The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.     

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.     

Alleviation of Both Water and Nutrient Limitations is Necessary to Accelerate Ecological Restoration of Waste Rock Tips

Hard rock quarries are commonly located close to national parks and special areas of conservation and are generally regarded as visually intrusive. Consequently, restoration strategies that effectively accelerate natural plant regeneration processes are required. Slate waste tips present extreme conditions for plant establishment with multiple potential limiting factors (e.g., lack of organic matter, nutrients, and poor water retention). In this study, we investigated ecological strategies to accelerate natural regeneration at the largest slate quarry in Europe. A field experiment was conducted to assess ecosystem restoration using a contrasting set of native woody species. Treatments included amendments of waste tips with: polyacrylamide gel to increase water‐holding capacity; mineral fertilizer to increase nutrient supply; and two treatments that increased both (organic waste or boulder clay addition). Ecosystem recovery was evaluated through above‐ and below‐ground productivity (plant and microbial, respectively) and soil analyses. Neither increasing nutrient supply (with mineral fertilizer) nor water‐holding capacity (with polyacrylamide gel) was sufficient, alone, to improve plant establishment. However, both boulder clay and organic waste amendment significantly enhanced plant growth. There was a marked positive interaction in the effects on tree growth of the amendment with organic waste and boulder clay. Large interactions occurred between tree species and substrate amendments. The growth of N₂‐fixing species was strongly favored over non‐fixers where there was no addition of material increasing soil nitrogen supply, whereas the growth advantage of pioneer species over non‐pioneers was greatest with fertilizer, organic waste, or clay additions. Organic waste addition had the greatest positive impact on soil processes.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

Isolation of Naturally Occurring Viruses of the Murine Leukemia Virus Group in Tissue Culture

A tissue culture cell system for isolation and identification of members of the murine leukemia virus group (the complement fixation for murine leukemia test) was modified to permit the isolation of naturally occurring virus from leukemic and normal mice. The important factors for increasing the sensitivity of the test were the use of National Institutes of Health (NIH) strain Webster Swiss embryo cell cultures and the selection of rat-immune sera having complement-fixing antibodies to tissue culture antigens of both the Gross and FMR subgroups. In all, 163 strains of mouse leukemia virus, from 11 inbred mouse strains, have been isolated. Representative virus isolates were shown to possess the properties of the murine leukemia virus group; i.e., they were chloroform-sensitive, noncytopathic agents which replicated in mouse embryo tissue culture and produced group-reactive, complement-fixing antigen and budding C-type particles visible by electron microscopy. These viruses could serve as helpers in the rescue of Moloney sarcoma virus genome from non-producer hamster sarcoma cells, yielding pseudotypes. All of the 19 field isolates tested were neutralized by Gross passage A antiserum but not by potent antisera to the Moloney, Rauscher, and Friend strains. Virus was recovered regularly from embryos and from the plasma and spleen of adult mice of high leukemic strains. In low leukemic mouse strains, different patterns of virus detection were observed. In C3H/He mice, virus was occasionally present in embryos and was found in 40% of adult spleens. BALB/c mice were virus-negative as fetuses or weanlings, but spleens of more than half of the mice over 6 months of age yielded virus. NIH mice have never yielded virus. In reciprocal matings between AKR and BALB/c mice, virus recovery from embryos was maternally determined. The development of tissue culture isolation procedures made possible for the first time the application of classical infectious disease methods to the study of the natural history of murine leukemia virus infection.     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

Production of Vero cytotoxin by strains of Escherichia coli as related to the availability of iron

Abstract Vero cytotoxin (VT) producing strains of Escherichia coli (VTEC), including isolates from cases of haemolytic uraemic syndrome and infantile diarrhoea, were used to determine the effect of iron availability on the production of intra- and extracellular VT, with particular interest in elevating toxin production by low-level toxin producing VTEC. Culturing bacteria under iron restriction resulted in growth retardation and a decrease in the production of VT. For the routine detection of both high- and low-level VT-producing E. coli , there was no advantage to be gained by growing bacteria under iron restriction or using disrupted bacterial cell preparations; on the contrary, testing culture supernatants from bacteria grown in iron-replete media for approximately 14 h proved to be the most sensitive and accurate method for detecting VT and the resultant identification of VTEC.