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OBJECTIVE: Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of complex autoimmune conditions characterized by inflammation in skeletal muscle and extramuscular compartments, and interferon (IFN) system activation. We undertook this study to examine the contribution of genetic variation to disease susceptibility and to identify novel avenues for research in IIMs.METHODS: Targeted DNA sequencing was used to mine coding and potentially regulatory single nucleotide variants from ~1,900 immune-related genes in a Scandinavian case-control cohort of 454 IIM patients and 1,024 healthy controls. Gene-based aggregate testing, together with rare variant- and gene-level enrichment analyses, was implemented to explore genotype-phenotype relations.RESULTS: Gene-based aggregate tests of all variants, including rare variants, identified IFI35 as a potential genetic risk locus for IIMs, suggesting a genetic signature of type I IFN pathway activation. Functional annotation of the IFI35 locus highlighted a regulatory network linked to the skeletal muscle-specific gene PTGES3L, as a potential candidate for IIM pathogenesis. Aggregate genetic associations with AGER and PSMB8 in the major histocompatibility complex locus were detected in the antisynthetase syndrome subgroup, which also showed a less marked genetic signature of the type I IFN pathway. Enrichment analyses indicated a burden of synonymous and noncoding rare variants in IIM patients, suggesting increased disease predisposition associated with these classes of rare variants.CONCLUSION: Our study suggests the contribution of rare genetic variation to disease susceptibility in IIM and specific patient subgroups, and pinpoints genetic associations consistent with previous findings by gene expression profiling. These features highlight genetic profiles that are potentially relevant to disease pathogenesis.

The propensity forSaccharomyces cerevisiaeyeast to ferment sugars into ethanol and CO2has long been useful in the production of a wide range of food and drink. In the production of alcoholic beverages, the yeast strain selected for fermentation is crucial because not all strains are equally proficient in tolerating fermentation stresses. One potential mechanism by which domesticated yeast may have adapted to fermentation stresses is through changes in the expression of stress response genes.MED15is a general transcriptional regulator and RNA Pol II Mediator complex subunit which modulates the expression of many metabolic and stress response genes. In this study, we explore the role ofMED15in alcoholic fermentation. In addition, we ask whetherMED15alleles from wine, sake or palm wine yeast improve fermentation activity and grape juice fermentation stress responses. And last, we investigate to what extent any differences in activity are due to allelic differences in the lengths of three polyglutamine tracts inMED15. We find that strains lackingMED15are deficient in fermentation and fermentation stress responses and thatMED15alleles from alcoholic beverage yeast strains can improve both the fermentation capacity and the response to ethanol stresses when transplanted into a standard laboratory strain. Finally, we find that polyglutamine tract length in the Med15 protein is one determinant in the efficiency of the alcoholic fermentation process. These data lead to a working model in which polyglutamine tract length and other types of variability within transcriptional hubs like the Mediator subunit, Med15, may contribute to a reservoir of transcriptional profiles that may provide a fitness benefit in the face of environmental fluctuations.

Objectives To describe the prevalence and clinical associations of autoantibodies to a novel autoantigen, eukaryotic initiation factor 3 (eIF3), detected in idiopathic inflammatory myositis. Methods Sera or plasma from 678 PM patients were analysed for autoantigen specificity by radio-labelled protein immunoprecipitation (IPP). Samples immunoprecipitating the same novel autoantigens were further analysed by indirect immunofluorescence and IPP using pre-depleted cell extracts. The autoantigen was identified through a combination of IPP and MALDI-TOF mass spectrometry, and confirmed using commercial antibodies and IPP-western blots. Additional samples from patients with DM (668), DM-overlap (80), PM-overlap (191), systemic sclerosis (150), systemic lupus erythematosus (200), Sjogren’s syndrome (40), rheumatoid arthritis (50) and healthy controls (150) were serotyped by IPP as disease or healthy controls. Results IPP revealed a novel pattern in three PM patients (0.44%) that was not found in disease-specific or healthy control sera. Indirect immunofluorescence demonstrated a fine cytoplasmic speckled pattern for all positive patients. Mass spectrometry analysis of the protein complex identified the target autoantigen as eIF3, a cytoplasmic complex with a role in the initiation of translation. Findings were confirmed by IPP-Western blotting. The three anti-eIF3-positive patients had no history of malignancy or interstitial lung disease, and had a favourable response to treatment. Conclusion We report a novel autoantibody in 0.44% of PM patients directed against a cytoplasmic complex of proteins identified as eIF3. Although our findings need further confirmation, anti-eIF3 appears to correlate with a good prognosis and a favourable response to treatment.

We measured the stable isotope ratios (C, N, S) of modern human hair collected from 49 people in rural southern Ethiopia to characterize and compare the stable isotope signatures of interacting and ethnically distinct populations with differing economic practices (farmers, pastoralists, fishers) in order to determine if the dietary differences are visible and measurable in their hair isotope values. We found that there were significant differences in all three elements that can distinguish these economic practices (carbon: Χ2 = 8.523, p = 0.014; nitrogen: Χ2 = 35.372, p

