Folate as a potential treatment for lethal ventricular arrhythmias in TANGO2-deficiency disorder.

TANGO2-deficiency disorder (TDD) is an autosomal-recessive genetic disease caused by biallelic loss-of-function variants in the TANGO2 gene. TDD-associated cardiac arrhythmias are recalcitrant to standard antiarrhythmic medications and constitute the leading cause of death. Disease modeling for TDD has been primarily carried out using human dermal fibroblast and, more recently, in Drosophila by multiple research groups. No human cardiomyocyte system has been reported, which greatly hinders the investigation and understanding of TDD-associated arrhythmias. Here, we established potentially novel patient-derived induced pluripotent stem cell differentiated cardiomyocyte (iPSC-CM) models that recapitulate key electrophysiological abnormalities in TDD. These electrophysiological abnormalities were rescued in iPSC-CMs with either adenoviral expression of WT-TANGO2 or correction of the pathogenic variant using CRISPR editing. Our natural history study in patients with TDD suggests that the intake of multivitamin/B complex greatly diminished the risk of cardiac crises in patients with TDD. In agreement with the clinical findings, we demonstrated that high-dose folate (vitamin B9) virtually abolishes arrhythmias in TDD iPSC-CMs and that folate's effect was blocked by the dihydrofolate reductase inhibitor methotrexate, supporting the need for intracellular folate to mediate antiarrhythmic effects. In summary, data from TDD iPSC-CM models together with clinical observations support the use of B vitamins to mitigate cardiac crises in patients with TDD, providing potentially life-saving treatment strategies during life-threatening events.

Characteristics of logs with signs of oviposition by the polyphagous xylophage Asian longhorned beetle (Coleoptera: Cerambycidae).

During the eradication program undertaken against Anoplophora glabripennis (Motschulsky) in the Greater Toronto Area, information was collected on the numerous signs of injury found on wounded trees. Herein, we used a portion of this information to assess the characteristics of logs with signs of oviposition (i.e., pits). Specifically, we related the basal diameter, type (log bole vs. log branch), height above ground, and branch hierarchy level of logs with pits to tree size (i.e., height and diameter at breast height) and level of infestation intensity. In general, pits were concentrated on logs from the bole and branches that were 8-14 cm in diameter in the lower 8 m of the bole and in the first 2 levels of the branching hierarchy. Oviposition pit location was strongly influenced by tree size-both height and diameter at breast height, with more pits on the lower bole in small trees and then higher on the bole and into the branches as tree size increased. As tree-level infestation intensity increased, pits were found on both larger and smaller diameter portions of the trees, presumably as preferred oviposition sites became saturated. These findings can improve the efficacy of surveillance activities for A. glabripennis.

Adaptive immune responses to two-dose COVID-19 vaccine series in healthy Canadian adults ≥ 50 years: a prospective, observational cohort study.

To evaluate immune responses to COVID-19 vaccines in adults aged 50 years and older, spike protein (S)-specific antibody concentration, avidity, and function (via angiotensin-converting enzyme 2 (ACE2) inhibition surrogate neutralization and antibody dependent cellular phagocytosis (ADCP)), as well as S-specific T cells were quantified via activation induced marker (AIM) assay in response to two-dose series. Eighty-four adults were vaccinated with either: mRNA/mRNA (mRNA-1273 and/or BNT162b2); ChAdOx1-S/mRNA; or ChAdOx1-S/ChAdOx1-S. Anti-S IgG concentrations, ADCP scores and ACE2 inhibiting antibody concentrations were highest at one-month post-second dose and declined by four-months post-second dose for all groups. mRNA/mRNA and ChAdOx1-S/mRNA schedules had significantly higher antibody responses than ChAdOx1-S/ChAdOx1-S. CD8+ T-cell responses one-month post-second dose were associated with increased ACE2 surrogate neutralization. Antibody avidity (total relative avidity index) did not change between one-month and four-months post-second dose and did not significantly differ between groups by four-months post-second dose. In determining COVID-19 correlates of protection, a measure that considers both antibody concentration and avidity should be considered.

Selective CDK7 Inhibition Suppresses Cell Cycle Progression and MYC Signaling While Enhancing Apoptosis in Therapy-resistant Estrogen Receptor-positive Breast Cancer.

PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.

Cancer-Specific Alterations in Nuclear Matrix Proteins Determined by Multi-omics Analyses of Ductal Carcinoma in Situ.

