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Group 1 innate lymphoid cells (ILCs), which comprise both natural killer (NK) cells and ILC1s, are important innate effectors that can also positively and negatively influence adaptive immune responses. The latter function is generally ascribed to the ability of NK cells to recognize and kill activated T cells. Here, we used multiphoton intravital microscopy in mouse models of hepatitis B to study the intrahepatic behavior of group 1 ILCs and their cross-talk with hepatitis B virus (HBV)–specific CD8+T cells. We found that hepatocellular antigen recognition by effector CD8+T cells triggered a prominent increase in the number of hepatic NK cells and ILC1s. Group 1 ILCs colocalized and engaged in prolonged interactions with effector CD8+T cells undergoing hepatocellular antigen recognition; however, they did not induce T cell apoptosis. Rather, group 1 ILCs constrained CD8+T cell proliferation by controlling local interleukin-2 (IL-2) availability. Accordingly, group 1 ILC depletion, or genetic removal of their IL-2 receptor a chain, considerably increased the number of intrahepatic HBV-specific effector CD8+T cells and the attendant immunopathology. Together, these results reveal a role for group 1 ILCs in controlling T cell–mediated liver immunopathology by limiting local IL-2 concentration and have implications for the treatment of chronic HBV infection.

Purpose: To describe the physical activity patterns of patients in the first week after lumbar surgery, and to investigate factors that potentially limit walking time early after surgery.Ma...

Eukaryotic cells integrate layers of gene regulation to coordinate complex cellular processes; however, mechanisms of post-transcriptional gene regulation remain poorly studied. The human fungal pathogen Histoplasma capsulatum (Hc) responds to environmental or host temperature by initiating unique transcriptional programs to specify multicellular (hyphae) or unicellular (yeast) developmental states that function in infectivity or pathogenesis, respectively. Here we used recent advances in next-generation sequencing to uncover a novel re-programming of transcript length between Hc developmental cell types. We found that ~2% percent of Hc transcripts exhibit 5’ leader sequences that differ markedly in length between morphogenetic states. Ribosome density and mRNA abundance measurements of differential leader transcripts revealed nuanced transcriptional and translational regulation. One such class of regulated longer leader transcripts exhibited tight transcriptional and translational repression. Further examination of these dually repressed genes revealed that some control Hc morphology and that their strict regulation is necessary for the pathogen to make appropriate developmental decisions in response to temperature., Author Summary Eukaryotic cells alter their developmental programs in response to environmental signals. Histoplasma capsulatum (Hc), a ubiquitous fungal pathogen of humans, establishes unique transcriptional programs to specify growth in either a multicellular hyphal form or unicellular yeast form in response to temperature. Since hyphae and yeast are specialized to function in infectivity or pathogenesis, respectively, Hc provides a clinically relevant system in which to query eukaryotic regulatory processes. Here we used next-generation sequencing approaches to annotate the transcriptomes of four distinct Hc strains in response to temperature. We found that a fraction of Hc transcripts have differential transcript architecture in hyphae and yeast, exhibiting 5’ leader sequences that differ markedly in length between morphogenetic states. To begin to understand the effect of these differential leader sequences on expression, we performed the first ribosome density and mRNA abundance measurements in Hc, thereby uncovering transcriptional and translational control that contribute to cell-type regulation.

Purpose: Measuring step count using activity monitors is an increasingly popular method of quantifying physical activity, however it is unknown whether gait irregularities or the use of gait aids affect the accuracy of these devices. This study evaluates the validity of the ActivPAL3, Fitbit Flex, and Jawbone UP Move activity monitors for measuring step count in hospital inpatients after lumbar fusion. Methods: The ActivPAL3 was tested on the thigh, the Fitbit, and the Jawbone were tested on the wrist and thigh, each monitor was tested 20 times. Validity was examined by calculating the percentage of steps detected by each monitor compared to the criterion measure of observed step count, the Standard Error of Measurement, and the Intraclass Correlation Coefficient. Results: The ActivPAL3 detected 85% (SD 27%) of observed steps. On the wrist, the Fitbit detected 24% (SD 34%) and the Jawbone detected 17% (SD 40%) of observed steps. On the thigh, the Fitbit detected 66% (SD 42%) and the Jawbone detected 22% (SD 35%) of observed steps. Conclusion: The ActivPAL3 activity monitor is a sufficiently valid tool to detect step count immediately after lumbar fusion. Wrist worn monitors are not recommended in this population, particularly with patients using gait aids.Implications for RehabilitationWalking may be quantified using activity monitoring, as both an assessment tool and as an adjunct to treatmentThe ActivPAL3 is sufficiently valid for use after lumbar fusion surgeryThe Fitbit Flex and Jawbone UP Move had low accuracy, particularly when a gait aid was used Walking may be quantified using activity monitoring, as both an assessment tool and as an adjunct to treatment The ActivPAL3 is sufficiently valid for use after lumbar fusion surgery The Fitbit Flex and Jawbone UP Move had low accuracy, particularly when a gait aid was used

