Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

Biophysical analysis of HTLV-1 particles reveals novel insights into particle morphology and Gag stoichiometry

Background

The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT).

Results

We show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin.

Conclusion

Our data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.     

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity

Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j–dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.     

From microbial gene essentiality to novel antimicrobial drug targets

Background

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result

Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion

Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.     

Fragile-X-associated tremor/ataxia syndrome (FXTAS) in females with the FMR1 premutation

We describe five female carriers of the FMR1 premutation who presented with symptoms of tremor and ataxia and who received a diagnosis of definite or probable fragile-X-associated tremor/ataxia syndrome (FXTAS). Unlike their male counterparts with FXTAS, none of the women had dementia. Females had not been reported in previous studies of FXTAS, suggesting that they may be relatively protected from this disorder. Brain tissue was available from one of the five subjects, a women who died at age 85 years; microscopic examination revealed intranuclear neuronal and astrocytic inclusions, in accord with the findings previously reported in males with FXTAS. The work-up of families with the FMR1 mutation should include questions regarding neurological symptoms in both older male and female carriers, with the expectation that females may also manifest the symptoms of FXTAS, although more subtly and less often than their male counterparts.     

Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx

We report the genomic organization and deduced protein sequence of a cephalochordate member of the Otx homeobox gene family (AmphiOtx) and show its probable single-copy state in the genome. We also present molecular phylogenetic analysis indicating that there was single ancestral Otx gene in the first chordates which was duplicated in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1 descendant genes.