The rockefeller university

"What I cannot create, I do not understand." Richard Feynman may have championed reasoning from first principles in his famous blackboard missive, but he could just as well have been referring to the plight of a molecular virologist. What cannot be grown in a controlled laboratory setting, we cannot fully understand. The story of the laboratory domestication of hepatitis C virus (HCV) is now a classic example of virologists applying all manner of inventive skill to create cell-based models of infection in order to clarify prospective drug targets. In this review, we highlight key successes and failures that were instructive in achieving cell-based models for HCV studies and drug development. We also emphasize the lessons learned from the 40 year saga that may be applicable to viruses yet unknown and uncultured.

PurposeThe presence of Propionibacterium acnes in a substantial component of resected disc specimens obtained from patients undergoing discectomy or microdiscectomy has led to the suggestion that this prominent human skin and oral commensal may exacerbate the pathology of degenerative disc disease. This hypothesis, therefore, raises the exciting possibility that antibiotics could play an important role in treating this debilitating condition. To date, however, little information about antibiotic penetration into the intervertebral disc is available. MethodsIntervertebral disc tissue obtained from 54 microdiscectomy patients given prophylactic cefazolin (n=25), clindamycin (n=17) or vancomycin (n=12) was assayed by high-performance liquid chromatography, with cefaclor as an internal standard, to determine the concentration of antibiotic penetrating into the disc tissue.ResultsIntervertebral disc tissues from patients receiving the positively charged antibiotic clindamycin contained a significantly greater percentage of the antibacterial dose than the tissue from patients receiving negatively charged cefazolin (P<0.0001) and vancomycin, which has a slight positive charge (P<0.0001).ConclusionPositively charged antibiotics appear more appropriate for future studies investigating potential options for the treatment of low-virulence disc infections. [GRAPHICS]

Foraging strategies emerge from genetically encoded programs that are similar across animal species. Here, we examine circuits that control a conserved foraging state, local search behavior after food removal, in Caenorhabditis elegans. We show that local search is triggered by two parallel groups of chemosensory and mechanosensory glutamater-gic neurons that detect food-related cues. Each group of sensory neurons suppresses distinct integrating neurons through a G protein-coupled metabotropic glutamate receptor, MGL-1, to release local search. The chemosensory and mechanosensory modules are separate and redundant; glutamate release from either module can drive the full behavior. A transition from local search to global search over several minutes after food removal is associated with two changes in circuit function. First, the spontaneous activity of sensory neurons falls. Second, the motor pattern generator for local search becomes less responsive to sensory input. This multimodal, distributed short-term food memory provides robust control of an innate behavior.

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease. Author summary Identifying the genetic basis of rare disorders can provide insight into gene function, susceptibility to disease, guide the development of new therapeutics, improve opportunities for genetic counseling, and help clinicians evaluate and potentially treat complicated clinical presentations. However, it is estimated that the genetic basis of approximately one-half of all rare genetic disorders remains unknown. We describe one such rare disorder based on genetic and clinical evaluations of individuals from 3 unrelated consanguineous families with a similar constellation of features including short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations including stroke, among other findings. We discovered that these features were due to deficiency of the PIK3C2A enzyme. PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of the lipids phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2 that are essential for a variety of cellular processes including cilia formation and vesicle trafficking. This syndrome is the first monogenic disorder caused by mutations in a class II PI3K family member and thus sheds new light on their role in human development.

The replisome, the molecular machine dedicated to copying DNA, encounters a variety of obstacles during S phase. Without a proper response to this replication stress, the genome becomes unstable, leading to disease, including cancer. The immediate response is localized to the stalled replisome and includes protection of the nascent DNA. A number of recent studies have provided insight into the factors recruited to and responsible for protecting stalled replication forks. In response to replication stress, the SNF2 family of DNA translocases has emerged as being responsible for remodeling replication forks in vivo. The protection of stalled replication forks requires the cooperation of RAD51, BRCA1, BRCA2, and many other DNA damage response proteins. In the absence of these fork protection factors, fork remodeling renders them vulnerable to degradation by nucleases and helicases, ultimately compromising genome integrity. In this review, we focus on the recent progress in understanding the protection, processing, and remodeling of stalled replication forks in mammalian cells.

