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Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Xpert MTB/RIF Ultra (Xpert Ultra) might have higher sensitivity than its predecessor, Xpert MTB/RIF (Xpert), but its role in tuberculous meningitis diagnosis is uncertain. We aimed to compare Xpert Ultra with Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults. In this prospective, randomised, diagnostic accuracy study, adults (≥16 years) with suspected tuberculous meningitis from a single centre in Vietnam were randomly assigned to cerebrospinal fluid testing by either Xpert Ultra or Xpert at baseline and, if treated for tuberculous meningitis, after 3–4 weeks of treatment. Test performance (sensitivity, specificity, and positive and negative predictive values) was calculated for Xpert Ultra and Xpert and compared against clinical and mycobacterial culture reference standards. Analyses were done for all patients and by HIV status. Between Oct 16, 2017, and Feb 10, 2019, 205 patients were randomly assigned to Xpert Ultra (n=103) or Xpert (n=102). The sensitivities of Xpert Ultra and Xpert for tuberculous meningitis diagnosis against a reference standard of definite, probable, and possible tuberculous meningitis were 47·2% (95% CI 34·4–60·3; 25 of 53 patients) for Xpert Ultra and 39·6% (27·6–53·1; 21 of 53) for Xpert (p=0·56); specificities were 100·0% (95% CI 92·0–100·0; 44 of 44) and 100·0% (92·6–100·0; 48 of 48), respectively. In HIV-negative patients, the sensitivity of Xpert Ultra was 38·9% (24·8–55·1; 14 of 36) versus 22·9% (12·1–39·0; eight of 35) by Xpert (p=0·23). In HIV co-infected patients, the sensitivities were 64·3% (38·8–83·7; nine of 14) for Xpert Ultra and 76·9% (49·7–91·8; ten of 13) for Xpert (p=0·77). Negative predictive values were 61·1% (49·6–71·5) for Xpert Ultra and 60·0% (49·0–70·0) for Xpert. Against a reference standard of mycobacterial culture, sensitivities were 90·9% (72·2–97·5; 20 of 22 patients) for Xpert Ultra and 81·8% (61·5–92·7; 18 of 22) for Xpert (p=0·66); specificities were 93·9% (85·4–97·6; 62 of 66) and 96·9% (89·5–91·2; 63 of 65), respectively. Six (22%) of 27 patients had a positive test by Xpert Ultra after 4 weeks of treatment versus two (9%) of 22 patients by Xpert. Xpert Ultra was not statistically superior to Xpert for the diagnosis of tuberculous meningitis in HIV-uninfected and HIV-infected adults. A negative Xpert Ultra or Xpert test does not rule out tuberculous meningitis. New diagnostic strategies are urgently required. Wellcome Trust and the Foundation for Innovative New Diagnostics.

Somatic mutations in te t methylcytosine dioxygenase 2 ( TET2 ), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies 1 – 7 . In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage 1 , 4 , 8 , 9 . However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2 −/− mice 8 , 9 and humans with TET2 mutations 1 , 3 , 5 – 7 , suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2 −/− mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2 −/− mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2 −/− mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies. Microbial signals are crucial to the development of pre-leukaemic myeloproliferation, which can be induced by disrupting the intestinal barrier or by introducing systemic bacterial stimuli in Tet2 -deficient mice.

SIRT1 activators in diabetes SIRT1, an NAD + -dependent deacetylase that acts on proteins involved in cellular regulation, has been implicated in longevity and as a mediator of the beneficial effects of calorie restriction. A new screening programme has identified a series of small-molecule SIRT1 activators that are structurally unlike, and 1,000-fold more potent than, resveratrol, the well-known SIRT1 activator found in red wine. These new compounds improve metabolic function in animal models of diabetes and obesity, suggesting that they may have therapeutic potential in type 2 diabetes and insulin resistance. This work describes the identification and characterization of novel small molecule activators of SIRT1, an NAD + -dependent deacetylase that mediates the beneficial effects of caloric restriction. These small molecules are structurally unrelated to, and much more potent than, resveratrol, and improve metabolic function in animal models of diabetes and obesity. Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes 1 , 2 . SIRT1, an NAD + -dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity 3 , 4 , 5 , 6 , 7 , 8 , 9 . Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival 10 , 11 , 12 , 13 , 14 . Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme–peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

