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The kappa-opioid receptor (KOR) / dynorphin system is implicated with behavioral and neurobiological effects of stress exposure (including heavy exposure to drugs of abuse) in translational animal models. Thus some KOR-antagonists can decrease the aversive, depressant-like and anxiety-like effects caused by stress exposure. The first generation of selective KOR-antagonists have slow onsets (hours) and extremely long durations of action (days-weeks), in vivo. A new generation of KOR antagonists with rapid onset and shorter duration of action can potentially decrease the effects of stress exposure in translational models, and may be of interest for medication development. This study examined the rapid onset anti-stress effects of one of the shorter acting novel KOR-antagonists (LY2795050, (3-chloro-4-(4-(((2S)-2-pyridin-3-ylpyrrolidin-1-yl)methyl) phenoxy)benzamide)) in a single-session open space swim (OSS) stress paradigm (15 min duration), in adult male and female C57BL/6 J mice. LY2795050 (0.32 mg/kg, i.p.) had rapid onset (within 15 min) and short duration (<3 h) of KOR-antagonist effects, based on its blockade of the locomotor depressant effects of the KOR-agonist U50,488 (10 mg/kg). LY2795050 (0.32 mg/kg), when administered only 1 min prior to the OSS stress paradigm, decreased immobility in males, but not females. With a slightly longer pretreatment time (15 min), this dose of LY2795050 decreased immobility in both males and females. A 10-fold smaller dose of LY2795050 (0.032 mg/kg) was inactive in the OSS, showing dose-dependence of this anti-stress effect. Overall, these studies show that a novel KOR-antagonist can produce very rapid onset anti-immobility effects in this model of acute stress exposure.

Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here, we devise the template-, reference- and selection-free (TRSF) cryo-EM pipeline to simultaneously reconstruct cryo-EM structures of protein complexes from interphase or metaphase chromosomes. The reconstructed interphase and metaphase nucleosome structures are on average indistinguishable from canonical nucleosome structures, despite DNA sequence heterogeneity, cell-cycle-specific posttranslational modifications, and interacting proteins. Nucleosome structures determined by a decoy-classifying method and structure variability analyses reveal the nucleosome structural variations in linker DNA, histone tails, and nucleosome core particle configurations, suggesting that the opening of linker DNA, which is correlated with H2A C-terminal tail positioning, is suppressed in chromosomes. High-resolution (3.4-3.5 A degrees) nucleosome structures indicate DNA-sequence-independent stabilization of superhelical locations +/- 0-1 and +/- 3.5-4.5. The linker histone H1.8 preferentially binds to metaphase chromatin, from which chromatosome cryo-EM structures with H1.8 at the on-dyad position are reconstituted. This study presents the structural characteristics of nucleosomes in chromosomes.

K-ATP channels are metabolic sensors that translate intracellular ATP/ADP balance into membrane excitability. The molecular composition of K-ATP includes an inward-rectifier potassium channel (Kir) and an ABC transporter-like sulfonylurea receptor (SUR). Although structures of K-ATP have been determined in many conformations, in all cases, the pore in Kir is closed. Here, we describe human pancreatic K-ATP (hK(ATP)) structures with an open pore at 3.1- to 4.0-angstrom resolution using single-particle cryo-electron microscopy (cryo-EM). Pore opening is associated with coordinated structural changes within the ATP-binding site and the channel gate in Kir. Conformational changes in SUR are also observed, resulting in an area reduction of contact surfaces between SUR and Kir. We also observe that pancreatic hK(ATP) exhibits the unique (among inward-rectifier channels) property of PIP2-independent opening, which appears to be correlated with a docked cytoplasmic domain in the absence of PIP2.

The importance of histone variant H2A.Z in transcription regulation has been well established, yet its mechanism-of-action remains enigmatic. Conflicting evidence exists in support of both an activating and a repressive role of H2A.Z in transcription. Here we report cryo-electron microscopy (cryo-EM) structures of nucleosomes and chromatin fibers containing H2A.Z and those containing canonical H2A. The structures show that H2A.Z incorporation results in substantial structural changes in both nucleosome and chromatin fiber. While H2A.Z increases the mobility of DNA terminus in nucleosomes, it simultaneously enables nucleosome arrays to form a more regular and condensed chromatin fiber. We also demonstrated that H2A.Z's ability to enhance nucleosomal DNA mobility is largely attributed to its characteristic shorter C-terminus. Our study provides the structural basis for H2A.Z-mediated chromatin regulation, showing that the increase flexibility of the DNA termini in H2A.Z nucleosomes is central to its dual-functions in chromatin regulation and in transcription.

Glutathione (GSH) is a small-molecule thiol that is abundant in all eukaryotes and has key roles in oxidative metabolism(1). Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions(2). GSH is synthesized exclusively in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remains unknown. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, as a regulator of GSH transport into mitochondria. Loss of SLC25A39 reduces mitochondrial GSH import and abundance without affecting cellular GSH levels. Cells lacking both SLC25A39 and its paralogue SLC25A40 exhibit defects in the activity and stability of proteins containing iron-sulfur clusters. We find that mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enables mitochondrial GSH production and ameliorates the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH-import machinery.