Study objective To determine whether risk stratification in the out-of-hospital setting could identify patients with chest pain who are at low and high risk to avoid admission or aid direct transfer to cardiac centers. Methods Paramedics prospectively enrolled patients with suspected acute coronary syndrome without diagnostic ST-segment elevation on the ECG. The History, ECG, Age and Risk Factors (HEAR) score was recorded contemporaneously, and out-of-hospital samples were obtained to measure cardiac Troponin I (cTnI) level on a point-of-care device, to allow calculation of the History, ECG, Age, Risk Factors, and Troponin (HEART) score. HEAR and HEART scores less than or equal to 3 and greater than or equal to 7 were defined as low and high risk for major adverse cardiac events at 30 days. Results Of 1,054 patients (64 years [SD 15 years]; 42% women), 284 (27%) experienced a major adverse cardiac event at 30 days. The HEAR score was calculated in all patients, with point-of-care cTnI testing available in 357 (34%). A HEAR score less than or equal to 3 identified 32% of patients (334/1,054) as low risk, with a sensitivity of 84.9% (95% confidence interval [CI] 80.7% to 89%), whereas a score greater than or equal to 7 identified just 3% of patients (30/1,054) as high risk, with a specificity of 98.7% (95% CI 97.9% to 99.5%). A point-of-care HEART score less than or equal to 3 identified a similar proportion as low risk (30%), with a sensitivity of 87.0% (95% CI 80.7% to 93.4%), whereas a score greater than or equal to 7 identified 14% as high risk, with a specificity of 94.8% (95% CI 92.0% to 97.5%). Conclusion Paramedics can use the HEAR score to discriminate risk, but even when used in combination with out-of-hospital point-of-care cTnI testing, the HEART score does not safely rule out major adverse cardiac events, and only a small proportion of patients are identified as high risk.

Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656–666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3–Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3–Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3–Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3–Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3–15-fold with three of them by 10–15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3–Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.

Micromanipulators, more than any other instrument, opened the early doors to developing the powerful genetics of yeast that underlies much of the molecular work today. The ability to separate the spores of a tetrad and analyze their phenotypes generated the genetic maps and biology upon which subsequent cloning, sequencing, cutting edge molecular and cell biology depended. This work describes the development of those micromanipulators from garage to barn to factory and the developer of the sophisticated instruments we use today. For more than 30 years Carl Singer and his family were staunch and generous supporters of the International Conferences on Yeast Genetics and Molecular Biology meetings both in Europe and America. Carl Singer's displays at meetings became a traditional fixture and engaged the appetites of many students and advanced researchers to employ a technique that many perceived as too complicated or difficult, but which he made simple and easy to learn. His experiences also document a sketch of the international yeast meetings, their venues and how they developed through the years.

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array., Protein HilD of Salmonella Typhimurium coordinates motility and host cell invasion by upregulating flagellar genes and a secretion system. Here, Cooper et al. show that HilD also modulates swimming behaviour by upregulating a subunit of the chemotactic receptor array, and this is important for invasion of epithelial cells.

We report here that the neuronal (pro)renin receptor (PRR), a key component of the brain renin-angiotensin system (RAS), plays a critical role in the central regulation of high-fat-diet (HFD)-induced metabolic pathophysiology. The neuronal PRR is known to mediate formation of the majority of angiotensin (ANG) II, a key bioactive peptide of the RAS, in the central nervous system and to regulate blood pressure and cardiovascular function. However, little is known about neuronal PRR function in overnutrition-related metabolic physiology. Here, we show that PRR deletion in neurons reduces blood pressure, neurogenic pressor activity, and fasting blood glucose and improves glucose tolerance without affecting food intake or body weight following a 16-wk HFD. Mechanistically, we found that a HFD increases levels of the PRR ligand (pro)renin in the circulation and hypothalamus and of ANG II in the hypothalamus, indicating activation of the brain RAS. Importantly, PRR deletion in neurons reduced astrogliosis and activation of the astrocytic NF-κB p65 (RelA) in the arcuate nucleus and the ventromedial nucleus of the hypothalamus. Collectively, our findings indicate that the neuronal PRR plays essential roles in overnutrition-related metabolic pathophysiology.

Beachrocks are coastal sediments that are lithified through the precipitation of carbonate cements. It is widely acknowledged that lithofacies in beachrocks are variable and their interpretation is useful when using beachrock as a sea level indicator or when studying shoreline evolution over the centurial to millennial scales. Surprisingly however, the facies variability of beachrocks remains understudied as they are almost exclusively described as seaward dipping, slab-shaped outcrop forming in low energy dissipative beach environments. The Mission Rocks coastline of north-eastern South Africa is in stark contrast. Here the coast comprises an up to 3 m thick raised shore platform of beachrock, where a variety of sedimentological facies are observed. These comprise seaward-dipping planar bedded sandstones and conglomeratic units, often interbedded with bimodally-orientated trough cross bedded sandstones. In our study we aim to use sedimentological facies analysis, petrography and cathodoluminescence to unravel the deposition- and cementation processes of this beachrock facies.In particular, an unusual beachrock breccia interposed amongst the breakdown remnants of the platform forms the basis of this paper. The breccia documents a cycle of simultaneous erosional breakdown and depositional buildup of the beachrock platform, a yet undescribed process for the development of beachrock. Since it forms as a thin veneer (< 0.10 m), with a slightly thicker infill (≤ 0.5 m) amidst erosional hollows and gullies of the + 2 m high rocky platform, it raises into question the necessity of a thick sedimentary overburden, that is typically considered the requirement for beachrock cementation in the mixing zone. Timing of beachrock formation is constrained by recent anthropogenic activities, as the underlaying platform was mined for building purposes during WWII and it is in these quarry slots and crack that the beachrock is found. While it is generally suspected that beachrocks may form at the centennial scale, evidence for this remains weak. Not only can the interpretation of this facies contribute to our understanding of the long term processes that form and break down beachrocks on high energetic coastlines, it provides insight into rapid beachrock formation and as such its utility as a sea level index point.