Breast cancer (BC) is the most common cancer affecting women in the United States. Ductal carcinoma in situ (DCIS) is the earliest identifiable pre-invasive BC lesion. Estimates show that 14 to 50% of DCIS cases progress to invasive BC. Our objective was to identify nuclear matrix proteins (NMP) with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast reduction mammoplasty and a contralateral breast explant using mass spectrometry and RNA sequencing to accurately identify aggressive DCIS. Sixty NMPs were significantly differentially expressed between the DCIS and non-diseased breast epithelium in an isogenic contralateral pair of patient-derived extended explants. Ten of the sixty showed significant mRNA expression level differences that matched the protein expression. These 10 proteins were similarly expressed in non-diseased breast reduction cells. Three NMPs (RPL7A, RPL11, RPL31) were significantly upregulated in DCIS and all other BC stages compared to the matching contralateral breast culture and an unrelated non-diseased breast reduction culture. RNA sequencing analyses showed that these three genes were upregulated increasingly with BC progression. Finally, we identified three NMPs (AHNAK, CDC37 and DNAJB1) that were significantly downregulated in DCIS and all other BC stages compared to the isogenically matched contralateral culture and the non-diseased breast reduction culture using both proteomics and RNA sequencing techniques.

PI3Kγ maintains the self-renewal of acute myeloid leukemia stem cells by regulating the pentose phosphate pathway.

ABSTRACT: Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating from transformed hematopoietic stem or progenitor cells. AML prognosis remains poor owing to resistance and relapse driven by leukemia stem cells (LSCs). Targeting molecules essential for LSC function is a promising therapeutic approach. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is often dysregulated in AML. We found that although PI3Kγ is highly enriched in LSCs and critical for self-renewal, it was dispensable for normal hematopoietic stem cells. Mechanistically, PI3Kγ-AKT signaling promotes nuclear factor erythroid 2-related factor 2 (NRF2) nuclear accumulation, which induces 6-phosphogluconate dehydrogenase (PGD) and the pentose phosphate pathway, thereby maintaining LSC stemness. Importantly, genetic or pharmacological inhibition of PI3Kγ impaired expansion and stemness of murine and human AML cells in vitro and in vivo. Together, our findings reveal a key role for PI3Kγ in selectively maintaining LSC function by regulating AKT-NRF2-PGD metabolic pathway. Targeting the PI3Kγ pathway may, therefore, eliminate LSCs without damaging normal hematopoiesis, providing a promising therapeutic strategy for AML.

Generation of five induced pluripotent stem cell lines from patients with MECP2 Duplication Syndrome.

MECP2 Duplication Syndrome (MDS) is a rare, severe neurodevelopmental disorder arising from duplications in the Xq28 region containing the MECP2 gene that predominantly affects males. We generated five human induced pluripotent stem cell (iPSC) lines from the fibroblasts of individuals carrying between 0.355 and 11.2 Mb size duplications in the chromosomal locus containing MECP2. All lines underwent extensive testing to confirm MECP2 duplication and iPSC-related features such as morphology, pluripotency markers, and trilineage differentiation potential. These lines are a valuable resource for molecular and functional studies of MDS as well as screening for a variety of therapeutic approaches.

Potential side effects of currently available pharmacotherapies in male lower urinary tract symptoms suggestive of benign prostatic hyperplasia.

INTRODUCTION: The drug classes of α1-adrenoceptor antagonists, 5α-reductase inhibitors, and phosphodiesterase type 5 inhibitors are guideline-recommended treatments of lower urinary tract symptoms suggestive of benign prostatic hyperplasia; muscarinic receptor antagonists and ß3-adrenoceptor agonists are also recommended if storage symptoms are insufficiently addressed with one of the other three drug classes. AREAS COVERED: We provide a narrative review (no formalized literature searches performed) of the tolerability of these drug classes with emphasis on the more recently introduced medications, on combination treatment, and on more lately emerging risks. EXPERT OPINION/COMMENTARY: The tolerability profiles are distinct between drug classes but, with few exceptions, similar within a drug class. Within a drug, formulations with longer duration of action tend to have better tolerability. Efficacy gains using combination treatment at least partly come at a cost of lesser tolerability. Greater susceptibility to experience adverse events based on age, comorbidities, and comedications appears conceptually important but remains under-investigated in this therapeutic area.

Several classes of medicines are available to treat male lower urinary tract symptoms that are believed to result from an enlarged prostate. These include α₁-adrenoceptor antagonists (α-blockers), 5α-reductase inhibitors (ARI), and phosphodiesterase type 5 inhibitors (PDEI); muscarinic receptor antagonists and ß₃-adrenoceptor agonists are additionally used in men that have persisting storage symptoms upon treatment with the former three drug classes. Each drug class has a distinct tolerability profile. Within a drug class, medicines with a longer duration of action, either intrinsically or due to specific drug formulations, tend to have better tolerability. Men with greater age, comorbidities, and comedications may be at greater risk of experiencing side effects when medically treating their lower urinary tract symptoms. While combination of members of multiple drug classes may increase efficacy, this often comes at the price of experiencing more side effects. The relative benefit/risk ratio needs to be individually analyzed in each patient.