Expected treatment effectiveness from medications can be diminished due to suboptimal adherence. Medication nonadherence has been linked to pill burden from the quantity of medications; however, medication regimens with similar quantities of medications vary in complexity due to multiple dosage forms, frequency of dosing, and additional usage directions. Thus, a simple medication count ignores medication regimen complexity, especially as it pertains to a patient-level perspective that includes prescription and over-the-counter medications. A gap exists in the study of a patient-level medication regimen complexity metric across disease-specific populations. The goal of this study was to implement the quantitative Medication Regimen Complexity Index (MRCI) at the patient level in defined populations with chronic disease (geriatric depression, HIV, diabetes mellitus, and hypertension). Patient-level medication regimen complexity included all prescribed medications and over-the-counter medications documented in the electronic medication list. Using electronic medical records at the University of Colorado Hospital ambulatory clinics, we sampled 4 retrospective cohorts of adult patients in active care in 2011 with a qualifying medical diagnosis and prescribed disease-specific medication. Samples were randomly selected from all qualifying patients; de-identified information was coded using the MRCI. Cohort-defining disease-specific prescription medications (eg, antidepressants for the depression-defined cohort) contributed <20% to the total patient-level complexity MRCI score; the MRCI score was dominated by complexity associated with all other prescription medications. Within disease-specific cohorts, MRCI scores differentiated patients with the highest and lowest medication counts, comorbidity counts, and the Charlson comorbidity index scores. For example, geriatric depression patients had a highest quartile mean MRCI score of 41 and a lowest quartile mean MRCI score of 13. Between disease-specific cohorts, high and low MRCI scores differed because each cohort had its own MRCI ranges. For example, highest quartile MRCI scores varied from a mean MRCI score of 41 (geriatric depression) to 30 (hypertension); lowest quartile scores ranged from a mean MRCI score of 7 (hypertension and HIV) to 13 (geriatric depression). MRCI components of dosing frequency and prescribed medications outside of the cohort-defining disease medications contributed the most to the patient-level scores. Thus, chronic disease management programs may want to consider all medications that patients are taking and examine ways to reduce complexity, such as reducing multiple dosing frequencies when possible. MRCI scores differentiated high and low patient-level complexity measures, representing possible utility as a prospective tool to identify target patients for intervention. Future work includes simplifying the MRCI and enhancing the scores with medication risk factors, as well as explicitly linking to adherence and health services.

Protein synthesis is frequently deregulated during tumorigenesis. However, the precise contexts of selective translational control and the regulators of such mechanisms in cancer is poorly understood. Here, we uncovered CNOT3, a subunit of the CCR4-NOT complex, as an essential modulator of translation in myeloid leukemia. Elevated CNOT3 expression correlates with unfavorable outcomes in patients with acute myeloid leukemia (AML). CNOT3 depletion induces differentiation and apoptosis and delayed leukemogenesis. Transcriptomic and proteomic profiling uncovers c-MYC as a critical downstream target which is translationally regulated by CNOT3. Global analysis of mRNA features demonstrates that CNOT3 selectively influences expression of target genes in a codon usage dependent manner. Furthermore, CNOT3 associates with the protein network largely consisting of ribosomal proteins and translation elongation factors in leukemia cells. Overall, our work elicits the direct requirement for translation efficiency in tumorigenesis and propose targeting the post-transcriptional circuitry via CNOT3 as a therapeutic vulnerability in AML. Here the authors uncovered CNOT3, a subunit of the CCR4-NOT complex, as an essential modulator of translation in leukemia. The work pointed to the potential of targeting the posttranscriptional circuitry via CNOT3 as a therapeutic vulnerability in acute myeloid leukemia.