Persistence and intermittent fecal shedding, hallmarks of Salmonella infections, are critical for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invade intestinal epithelial cells and survive in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol, but replicate normally in the SCV, to examine the role of cytosolic STm in persistence and shedding. Intestinal expansion of these STm was impaired in an acute enterocolitis infection model, while fecal shedding of STm was eliminated in a mouse model of persistent infection. Thus, the cytosol is a critical reservoir of intestinal STm that maintains the high luminal density required for fecal shedding and transmission. This reveals the requirement of an intracellular niche for pathogen transmission and presents new targets for disease control.

The idiopathic inflammatory myopathies (IIM), often referred to collectively but less specifically as “myositis,” have an estimated prevalence of ~14 per 100,000 with an annual incidence of ~8 per 1,000,000 population, which appears to be increasing over time. Any age group can be affected, but myositis commonly presents between 30 and 60 years of age. Differences are seen based on autoantibody subtypes as, for example, anti-signal recognition particle (SRP) seems more common in younger adults. Females are affected about twice as often as males, but again there are important differences when considering specific clinical subtypes as inclusion body myositis (IBM) is more common in males. Current research is focussed on understanding the complex interactions between genetic susceptibility and exposures to environmental triggers. The majority of genetic risk factors identified to date lie within the major histocompatibility complex (MHC), a region which contains genes responsible for antigen presentation, but a small number of genetic risk factors have been found outside this region and implicated in innate and adaptive immune functions. Important environmental associations have been already identified, including exposures to ultraviolet radiation, smoking and certain medications such as statins.

Persistence and intermittent fecal shedding, hallmarks of Salmonella infections, are critical for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicates rapidly, expresses invasion factors, and induces extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destructs in the cytosol (STm(CytoKill)), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STm(CytoKill) are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication and release of invasive STm from extruded IECs, fuels the high luminal density required for fecal shedding.

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex™. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.

Key Clinical Message Insulinomas are uncommon tumors, and in patients with diabetes mellitus they are extremely rare. We describe a patient with type 1 diabetes who developed malignant insulinoma. When hypoglycemic episodes persist in a patient with diabetes and treatment‐induced and other causes of hypoglycemia have been ruled out, an insulin‐producing tumor should be considered.

Nitrogen catabolite repression (NCR), the ability of Saccharomyces cerevisiae to use good nitrogen sources in preference to poor ones, derives from nitrogen-responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen-replete conditions, the GATA factors are cytoplasmic and NCR-sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear, dramatically increasing GATA factor-mediated transcription. This regulation was originally attributed to mechanistic Tor protein kinase complex 1 (mTorC1)-mediated control of Gln3. However, we recently showed that two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration, only one of which is mTorC1. Present experiments demonstrate that the other previously elusive component is uncharged transfer RNA-activated, Gcn2 protein kinase-mediated general amino acid control (GAAC). Gcn2 and Gcn4 are required for NCR-sensitive nuclear Gln3-Myc13 localization, and from epistasis experiments Gcn2 appears to function upstream of Ure2. Bmh1/2 are also required for nuclear Gln3-Myc13 localization and appear to function downstream of Ure2. Overall, Gln3 phosphorylation levels decrease upon loss of Gcn2, Gcn4, or Bmh1/2. Our results add a new dimension to nitrogen-responsive GATA-factor regulation and demonstrate the cumulative participation of the mTorC1 and GAAC pathways, which respond oppositely to nitrogen availability, in the nitrogen-responsive control of catabolic gene expression in yeast.

This article updates on the concept that muscle-derived cytokines (myokines) play important roles in muscle health and disease.Interleukin-6 (IL-6) is released from normal skeletal muscle in response to exercise, mediating both anti-inflammatory responses and metabolic adaptations, actions contradictory to the prevailing view that IL-6 is a proinflammatory cytokine that is inducing and propagating disease. The anti-inflammatory effects of IL-6 result from its trans-membrane signalling capability, via membrane-bound receptors, whereas its proinflammatory effects result instead from signalling via the soluble IL-6 receptor and gp130. IL-15 is elevated following exercise, promoting muscle fibre hypertrophy in some circumstances, while inducing fibre apoptosis in others. This functional divergence appears because of variations in expression of IL-15 receptor isoforms. Decorin, a recently described myokine, is also elevated following exercise in normal muscle, and promotes muscle fibre hypertrophy by competitively binding to, and thus inhibiting, myostatin, a negative regulator of muscle protein synthesis. Exercise-induced myostatin downregulation thus promotes muscle fibre growth, prompting recent trials of a biological myostatin inhibitor in inclusion body myositis.Myokines appear to exert diverse beneficial effects, though their mechanistic roles in myositis and other myopathologies remain poorly understood.

Objectives To characterise the 10 year relationship between anti-transcriptional intermediary factor 1 antibody (anti-TIF1-Ab) positivity and cancer onset in a large UK-based adult dermatomyositis (DM) cohort. Methods Data from anti-TIF1-Ab positive/negative adults with verified diagnoses of DM from UKMYONET were analysed. Each patient was followed up until they developed cancer. Kaplan-Meier methods and Cox-proportional hazard modelling were employed to estimate cumulative cancer incidence. Results Data from 263 DM cases were analysed, with a total of 3,252 person-years and a median 11 years follow-up; 55 (21%) of DM cases were anti-TIF1-Ab positive. After 10 years of follow up, a higher proportion of anti-TIF1-Ab positive cases developed cancer, compared with anti-TIF1-Ab negative cases: 38% vs 15% (hazard ratio 3.4 [95% CI 2.2, 5.4]). All the detected malignancy cases in the anti-TIF1-Ab positive cohort occurred between 3 years prior to and 2.5 years after DM onset. No cancer cases were detected within the following 7.5 years in this group, whereas cancers were detected during this period in the anti-TIF1-Ab negative cases. Ovarian cancer was more common in the anti-TIF1-Ab positive vs negative cohort – 19% versus 2% respectively (p-value