Objective- Psoriasis is an inflammatory skin disease which heightens the

Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles. Transmitted/founder (T/F) viruses are particularly important to study since they represent viruses that initiated primary HIV-1 infections and may have unique attributes. Here we combined in vivo competition and rational design to develop novel subtype C SHIVs containing T/F envelopes. We successfully generated 19 new, infectious subtype C SHIVs, which were tested in multiple combinatorial pools in Indian-origin rhesus macaques. Infected animals attained peak viremia within 5 weeks ranging from 10(3) to 10(7) vRNA copies/mL. Sequence analysis during primary infection revealed 7 different SHIVs replicating in 8 productively infected animals with certain clones prominent in each animal. We then generated 5 variants each of 6 SHIV clones (3 that predominated and 3 undetectable after pooled in vivo inoculations), converting a serine at Env375 to methionine, tyrosine, histidine, tryptophan or phenylalanine. Overall, most Env375 mutants replicated better in vitro and in vivo than wild type with both higher and earlier peak viremia. In 4 of these SHIV clones (with and without Env375 mutations) we also created mutations at position 281 to include serine, alanine, valine, or threonine. Some Env281 mutations imparted in vitro replication dynamics similar to mutations at 375; however, clones with both mutations did not exhibit incremental benefit. Therefore, we identified unique subtype C T/F SHIVs that replicate in rhesus macaques with improved acute phase replication kinetics without altering phenotype. In vivo competition and rational design can produce functional SHIVs with globally relevant HIV-1 Envs to add to the growing number of SHIV clones for HIV-1 research in NHPs. Author summary Nonhuman primates provide useful models for studying HIV transmission, pathogenesis and cure strategies. Due to species-specific antiviral factors, however, HIV cannot replicate in Asian macaques directly. Some chimeric viruses incorporating HIV Envelope genes in simian immunodeficiency virus (SIV) backbone can replicate to sufficient levels in Asian macaques to permit evaluation of anti-HIV interventions. Here we describe the generation of new SHIV clones unique to the field in 4 important ways. First, these clones were generated from the globally relevant HIV-1 subtype C, which is the most prevalent form of HIV globally and is found predominately in sub-Saharan Africa where the pandemic is particularly devastating but is poorly represented among SHIVs studied to date. Second, we utilized Envelope genes from viruses that established primary infection, making these clones particularly useful in transmission studies. Third, these clones were not generated by animal passage, which may alter some of the unique properties of these Envelopes. Finally, we used direct within animal competition studies and two targeted mutations to select highly replicative clones. We provide here both the discovery of new SHIV clones, and also a process to generate additional clones in the future.

Approximately 500 monogenic causes of chronic kidney disease (CKD) have been identified, mainly in pediatric populations. The frequency of monogenic causes among adults with CKD has been less extensively studied. To determine the likelihood of detecting monogenic causes of CKD in adults presenting to nephrology services in Ireland, we conducted whole exome sequencing (WES) in a multicentre cohort of 114 families including 138 affected individuals with CKD. Affected adults were recruited from 78 families with a positive family history, 16 families with extra-renal features, and 20 families with neither a family history nor extra-renal features. We detected a pathogenic mutation in a known CKD gene in 42 of 114 families (37%). A monogenic cause was identified in 36% of affected families with a positive family history of CKD, 69% of those with extra-renal features, and only 15% of those without a family history or extra-renal features. There was no difference in the rate of genetic diagnosis in individuals with childhood versus adult onset CKD. Among the 42 families in whom a monogenic cause was identified, WES confirmed the clinical diagnosis in 17 (40%), corrected the clinical diagnosis in 9 (22%), and established a diagnosis for the first time in 16 families referred with CKD of unknown etiology (38%). In this multi-centre study of adults with CKD, a molecular genetic diagnosis was established in over one-third of families. In the evolving era of precision medicine, WES may be an important tool to identify the cause of CKD in adults.

How cells correct deviations from a mean cell size at mitosis remains uncertain. Classical cell-size homeostasis models are the sizer, timer, and adder [1]. Sizers postulate that cells divide at some threshold size; timers, that cells grow for a set time; and adders, that cells add a constant volume before division. Here, we show that a size-based probabilistic model of cell-size control at the G2/M transition (P(Div)) can generate realistic cell-size homeostasis in silico. In fission yeast cells, Cyclin B-Cdc13 scales with size, and we propose that this increases the like-lihood of mitotic entry, while molecular noise in its expression adds a probabilistic component to the model. Varying Cdc13 expression levels exogenously using a newly developed tetracycline inducible promoter shows that both the level and variability of its expression influence cell size at division. Our results demonstrate that as cells grow larger, their probability of dividing increases, and this is sufficient to generate cell-size homeostasis. Size-correlated Cdc13 expression forms part of the molecular circuitry of this system.

DOCK2 is a guanine-nucleotide-exchange factor for Rac proteins. Activated Rac serves various cellular functions including the reorganization of the actin cytoskeleton in lymphocytes and neutrophils and production of reactive oxygen species in neutrophils. Since 2015, six unrelated patients with combined immunodeficiency and early-onset severe viral infections caused by bi-allelic loss-of-function mutations in DOCK2 have been described. Until now, the function of phagocytes, specifically neutrophils, has not been assessed in human DOCK2 deficiency. Here, we describe a new kindred with four affected siblings harboring a homozygous splice-site mutation (c.2704-2 A > C) in DOCK2. The mutation results in alternative splicing and a complete loss of DOCK2 protein expression. The patients presented with leaky severe combined immunodeficiency or Omenn syndrome. The novel mutation affects EBV-B cell migration and results in NK cell dysfunction similar to previous observations. Moreover, both cytoskeletal rearrangement and reactive oxygen species production are partially impaired in DOCK2-deficient neutrophils.