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Auditory transduction is fast and sensitive owing to the direct detection of mechanical stimuli by hair cells, the sensory receptors of the internal ear. A study by Dionne et al. (2018) in this issue of Neuron suggests how signals propagate through tip links, the cadherin-based strands that gate mechanically sensitive channels.

To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Cross-linking and immunoprecipitation (CLIP) serves this purpose by exploiting covalent protein-RNA cross-linking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare complementary DNA (cDNA) libraries for sequencing. Here we describe the core steps of CLIP, its primary variations, and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice and discuss the mechanistic and physiologic insights that have already been gained from studies using CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms while providing important biologic and clinically relevant insights.

Mendelian suceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency predisposing to severe disease caused by mycobacteria and other intracellular pathogens. Delay in diagnosis can have an impact on the patient's prognosis. We evaluated the IFN-gamma circuit by studying IFN-gamma production after mycobacterial challenge as well as IL-12R beta 1 expression and STAT4 phosphorylation in response to IL-12p70 stimulation in whole blood of a 6-year-old Peruvian girl with disseminated recurrent mycobacterial infection diagnosed as multidrug-resistant tuberculosis. Genetic studies with Sanger sequencing were used to identify the causative mutation. Microbiological studies based on PCR reactions were used to diagnose the specific mycobacterial species. We identified a homozygous mutation in the IL12RB1 gene (p. Arg211*) causing abolished expression of IL-12R beta 1 and IL-12 response. MSMD diagnosis led to a microbiological reevaluation of the patient, revealing a BCG vaccine-related infection instead of tuberculosis. Treatment was then adjusted, with good response. We report the first Peruvian patient with IL-12R beta 1 deficiency. Specific mycobacterial species diagnosis within Mycobacterium tuberculosis complex is still challenging in countries with limited access to PCR-based microbiological diagnostic techniques. Awareness of MSMD warning signs and accurate microbiological diagnosis of mycobacterial infections are of the utmost importance for optimal diagnosis and management of affected patients.

A critical factor in the maturation of influenza vaccine responses is the nearly inevitable binding of vaccine antigens by exiting anti-influenza IgGs. These antigen-IgG immune complexes direct the response to immunization by modulating cellular processes that determine antibody and T-cell repertoires: maturation of dendritic cells, processing and presentation of antigens to T cells, trafficking of antigens to the germinal center, and selection of B cells for antibody production. By focusing on the recent advances in the study of the immunomodulatory processes mediated by IgG immune complexes upon influenza vaccination, we discuss a pathway that is critical for modulating the breadth and potency of anti HA antibody responses and has previously led to the development of strategies to improve influenza vaccine efficacy.

Background: Alternative RNA processing plays an essential role in shaping cell identity and connectivity in the central nervous system. This is believed to involve differential regulation of RNA processing in various cell types. However, in vivo study of cell type-specific post-transcriptional regulation has been a challenge. Here, we describe a sensitive and stringent method combining genetics and CLIP (crosslinking and immunoprecipitation) to globally identify regulatory interactions between NOVA and RNA in the mouse spinal cord motoneurons. Results: We developed a means of undertaking motoneuron-specific CLIP to explore motoneuron-specific protein- RNA interactions relative to studies of the whole spinal cord in mouse. This allowed us to pinpoint differential RNA regulation specific to motoneurons, revealing a major role for NOVA in regulating cytoskeleton interactions in motoneurons. In particular, NOVA specifically promotes the palmitoylated isoform of the cytoskeleton protein Septin 8 in motoneurons, which enhances dendritic arborization. Conclusions: Our study demonstrates that cell type-specific RNA regulation is important for fine tuning motoneuron physiology and highlights the value of defining RNA processing regulation at single cell type resolution.

Results of a search for the standard model Higgs boson produced in association with a top quark pair (t (t) over barH) in final states with electrons, muons, and hadronically decaying tau leptons are presented. The analyzed data set corresponds to an integrated luminosity of 35.9 fb(-1) recorded in proton-proton collisions at root s = 13 TeV by the CMS experiment in 2016. The sensitivity of the search is improved by using matrix element and machine learning methods to separate the signal from backgrounds. The measured signal rate amounts to 1.23(-0.43)(+0.45) times the production rate expected in the standard model, with an observed (expected) significance of 3.2 sigma (2.8 sigma), which represents evidence for t (t) over barH production in those final states. An upper limit on the signal rate of 2.1 times the standard model production rate is set at 95% confidence level.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZA (TM), has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

In experimental cultures, when bacteria are mixed with lytic ( virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, lambda(VIR), and Escherichia coli in serial transfer cultures. We show that, following the ascent of lambda(VIR) resistant bacteria, lambda(VIR) is maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of lambda(VIR) in these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility-a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria-their "existence conditions."

This paper reviews early evidence for the existence of rapid, non-genomic effects of estrogens on neurons, and, further, proposes that these rapid effects are often synergistic with later, genomic effects. Finally, suggestions about potential molecular mechanisms underlying the rapid effects of estrogens are offered. A mechanistic step we propose to be common among rapid estrogenic actions includes membrane ER's binding to histamine, and NMDA receptors and subsequent dimerization, and clustering (respectively) in a manner that enhances histamine and NMDA actions.

Multidrug-resistant bacteria pose a major threat to effective antibiotics and alternatives to fight multidrug-resistant pathogens are needed. We synthetized molybdenum oxide (MoO3) nanoparticles (NP) and determined their antibacterial activity against 39 isolates: (i) eight Staphylococcus aureus, including representatives of methicillin-resistant S. aureus epidemic clones; (ii) six enterococci, including vancomycin-resistant isolates; and (iii) 25 Gram-negative isolates (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae), including extended spectrum beta-lactamases and carbapenemases producers. All isolates showed a MoO3 NP MIC of 700-800 mg l(-1). MoO3 NP produced a clear inhibition zone for S. aureus and all Gram-negative isolates at concentrations >= 25 mg ml(-1) and >= 50 mg ml(-1) for enterococci. When the NP solutions were adjusted to pH similar to 7, the biocidal activity was completely abolished. MoO3 NP create an acidic pH and show a universal antimicrobial activity against susceptible and resistant isolates belonging to the most relevant bacterial species responsible for hospital-acquired infections.