The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Nitrogen Cycles: Past, Present, and Future

This paper contrasts the natural and anthropogenic controls on the conversion of unreactive N₂ to more reactive forms of nitrogen (Nr). A variety of data sets are used to construct global N budgets for 1860 and the early 1990s and to make projections for the global N budget in 2050. Regional N budgets for Asia, North America, and other major regions for the early 1990s, as well as the marine N budget, are presented to Highlight the dominant fluxes of nitrogen in each region. Important findings are that human activities increasingly dominate the N budget at the global and at most regional scales, the terrestrial and open ocean N budgets are essentially disconnected, and the fixed forms of N are accumulating in most environmental reservoirs. The largest uncertainties in our understanding of the N budget at most scales are the rates of natural biological nitrogen fixation, the amount of Nr storage in most environmental reservoirs, and the production rates of N₂ by denitrification.     

Myofilament sensitivity to Ca2+ in ventricular myocytes from the Goto–Kakizaki diabetic rat

The chronic effects of type 2 diabetes mellitus on myofilament sensitivity to Ca(2+) in ventricular myocytes from the Goto-Kakizaki (GK) rat have been investigated. Experiments were performed in ventricular myocytes isolated from 17-month GK rats and age-matched Wistar controls. Myocytes were loaded with fura-2 (an indicator for intracellular Ca(2+) concentration) and the fura-2 ratio (340/380 nm), and shortening were measured simultaneously in electrically stimulated myocytes. Myofilament sensitivity to Ca(2+) was assessed from phase-plane diagrams of fura-2 versus cell length by measuring the gradient of the fura-2-cell length trajectory during late relaxation of the twitch contraction. Non-fasting and fasting blood glucose were elevated in GK rats compared to controls. Fasting blood glucose was 151.5 +/- 15.3 mg/dl (n = 8) in GK rats compared to 72.1 +/- 3.6 mg/dl (n = 9) in controls. At 120 min after intraperitoneal injection of glucose (2 g/kg body weight), blood glucose was 570.8 +/- 36.8 mg/dl in GK rats compared to 148 +/- 8.6 mg/dl in controls. Amplitude of shortening was significantly increased in myocytes from GK rats (6.56 +/- 0.54%, n = 31) compared to controls (5.05 +/- 0.43%, n = 36), and the amplitude of the Ca(2+) transient was decreased in myocytes from GK rats (0.23 +/- 0.02 RU, n = 31) compared to controls (0.30 +/- 0.02 RU, n = 36). The fura-2-cell length trajectory during the late stages of relaxation of the twitch contraction was steeper in myocytes from GK rats (89.2 +/- 16.6 microm/RU, n = 27) compared to controls (31.9 +/- 5.9 microm/RU, n = 35). Increased amplitude of shortening, accompanied by a decrease in amplitude of the Ca(2+) transient, might be explained by an increased myofilament sensitivity to Ca(2+).     

Genetic divergence of Connecticut Melanoplus femurrubrum populations

We surveyed Melanoplus femurrubrum populations within the state of Connecticut for genetic diversity at multiple genetic markers, including three mitochondrial [cytochrome oxidase subunit 1 (COI), reduced form of nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2), and AT rich] and one nuclear [internal transcribed spacers of the ribosomal DNA cluster (ITS1)] gene regions. All markers were variable, and the AT-rich gene showed the highest sequence divergence. Analysis of molecular variance (AMOVA), fixation index (Fst) analysis, and phylogeographic patterns showed little divergence between northern and southern regions. Estimates of genetic diversity (pi) showed higher mitochondrial diversity in the northern region but nearly equal diversity for the ITS1 gene. This study shows for the first time in Melanoplus genetic variation for the ND2, AT rich, and ITS genes within a small geographic area. Our methods and results should be useful for other researchers interested in conducting population-level studies on closely related species.     

Validation of a rodent model of source memory

Source memory represents the origin (source) of information. Recently, we proposed that rats (Rattus norvegicus) remember the source of information. However, an alternative to source memory is the possibility that rats selectively encoded some, but not all, information rather than retrieving an episodic memory. We directly tested this ‘encoding failure’ hypothesis. Here, we show that rats remember the source of information, under conditions that cannot be attributed to encoding failure. Moreover, source memory lasted at least seven days but was no longer present 14 days after studying. Our findings suggest that long-lasting source memory may be modelled in non-humans. Our model should facilitate attempts to elucidate the biological underpinnings of source memory impairments in human memory disorders such as Alzheimer''s disease.     

HSP70 interacting protein prevents the accumulation of inclusions in polyglutamine disease1

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over‐expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co‐expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over‐expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.     

IL-6 induced STAT3 signalling is associated with the proliferation of human muscle satellite cells following acute muscle damage

Background

Although the satellite cell (SC) is a key regulator of muscle growth during development and muscle adaptation following exercise, the regulation of human muscle SC function remains largely unexplored. STAT3 signalling mediated via interleukin-6 (IL-6) has recently come to the forefront as a potential regulator of SC proliferation. The early response of the SC population in human muscle to muscle-lengthening contractions (MLC) as mediated by STAT3 has not been studied.

Methodology/Principal Findings

Twelve male subjects (21±2 y; 83±12 kg) performed 300 maximal MLC of the quadriceps femoris at 180°•s⁻¹ over a 55° range of motion with muscle samples (vastus lateralis) and blood samples (antecubital vein) taken prior to exercise (PRE), 1 hour (T1), 3 hours (T3) and 24 hours (T24) post-exercise. Cytoplasmic and nuclear fractions of muscle biopsies were purified and analyzed for total and phosphorylated STAT3 (p-STAT3) by western blot. p-STAT3 was detected in cytoplasmic fractions across the time course peaking at T24 (p<0.01 vs. PRE). Nuclear total and p-STAT3 were not detected at appreciable levels. However, immunohistochemical analysis revealed a progressive increase in the proportion of SCs expressing p-STAT3 with ∼60% of all SCs positive for p-STAT3 at T24 (p<0.001 vs. PRE). Additionally, cMyc, a STAT3 downstream gene, was significantly up-regulated in SCs at T24 versus PRE (p<0.05). Whole muscle mRNA analysis revealed induction of the STAT3 target genes IL-6, SOCS3, cMyc (peaking at T3, p<0.05), IL-6Rα and GP130 (peaking at T24, p<0.05). In addition, Myf5 mRNA was up-regulated at T24 (p<0.05) with no appreciable change in MRF4 mRNA.

Conclusions/Significant Findings

We demonstrate that IL-6 induction of STAT3 signaling occurred exclusively in the nuclei of SCs in response to MLC. An increase in the number of cMyc+ SCs indicated that human SCs were induced to proliferate under the control of STAT3 signaling.