Scalable Apparatus to Measure Posture and Locomotion (SAMPL): a high-throughput solution to study unconstrained vertical behavior in small animals.

Balance and movement are impaired in a wide variety of neurological disorders. Recent advances in behavioral monitoring provide unprecedented access to posture and locomotor kinematics, but without the throughput and scalability necessary to screen candidate genes / potential therapeutics. We present a powerful solution: a Scalable Apparatus to Measure Posture and Locomotion (SAMPL).

Graded impact of obstacle size on scanning by RNase E.

In countless bacterial species, the lifetimes of most mRNAs are controlled by the regulatory endonuclease RNase E, which preferentially degrades RNAs bearing a 5' monophosphate and locates cleavage sites within them by scanning linearly from the 5' terminus along single-stranded regions. Consequently, its rate of cleavage at distal sites is governed by any obstacles that it may encounter along the way, such as bound proteins or ribosomes or base pairing that is coaxial with the path traversed by this enzyme. Here, we report that the protection afforded by such obstacles is dependent on the size and persistence of the structural discontinuities they create, whereas the molecular composition of obstacles to scanning is of comparatively little consequence.

A giant virus genome is densely packaged by stable nucleosomes within virions.

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes.

Longitudinal in Utero Analysis of Engrailed-1 Knockout Mouse Embryonic Phenotypes Using High-Frequency Ultrasound.

Large-scale international efforts to generate and analyze loss-of-function mutations in each of the approximately 20,000 protein-encoding gene mutations are ongoing using the "knockout" mouse as a model organism. Because one-third of gene knockouts are expected to result in embryonic lethality, it is important to develop non-invasive in utero imaging methods to detect and monitor mutant phenotypes in mouse embryos. We describe the utility of 3-D high-frequency (40-MHz) ultrasound (HFU) for longitudinal in utero imaging of mouse embryos between embryonic days (E) 11.

Plexin D1 negatively regulates zebrafish lymphatic development.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish.

Independent regulation of mitochondrial DNA quantity and quality in primordial germ cells.

Mitochondria harbor an independent genome, called mitochondrial DNA (mtDNA), which contains essential metabolic genes. Although mtDNA mutations occur at high frequency, they are inherited infrequently, indicating that germline mechanisms limit their accumulation. To determine how germline mtDNA is regulated, we examined the control of mtDNA quantity and quality in primordial germ cells (PGCs).

Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney.

Chronic kidney disease (CKD), characterized by sustained inflammation and progressive fibrosis, is highly prevalent and can eventually progress to end-stage kidney disease. However, current treatments to slow CKD progression are limited. Sphingosine 1-phosphate (S1P), a product of sphingolipid catabolism, is a pleiotropic mediator involved in many cellular functions, and drugs targeting S1P signaling have previously been studied particularly for autoimmune diseases.

Structures and mechanism of the plant PIN-FORMED auxin transporter.

Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood.

Viral histones: pickpocket's prize or primordial progenitor?

The common histones H2A, H2B, H3, and H4 are the characteristic components of eukaryotic nucleosomes, which function to wrap DNA and compact the genome as well as to regulate access to DNA for transcription and replication in all eukaryotes. In the past two decades, histones have also been found to be encoded in some DNA viruses, where their functions and properties are largely unknown, though recently histones from two related viruses have been shown to form nucleosome-like structures in vitro. Viral histones can be highly similar to eukaryotic histones in primary sequence, suggesting they have been recently picked up from eukaryotic hosts, or they can be radically divergent in primary sequence and may occur as conjoined histone doublets, triplets, or quadruplets, suggesting ancient origins prior to the divergence of modern eukaryotes.

A cationic amino acid polymer nanocarrier synthesized in supercritical CO for co-delivery of drug and gene to cervical cancer cells.

The present study was undertaken to investigate the ability of a drug curcumin-loaded polymer to inhibit the growth of cervical cancer cells by enhancing the anti-cancer efficiency of curcumin. We synthesized poly(methacryloyl beta-alanine) (PMBA) as a nanocarrier by radical polymerization in supercritical CO. The results showed that the curcumin encapsulated and folic acid (FA)-treated PMBA (Poly@Cur-FA) for 24 h activated the reactive oxygen species-mediated programmed cell death machinery in HeLa cells.

Focal adhesion-mediated cell anchoring and migration: from in vitro to in vivo.

Cell-extracellular matrix interactions have been studied extensively using cells cultured in vitro. These studies indicate that focal adhesion (FA)-based cell-extracellular matrix interactions are essential for cell anchoring and cell migration. Whether FAs play a similarly important role in vivo is less clear.