The CARD-BCL10-MALT1 (CBM) complex is critical for the proper assembly of human immune responses. The clinical and immunological consequences of deficiencies in some of its components such as CARD9, CARD11, and MALT1 have been elucidated in detail. However, the scarcity of BCL10 deficient patients has prevented gaining detailed knowledge about this genetic disease. Only two patients with BCL10 deficiency have been reported to date. Here we provide an in-depth description of an additional patient with autosomal recessive complete BCL10 deficiency caused by a nonsense mutation that leads to a loss of expression (K63X). Using mass cytometry coupled with unsupervised clustering and machine learning computational methods, we obtained a thorough characterization of the consequences of BCL10 deficiency in different populations of leukocytes. We showed that in addition to the near absence of memory B and T cells previously reported, this patient displays a reduction in NK, gamma delta T, Tregs, and T-FH cells. The patient had recurrent respiratory infections since early childhood, and showed a family history of lethal severe infectious diseases. Fortunately, hematopoietic stem-cell transplantation (HSCT) cured her. Overall, this report highlights the importance of early genetic diagnosis for the management of BCL10 deficient patients and HSCT as the recommended treatment to cure this disease.

RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states. RNA Polymerase II (Pol II) recycling can influence transcription efficiency. Here the authors describe an approach aimed at facilitating the identification of factors involved in Pol II recycling and identify PAF1 complex components as mediators of recycling.

CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage lambda uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that lambda Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.

Allogeneic hematopoietic cell transplantation (HCT) offers a potentially curative therapy in patients with hematologic malignancies; however, nonrelapse mortality (NRM) remains a concern. Strategies to improve neutrophil recovery and immune reconstitution are needed to decrease NRM. Murine models of allogeneic HCT suggest that fractionated hematopoietic progenitor cell (HPC) infusion may improve engraftment through improved access of HPCs to a viable hematopoietic niche. The primary objective of the present study was to determine the impact of fractionated infusion versus unfractionated (bulk) infusion of HPCs on the time to achieve neutrophil engraftment. Secondary objectives included the effect of fractionated versus bulk infusion of HPCs on platelet engraftment, immune reconstitution, the incidence of acute graft-versus-host disease (GVHD) grade NRM, and overall survival (OS). In this randomized phase 2 study, patients with hematologic malignancies undergoing allogeneic HCT were randomized to receive HPC infusion as a bulk (bulk arm) or in fractions (fractionated arm): 4 x 10(6) CD34(+) cells/kg recipient weight infused on day 0, with the remaining HPCs CD34' cell-selected then infused in equally distributed aliquots on days 2, 4, and 6 post-HCT. Randomization was stratified by type of transplant, unmodified (i.e. T cell-replete graft) versus CD34(+) cell-selected (T cell-depleted graft). Patients whose donor failed to collect at least 7 x 10(6) CD34(+) cells/kg of recipient weight received bulk HPC infusions regardless of randomization, for safety. These patients continued the HG process on study but were replaced until each arm reached the prespecified accrual target. Per protocol, these patients were not included in this modified intention-to-treat analysis. A total of 116 patients were enrolled. Donors of 42 patients failed to mobilize the minimum CD34(+) cell dose (7 x 106 cells/kg recipient weight) and were excluded from the analysis. The 74 evaluable patients included 38 randomized to the bulk arm and 36 randomized to the fractionated arm. All patients engrafted. The median time to an absolute neutrophil count of >0.5 x 10(9)/L was 11 days on both arms. The day +180 median CD4* cell count was 179 cells/AL in the bulk arm and 111 cells/AL in the fractionated arm (P =.779). The cumulative incidence of grade II-IV acute GVHD on post-transplant day +100 was 32% in the bulk arm and 17% in the fractionated arm (P =.131). Two patients in the bulk arm, but none in the fractionated arm, experienced grade III-IV GVHD. The 4-year OS was 60% in the bulk arm and 62% in the fractionated arm (P =.414), whereas the 4-year cumulative incidences of NRM and relapse were similar in the 2 arms. Fractionated infusion of HPCs in allogeneic HG recipients did not impact neutrophil or CD4(+) cell recovery, NRM, relapse, or OS when compared with bulk HPC infusion. We also observed that with current mobilization techniques, it was unlikely that more than 60% of healthy donors would be able to collect >7 x 10(6) CD34(+) cells/kg recipient weight for adult recipients. (C) 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. (C) 2021 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE 'seeds' featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCin GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions. Elemento, Melnick and colleagues examine the chromatin and transcriptional changes that occur during differentiation of human primary B cells into antibody-secreting cells. In naive B cells, the transcription factor OCT2 is preloaded at high-affinity super-enhancer sites present in repressed 'silent' chromatin; upon activation, OCis recruited to these regions, where it facilitates arrays of OCT2 binding to lower-affinity octamer motifs, leading to active formation of germinal center B cell-specific super-enhancers.