Although one of the defining characteristics of Alzheimer’s disease is the presence of amyloid-beta (Aβ) plaques, the early accumulation of soluble Aβ oligomers (AβOs) may disrupt synaptic function and trigger cognitive impairments long before the appearance of plaques. Furthermore, murine models aimed at understanding how AβOs alter formation and retrieval of associative memories are conducted using human Aβ species, which are more neurotoxic in the mouse brain than the native murine species. Unfortunately, there is currently a lack of attention in the literature as to what the murine version of the peptide (mAβ) does to synaptic function and how it impacts the consolidation and retrieval of associative memories. In the current study, adult mice were infused with mAβ 0, 2, 6, or 46 h after contextual-fear conditioning, and were tested 2–48 h later. Interestingly, only mAβ infusions within 2 h of training reduced freezing behavior at test, indicating that mAβ disrupted the consolidation, but not retrieval of fear memory. This consolidation deficit coincided with increased IL-1β and reduced synaptophysin mRNA levels, without disrupting other synaptic signaling-related genes here examined. Despite differences between murine and human Aβ, the deleterious functional outcomes of early-stage synaptic oligomer presence are similar. Thus, models utilizing or inducing the production of mAβ in non-transgenic animals are useful in exploring the role of dysregulated synaptic plasticity and resultant learning deficits induced by Aβ oligomers.

ObjectivesIdiopathic inflammatory myopathies (IIM) are a spectrum of rare autoimmune diseases characterised clinically by muscle weakness and heterogeneous systemic organ involvement. The strongest genetic risk is within the major histocompatibility complex (MHC). Since autoantibody presence defines specific clinical subgroups of IIM, we aimed to correlate serotype and genotype, to identify novel risk variants in the MHC region that co-occur with IIM autoantibodies.MethodsWe collected available autoantibody data in our cohort of 2582 Caucasian patients with IIM. High resolution human leucocyte antigen (HLA) alleles and corresponding amino acid sequences were imputed using SNP2HLA from existing genotyping data and tested for association with 12 autoantibody subgroups.ResultsWe report associations with eight autoantibodies reaching our study-wide significance level of p–5. Associations with the 8.1 ancestral haplotype were found with anti-Jo-1 (HLA-B*08:01, p=2.28×10–53 and HLA-DRB1*03:01, p=3.25×10–9), anti-PM/Scl (HLA-DQB1*02:01, p=1.47×10–26) and anti-cN1A autoantibodies (HLA-DRB1*03:01, p=1.40×10–11). Associations independent of this haplotype were found with anti-Mi-2 (HLA-DRB1*07:01, p=4.92×10–13) and anti-HMGCR autoantibodies (HLA-DRB1*11, p=5.09×10–6). Amino acid positions may be more strongly associated than classical HLA associations; for example with anti-Jo-1 autoantibodies and position 74 of HLA-DRB1 (p=3.47×10–64) and position 9 of HLA-B (p=7.03×10–11). We report novel genetic associations with HLA-DQB1 anti-TIF1 autoantibodies and identify haplotypes that may differ between adult-onset and juvenile-onset patients with these autoantibodies.ConclusionsThese findings provide new insights regarding the functional consequences of genetic polymorphisms within the MHC. As autoantibodies in IIM correlate with specific clinical features of disease, understanding genetic risk underlying development of autoantibody profiles has implications for future research.

Precise neural sequences are associated with the production of well-learned skilled behaviors. Yet, how neural sequences arise in the brain remains unclear. In songbirds, premotor projection neurons in the cortical song nucleus HVC are necessary for producing learned song and exhibit precise sequential activity during singing. Using cell-type specific calcium imaging we identify populations of HVC premotor neurons associated with the beginning and ending of singing-related neural sequences. We characterize neurons that bookend singing-related sequences and neuronal populations that transition from sparse preparatory activity prior to song to precise neural sequences during singing. Recordings from downstream premotor neurons or the respiratory system suggest that pre-song activity may be involved in motor preparation to sing. These findings reveal population mechanisms associated with moving from non-vocal to vocal behavioral states and suggest that precise neural sequences begin and end as part of orchestrated activity across functionally diverse populations of cortical premotor neurons., eLife, 8, ISSN:2050-084X

The Mediator complex is required for basal activity of the RNA polymerase (Pol) II transcriptional apparatus and for responsiveness to some activator proteins. Med15, situated in the Mediator tail, plays a role in transmitting regulatory information from distant DNA-bound transcription factors to the transcriptional apparatus poised at promoters. Yeast Med15 and its orthologs share an unusual, glutamine-rich amino acid composition. Here, we discuss this sequence feature and the tendency of polyglutamine tracts to vary in length among strains of Saccharomyces cerevisiae, and we propose that different polyglutamine tract lengths may be adaptive within certain domestication habitats.