Phenotypic switching between 2 opposing cellular states is a fundamental aspect of biology, and fungi provide facile systems to analyze the interactions between regulons that control this type of switch. A long-standing mystery in fungal pathogens of humans is how thermally dimorphic fungi switch their developmental form in response to temperature. These fungi, including the subject of this study, Histoplasma capsulatum, are temperature-responsive organisms that utilize unknown regulatory pathways to couple their cell shape and associated attributes to the temperature of their environment. H. capsulatum grows as a multicellular hypha in the soil that switches to a pathogenic yeast form in response to the temperature of a mammalian host. These states can be triggered in the laboratory simply by growing the fungus either at room temperature (RT; which promotes hyphal growth) or at 37 °C (which promotes yeast-phase growth). Prior worked revealed that 15% to 20% of transcripts are differentially expressed in response to temperature, but it is unclear which transcripts are linked to specific phenotypic changes, such as cell morphology or virulence. To elucidate temperature-responsive regulons, we previously identified 4 transcription factors (required for yeast-phase growth [Ryp]1–4) that are required for yeast-phase growth at 37 °C; in each ryp mutant, the fungus grows constitutively as hyphae regardless of temperature, and the cells fail to express genes that are normally induced in response to growth at 37 °C. Here, we perform the first genetic screen to identify genes required for hyphal growth of H. capsulatum at RT and find that disruption of the signaling mucin MSB2 results in a yeast-locked phenotype. RNA sequencing (RNAseq) experiments reveal that MSB2 is not required for the majority of gene expression changes that occur when cells are shifted to RT. However, a small subset of temperature-responsive genes is dependent on MSB2 for its expression, thereby implicating these genes in the process of filamentation. Disruption or knockdown of an Msb2-dependent mitogen-activated protein (MAP) kinase (HOG2) and an APSES transcription factor (STU1) prevents hyphal growth at RT, validating that the Msb2 regulon contains genes that control filamentation. Notably, the Msb2 regulon shows conserved hyphal-specific expression in other dimorphic fungi, suggesting that this work defines a small set of genes that are likely to be conserved regulators and effectors of filamentation in multiple fungi. In contrast, a few yeast-specific transcripts, including virulence factors that are normally expressed only at 37 °C, are inappropriately expressed at RT in the msb2 mutant, suggesting that expression of these genes is coupled to growth in the yeast form rather than to temperature. Finally, we find that the yeast-promoting transcription factor Ryp3 associates with the MSB2 promoter and inhibits MSB2 transcript expression at 37 °C, whereas Msb2 inhibits accumulation of Ryp transcripts and proteins at RT. These findings indicate that the Ryp and Msb2 circuits antagonize each other in a temperature-dependent manner, thereby allowing temperature to govern cell shape and gene expression in this ubiquitous fungal pathogen of humans., This study reveals how temperature controls cell shape and gene expression in the thermally dimorphic fungal pathogen Histoplasma capsulate, uncovering the interlocking circuitry that allows temperature to trigger a critical developmental choice in these organisms.

Background Physiotherapeutic management of patients immediately following lumbar spinal surgery is common. However, there is considerable variability in the interventions provided. Objectives To assess the effect of peri-operative physiotherapeutic intervention in adults undergoing surgery for the management of degenerative lumbar conditions. Data sources The Cochrane Library, Medline, Embase, CINAHL and PEDro were searched from inception to August 2012. Study selection Randomised controlled trials investigating physiotherapeutic interventions prior to and immediately following surgery for degenerative lumbar conditions were included. Data extraction and synthesis Two reviewers extracted data independently using a standardised form. Risk of bias was assessed using a modified version of the Cochrane Collaboration tool. The quality of evidence was assessed using the GRADE approach, and the treatment effect size was calculated where comparable outcome measures were used across multiple trials. Results Four studies were included. There is very-low-quality evidence that pre- and post-operative exercise in addition to standard physiotherapeutic care may reduce pain, time taken to achieve post-operative functional milestones, and post-operative time off work. Results from one study indicated that there is no clear benefit or risk of harm from performing either prone or side-lying transfers. Conclusion and implications of key findings Very-low-quality evidence suggests that physiotherapy may improve pain and function following lumbar surgery. Due to low numbers of included studies and variation in the interventions assessed, the current evidence provides limited guidance for physiotherapeutic practice. Further research is required to determine the effectiveness of physiotherapeutic interventions in this population.