MYD88 signalling in cancer RNA interference screening and high-throughput RNA resequencing have been used to reveal oncogenic mutations in the signalling adapter MYD88 in human lymphomas. One amino acid substitution, L265P, was found in 29% of biopsies from patients with the activated B-cell-like subtype of diffuse large B-cell lymphoma. The same mutation was observed with lower frequency in mucosa-associated lymphoid tissue lymphomas. MYD88 mediates signalling by Toll-like receptors, and the mutations, most of which affect the same amino acid, were shown to activate the pathway and promote cancer cell survival. This study finds frequent mutations in MYD88 in the activated B-cell-like subtype of diffuse large B-cell lymphoma and, with lower frequency, in mucosa-associated lymphoid tissue lymphomas. MYD88 mediates signalling by Toll-like receptors, and the mutations, most of which affect the same amino acid, are shown to activate the pathway and promote cancer cell survival. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy 1 . Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling 2 , 3 , and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt’s lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

B-cell receptors and lymphoma A link between B-cell-receptor (BCR) signalling and human B-cell lymphomas has been inferred from the study of immunoglobulin genes in human lymphomas and from work on mouse models, but more evidence is required to confirm an oncogenic role. New work from Davis et al . shows that chronic activation of BCR signalling is required for the survival of the ABC DLBCL subset of B-cell lymphomas. Somatic mutations in CD29A and CD79B found in these lymphomas appear to contribute to BCR activation. The role of B-cell-receptor (BCR) signalling in human B cell lymphomas has been a long-standing question, with genetic and functional evidence for its oncogenic role in human lymphomas lacking. Here, a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like subtype of diffuse large B-cell lymphoma is described and analysed, with potential implications for future therapeutic strategies. A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas 1 , 2 and by engineering mouse models 3 , but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of ‘chronic active’ BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-κB pathway activity and survival in ABC DLBCL 4 . Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-κB 5 , but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton’s tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-κ, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules 6 of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt’s lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Blast traumatic brain injury (bTBI) presents a serious threat to military personnel and often results in psychiatric conditions related to limbic system dysfunction. In this study, the functional outcomes for anxiety- and depressive-like behaviors and neuronal activation were evaluated in male and female mice after exposure to an Advanced Blast Simulator (ABS) shock wave. Mice were placed in a ventrally exposed orientation inside of the ABS test section and received primary and tertiary shock wave insults of approximately 15 psi peak pressure. Evans blue staining indicated cases of evident blood-brain barrier breach in the superficial cerebral cortex four, but not 24 hours after blast, but sampling suggested the severity was variable. Behavioral testing with the elevated plus maze (EPM) or elevated zero maze (EZM), sucrose preference test (SPT), and tail suspension test (TST) or forced swim test (FST) were conducted 8 days ̶ 3.5 weeks after shock wave exposure. There was a sex difference, but no injury effect, for distance travelled in the EZM where female mice travelled significantly farther than males. The SPT and FST did not indicate group differences; however, injured mice were less immobile than sham mice during the TST. In summary, mice did not display significantly increased anxiety- or depressive-like behavior after single shock wave exposure, but demonstrated less immobility during the TST; possibly indicating more agitated behavior. In a separate cohort of animals, the expression of the immediate early gene, c-Fos, was detected four hours after undergoing the same procedures. No differences in c-Fos expression were apparent in the cerebral cortex, but female mice in general displayed enhanced c-Fos activation in the paraventricular nucleus of the thalamus (PVT) compared to male mice. In the amygdala, more c-Fos-positive cells were observed in injured animals compared to sham mice. The observed sex differences in the PVT and c-Fos activation in the amygdala may correlate with the reported hyperactivity of females post-injury. This study demonstrates, albeit with mild effects, behavioral and neuronal activation correlates in female rodents after blast injury that could be relevant to the incidence of increased post-traumatic stress disorder in women.