Strands of human hair excavated from Basketmaker II Turkey Pen Ruins in Utah were examined using stable carbon and nitrogen isotope analysis and show that these individuals were heavily reliant on maize but their diet fluctuated over a period of months. Through serial bulk carbon and nitrogen analysis and serial single amino acid carbon isotope analysis of individual strands of hair, this study addresses the questions of how human diets at the site varied over a period of months and if domesticated turkeys ( Meleagris gallopavo ) were part of the diet during any point of the year. Hair carbon isotope values range from − 9.2‰ to − 12.4‰ with an average of − 10.8 ± 1.2, and nitrogen isotope values range from 6.0‰ to 7.3‰ with an average of 6.6‰ ± 0.5. There was also variability in the single amino acid δ 13 C values, with an overall pattern of terrestrial C 4 signatures of individual amino acids ( δ 13 C for leucine averaged − 17.8‰ ± 1.7). Δ 13 C valine-phenylalanine values ranging from − 1.6‰ to 2.6‰ also point strongly to a terrestrial diet. From the stable isotope data presented here, we suggest that BMII diet, while being heavily maize based, did vary during the year, and that turkeys were never a diet staple.

The aim of the study was to evaluate geometrical parameters of tibiotarsal bones in geese depending on age, sex and place in the bone and conditions of breeding. The inner structure of tibiotarsal bones of White Kołuda® geese (W31) subjected to intensive breeding was analyzed using peripheral quantitative computed tomography (pQCT ). The analyzed bones were derived from birds representing five age groups: one-day-old as well as birds of 2, 4, 6 and 8 wk of age. Bones of 10 males and 10 females were isolated from each group. The analysis was conducted at two levels of the bone: the area of the proximal metaphysis and at half the length of the diaphysis. The proximal metaphysis was selected for the research as it had higher content of the cancellous bone than the distal metaphysis. It was known that the cancellous bone was characterized by a higher rate of metabolic changes. The following geometric parameters were determined: total bone area (TOT _A), trabecular area (TRAB _A), cortical area (CRT _A), cortical thickness (CRT _TH K_C), periosteal circumference (PERI C), endocortical circumference (ENDO _C) and Strength Strain Index (SSI ). It was found that 6 wk of life may be considered as critical in the postnatal development of geese. In the group of 6 wk males in the shafts of the tibial bones, there was a decrease in the values of the following parameters: TOT _A, TRAB _A, PERI _C, ENDO _C. Whereas in 8 wk, an attenuation of the values of all geometric parameters of tibiotarsal bones was observed in both sexes (with the exception of CRT _TH K_C), which may have a negative influence on bone resistance to fractures. In this period, a significant decrease in the values of geometric parameters of tibiotarsal bones could be observed, negatively affecting the resistance of the bone to fractures in 8 wk. The values of SSI parameter at the proximal metaphysis of the tibiotarsal bone decreased in both males and females in 8 wk. In turn, at mid-diaphysis, the SSI values decreased only in males in 8 wk. The achieved results and observations justify the use of optimal environmental and nutritional conditions in geese in order to improve the quality of their skeletal system and to minimize the risk of the occurrence of deformities and fractures of the pelvic limb during the breeding.

Background Serratia Marcescens (SM) is often an opportunist that has been associated as a cause of healthcare-associated infection and in some people who inject drugs (PWID). Decisions about the treatment of SM infections are difficult given the small clinical studies available and concerns for multidrug resistance. SM has the ability to produce inducible AmpC b-lactamase and may acquire extended-spectrum b-lactamase (ESBL). Evidence-based guidance is lacking in terms of identifying preferred antimicrobial therapy of SM bacteremia and endocarditis. Compared to other reports, our hospital has one of the largest SM data sets to compare. Methods This observation study included adult patients admitted to our hospital (2016–2019) with SM bloodstream infections, including endocarditis. Patients were excluded from the analysis, if they had a concomitant infection with another gram-negative organism. Our evaluation was designed to: compare outcomes associated with different antibiotic regimens, evaluate how care differed in PWID patients versus others, and identify factors associated with obtaining infectious diseases expert consultations (ID Consult). Results Forty-three patients met study inclusion/exclusion. Twenty-eight patients (65.1%) had ID Consults. Twenty-four (55.8%) were PWID. Endocarditis was diagnosed in 30.2% of patients. The most common regimen was cefepime +/- aminoglycoside, followed by a carbapenem +/- aminoglycoside. Combination therapy was only recommended during ID Consult. Piperacillin-tazobactam was used in 11.6% of patients. No regimen displayed an efficacy or safety advantage over another. Most patients (90.7%) cleared their blood stream within 48 hours of antibiotic start. Phenotypic susceptibility testing did not identify either ESBL or AmpC production in any of the isolates, including recurrences. Multi-drug resistance was not appreciated. Significant factors associated with obtaining ID Consult were: PWID (p=0.004), endocarditis (p=0.0002), sepsis (p=0.022), surgical intervention (p=0.003). Conclusion We could not identify an advantage with any particular antibiotic treatment regimen in this study. Induction of AmpC or selection of ESBL organisms was not displayed by any of the organisms. Disclosures All Authors: No reported disclosures

Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation and mitochondrial abnormalities, and is postulated as a potential mechanism involved in muscle weakness in myositis, an acquired autoimmune neuromuscular disease. This study investigates the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 h) in the presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. Tunicamycin induced maladaptive ER stress, which was mitigated by EUK-134 at the transcriptional level. ER stress promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as a reduction in respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. Tunicamycin induced ROS-mediated biogenesis and fusion of mitochondria, which, however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation was observed under ER stress that was prevented by EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function, and therefore, in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction.

