Experimental selection of long‐term intracellular mycobacteria

Some intracellular bacteria are known to cause long‐term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long‐term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re‐infection and also with the specific expression of stress‐ and survival‐related genes. Our findings identify bacterial traits implicated in the establishment of long‐term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.     

DDESC: Dragon database for exploration of sodium channels in human

Background

Hepcidin/LEAP-1 is an iron regulatory hormone originally identified as an antimicrobial peptide. As part of a systematic analysis of the evolution of host defense peptides in primates, we have sequenced the orthologous gene from 14 species of non-human primates.

Results

The sequence of the mature peptide is highly conserved amongst all the analyzed species, being identical to the human one in great apes and gibbons, with a single residue conservative variation in Old-World monkeys and with few substitutions in New-World monkeys.

Conclusion

Our analysis indicates that hepcidin's role as a regulatory hormone, which involves interaction with a conserved receptor (ferroportin), may result in conservation over most of its sequence, with the exception of the stretch between residues 15 and 18, which in New-World monkeys (as well as in other mammals) shows a significant variation, possibly indicating that this structural region is involved in other functions.     

The use of correlation integrals in the study of localized muscle fatigue of elbow flexors during maximal efforts.

Innovative applications of non-linear time series analysis have recently been used to investigate physiological phenomena. In this study, we investigated the feasibility of using the correlation integral to monitor the localized muscle fatigue process in the biceps brachii during sustained maximal efforts. The subjects performed isometric maximum contractions until failure in elbow flexion (90 degrees from neutral). The median and the 70th percentile frequency of the Surface electromyography (SEMG) power spectrum, the integrated SEMG, and the Correlation Integral (CI) were evaluated during the trials. The linear correlation between these variables and the elbow torque production was used to quantify the ability of a parameter to follow the fatiguing process. The CI had the highest linear correlation with torque (0.77 (0.12SD)), while the spectral indices correlations with torque were much lower. The decreasing trend of the torque production was followed by the spectral indices for only the beginning part of the contraction, while the CI increased sharply after the torque production fell to about 0.60 of the MVC. This suggests that the CI is sensitive to different changes of the SEMG signal during fatigue than the spectral variables.     

Validation of a method for quantification of ketobemidone in human plasma with liquid chromatography-tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of the analgesic aminophenol ketobemidone in human plasma is presented. Two preparation methods for plasma samples containing ketobemidone were compared, liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Both methods showed good precision (n=10), 1.7% and 2.9%, respectively (0.04 micro M) and 1.1% and 2.5%, respectively (0.14 micro M). The accuracy was 98% and 103%, respectively (0.04 micro M) and 105% and 99%, respectively (0.14 micro M). Ketobemidone could be quantified at 0.43 nM, with a relative standard deviation of 17.5% (n=19) using LLE and 18.6% (n=10) using SPE. This level was an order of magnitude lower than earlier reported quantification limits. Quantitative data from plasma samples analyzed with LC-MS-MS were in good agreement with those obtained by gas chromatography with chemical ionization mass spectrometry (GC-CI/MS). This indicates that LC-MS-MS is a good alternative method to GC-MS as it is more sensitive and time-consuming derivatization can be avoided.     

QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling

The 6-O sulfation states of cell surface heparan sulfate proteoglycans (HSPGs) are dynamically regulated to control the growth and specification of embryonic progenitor lineages. However, mechanisms for regulation of HSPG sulfation have been unknown. Here, we report on the biochemical and Wnt signaling activities of QSulf1, a novel cell surface sulfatase. Biochemical studies establish that QSulf1 is a heparan sulfate (HS) 6-O endosulfatase with preference, in particular, toward trisulfated IdoA2S-GlcNS6S disaccharide units within HS chains. In cells, QSulf1 can function cell autonomously to remodel the sulfation of cell surface HS and promote Wnt signaling when localized either on the cell surface or in the Golgi apparatus. QSulf1 6-O desulfation reduces XWnt binding to heparin and HS chains of Glypican1, whereas heparin binds with high affinity to XWnt8 and inhibits Wnt signaling. CHO cells mutant for HS biosynthesis are defective in Wnt-dependent Frizzled receptor activation, establishing that HS is required for Frizzled receptor function. Together, these findings suggest a two-state "catch or present" model for QSulf1 regulation of Wnt signaling in which QSulf1 removes 6-O sulfates from HS chains to promote the formation of low affinity HS-Wnt complexes that can functionally interact with Frizzled receptors to initiate Wnt signal transduction.     