Voluntary Exercise Boosts Striatal Dopamine Release: Evidence for the Necessary and Sufficient Role of BDNF.

Physical exercise improves motor performance in individuals with Parkinson's disease and elevates mood in those with depression. Although underlying factors have not been identified, clues arise from previous studies showing a link between cognitive benefits of exercise and increases in brain-derived neurotrophic factor (BDNF). Here, we investigated the influence of voluntary wheel-running exercise on BDNF levels in the striatum of young male wild-type (WT) mice, and on the striatal release of a key motor-system transmitter, dopamine (DA).

Modeling the C. elegans germline stem cell genetic network using automated reasoning.

Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation.

PABLO-QA: A sensitive assay for quantifying monophosphorylated RNA 5' ends.

Generated by RNA deprotection or cleavage, 5' monophosphates trigger RNA degradation in all organisms. Here we describe PABLO-QA (Phosphorylation Assay By Ligation of Oligonucleotides and Quantitative Amplification), a sensitive, low-cost procedure for determining the percentage of specific RNA 5' ends that are monophosphorylated from their ability to undergo ligation to an oligonucleotide. Comparison to a cognate internal standard and a fully monophosphorylated control allows precise quantification of monophosphorylated 5' termini by RT-PCR, enabling the analysis of transcripts undetectable by blotting.

Waffle Method: A general and flexible approach for improving throughput in FIB-milling.

Cryo-FIB/SEM combined with cryo-ET has emerged from within the field of cryo-EM as the method for obtaining the highest resolution structural information of complex biological samples in-situ in native and non-native environments. However, challenges remain in conventional cryo-FIB/SEM workflows, including milling thick specimens with vitrification issues, specimens with preferred orientation, low-throughput when milling small and/or low concentration specimens, and specimens that distribute poorly across grid squares. Here we present a general approach called the 'Waffle Method' which leverages high-pressure freezing to address these challenges.

Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus.

Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA).

Rap1 regulates TIP60 function during fate transition between two-cell-like and pluripotent states.

In mammals, the conserved telomere binding protein Rap1 serves a diverse set of nontelomeric functions, including activation of the NF-kB signaling pathway, maintenance of metabolic function in vivo, and transcriptional regulation. Here, we uncover the mechanism by which Rap1 modulates gene expression. Using a separation-of-function allele, we show that Rap1 transcriptional regulation is largely independent of TRF2-mediated binding to telomeres and does not involve direct binding to genomic loci.

Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity.

Kinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner.

Rear traction forces drive adherent tissue migration in vivo.

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration.

Anatomy and development of the pectoral fin vascular network in the zebrafish.

The pectoral fins of teleost fish are analogous structures to human forelimbs, and the developmental mechanisms directing their initial growth and patterning are conserved between fish and tetrapods. The forelimb vasculature is crucial for limb function, and it appears to play important roles during development by promoting development of other limb structures, but the steps leading to its formation are poorly understood. In this study, we use high-resolution imaging to document the stepwise assembly of the zebrafish pectoral fin vasculature.

A distinct RNA recognition mechanism governs Np decapping by RppH.

Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np) RNA caps. Removal of this cap is critical for initiating 5' end-dependent degradation of those RNAs, potentially affecting bacterial adaptability to stress; however, the predominant Np decapping enzyme in proteobacteria, ApaH, is inactivated by the very conditions of disulfide stress that enable Np-capped RNAs to accumulate to high levels. Here, we show that, in cells experiencing such stress, the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts, preferring them as substrates over their triphosphorylated and diphosphorylated counterparts.

Automatic mapping of multiplexed social receptive fields by deep learning and GPU-accelerated 3D videography.

Social interactions powerfully impact the brain and the body, but high-resolution descriptions of these important physical interactions and their neural correlates are lacking. Currently, most studies rely on labor-intensive methods such as manual annotation. Scalable and objective tracking methods are required to understand the neural circuits underlying social behavior.

Stem cells at odds with telomere maintenance and protection.

Telomeres are distinctive structures that protect the ends of linear chromosomes and ensure genome stability. They are composed of long tracks of repetitive and G-rich DNA that is bound by shelterin, a dedicated six-subunit protein complex. In somatic cells, shelterin protects telomeres from the DNA damage response and regulates telomere length.

Differential Requirement for the Cell Wall Integrity Sensor Wsc1p in Diploids Versus Haploids.

The primary role of the Cell Wall Integrity Pathway (CWI) in is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that has a stringent requirement in zygotes and diploids, unlike haploids, and differing from 's role in shmoos.