Gut microbiome plays an increasingly recognized important role in human health. This study aims to evaluate the association among race, neighborhood disadvantage index (NDI), and gut microbiome in patients undergoing screening colonoscopy. Methods: The study includes 689 patients aged 50–80 years undergoing screening colonoscopy at University Hospitals Cleveland Medical Center. Each patient donated a stool sample 1–4 weeks prior to their colonoscopy examination. 16S ribosomal RNA genes in stools were sequenced and classified into 236 Operational Taxonomic Units (OTUs) to species level. Neighborhood Disadvantage Index (NDI) was derived from 17 variables reflecting neighborhood contextual environment. We first examined the association of race with microbiota, and race with NDI. We then examined further the association between NDI and each microbiota by linear mixed effect model to account for the correlation within census tract, with adjustment of age, sex, BMI, smoking, and sequencing batch. The significance of associations between microbiota and NDI are evaluated by likelihood ratio tests. Q-value was estimated for False Discovery Rate (FDR)-based multiple testing correction. For the microbiota associated with NDI with Q-value ⇐ 0.05, we additionally adjusted for genetic ancestry in the model in a subset of 222 patients who have available genome-wide ancestry informative SNP genotype data. Results: Of all the 172 microbiota that have minimum relative abundances >0.005, 13 microbiota were significantly associated with race. We speculate that such association with race could be at least partially driven by neighborhood socioeconomic environment, i.e., racialized neighborhood environment - given the high correlation of race and NDI (corr = 0.60, P = 3.38E-56). Multivariate-adjusted regression models showed that 5 microbiota were positively associated with NDI (Q-value < 0.05). Subset analysis of 222 patients with further controlling for genetic ethnicity shows that two microbiotas remain significantly associated with NDI. Conclusions: Residential neighborhood disadvantage was found to impact gut microbiota. Further studies to validate our findings and to delineate the functionality of these NDI-associated microbiota are warranted.

In patients with myositis, persistent skeletal muscle weakness in the absence of significant inflammatory cell infiltrates is a well-recognised, but poorly understood, cause of morbidity. This has led researchers to investigate cellular mechanisms independent of immune cells, which may contribute to this underlying muscle weakness. Chronic ER stress pathway activation is evident in the muscle of myositis patients, and is now a potential mediator of muscle weakness in the absence of inflammation. Abnormal ER stress pathway activation is associated with mitochondrial dysfunction, resulting in bioenergetic deficits and reactive oxygen species (ROS) generation, which in this context may potentially damage muscle proteins and thus impair contractile performance. This study examined whether treatment with the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17AAG) could mitigate these ER stress-induced changes. C2C12 myotubes were treated with the ER stress-inducing compound Tunicamycin, in the presence or absence of 17AAG. Myotubes were examined for changes relating to ER stress pathway activation, mitochondrial function, markers of oxidative damage and in myotubular dimensions. ER stress pathway activation caused mitochondrial dysfunction, as evidenced by reduced oxygen consumption and ATP generation and by increased gene expression levels of the bio-energetic regulator, uncoupling protein 3 (UCP-3), the latter indicative of electron transport chain uncoupling. ER stress pathway activation also caused increased gene expression of superoxide dismutase (SOD) 2 and peroxiredoxin (PRDX) 3, elevated H2O2 levels, and reduced total thiol pool levels and a significant diminution of myotubular dimensions. Exposure to 17AAG ameliorated these ER stress-induced changes. These findings, which suggest that 17AAG can reduce ER stress-induced mitochondrial dysfunction, oxidative damage and myotubular atrophy, have potential implications in the context of human myositis.

Gliosis and fibrosis after spinal cord injury (SCI) lead to formation of a scar that is an impediment to axonal regeneration. Fibrotic scarring is characterized by the accumulation of fibronectin, collagen, and fibroblasts at the lesion site. The mechanisms regulating fibrotic scarring after SCI and its effects on axonal elongation and functional recovery are not well understood. In this study, we examined the effects of eliminating an isoform of fibronectin containing the Extra Domain A domain (FnEDA) on both fibrosis and on functional recovery after contusion SCI using male and female FnEDA-null mice. Eliminating FnEDA did not reduce the acute fibrotic response but markedly diminished chronic fibrotic scarring after SCI. Glial scarring was unchanged after SCI in FnEDA-null mice. We found that FnEDA was important for the long-term stability of the assembled fibronectin matrix during both the subacute and chronic phases of SCI. Motor functional recovery was significantly improved, and there were increased numbers of axons in the lesion site compared to wildtype mice, suggesting that the chronic fibrotic response is detrimental to recovery. Our data provide insight into the mechanisms of fibrosis after SCI and suggest that disruption of fibronectin matrix stability by targeting FnEDA represents a potential therapeutic strategy for promoting recovery after SCI.

Despite advances in antihypertensive therapeutics, at least 15–20% of hypertensive patients have resistant hypertension through mechanisms that remain poorly understood. In this study, we provide a new mechanism for the regulation of blood pressure (BP) in the central nervous system (CNS) by the (pro)renin receptor (PRR), a recently identified component of the renin-angiotensin system that mediates ANG II formation in the CNS. Although PRR also mediates ANG II-independent signaling, the importance of these pathways in BP regulation is unknown. Here, we developed a unique transgenic mouse model overexpressing human PRR (hPRR) specifically in neurons (Syn-hPRR). Intracerebroventricular infusion of human prorenin caused increased BP in Syn-hPRR mice. This BP response was attenuated by a NADPH oxidase (NOX) inhibitor but not by antihypertensive agents that target the renin-angiotensin system. Using a brain-targeted genetic knockdown approach, we found that NOX4 was the key isoform responsible for the prorenin-induced elevation of BP in Syn-hPRR mice. Moreover, inhibition of ERK significantly attenuated the increase in NOX activity and BP induced by human prorenin. Collectively, our findings indicate that an ANG II-independent, PRR-mediated signaling pathway regulates BP in the CNS by a PRR-ERK-NOX4 mechanism. NEW & NOTEWORTHY This study characterizes a new transgenic mouse model with overexpression of the human (pro)renin receptor in neurons and demonstrated a novel angiotensin II-independent mechanism mediated by human prorenin and the (pro)renin receptor in the central regulation of blood pressure.

