The rockefeller university

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.

The HIV-1 envelope glycoprotein (Env) (gp120-gp41)(3) is the target for neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC). HIV-1 Env is flexible, sampling different conformational states. Before engaging CD4, Env adopts a closed conformation (State 1) that is largely antibody resistant. CD4 binding induces an intermediate state (State 2), followed by an open conformation (State 3) that is susceptible to engagement by antibodies that recognize otherwise occluded epitopes. We investigate conformational changes in Env that induce ADCC in the presence of a smallmolecule CD4-mimetic compound (CD4mc). We uncover an asymmetric Env conformation (State 2A) recognized by antibodies targeting the conserved gp120 inner domain and mediating ADCC. Sera from HIV+ individuals contain these antibodies, which can stabilize Env State 2A in combination with CD4mc. Additionally, triggering State 2A on HIV-infected primary CD4(+) T cells exposes epitopes that induce ADCC. Strategies that induce this Env conformation may represent approaches to fight HIV-1 infection.

Type III-A CRISPR-Cas systems employ the Cas10-Csm complex to destroy bacteriophages and plasmids, using a guide RNA to locate complementary RNA molecules from the invader and trigger an immune response that eliminates the infecting DNA. In addition, these systems possess the non-specific RNase Csm6, which provides further protection for the host. While the role of Csm6 in immunity during phage infection has been determined, how this RNase is used against plasmids is unclear. Here, we show that Staphylococcus epidermidis Csm6 is required for immunity when transcription across the plasmid target is infrequent, leading to impaired target recognition and inefficient DNA degradation by the Cas10-Csm complex. In these conditions, Csm6 causes growth arrest in the host and prevents further plasmid replication through the indiscriminate degradation of host and plasmid transcripts. In contrast, when plasmid target sequences are efficiently transcribed, Csm6 is dispensable and DNA degradation by Cas10 is sufficient for anti-plasmid immunity. Csm6 therefore provides robustness to the type III-A CRISPR-Cas immune response against difficult targets for the Cas10-Csm complex.

Objective: Toll-like receptor-3 agonist Poly-ICLC has been known to activate immune cells and induce HIV replication in pre-clinical experiments. In this study we investigated if Poly-ICLC could be used for disrupting HIV latency while simultaneously enhancing innate immune responses. Design: This was a randomized, placebo-controlled, double-blinded trial in aviremic, cART-treated HIV-infected subjects. Participants (n = 15) were randomized 3: 1 to receive two consecutive daily doses of Poly-ICLC (1.4mg subcutaneously) vs. placebo. Subjects were observed for adverse events, immune activation, and viral replication. Methods: Besides primary outcomes of safety and tolerability, several longitudinal immune parameters were evaluated including immune cell phenotype and function via flowcytometry, ELISA, and transcriptional profiling. PCR assays for plasma HIV-1 RNA, CD4+ T cell-associated HIV-1 RNA, and proviral DNA were performed to measure HIV reservoirs and latency. Results: Poly-ICLC was overall safe and well-tolerated. Poly-ICLC-related adverse events were Grade 1/2, with the exception of one Grade 3 neutropenia which was short-lived. Mild Injection site reactions were observed in nearly all participants in the Poly-ICLC arm. Transcriptional analyses revealed upregulation of innate immune pathways in PBMCs following Poly-ICLC treatment, including strong interferon signaling accompanied by transient increases in circulating IP-10 (CXCL10) levels. These responses generally peaked by 24-48 h after the first injection and returned to baseline by day 8. CD4(+) T cell number and phenotype were unchanged, plasma viral control was maintained and no significant effect on HIV reservoirs was observed. Conclusions: These finding suggest that Poly-ICLC could be safely used for inducing transient innate immune responses in treated HIV+ subjects indicating promise as an adjuvant for HIV therapeutic vaccines.

Linker histone H1 has been correlated with transcriptional inhibition, but the mechanistic basis of the inhibition and its reversal during gene activation has remained enigmatic. We report that H1-compacted chromatin, reconstituted in vitro, blocks transcription by abrogating core histone modifications by p300 but not activator and p300 binding. Transcription from H1-bound chromatin is elicited by the H1 chaperone NAP1, which is recruited in a genespecific manner through direct interactions with activator-bound p300 that facilitate core histone acetylation (by p300) and concomitant eviction of H1 and H2A-H2B. An analysis in B cells confirms the strong dependency on NAP1-mediated H1 eviction for induction of the silent CD40 gene and further demonstrates that H1 eviction, seeded by activator-p300-NAP1-H1 interactions, is propagated over a CCCTC-binding factor (CTCF)-demarcated region through a distinct mechanism that also involves NAP1. Our results confirm direct transcriptional inhibition by H1 and establish a gene-specific H1 eviction mechanism through an activator -> p300 -> NAP1 -> H1 pathway.