Physiotherapists are commonly involved in the management of patients immediately before and after spinal surgery, however there is currently little known about what constitutes physiotherapy intervention in the hospital setting. This research aimed to describe the current physiotherapy practice in Australia for the peri-operative management of adults undergoing lumbar spinal surgery. A telephone survey was conducted using a structured questionnaire format. All Australian hospitals that admit one or more patients per week for lumbar spinal surgery were invited to take part in the survey. Sixty-four interviews were conducted (response rate 79%). All participating hospitals provided a physiotherapy service for patients undergoing lumbar spinal surgery, with the majority commencing the day following surgery. Physiotherapy intervention consistently included mobility and functional task training, exercise prescription and provision of an educational handout. However, there was considerable variability in the type of exercises prescribed, the advice given regarding post-operative movement and activity restrictions, the use of outcome measurement tools, and referral to inpatient and outpatient physiotherapy services. This survey provides physiotherapists and rehabilitation service providers with information regarding current clinical practice, and identifies the key focus areas for future research into the effectiveness of specific physiotherapy interventions.

Regulation of iron acquisition genes is critical for microbial survival under both iron-limiting conditions (to acquire essential iron) and iron-replete conditions (to limit iron toxicity). In fungi, iron acquisition genes are repressed under iron-replete conditions by a conserved GATA transcriptional regulator. Here we investigate the role of this transcription factor, Sre1, in the cellular responses of the fungal pathogen Histoplasma capsulatum to iron. We showed that cells in which SRE1 levels were diminished by RNA interference were unable to repress siderophore biosynthesis and utilization genes in the presence of abundant iron and thus produced siderophores even under iron-replete conditions. Mutation of a GATA-containing consensus site found in the promoters of these genes also resulted in inappropriate gene expression under iron-replete conditions. Microarray analysis comparing control and SRE1 -depleted strains under conditions of iron limitation or abundance revealed both iron-responsive genes and Sre1-dependent genes, which comprised distinct but overlapping sets. Iron-responsive genes included those encoding putative oxidoreductases, metabolic and mitochondrial enzymes, superoxide dismutase, and nitrosative-stress-response genes; Sre1-dependent genes were of diverse functions. Genes regulated by iron levels and Sre1 included all of the siderophore biosynthesis genes, a gene involved in reductive iron acquisition, an iron-responsive transcription factor, and two catalases. Based on transcriptional profiling and phenotypic analyses, we conclude that Sre1 plays a critical role in the regulation of both traditional iron-responsive genes and iron-independent pathways such as regulation of cell morphology. These data highlight the evolving realization that the effect of Sre1 orthologs on fungal biology extends beyond the iron regulon.

Type I sulfatases require an unusual co- or post-translational modification for their activity in hydrolyzing sulfate esters. In eukaryotic sulfatases, an active site cysteine residue is oxidized to the aldehyde-containing C(alpha)-formylglycine residue by the formylglycine-generating enzyme (FGE). The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Here we describe the identification of a prokaryotic FGE from Mycobacterium tuberculosis. In addition, we solved the crystal structure of the Streptomyces coelicolor FGE homolog to 2.1 A resolution. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. Both biochemical and structural data indicate the presence of an oxidized cysteine modification in the active site that may be relevant to catalysis. In addition, we generated a mutant M. tuberculosis strain lacking FGE. Although global sulfatase activity was reduced in the mutant, a significant amount of residual sulfatase activity suggests the presence of FGE-independent sulfatases in this organism.

The monosaccharide N-acetylglucosamine (GlcNAc) is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc stimulates developmental pathways in the fungal pathogen Candida albicans, which is a commensal organism that colonizes the mammalian gut and causes disease in the setting of host immunodeficiency. Here we investigate GlcNAc signaling in thermally dimorphic human fungal pathogens, a group of fungi that are highly evolutionarily diverged from C. albicans and cause disease even in healthy individuals. These soil organisms grow as polarized, multicellular hyphal filaments that transition into a unicellular, pathogenic yeast form when inhaled by a human host. Temperature is the primary environmental cue that promotes reversible cellular differentiation into either yeast or filaments; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. We found GlcNAc to be a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. In addition to increasing the rate of filamentous growth, micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift that was independent of GlcNAc catabolism, indicating that fungal cells sense GlcNAc to promote filamentation. Whole-genome expression profiling to identify candidate genes involved in establishing the filamentous growth program uncovered two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes., Author Summary In stark contrast to most fungal pathogens, thermally dimorphic fungal pathogens cause systemic infections in immunocompetent humans. Thermally dimorphic fungi grow in the soil as a multicellular filamentous form specialized for replication in this particular environmental niche. Upon infection of a human, these fungi transition to a parasitic cell type that is adapted for replication and pathogenesis within a mammalian host. In this work, we examined factors that are important for growth of the infectious, environmental form of thermally dimorphic fungi. We discovered that N-acetylglucosamine (GlcNAc), a ubiquitous carbohydrate with cellular roles across all kingdoms of life, stimulated a switch to the environmental form for two thermally dimorphic fungal pathogens, Histoplasma capsulatum and Blastomyces dermatitidis. Analysis of how fungal cells respond to GlcNAc revealed that these fungi possess two GlcNAc transporters that are important for controlling their ability to switch between infectious and parasitic states. Overall, our work begins to elucidate the pathways that promote growth in the infectious form of these organisms, which is critical to our understanding of environmental signals that promote disease transmission of thermally dimorphic fungi.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.

