The rockefeller university

Editor Keloids are benign growths representing a disrupted wound healing process involving abnormal collagen proliferation. Keloids are most prevalent in African American (AA)1 and Asian populations,2 and greatly impact quality of life.3 Keloids show high recurrence rates and current therapies (intralesional steroids, bleomycin, surgical excision, etc.) have limited efficacy,4 thus, presenting a high unmet need for better treatments. Genetic studies have identified chromosome variants associated with keloid formation in AAs and Asians.1, 5 A genomic profiling study on lesional and non‐lesional keloid skin from three AA patients identified bone/cartilage abnormalities,6 but did not assess inflammatory pathways. Recently, atopic dermatitis (AD) has been indicated as an independent risk factor for developing keloids.7 Increases in IL‐4 and IL‐13 receptors have been detected through laser capture microdissection in lesional keloid skin, but increases in cognate ligands were not detected.

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8(+) T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8(+) T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.

CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR-Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5kbase, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps. Hence, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein.

Objective Morning stiffness is a hallmark symptom of rheumatoid arthritis (RA), but its etiology is poorly understood. This study was undertaken to determine whether any histologic features of synovium are associated with this symptom. Methods Data on patient-reported morning stiffness duration and severity, and Disease Activity Score in 28 joints (DAS28) were collected from 176 patients with RA undergoing arthroplasty. Synovium was scored for 10 histopathologic features: synovial lining hyperplasia, lymphocytes, plasma cells, Russell bodies, binucleate plasma cells, fibrin, synovial giant cells, detritus, neutrophils, and mucin. Fibrinolysis of clots seeded with various cell types was measured in turbidimetric lysis assays. Results Stiffness severity and morning stiffness duration were both significantly associated with DAS28 (P = 0.0001 and P = 0.001, respectively). None of the synovial features examined were associated with patient-reported stiffness severity. The presence of neutrophils and fibrin in RA synovial tissue were significantly associated (P < 0.0001) with patient-reported morning stiffness of >= 1 hour, such that 73% of patients with both synovial fibrin and neutrophils reported morning stiffness of >= 1 hour. Further, neutrophils and fibrin deposits colocalized along the synovial lining. In in vitro analyses, fibrin clots seeded with necrotic neutrophils were more resistant to fibrinolysis than those seeded with living neutrophils or no cells (P = 0.008). DNase I treatment of necrotic neutrophils abrogated the delay in fibrinolysis. Conclusion In RA, prolonged morning stiffness may be related to impaired fibrinolysis of neutrophil-enmeshed fibrin deposits along the synovial membrane. Our findings also suggest that morning stiffness severity and duration may reflect distinct pathophysiologic phenomena.

Cytoplasmic dynein is a highly complex motor protein that generates forces toward the minus end of microtubules. Using optical tweezers, we demonstrate that the low processivity (ability to take multiple steps before dissociating) of human dynein limits its force generation due to premature microtubule dissociation. Using a high trap stiffness whereby the motor achieves greater force per step, we reveal that the motor's true maximal force ("stall force") is similar to 2 pN. Furthermore, an average force versus trap stiffness plot yields a hyperbolic curve that plateaus at the stall force. We derive an analytical equation that accurately describes this curve, predicting both stall force and zero-load processivity.This theoretical model describes the behavior of a kinesin motor under low-processivity conditions. Our work clarifies the true stall force and processivity of human dynein and provides a new paradigm for under-standing and analyzing molecular motor force generation for weakly processive motors.

Background Prenatal exposure to ethanol (EtOH) has lasting effects on neuropeptide and neuroimmune systems in the brain alongside detrimental alcohol-related behaviors. At low-to-moderate doses, prenatal EtOH stimulates neurogenesis in lateral hypothalamus (LH) and increases neurons that express the orexigenic peptides hypocretin/orexin (Hcrt/OX) and melanin-concentrating hormone (MCH), and the proinflammatory chemokine CCL2, which through its receptor CCR2 stimulates cell differentiation and movement. Our recent studies demonstrated that CCL2 and CCR2 colocalize with MCH neurons and are involved in EtOH's stimulatory effect on their development but show no relation to Hcrt/OX. Here, we investigated another chemokine, CXCL12, and its receptor, CXCR4, which promote neurogenesis and neuroprogenitor cell proliferation, to determine if they also exhibit peptide specificity in their response to EtOH exposure. Methods Pregnant rats were intraorally administered a moderate dose of EtOH (2 g/kg/d) from embryonic day 10 (E10) to E15. Their embryos and postnatal offspring were examined using real-time quantitative PCR and immunofluorescence histochemistry, to determine if EtOH affects CXCL12 and CXCR4 and the colocalization of CXCR4 with Hcrt/OX and MCH neurons in the LH and with radial glia neuroprogenitor cells in the hypothalamic neuroepithelium (NEP). Results Prenatal EtOH strongly stimulated CXCL12 and CXCR4 in LH neurons of embryos and postnatal offspring. This stimulation was significantly stronger in Hcrt/OX than MCH neurons in LH and also occurred in radial glia neuroprogenitor cells dense in the NEP. These effects were sexually dimorphic, consistently stronger in females than males. Conclusions While showing prenatal EtOH exposure to have a sexually dimorphic, stimulatory effect on CXCL12 and CXCR4 in LH similar to CCL2 and its receptor, these results reveal their distinct relationship to the peptide neurons, with the former closely related to Hcrt/OX and the latter to MCH, and they link EtOH's actions in LH to a stimulatory effect on neuroprogenitor cells in the NEP.

Rationale Cocaine addiction is a chronic brain disease characterized by compulsive drug intake and dysregulation of brain reward systems. Few preclinical studies have modeled the natural longitudinal course of cocaine addiction. Extended access self-administration protocols are powerful tools for modeling the advanced stages of addiction; however, few studies have duration of drug access longer than 12 h/session, potentially limiting their construct validity. Identification of changes in cocaine intake patterns during the development of addictive-like states may allow better treatments for vulnerable subjects. The kappa opioid receptor (KOPr) system has been implicated in the neurobiological regulation of addictive states as well as mood and stress disorders, with selective KOPr antagonists proposed as possible pharmacotherapeutic agents. Chronic cocaine exposure increases the expression of KOPr and its endogenous agonists, the dynorphins, in several brain areas in rodents. Objectives To examine the behavioral pattern of intake during chronic (14 days) 18 h intravenous cocaine self-administration (0.5 mg/kg/infusion) and the effect of a novel short-acting KOPr antagonist LY2444296 HCl (3 mg/kg) administered during sessions 8 to 14 of chronic 18 h/day cocaine self-administration and prior to a single re-exposure session after 2 cocaine-free withdrawal days. Results Both daily and hourly cocaine intake patterns changed over 14 days of 18 h self-administration. LY pretreatment affected the pattern of self-administration across the second week of extended access cocaine self-administration and prevented the increase in cocaine intake during re-exposure. Conclusions Overall, the KOPr antagonist attenuated escalated cocaine consumption in a rat model of extended access cocaine self-administration.

Metastatic prostate cancer is characterized by recurrent genomic copy number alterations that are presumed to contribute to resistance to hormone therapy. We identified CHD1 loss as a cause of antiandrogen resistance in an in vivo small hairpin RNA (shRNA) screen of 730 genes deleted in prostate cancer. ATAC-seq and RNA-seq analyses showed that CHD1 loss resulted in global changes in open and closed chromatin with associated transcriptomic changes. Integrative analysis of this data, together with CRISPR-based functional screening, identified four transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) that contribute to antiandrogen resistance, with associated activation of non-luminal lineage programs. Thus, CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.