Here we describe the use of synthetic genetic elements to improve the predictability and tunability of episomal protein production in Salmonella. We used a multi-pronged approach, in which a series of variable-strength synthetic promoters were combined with a synthetic transcriptional terminator, and plasmid copy number variation. This yielded a series of plasmids that drive uniform production of fluorescent and endogenous proteins, over a wide dynamic range. We describe several examples where this system is used to fine-tune constitutive expression in Salmonella, providing an efficient means to titrate out toxic effects of protein production.

Philosophical debate about the proper role of athletics within the academy has reverberated through each era of collegiate sport, and a growing body of literature points toward an impending tipping point unless radical reform ensues. This study contributes perspective to a proposed reform model through investigating perceptions of National Collegiate Athletics Association Division I coaches (N = 661) about their roles as educators and how this role could be altered through structural and philosophical changes within the academy. Quantitative and qualitative data provided mixed findings related to coach support for an integrated organizational structure with high variance in all structural facets explored except for compensation, where coaches believed structures should not be uniform between athletic and academic units because of the perceived greater workload, hours, media attention, and pressure in athletics.

Autoimmune muscle diseases (myositis) comprise a group of complex phenotypes influenced by genetic and environmental factors. To identify genetic risk factors in patients of European ancestry, we conducted a genome-wide association study (GWAS) of the major myositis phenotypes in a total of 1710 cases, which included 705 adult dermatomyositis; 473 juvenile dermatomyositis; 532 polymyositis; and 202 adult dermatomyositis, juvenile dermatomyositis or polymyositis patients with anti-histidyl tRNA synthetase (anti-Jo-1) autoantibodies, and compared them with 4724 controls. Single-nucleotide polymorphisms showing strong associations (P < 5 × 10−8) in GWAS were identified in the major histocompatibility complex (MHC) region for all myositis phenotypes together, as well as for the four clinical and autoantibody phenotypes studied separately. Imputation and regression analyses found that alleles comprising the human leukocyte antigen (HLA) 8.1 ancestral haplotype (AH8.1) defined essentially all the genetic risk in the phenotypes studied. Although the HLA DRB1*03:01 allele showed slightly stronger associations with adult and juvenile dermatomyositis, and HLA B*08:01 with polymyositis and anti-Jo-1 autoantibody-positive myositis, multiple alleles of AH8.1 were required for the full risk effects. Our findings establish that alleles of the AH8.1haplotype comprise the primary genetic risk factors associated with the major myositis phenotypes in geographically diverse Caucasian populations.

The first step in executing the genetic program of a cell is production of mRNA. In yeast, almost every gene is transcribed as multiple distinct isoforms, differing at their 5′ and/or 3′ termini. However, the implications and functional significance of the transcriptome-wide diversity of mRNA termini remains largely unexplored. In this paper, we show that the GAT1 gene, encoding a transcriptional activator of nitrogen-responsive catabolic genes, produces a variety of mRNAs differing in their 5′ and 3′ termini. Alternative transcription initiation leads to the constitutive, low level production of 2 full length proteins differing in their N-termini, whereas premature transcriptional termination generates a short, highly nitrogen catabolite repression- (NCR-) sensitive transcript that, as far as we can determine, is not translated under the growth conditions we used, but rather likely protects the cell from excess Gat1.

The TorC1 protein kinase complex is a central component in a eukaryotic cell's response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g. proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor2dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3- Myc13 localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells' nitrogen environment., SCOPUS: ar.j, info:eu-repo/semantics/published

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.

β-catenin is an integral part of the Wnt signaling pathway and has been linked to tumorigenesis and multiple developmental processes. The high β-catenin expression with low tumor incidence in the human epididymis is thus intriguing. In the present study, the β-catenin gene and protein was found to be highly expressed in the murine caput epididymidis, and the protein mainly localized along the lateral plasma membranes of adjacent epithelial cells throughout both human and mouse epididymides. Furthermore, the adult mouse epididymis was found to express almost all the Wnt/β-catenin signaling pathway genes that were determined previously by our group in the human organ. Despite the differences in epididymal structure, the similar location of β-catenin and the high concordance of this pathway's components' gene expression in both the adult human and mouse epididymides make the mouse a suitable animal model for studying the anti-tumor mechanism of the epididymis. In addition, both the mRNA and protein expression of β-catenin shared a similar spatial expression as the mRNA of Ros1, a proto-oncogene and a key developmental regulator of the initial segment of the mouse epididymis. The observations on the parallel temporal expression of β-catenin and Ros1 during postnatal development raise the possibility that the canonical Wnt signaling pathway has an additional role in the postnatal development of mouse epididymis.

A leucine, leucyl-tRNA synthetase–dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of Gln3 and Gat1 to rapamycin inhibition of TorC1 and nitrogen starvation.

