV) care into primary care services is one strategy proposed to achieve universal access to antiretroviral treatment (ART) for HIV-positive patients in high burden countries. There is a need for controlled studies of programmes to integrate HIV care with details of the services being integrated.

Methods

A semi-quantitative questionnaire was developed in consultation with clinic staff, tested for internal consistency using Cronbach''s alpha coefficients and checked for inter-observer reliability. It was used to conduct four assessments of the integration of HIV care into referring primary care clinics (mainstreaming HIV) and into the work of all nurses within ART clinics (internal integration) and the integration of pre-ART and ART care during the Streamlining Tasks and Roles to Expand Treatment and Care for HIV (STRETCH) trial in South Africa. Mean total integration and four component integration scores at intervention and control clinics were compared using one way analysis of variance (ANOVA). Repeated measures ANOVA was used to analyse changes in scores during the trial.

Results

Cronbach''s alpha coefficients for total integration, pre-ART and ART integration and mainstreaming HIV and internal integration scores showed good internal consistency. Mean total integration, mainstreaming HIV and ART integration scores increased significantly at intervention clinics by the third assessment. Mean pre-ART integration scores were almost maximal at the first assessment and showed no further change. There was no change in mean internal integration score.

Conclusion

The questionnaire developed in this study is a valid tool with potential for monitoring integration of HIV care in other settings. The STRETCH trial interventions resulted in increased integration of HIV care, particularly ART care, by providing HIV care at referring primary care clinics, but had no effect on integrating HIV care into the work of all nurses with the ART clinic.     

Drug-Related Hospital Visits and Admissions Associated with Laboratory or Physiologic Abnormalities—A Systematic-Review

Countless studies have demonstrated that many emergency-room visits and hospital admissions are drug-related and that a significant proportion of these drug-related visits (DRVs) are preventable. It has not been previously studied which DRVs could be prevented through enhanced monitoring of therapy. The objective of the study was to determine the incidence of DRVs attributed to laboratory or physiologic abnormalities. Three authors independently performed comprehensive searches in relevant health care databases using pre-determined search terms. Articles discussing DRV associated with poisoning, substance abuse, or studied among existing in-patient populations were excluded. Study country, year, sample, design, duration, DRV identification method, proportion of DRVs associated with laboratory or physiologic abnormalities and associated medications were extracted. The three authors independently assessed selected relevant articles according to the Strengthening the reporting of observational studies in epidemiology (STROBE) as applicable according to the studies'' methodology. The initial literature search yielded a total of 1,524 articles of which 30 articles meeting inclusion criteria and reporting sufficient laboratory or physiologic data were included in the overall analysis. Half employed prospective methodologies, which included both chart review and patient interview; however, the overwhelming majority of identified studies assessed only adverse drug reactions (ADRs) as a drug-related cause for DRV. The mean (range) prevalence of DRVs found in all studies was 15.4% (0.44%–66.7%) of which an association with laboratory or physiologic abnormalities could be attributed to a mean (range) of 29.4% (4.3%–78.1%) of cases. Most laboratory-associated DRVs could be linked to immunosuppressant, antineoplastic, anticoagulant and diabetes therapy, while physiologic-associated DRVs were attributed to cardiovascular therapies and NSAIDs. Significant proportions of laboratory and physiologic abnormalities contribute to DRVs and are consistently linked to specific drugs. These therapies are potential targets for enhanced medication monitoring initiatives to proactively avert potential DRVs.     

Relative Biological Effectiveness of HZE Particles for Chromosomal Exchanges and Other Surrogate Cancer Risk Endpoints

The biological effects of high charge and energy (HZE) particle exposures are of interest in space radiation protection of astronauts and cosmonauts, and estimating secondary cancer risks for patients undergoing Hadron therapy for primary cancers. The large number of particles types and energies that makeup primary or secondary radiation in HZE particle exposures precludes tumor induction studies in animal models for all but a few particle types and energies, thus leading to the use of surrogate endpoints to investigate the details of the radiation quality dependence of relative biological effectiveness (RBE) factors. In this report we make detailed RBE predictions of the charge number and energy dependence of RBE’s using a parametric track structure model to represent experimental results for the low dose response for chromosomal exchanges in normal human lymphocyte and fibroblast cells with comparison to published data for neoplastic transformation and gene mutation. RBE’s are evaluated against acute doses of γ-rays for doses near 1 Gy. Models that assume linear or non-targeted effects at low dose are considered. Modest values of RBE (<10) are found for simple exchanges using a linear dose response model, however in the non-targeted effects model for fibroblast cells large RBE values (>10) are predicted at low doses <0.1 Gy. The radiation quality dependence of RBE’s against the effects of acute doses γ-rays found for neoplastic transformation and gene mutation studies are similar to those found for simple exchanges if a linear response is assumed at low HZE particle doses. Comparisons of the resulting model parameters to those used in the NASA radiation quality factor function are discussed.