Differential effects of Ydj1 and Sis1 on Hsp70-mediated clearance of stress granules in Saccharomyces cerevisiae.

Stress granules and P-bodies are conserved assemblies of nontranslating mRNAs in eukaryotic cells that can be related to RNA-protein aggregates found in some neurodegenerative diseases. Herein, we examine how the Hsp70/Hsp40 protein chaperones affected the assembly and disassembly of stress granules and P-bodies in yeast. We observed that Hsp70 and the Ydj1 and Sis1 Hsp40 proteins accumulated in stress granules and defects in these proteins led to decreases in the disassembly and/or clearance of stress granules.

The retromer complex in development and disease.

The retromer complex is a multimeric protein complex involved in recycling proteins from endosomes to the trans-Golgi network or plasma membrane. It thus regulates the abundance and subcellular distribution of its cargo within cells. Studies using model organisms show that the retromer complex is involved in specific developmental processes.

HUMORAL IMMUNITY. T cell help controls the speed of the cell cycle in germinal center B cells.

The germinal center (GC) is a microanatomical compartment wherein high-affinity antibody-producing B cells are selectively expanded. B cells proliferate and mutate their antibody genes in the dark zone (DZ) of the GC and are then selected by T cells in the light zone (LZ) on the basis of affinity. Here, we show that T cell help regulates the speed of cell cycle phase transitions and DNA replication of GC B cells.

PLANT MICROBIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa.

Immune systems distinguish "self" from "nonself" to maintain homeostasis and must differentially gate access to allow colonization by potentially beneficial, nonpathogenic microbes. Plant roots grow within extremely diverse soil microbial communities but assemble a taxonomically limited root-associated microbiome. We grew isogenic Arabidopsis thaliana mutants with altered immune systems in a wild soil and also in recolonization experiments with a synthetic bacterial community.

Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors.

Unlabelled: HIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified via in vitro mutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes.

The NIH BD2K center for big data in translational genomics.

The world's genomics data will never be stored in a single repository - rather, it will be distributed among many sites in many countries. No one site will have enough data to explain genotype to phenotype relationships in rare diseases; therefore, sites must share data. To accomplish this, the genetics community must forge common standards and protocols to make sharing and computing data among many sites a seamless activity.

Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora.

Novel mechanisms of action and new chemical scaffolds are needed to rejuvenate antibacterial drug discovery, and riboswitch regulators of bacterial gene expression are a promising class of targets for the discovery of new leads. Herein, we report the characterization of 5-(3-(4-fluorophenyl)butyl)-7,8-dimethylpyrido[3,4-b]quinoxaline-1,3(2H,5H)-dione (5FDQD)-an analog of riboflavin that was designed to bind riboswitches that naturally recognize the essential coenzyme flavin mononucleotide (FMN) and regulate FMN and riboflavin homeostasis. In vitro, 5FDQD and FMN bind to and trigger the function of an FMN riboswitch with equipotent activity.

HIF modulation of Wnt signaling regulates skeletal myogenesis in vivo.

Deeper insight into the molecular pathways that orchestrate skeletal myogenesis should enhance our understanding of, and ability to treat, human skeletal muscle disease. It is now widely appreciated that nutrients, such as molecular oxygen (O2), modulate skeletal muscle formation. During early stages of development and regeneration, skeletal muscle progenitors reside in low O2 environments before local blood vessels and differentiated muscle form.

Inhibition of β-catenin signaling respecifies anterior-like endothelium into beating human cardiomyocytes.

During vertebrate development, mesodermal fate choices are regulated by interactions between morphogens such as activin/nodal, BMPs and Wnt/β-catenin that define anterior-posterior patterning and specify downstream derivatives including cardiomyocyte, endothelial and hematopoietic cells. We used human embryonic stem cells to explore how these pathways control mesodermal fate choices in vitro. Varying doses of activin A and BMP4 to mimic cytokine gradient polarization in the anterior-posterior axis of the embryo led to differential activity of Wnt/β-catenin signaling and specified distinct anterior-like (high activin/low BMP) and posterior-like (low activin/high BMP) mesodermal populations.

Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor.

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously.

STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition.

Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation.

Draft Genome Sequences of Ciliovirus and Brinovirus from San Francisco Wastewater.

We report the draft genome sequences of ciliovirus and brinovirus, two members of a likely new family of RNA viruses assembled from San Francisco wastewater. Based on sequence alignments and a nonuniversal genetic code, we believe these to be the first described RNA viruses of ciliates; however, more work is necessary to confirm their host.

Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces.

We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6.

