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The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 angstrom-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 angstrom-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP a-subunit C-terminal domain (alpha CTD) with DNA, and we provide evidence that the alpha CTD may play a role in Mtb transcription regulation. Our results reveal the structure of an Actinobacteria-unique insert of the RNAP beta' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm sigma(A), which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

The function of the brain is unlikely to be understood without an accurate description of its output, yet the nature of movement elements and their organization remains an open problem. Here, movement elements are identified from dynamics of walking in flies, using unbiased criteria. On one time scale, dynamics of walking are consistent over hundreds of milliseconds, allowing elementary features to be defined. Over longer periods, walking is well described by a stochastic process composed of these elementary features, and a generative model of this process reproduces individual behavior sequences accurately over seconds or longer. Within elementary features, velocities diverge, suggesting that dynamical stability of movement elements is a weak behavioral constraint. Rather, long-term instability can be limited by the finite memory between these elementary features. This structure suggests how complex dynamics may arise in biological systems from elements whose combination need not be tuned for dynamic stability.

Hunger, driven by negative energy balance, elicits the search for and consumption of food. While this response is in part mediated by neurons in the hypothalamus, the role of specific cell types in other brain regions is less well defined. Here, we show that neurons in the dorsal raphe nucleus, expressing vesicular transporters for GABA or glutamate (hereafter, DRN Vgat and DRN VGLUT3 neurons), are reciprocally activated by changes in energy balance and that modulating their activity has opposite effects on feeding-DRN Vgat neurons increase, whereas DRN VGLUT3 neurons suppress, food intake. Furthermore, modulation of these neurons in obese (ob/ob) mice suppresses food intake and body weight and normalizes locomotor activity. Finally, using molecular profiling, we identify druggable targets in these neurons and show that local infusion of agonists for specific receptors on these neurons has potent effects on feeding. These data establish the DRN as an important node controlling energy balance.

Background: The spread of drug-resistant bacterial pathogens poses a major threat to global health. It is widely recognised that the widespread use of antibiotics has generated selective pressures that have driven the emergence of resistant strains. Methicillin-resistant Staphylococcus aureus (MRSA) was first observed in 1960, less than one year after the introduction of this second generation beta-lactam antibiotic into clinical practice. Epidemiological evidence has always suggested that resistance arose around this period, when the mecA gene encoding methicillin resistance carried on an SCCmec element, was horizontally transferred to an intrinsically sensitive strain of S. aureus. Results: Whole genome sequencing a collection of the first MRSA isolates allows us to reconstruct the evolutionary history of the archetypal MRSA. We apply Bayesian phylogenetic reconstruction to infer the time point at which this early MRSA lineage arose and when SCCmec was acquired. MRSA emerged in the mid-1940s, following the acquisition of an ancestral type I SCCmec element, some 14 years before the first therapeutic use of methicillin. Conclusions: Methicillin use was not the original driving factor in the evolution of MRSA as previously thought. Rather it was the widespread use of first generation beta-lactams such as penicillin in the years prior to the introduction of methicillin, which selected for S. aureus strains carrying the mecA determinant. Crucially this highlights how new drugs, introduced to circumvent known resistance mechanisms, can be rendered ineffective by unrecognised adaptations in the bacterial population due to the historic selective landscape created by the widespread use of other antibiotics.

Background: Germline heterozygous mutations in human signal transducer and activator of transcription 1 (STAT1) can cause loss of function (LOF), as in patients with Mendelian susceptibility to mycobacterial diseases, or gain of function (GOF), as in patients with chronic mucocutaneous candidiasis. LOF and GOF mutations are equally rare and can affect the same domains of STAT1, especially the coiled-coil domain (CCD) and DNA-binding domain (DBD). Moreover, 6% of patients with chronic mucocutaneous candidiasis with a GOF STAT1 mutation have mycobacterial disease, obscuring the functional significance of the identified STAT1 mutations. Current computational approaches, such as combined annotation-dependent depletion, do not distinguish LOF and GOF variants. Objective: We estimated variations in the CCD/DBD of STAT1. Methods: We mutagenized 342 individual wild-type amino acids in the CCD/DBD (45.6% of full-length STAT1) to alanine and tested the mutants for STAT1 transcriptional activity. Results: Of these 342 mutants, 201 were neutral, 30 were LOF, and 111 were GOF mutations in a luciferase assay. This assay system correctly estimated all previously reported LOF mutations (100%) and slightly fewer GOF mutations (78.1%) in the CCD/DBD of STAT1. We found that GOF alanine mutants occurred at the interface of the antiparallel STAT1 dimer, suggesting that they destabilize this dimer. This assay also precisely predicted the effect of 2 hypomorphic and dominant negative mutations, E157K and G250E, in the CCD of STAT1 that we found in 2 unrelated patients with Mendelian susceptibility to mycobacterial diseases. Conclusion: The systematic alanine-scanning assay is a useful tool to estimate the GOF or LOF status and the effect of heterozygous missense mutations in STAT1 identified in patients with severe infectious diseases, including mycobacterial and fungal diseases.

Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. Here, we map the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, we quantified the behavioral effects of activating 2,204 genetically targeted populations of neurons. We combined a novel quantification of anatomy with our behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, we generated hypotheses of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. Our maps directly specify genetic tools to target these regions, which we used to identify a small population of neurons with a role in the control of walking.

Many animals keep track of their angular heading over time while navigating through their environment. However, a neural-circuit architecture for computing heading has not been experimentally defined in any species. Here we describe a set of clockwise-and anticlockwise-shifting neurons in the Drosophila central complex whose wiring and physiology provide a means to rotate an angular heading estimate based on the fly's angular velocity. We show that each class of shifting neurons exists in two subtypes, with spatiotemporal activity profiles that suggest different roles for each subtype at the start and end of tethered-walking turns. Shifting neurons are required for the heading system to properly track the fly's heading in the dark, and stimulation of these neurons induces predictable shifts in the heading signal. The central features of this biological circuit are analogous to those of computational models proposed for head-direction cells in rodents and may shed light on how neural systems, in general, perform integration.

We compare several major white-matter tracts in human and macaque occipital lobe using diffusion magnetic resonance imaging. The comparison suggests similarities but also significant differences in the tracts. There are several apparently homologous tracts in the 2 species, including the vertical occipital fasciculus (VOF), optic radiation, forceps major, and inferior longitudinal fasciculus (ILF). There is one large human tract, the inferior fronto-occipital fasciculus, with no corresponding fasciculus in macaque. We could identify the macaque VOF (mVOF), which has been little studied. Its position is consistent with classical invasive anatomical studies by Wernicke. VOF homology is supported by similarity of the endpoints in V3A and ventral V4 across species. The mVOF fibers intertwine with the dorsal segment of the ILF, but the human VOF appears to be lateral to the ILF. These similarities and differences between the occipital lobe tracts will be useful in establishing which circuitry in the macaque can serve as an accurate model for human visual cortex.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA: DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA: DNA hybrid, brought telomeric RNA: DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA: DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription.

Type III CRISPR-Cas systems have a unique targeting mechanism that requires the transcription of the DNA target and results in the degradation of not only the genome of the invader but also its transcripts. Here we discuss the most recent studies describing dual DNA and RNA targeting by these systems, as well as the implications of this complex molecular mechanism for immunity in vivo.