Power and work produced in different leg muscle groups when rising from a chair

The aim of this study was to determine the power output and work done by different muscle groups at the hip and knee joints during a rising movement, to be able to tell the degree of activation of the muscle groups and the relationship between concentric and eccentric work. Nine healthy male subjects rose from a chair with the seat at knee level. The moments of force about the hip and knee joints were calculated semidynamically. The power output (P) and work in the different muscle groups surrounding the joints was calculated as moment of force times joint angular velocity. Work was calculated as: work = f Pdt. The mean peak concentric power output was for the hip extensors 49.9 W, hip flexors 7.9 W and knee extensor 89.5 W. This power output corresponded to a net concentric work of 20.7 J, 1.0 J and 55.6 J, respectively. There was no concentric power output from the knee flexor muscles. Energy absorption through eccentric muscle action was produced by the hip extensors and hip flexors with a mean peak power output of 4.8 W and 7.4 W, respectively. It was concluded that during rising, the hip and knee muscles mainly worked concentrically and that the greatest power output and work were produced during concentric contraction of the knee and hip extensor muscles. There was however also a demand for eccentric work by the hip extensors as well as both concentric and eccentric work by the hip flexors. The knee flexor muscles were unloaded.     

Folding of immunogenic peptide fragments of proteins in water solution. II. The nascent helix

1H nuclear magnetic resonance experiments indicate formation of secondary structures in water solutions of a synthetic immunogenic peptide of sequence EVVPHKKMHKDFLEKIGGL corresponding to the C-helix (residues 69 to 87) of myohemerythrin. The conformational ensemble consists of a set of turn-like structures, distributed over the C-terminal half of the peptide and rapidly interconverting by way of unfolded states. These structures, termed nascent helix, are stabilized into helical structure with long-range order in water/trifluorethanol mixtures. Circular dichroism measurements confirm the presence of 50% helix in water/trifluoroethanol but show no evidence of helicity in water solutions of the peptide. It is apparent that no one member of the transient set of helical conformations which constitutes the nascent helix is sufficiently long to be detectable by circular dichroism experiments. No preferred conformations could be detected by nuclear magnetic resonance in the N-terminal half of the peptide, either in water or water/trifluoroethanol mixtures. This region of the peptide is stabilized in helix by long-range interactions in the folded protein. The possible role of nascent secondary structure in induction of antipeptide antibodies and in initiation of protein folding is discussed.     

Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APCMin/+ mice

Tumor progression in the colon moves from aberrant crypt foci to adenomatous polyps to invasive carcinomas. The composition of the tumor-infiltrating leukocyte population affects the ability of the immune system to fight the tumor. T cell infiltration into colorectal adenocarcinomas, particularly T helper 1 (Th1) type T cells as well as increased regulatory T cell (Treg) frequencies, is correlated with improved prognosis. However, whether Th1 cells and Tregs are already present at the adenoma stage is not known. In this study, the APC^Min/+ mouse model of intestinal adenomatous polyposis was used to investigate tumor-associated lymphocyte subsets and the mechanisms of their accumulation into gastrointestinal adenomas. Compared to unaffected tissue, adenomas accumulated CD4⁺FoxP3⁺ putative Treg in parallel with lower frequencies of conventional T cells and B cells. The accumulation of Treg was also observed in human adenomatous polyps. Despite high Treg numbers, the function of conventional T cells present in the APC^Min/+ adenomas was not different from those in the unaffected tissue. Adenomas displayed an altered chemokine balance, with higher CCL17 and lower CXCL11 and CCL25 expression than in the unaffected tissue. In parallel, CXCR3⁺ Tregs were largely absent from adenomas. The data indicate that already in early stages of tumor development, the balance of lymphocyte-recruiting chemokines is altered possibly contributing to the observed shift toward higher frequencies of Treg.