FliO Regulation of FliP in the Formation of the Salmonella enterica Flagellum

The type III secretion system of the Salmonella flagellum consists of 6 integral membrane proteins: FlhA, FlhB, FliO, FliP, FliQ, and FliR. However, in some other type III secretion systems, a homologue of FliO is apparently absent, suggesting it has a specialized role. Deleting the fliO gene from the chromosome of a motile strain of Salmonella resulted in a drastic decrease of motility. Incubation of the ΔfliO mutant strain in motility agar, gave rise to pseudorevertants containing extragenic bypass mutations in FliP at positions R143H or F190L. Using membrane topology prediction programs, and alkaline phosphatase or GFPuv chimeric protein fusions into the FliO protein, we demonstrated that FliO is bitopic with its N-terminus in the periplasm and C-terminus in the cytoplasm. Truncation analysis of FliO demonstrated that overexpression of FliO_43–125 or FliO_1–95 was able to rescue motility of the ΔfliO mutant. Further, residue leucine 91 in the cytoplasmic domain was identified to be important for function. Based on secondary structure prediction, the cytoplasmic domain, FliO_43–125, should contain beta-structure and alpha-helices. FliO_43–125-Ala was purified and studied using circular dichroism spectroscopy; however, this domain was disordered, and its structure was a mixture of beta-sheet and random coil. Coexpression of full-length FliO with FliP increased expression levels of FliP, but coexpression with the cytoplasmic domain of FliO did not enhance FliP expression levels. Overexpression of the cytoplasmic domain of FliO further rescued motility of strains deleted for the fliO gene expressing bypass mutations in FliP. These results suggest FliO maintains FliP stability through transmembrane domain interaction. The results also demonstrate that the cytoplasmic domain of FliO has functionality, and it presumably becomes structured while interacting with its binding partners.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Expression and overproduction of glucose oxidase in Aspergillus niger

Summary This report describes the expression of cloned glucose oxidase gene (god) in glucose-oxidase-deficient mutants (God^–) of Aspergillus niger NRRL-3, the use of this gene for the elevation of glucose oxidase (GOD) productivity in the parental strain, and the further improvement of GOD production by subjecting the transformants to nitrous acid mutagenesis.Correspondence to: F. A. Sharif     

Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.     

New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

Environmental stresses inhibit splicing in the aquatic fungus Blastocladiella emersonii

Background  

Exposure of cells to environmental stress conditions can lead to the interruption of several intracellular processes, in particular those performed by macromolecular complexes such as the spliceosome.     

Mesenchymal stem cell transfusion for desensitization of positive lymphocyte cross‐match before kidney transplantation: outcome of 3 cases

Objectives

Donor specific antibodies (DSA) and a positive cross‐match are contraindications for kidney transplantation. Trials of allograft transplantation across the HLA barrier have employed desensitization strategies, including the use of plasmapheresis, intravenous immunoglobulins, anti‐B‐cell monoclonal antibodies and splenectomy, associated with high‐intensity immunosuppressive regimens. Our case 1 report suffered from repeatedly positive lymphocyte cross match after 1st renal transplantation. Graft nephrectomy could not correct the state of sensitization. Splenectomy was done in a trial to get rid of the antibody producing clone. Furthermore plasmapheresis with low dose IVIG could not as well revert the state of sensitization for the patient.

Material and methods

About 50 millions donor specific MSCs were injected to the patient.

Results

MSCs transfusion proved to be the only procedure which could achieve successful desensitization before performing the second transplantation owing to their immunosuppressive properties.

Conclusion

This case indicates that DS‐MSCs is a potential option for anti‐HLA desensitization. In cases 2 and 3 IV DS‐MSCs transfusion was selected from the start as a successful line of treatment for pre renal transplantation desensitization to save other unnecessary lines of treatment that were tried in case 1.

     

Exercise intensity influences cardiac baroreflex function at the onset of isometric exercise in humans.

We sought to examine the influence of exercise intensity on carotid baroreflex (CBR) control of heart rate (HR) and mean arterial pressure (MAP) at the onset of exercise in humans. To accomplish this, eight subjects performed multiple 1-min bouts of isometric handgrip (HG) exercise at 15, 30, 45 and 60% maximal voluntary contraction (MVC), while breathing to a metronome set at eupneic frequency. Neck suction (NS) of -60 Torr was applied for 5 s at end expiration to stimulate the CBR at rest, at the onset of HG (<1 s), and after approximately 40 s of HG. Beat-to-beat measurements of HR and MAP were recorded throughout. Cardiac responses to NS at onset of 15% (-12 +/- 2 beats/min) and 30% (-10 +/- 2 beats/min) MVC HG were similar to rest (-10 +/- 1 beats/min). However, HR responses to NS were reduced at the onset of 45% and 60% MVC HG (-6 +/- 2 and -4 +/- 1 beats/min, respectively; P < 0.001). In contrast to HR, MAP responses to NS were not different from rest at exercise onset. Furthermore, both HR and MAP responses to NS applied at approximately 40s of HG were similar to rest. In summary, CBR control of HR was transiently blunted at the immediate onset of high-intensity HG, whereas MAP responses were preserved demonstrating differential baroreflex control of HR and blood pressure at exercise onset. Collectively, these results suggest that carotid-cardiac baroreflex control is dynamically modulated throughout isometric exercise in humans, whereas carotid baroreflex regulation of blood pressure is well-maintained.     

Prediction of enteric methane emissions from cattle

Agriculture has a key role in food production worldwide and it is a major component of the gross domestic product of several countries. Livestock production is essential for the generation of high quality protein foods and the delivery of foods in regions where animal products are the main food source. Environmental impacts of livestock production have been examined for decades, but recently emission of methane from enteric fermentation has been targeted as a substantial greenhouse gas source. The quantification of methane emissions from livestock on a global scale relies on prediction models because measurements require specialized equipment and may be expensive. The predictive ability of current methane emission models remains poor. Moreover, the availability of information on livestock production systems has increased substantially over the years enabling the development of more detailed methane prediction models. In this study, we have developed and evaluated prediction models based on a large database of enteric methane emissions from North American dairy and beef cattle. Most probable models of various complexity levels were identified using a Bayesian model selection procedure and were fitted under a hierarchical setting. Energy intake, dietary fiber and lipid proportions, animal body weight and milk fat proportion were identified as key explanatory variables for predicting emissions. Models here developed substantially outperformed models currently used in national greenhouse gas inventories. Additionally, estimates of repeatability of methane emissions were lower than the ones from the literature and multicollinearity diagnostics suggested that prediction models are stable. In this context, we propose various enteric methane prediction models which require different levels of information availability and can be readily implemented in national greenhouse gas inventories of different complexity levels. The utilization of such models may reduce errors associated with prediction of methane and allow a better examination and representation of policies regulating emissions from cattle.