Tissue-specific roles of Fgfr2 in development of the external genitalia.

Congenital anomalies frequently occur in organs that undergo tubulogenesis. Hypospadias is a urethral tube defect defined by mislocalized, oversized, or multiple openings of the penile urethra. Deletion of Fgfr2 or its ligand Fgf10 results in severe hypospadias in mice, in which the entire urethral plate is open along the ventral side of the penis.

Regulation of the Rev1-pol ζ complex during bypass of a DNA interstrand cross-link.

DNA interstrand cross-links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL ("approach"), a nucleotide is inserted across from the unhooked lesion ("insertion"), and the leading strand is extended beyond the lesion ("extension"). How DNA polymerases bypass the ICL is incompletely understood.

Widespread occurrence of N6-methyladenosine in bacterial mRNA.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m(6)A as well as m(6)A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m(6)A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive.

Basal exon skipping and genetic pleiotropy: A predictive model of disease pathogenesis.

Genetic pleiotropy, the phenomenon by which mutations in the same gene result in markedly different disease phenotypes, has proven difficult to explain with traditional models of disease pathogenesis. We have developed a model of pleiotropic disease that explains, through the process of basal exon skipping, how different mutations in the same gene can differentially affect protein production, with the total amount of protein produced correlating with disease severity. Mutations in the centrosomal protein of 290 kDa (CEP290) gene are associated with a spectrum of phenotypically distinct human diseases (the ciliopathies).

Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells.

Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, is hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells.

Rett syndrome: disruption of epigenetic control of postnatal neurological functions.

Loss-of-function mutations in the X-linked gene Methyl-CpG-binding protein 2 (MECP2) cause a devastating pediatric neurological disorder called Rett syndrome. In males, these mutations typically result in severe neonatal encephalopathy and early lethality. On the other hand, owing to expression of the normal allele in ∼50% of cells, females do not suffer encephalopathy but instead develop Rett syndrome.

A Genome-Wide Screen Reveals that the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase System Modulates Virulence Gene Expression.

Diverse environmental stimuli and a complex network of regulatory factors are known to modulate expression of Vibrio cholerae's principal virulence factors. However, there is relatively little known about how metabolic factors impinge upon the pathogen's well-characterized cascade of transcription factors that induce expression of cholera toxin and the toxin-coregulated pilus (TCP). Here, we used a transposon insertion site (TIS) sequencing-based strategy to identify new factors required for expression of tcpA, which encodes the major subunit of TCP, the organism's chief intestinal colonization factor.

The role of ciliary trafficking in Hedgehog receptor signaling.

Defects in the biogenesis of or transport through primary cilia affect Hedgehog protein signaling, and many Hedgehog pathway components traffic through or accumulate in cilia. The Hedgehog receptor Patched negatively regulates the activity and ciliary accumulation of Smoothened, a seven-transmembrane protein that is essential for transducing the Hedgehog signal. We found that this negative regulation of Smoothened required the ciliary localization of Patched, as specified either by its own cytoplasmic tail or by provision of heterologous ciliary localization signals.

Memory. Engram cells retain memory under retrograde amnesia.

Memory consolidation is the process by which a newly formed and unstable memory transforms into a stable long-term memory. It is unknown whether the process of memory consolidation occurs exclusively through the stabilization of memory engrams. By using learning-dependent cell labeling, we identified an increase of synaptic strength and dendritic spine density specifically in consolidated memory engram cells.

Programmable probiotics for detection of cancer in urine.

Rapid advances in the forward engineering of genetic circuitry in living cells has positioned synthetic biology as a potential means to solve numerous biomedical problems, including disease diagnosis and therapy. One challenge in exploiting synthetic biology for translational applications is to engineer microbes that are well tolerated by patients and seamlessly integrate with existing clinical methods. We use the safe and widely used probiotic Escherichia coli Nissle 1917 to develop an orally administered diagnostic that can noninvasively indicate the presence of liver metastasis by producing easily detectable signals in urine.

The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs.

The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone.

Electrical stimulation of low-threshold afferent fibers induces a prolonged synaptic depression in lamina II dorsal horn neurons to high-threshold afferent inputs in mice.

Electrical stimulation of low-threshold Aβ-fibers (Aβ-ES) is used clinically to treat neuropathic pain conditions that are refractory to pharmacotherapy. However, it is unclear how Aβ-ES modulates synaptic responses to high-threshold afferent inputs (C-, Aδ-fibers) in superficial dorsal horn. Substantia gelatinosa (SG) (lamina II) neurons are important for relaying and modulating converging spinal nociceptive inputs.