The rockefeller university

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

A search is described for the production of a pair of bottom-type vectorlike quarks (B VLQs) with mass greater than 1000 GeV. Each B VLQ decays into a b quark and a Higgs boson, a b quark and a Z boson, or a t quark and a W boson. This analysis considers both fully hadronic final states and those containing a charged lepton pair from a Z boson decay. The products of the H -> bb boson decay and of the hadronic Z or W boson decays can be resolved as two distinct jets or merged into a single jet, so the final states are classified by the number of reconstructed jets. The analysis uses data corresponding to an integrated luminosity of 138 fb(-1) collected in proton-proton collisions at root s = 13 TeV with the CMS detector at the LHC from 2016 to 2018. No excess over the expected background is observed. Lower limits are set on the B VLQ mass at the 95% confidence level. These depend on the B VLQ branching fractions and are 1570 and 1540 GeV for 100% B -> bH and 100% B -> bZ, respectively. In most cases, the mass limits obtained exceed previous limits by at least 100 GeV.

A 13-year-old boy was admitted with severe meningococcal meningitis. Immunologic workup revealed a properdin deficiency, and genetic sequencing of CFP identified a novel, private and predicted pathogenic variant in exon 8. The patient received broad immunizations and penicillin prophylaxis. Children with invasive meningococcal disease should be tested for complement deficiency.

Spontaneous and sensory-evoked activity sculpts developing circuits. Yet, how these activity patterns intersect with cellular programs regulating the differentiation of neuronal subtypes is not well understood. Through electrophysiological and in vivo longitudinal analyses, we show that C-X-C motif chemokine ligand 14 (Cxcl14), a gene previously characterized for its association with tumor invasion, is expressed by single- bouquet cells (SBCs) in layer I (LI) of the somatosensory cortex during development. Sensory deprivation at neonatal stages markedly decreases Cxcl14 expression. Additionally, we report that loss of function of this gene leads to increased intrinsic excitability of SBCs-but not LI neurogliaform cells-and augments neuronal complexity. Furthermore, Cxcl14 loss impairs sensory map formation and compromises the in vivo recruitment of superficial interneurons by sensory inputs. These results indicate that Cxcl14 is required for LI differentiation and demonstrate the emergent role of chemokines as key players in cortical network development.

Mixed lineage leukemia-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce MLL through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here, we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II in HEL, a human erythroleukemia cell line without MLL1 rearrangement. MLL1 and AF9 only coregulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely believed elongation.

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

Introduction: To assess the effectiveness of novel HIV curative strategies, "cure" trials require periods of closely monitored antiretroviral therapy (ART) analytical treatment interruptions (ATIs). We performed a systematic review and meta-analysis to identify the impact of ATI with or without novel therapeutics in cure-related studies on the time to viral re-suppression following ART restart. Methods: Medline, Embase and Web of Science databases were searched for human studies involving ATIs from 1 January 2015 till 22 April 2024. The primary outcome was time to first viral re-suppression (plasma HIV viral load [VL] <50 copies/ml) stratified by receipt of interventional drug with ATI (IA) or ATI-only groups. Random-effects proportional meta-analysis and multivariable Cox proportional hazards analysis were performed using R. Results: Of 1073 studies screened, 13 were included that met the inclusion criteria with VL data available after restarting ART (n = 213 participants). There was no difference between time to viral suppression in IA or ATI-only cohorts (p = 0.22). For 87% of participants, viral suppression within 12 weeks of ART restart was achieved, and all eventually had at least one VL <50 copies/ml during follow-up. After adjusting for covariables, while participants in the IA cohort were associated with less rapid suppression (adjusted hazard ratio [aHR] 0.61, 95% CI 0.40-0.94, p = 0.026), other factors include greater log VL at ART restart (aHR 0.56, 95% CI 0.46-0.68, p<0.001), duration since HIV diagnosis (aHR 0.93, 95% CI 0.89-0.96) and longer intervals between HIV VL monitoring (aHR 0.66, 95% CI 0.59-0.74, p<0.001). However, the use of integrase inhibitors was associated with more rapid viral suppression (aHR 1.74, 95% CI 1.16-2.59). Discussion: When designing studies involving ATIs, information on time to viral re-suppression after restarting ART is important to share with participants, and should be regularly monitored and reported, to assess the impact and safety of specific trial interventions in ATI studies. Conclusions: The majority of participants achieved viral suppression after restarting ART in ATI studies. ART regimens containing integrase inhibitors and frequent VL monitoring should be offered for people restarting ART after ATI studies to ensure rapid re-suppression.

The proliferating cell nuclear antigen (PCNA) clamp encircles DNA to hold DNA polymerases (Pols) to DNA for processivity. The Ctf18-RFC PCNA loader, a replication factor C (RFC) variant, is specific to the leading-strand Pol (Pol epsilon). We reveal here the underlying mechanism of Ctf18-RFC specificity to Pol epsilon using cryo-electron microscopy and biochemical studies. We found that both Ctf18-RFC and Pol epsilon contain specific structural features that direct PCNA loading onto DNA. Unlike other clamp loaders, Ctf18-RFC has a disordered ATPase associated with a diverse cellular activities (AAA+) motor that requires Pol epsilon to bind and stabilize it for efficient PCNA loading. In addition, Ctf18-RFC can pry prebound Pol epsilon off of DNA, then load PCNA onto DNA and transfer the PCNA-DNA back to Pol epsilon. These elements in both Ctf18-RFC and Pol epsilon provide specificity in loading PCNA onto DNA for Pol epsilon.

Neurons from multiple prefrontal areas encode several key variables of social gaze interaction. To explore the causal roles of the primate prefrontal cortex in real-life gaze interaction, we applied weak closed-loop micro- stimulations that were precisely triggered by specific social gaze events. Microstimulations of the orbitofrontal cortex, but not the dorsomedial prefrontal cortex or the anterior cingulate cortex, enhanced momentary dynamic social attention in the spatial dimension by decreasing the distance of fixations relative to a partner's eyes and in the temporal dimension by reducing the inter-looking interval and the latency to reciprocate the other's directed gaze. By contrast, on a longer timescale, microstimulations of the dorsomedial prefrontal cortex modulated inter-individual gaze dynamics relative to one's own gaze positions. These findings demonstrate that multiple regions in the primate prefrontal cortex may serve as functionally accessible nodes in controlling different aspects of dynamic social attention and suggest their potential for a therapeutic brain interface.

During neural tube (NT) development, the notochord induces an organizer, the floorplate, which secretes Sonic Hedgehog (SHH) to pattern neural progenitors. Conversely, NT organoids (NTOs) from embryonic stem cells (ESCs) spontaneously form floorplates without the notochord, demonstrating that stem cells can self-organize without embryonic inducers. Here, we investigated floorplate self-organization in clonal mouse NTOs. Expression of the floorplate marker FOXA2 was initially spatially scattered before resolving into multiple clusters, which underwent competition and sorting, resulting in a stable "winning"floorplate. We identified that BMP signaling governed long-range cluster competition. FOXA2+ + clusters expressed BMP4, suppressing FOXA2 in receiving cells while simultaneously expressing the BMP-inhibitor NOGGIN, promoting cluster persistence. Noggin mutation perturbed floorplate formation in NTOs and in the NT in vivo at mid/hindbrain regions, demonstrating how the floorplate can form autonomously without the notochord. Identifying the pathways governing organizer self-organization is critical for harnessing the developmental plasticity of stem cells in tissue engineering.