Molecular characterization of pathogens such as the malaria parasite can lead to improved biological understanding and novel treatment strategies. However, the distinctive biology of the Plasmodium parasite, including its repetitive genome and the requirement for growth within a host cell, hinders progress toward these goals. Untargeted metabolomics is a promising approach to learn about pathogen biology. By measuring many small molecules in the parasite at once, we gain a better understanding of important pathways that contribute to the parasite’s response to perturbations such as drug treatment. Although increasingly popular, approaches for intracellular parasite metabolomics and subsequent analysis are not well explored. The findings presented in this report emphasize the critical need for improvements in these areas to limit misinterpretation due to host metabolites and to standardize biological interpretation. Such improvements will aid both basic biological investigations and clinical efforts to understand important pathogens., Metabolomics is increasingly popular for the study of pathogens. For the malaria parasite Plasmodium falciparum, both targeted and untargeted metabolomics have improved our understanding of pathogenesis, host-parasite interactions, and antimalarial drug treatment and resistance. However, purification and analysis procedures for performing metabolomics on intracellular pathogens have not been explored. Here, we purified in vitro-grown ring-stage intraerythrocytic P. falciparum parasites for untargeted metabolomics studies; the small size of this developmental stage amplifies the challenges associated with metabolomics studies as the ratio between host and parasite biomass is maximized. Following metabolite identification and data preprocessing, we explored multiple confounding factors that influence data interpretation, including host contamination and normalization approaches (including double-stranded DNA, total protein, and parasite numbers). We conclude that normalization parameters have large effects on differential abundance analysis and recommend the thoughtful selection of these parameters. However, normalization does not remove the contribution from the parasite’s extracellular environment (culture media and host erythrocyte). In fact, we found that extraparasite material is as influential on the metabolome as treatment with a potent antimalarial drug with known metabolic effects (artemisinin). Because of this influence, we could not detect significant changes associated with drug treatment. Instead, we identified metabolites predictive of host and medium contamination that could be used to assess sample purification. Our analysis provides the first quantitative exploration of the effects of these factors on metabolomics data analysis; these findings provide a basis for development of improved experimental and analytical methods for future metabolomics studies of intracellular organisms. IMPORTANCE Molecular characterization of pathogens such as the malaria parasite can lead to improved biological understanding and novel treatment strategies. However, the distinctive biology of the Plasmodium parasite, including its repetitive genome and the requirement for growth within a host cell, hinders progress toward these goals. Untargeted metabolomics is a promising approach to learn about pathogen biology. By measuring many small molecules in the parasite at once, we gain a better understanding of important pathways that contribute to the parasite’s response to perturbations such as drug treatment. Although increasingly popular, approaches for intracellular parasite metabolomics and subsequent analysis are not well explored. The findings presented in this report emphasize the critical need for improvements in these areas to limit misinterpretation due to host metabolites and to standardize biological interpretation. Such improvements will aid both basic biological investigations and clinical efforts to understand important pathogens.

What follows are snapshots of my career in chicken eyes, yeast and Rhodospirillum rubrum, castor beans, Escherichia coli and finally yeast again. In contrast, only a few of the failures that realistically make up a career are included. It is a tale of the generosity and influences of those who shaped what I am and what I learned in a wonderful profession. The science described is only that which I was lucky enough to do or was performed in my laboratory by those who really deserve the credit for any success that I've enjoyed. Not mentioned for lack of space are the critical contributions of many impressive investigators in the field of nitrogen-responsive regulation for no scientific investigation occurs in isolation.

Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.

Summary In a study aimed to determine the histopathology, component parasite communities and level of selected heavy metals, African catfish Clarias gariepinus from three rivers in Zimbabwe (Gwebi, Manyame and Mukuvisi) were analysed for heavy metals (Cd, Cr, Cu, Fe, Ni, Pb, Zn) in the gills, liver, kidney and muscles. The histopathology of these tissues was assessed by microscopic examination of stained thin sections. Metazoan parasite diversity and species composition in fish along different sites of the rivers were determined and compared. Levels of Cd, Fe, Pb and Zn were lowest in the Gwebi, Cr and Cu in the Manyame, and Ni in the Mukuvisi River. There were significant differences (P

Objective. To identify new genetic associations with juvenile and adult dermatomyositis (DM). Methods. We performed a genome-wide association study (GWAS) of adult and juvenile DM patients of European ancestry (n = 1178) and controls (n = 4724). To assess genetic overlap with other autoimmune disorders, we examined whether 141 single nucleotide polymorphisms (SNPs) outside the major histocompatibility complex (MHC) locus, and previously associated with autoimmune diseases, predispose to DM. Results. Compared to controls, patients with DM had a strong signal in the MHC region consisting of GWAS-level significance (P < 5x10(-8) ) at 80 genotyped SNPs. An analysis of 141 non-MHC SNPs previously associated with autoimmune diseases showed that three SNPs linked with three genes were associated with DM, with a false discovery rate (FDR) < 0.05. These genes were phospholipase C like 1 (PLCL1, rs6738825, FDR=0.00089), B lymphoid tyrosine kinase (BLK, rs2736340, FDR=0.00031), and chemokine (C-C motif) ligand 21 (CCL21, rs951005, FDR=0.0076). None of these genes was previously reported to be associated with DM. Conclusion. Our findings confirm the MHC as the major genetic region associated with DM and indicate that DM shares non-MHC genetic features with other autoimmune diseases, suggesting the presence of additional novel risk loci. This first identification of autoimmune disease genetic predispositions shared with DM may lead to enhanced understanding of pathogenesis and novel diagnostic and therapeutic approaches. � 2013 American College of Rheumatology.

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. Background: Five different conditions have been employed interchangeably to down-regulate TorC1. Results: Four of the five conditions exhibit different, hierarchially related phosphatase requirements. Gln3 responses to long-term nitrogen starvation and Msx treatment exhibit the same phosphatase requirements. Conclusion: Previous conditions for down-regulating TorC1 are not physiologically equivalent. Significance: The cellular responses to varying nitrogen supplies occur via multiple, distinguishable regulatory pathways.