Background: Sulfolipid-1 (SL-1) is a Mycobacterium tuberculosis outer membrane lipid whose biosynthesis is not fully understood. Results: Chp1 catalyzes two acyl transfer reactions to form SL-1. Sap modulates SL-1 levels and transmembrane transport. Conclusion: The activities of Chp1 and Sap complete the SL-1 pathway. Significance: Lipid biosynthesis and transport are coupled at the membrane interface by multiple proteins that may regulate substrate specificity and flux., Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces unique sulfated metabolites associated with virulence. One such metabolite from M. tuberculosis lipid extracts, S881, has been shown to negatively regulate the virulence of M. tuberculosis in mouse infection studies, and its cell-surface localization suggests a role in modulating host-pathogen interactions. However, a detailed structural analysis of S881 has remained elusive. Here we use high-resolution, high-mass-accuracy, and tandem mass spectrometry to characterize the structure of S881. Exact mass measurements showed that S881 is highly unsaturated, tandem mass spectrometry indicated a polyisoprene-derived structure, and characterization of synthetic structural analogs confirmed that S881 is a previously undescribed sulfated derivative of dihydromenaquinone-9, the primary quinol electron carrier in M. tuberculosis. To our knowledge, this is the first example of a sulfated menaquinone produced in any prokaryote. Together with previous studies, these findings suggest that this redox cofactor may play a role in mycobacterial pathogenesis.

SummaryTo understand the adaptation of Mycobacterium tuberculosis to the intracellular environment, we used comprehensive metabolite profiling to identify the biochemical pathways utilized during growth on cholesterol, a critical carbon source during chronic infection. Metabolic alterations observed during cholesterol catabolism centered on propionyl-CoA and pyruvate pools. Consequently, growth on this substrate required the transcriptional induction of the propionyl-CoA-assimilating methylcitrate cycle (MCC) enzymes, via the Rv1129c regulatory protein. We show that both Rv1129c and the MCC enzymes are required for intracellular growth in macrophages and that the growth defect of MCC mutants is largely attributable to the degradation of host-derived cholesterol. Together, these observations define a coordinated transcriptional and metabolic adaptation that is required for scavenging carbon during intracellular growth.

Physiotherapists are commonly involved in the management of patients immediately following lumbar spinal surgery. There is however, very little research to guide physiotherapy intervention in the acute post-operative period, and the advice provided to patients regarding post-operative walking and physical activity has been shown to be highly variable. The primary aim of this research is to establish whether the amount of walking patients perform in the week following lumbar spinal surgery predicts improvement in function at 6 months. This study will be a prospective cohort study design, with a projected sample size of 250 participants. Patients undergoing surgery for the management of a disc prolapse, degenerative disc disease, lumbar spinal stenosis and/or degenerative spondylolysthesis will be invited to participate in this study. Outcome measurement will take place pre-operatively and at six months post-operatively. The primary outcome variable will be self-reported function, measured using the Modified Oswestry Disability Questionnaire and the physical component summary of the SF-36. Each participant will be fitted with an activPAL3 accelerometer to be worn for the first seven post-operative days. This accelerometer will record time spent in active versus sedentary postures, step count and time spent walking. Multivariable logistic regression analysis will be used to investigate the relationship between the total time spent walking over the first seven post-operative days, and outcome at six months. The results from this research will help to guide patient management during the inpatient phase, by identifying patients who are at risk of poorer outcome due to limited walking time. These patients may benefit from ongoing rehabilitation and outpatient physiotherapy services. This information will also provide a foundation for further research into interventions designed to optimise post-operative activity. ACTRN12616000747426 , retrospectively registered 7th June 2016.