The rockefeller university

The incidence of nephrolithiasis continues to rise. Previously, we showed that a monogenic cause could be detected in 11.4% of individuals with adult-onset nephrolithiasis or nephrocalcinosis and in 16.7-20.8% of individuals with onset before 18 years of age, using gene panel sequencing of 30 genes known to cause nephrolithiasis/nephrocalcinosis. To overcome the limitations of panel sequencing, we utilized whole exome sequencing in 51 families, who presented before age 25 years with at least one renal stone or with a renal ultrasound finding of nephrocalcinosis to identify the underlying molecular genetic cause of disease. In 15 of 51 families, we detected a monogenic causative mutation by whole exome sequencing. A mutation in seven recessive genes (AGXT, ATP6V1B1, CLDN16, CLDN19, GRHPR, SLC3A1, SLC12A1), in one dominant gene (SLC9A3R1), and in one gene (SLC34A1) with both recessive and dominant inheritance was detected. Seven of the 19 different mutations were not previously described as disease-causing. In one family, a causative mutation in one of 117 genes that may represent phenocopies of nephrolithiasis-causing genes was detected. In nine of 15 families, the genetic diagnosis may have specific implications for stone management and prevention. Several factors that correlated with the higher detection rate in our cohort were younger age at onset of nephrolithiasis/nephrocalcinosis, presence of multiple affected members in a family, and presence of consanguinity. Thus, we established whole exome sequencing as an efficient approach toward a molecular genetic diagnosis in individuals with nephrolithiasis/nephrocalcinosis who manifest before age 25 years.

The flippase MurJ is responsible for transporting the cell wall intermediate lipid II from the cytoplasm to the outside of the cell. While essential for the survival of bacteria, it remains an underexploited target for antibacterial therapy. The humimycin antibiotics are lipid II flippase (MurJ) inhibitors that were synthesized on the basis of bioinformatic predictions derived from secondary metabolite gene clusters found in the human microbiome. Here, we describe an SAR campaign around humimycin A that produced humimycin 17S. Compared to humimycin A, 17S is a more potent beta lactam potentiator, has a broader spectrum of activity, which now includes both methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus faecalis (VRE), and did not lead to any detectable resistance when used in combination with a beta-lactam. Combinations of beta-lactam and humimycin 17S provide a potentially useful long-term MRSA regimen.

The clonal complexity of adult stem cell pools is progressively lost during homeostatic turnover in several tissues, suggesting a decrease in the number of stem cells with distinct clonal origins. The functional impact of reduced complexity on stem cell pools, and how different tissue microenvironments may contribute to such a reduction, are poorly understood. Here, we performed clonal multicolor lineage tracing of skeletal muscle stem cells (MuSCs) to address these questions. We found that MuSC clonal complexity is maintained during aging despite heterogenous reductions in proliferative capacity, allowing aged muscle to mount a clonally diverse, albeit diminished, response to injury. In contrast, repeated bouts of tissue repair cause a progressive reduction in MuSC clonal complexity indicative of neutral drift. Consistently, biostatistical modeling suggests that MuSCs undergo symmetric expansions with stochastic fate acquisition during tissue repair. These findings establish distinct principles that underlie stem cell dynamics during homeostatic aging and muscle regeneration.

The peroxisome-proliferator receptor-gamma (PPAR gamma) is expressed in multiple cancer types. Recently, our group has shown that PPAR gamma is phosphorylated on serine 273 (S273), which selectively modulates the transcriptional program controlled by this protein. PPAR gamma ligands, including thiazolidinediones (TZDs), block S273 phosphorylation. This activity is chemically separable from the canonical activation of the receptor by agonist ligands and, importantly, these noncanonical agonist ligands do not cause some of the known side effects of TZDs. Here, we show that phosphorylation of S273 of PPAR gamma occurs in cancer cells on exposure to DNA damaging agents. Blocking this phosphorylation genetically or pharmacologically increases accumulation of DNA damage, resulting in apoptotic cell death. A genetic signature of PPAR gamma phosphorylation is associated with worse outcomes in response to chemotherapy in human patients. Non-canonical agonist ligands sensitize lung cancer xenografts and genetically induced lung tumors to carboplatin therapy. Moreover, inhibition of this phosphorylation results in deregulation of p53 signaling, and biochemical studies show that PPAR gamma physically interacts with p53 in a manner dependent on S273 phosphorylation. These data implicate a role for PPAR gamma in modifying the p53 response to cytotoxic therapy, which can be modulated for therapeutic gain using these compounds.

Understanding cell fate patterning and morphogenesis in the mammalian embryo remains a formidable challenge. Recently, in vivo models based on embryonic stem cells (ESCs) have emerged as complementary methods to quantitatively dissect the physical and molecular processes that shape the embryo. Here we review recent developments in using ESCs to create both two-and three-dimensional culture models that shed light on mammalian gastrulation.

Studying the neurological, genetic, and evolutionary basis of human vocal communication mechanisms using animal vocalization models is an important field of neuroscience. The datasets typically comprise structured sequences of syllables or songs produced by animals from different genotypes under different social contexts. It has been difficult to come up with sophisticated statistical methods that appropriately model animal vocal communication syntax. We address this need by developing a novel Bayesian semiparametric framework for inference in such datasets. Our approach is built on a novel class of mixed effects Markov transition models for the songs that accommodate exogenous influences of genotype and context as well as animal-specific heterogeneity. Crucial advantages of the proposed approach include its ability to provide insights into key scientific queries related to global and local influences of the exogenous predictors on the transition dynamics via automated tests of hypotheses. The methodology is illustrated using simulation experiments and the aforementioned motivating application in neuroscience. Supplementary materials for this article, including a standardized description of the materials available for reproducing the work, are available as an online supplement.

Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems(1) or in living cells(2). Previously, synthetic nucleic acid motors(3-5) and modified natural protein motors(6-10) have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors(11-15). Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure(7,9). We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing(16). Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement(17) reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s(-1). Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

TET enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish owing to challenges in distinguishing global versus locus-specific effects. Here we show that TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding decrease in gene expression in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find that increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taking these findings together with further genome-wide methylation and TET1 and DNMT3B ChIP-seq analyses, we conclude that TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.

Mobile genetic elements (MGEs) are the genetic material often involved in the interspecies and intraspecies genetic transduction in bacteria. However, little is known about MGEs in the Anginosus group of streptococci (AGS), one of the streptococcal groups found in the oral cavity of humans. We looked for the presence of MGEs in Streptococcus anginosus subsp. anginosus (SAA), a representative species belonging to AGS, and found a novel plasmid from SAA strain 0430-08. This plasmid was 7038 by and similar to 31% G/C content which we named pSAA0430-08, and examined its genetic structure and characteristics. Open reading frame (ORF) prediction revealed that pSAA0430-08 was composed of 10 ORFs including a putative plasmid replication protein (ORF1) and a putative toxin-antitoxin system (ORF9 and ORF10). Between ORF10 and ORF 1, four tandem repeats of 22 bp each, generally termed as iteron, were also observed. Using variant plasmids of pSAA0430-08, we confirmed that both ORF1 and heron were necessary for replication in host cells. Interestingly, the region from ORF4 to ORF7 showed homology with a genomic DNA segment of S. gordonii strains. Thus, this plasmid may travel between the different species in Streptococci, i.e., S. gordonii and S. anginosus.