How does the lower urinary tract contribute to bladder sensation? ICI-RS 2023.

AIM: Bladder sensation is critical for coordinating voluntary micturition to maintain healthy bladder function. Sensations are initiated by the activation of sensory afferents that innervate throughout the bladder wall. However, the physiological complexity that underlies the initiation of bladder sensory signaling in health and disease remains poorly understood. This review summarises the latest knowledge of the mechanisms underlying the generation of bladder sensation and identifies key areas for future research. METHODS: Experts in bladder sensory signaling reviewed the literature on how the lower urinary tract contributes to bladder sensation and identified key research areas for discussion at the 10th International Consultation on Incontinence-Research Society. RESULTS: The importance of bladder sensory signals in maintaining healthy bladder function is well established. However, better therapeutic management of bladder disorders with exaggerated bladder sensation, including overactive bladder syndrome (OAB) and interstitial cystitis/bladder pain syndrome (IC/BPS) is limited by a lack of knowledge in a number of key research areas including; the contribution of different nerves (pudendal, pelvic, hypogastric) to filling sensations in health and disease; the relative contribution of stretch sensitive (muscular) and stretch-insensitive (mucosal) afferents to bladder sensation in health and disease; the direct and indirect contributions of the muscularis mucosae to bladder contraction and sensation; and the impact of manipulating urothelial release factors on bladder sensation. CONCLUSION: Disturbances in bladder sensory signaling can have severe consequences for bladder sensation and function including the development of OAB and IC/BPS. Advancing therapeutic treatments for OAB and IC/BPS requires a deeper understanding of the mechanisms underlying the generation of bladder sensation, and key areas for future research have been identified.

A novel pathogenic mutation of MeCP2 impairs chromatin association independent of protein levels.

Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.

Lysine vitcylation is a novel vitamin C-derived protein modification that enhances STAT1-mediated immune response.

Vitamin C (vitC) is a vital nutrient for health and also used as a therapeutic agent in diseases such as cancer. However, the mechanisms underlying vitC's effects remain elusive. Here we report that vitC directly modifies lysine without enzymes to form vitcyl-lysine, termed "vitcylation", in a dose-, pH-, and sequence-dependent manner across diverse proteins in cells. We further discover that vitC vitcylates K298 site of STAT1, which impairs its interaction with the phosphatase PTPN2, preventing STAT1 Y701 dephosphorylation and leading to increased STAT1-mediated IFN pathway activation in tumor cells. As a result, these cells have increased MHC/HLA class-I expression and activate immune cells in co-cultures. Tumors collected from vitC-treated tumor-bearing mice have enhanced vitcylation, STAT1 phosphorylation and antigen presentation. The identification of vitcylation as a novel PTM and the characterization of its effect in tumor cells opens a new avenue for understanding vitC in cellular processes, disease mechanisms, and therapeutics.

Interventions to improve outcomes for caregivers of patients with advanced cancer: a meta-analysis.

BACKGROUND: Family caregivers of patients with advanced cancer often have poor quality of life (QOL) and mental health. We examined the effectiveness of interventions offering support for caregivers of patients with advanced cancer on caregiver QOL and mental health outcomes. METHODS: We searched Ovid MEDLINE, EMBASE, Cochrane CENTRAL, and Cumulative Index to Nursing and Allied Health Literature databases from inception through June 2021. Eligible studies reported on randomized controlled trials for adult caregivers of adult patients with advanced cancer. Meta-analysis was conducted for primary outcomes of QOL, physical well-being, mental well-being, anxiety, and depression, from baseline to follow-up of 1-3 months; secondary endpoints were these outcomes at 4-6 months and additional caregiver burden, self-efficacy, family functioning, and bereavement outcomes. Random effects models were used to generate summary standardized mean differences (SMD). RESULTS: Of 12 193 references identified, 56 articles reporting on 49 trials involving 8554 caregivers were eligible for analysis; 16 (33%) targeted caregivers, 19 (39%) patient-caregiver dyads, and 14 (29%) patients and their families. At 1- to 3-month follow-up, interventions had a statistically significant effect on overall QOL (SMD = 0.24, 95% confidence interval [CI] = 0.10 to 0.39); I2 = 52.0%), mental well-being (SMD = 0.14, 95% CI = 0.02 to 0.25; I2 = 0.0%), anxiety (SMD = 0.27, 95% CI = 0.06 to 0.49; I2 = 74.0%), and depression (SMD = 0.34, 95% CI = 0.16 to 0.52; I2 = 64.4) compared with standard care. In narrative synthesis, interventions demonstrated improvements in caregiver self-efficacy and grief. CONCLUSIONS: Interventions targeting caregivers, dyads, or patients and families led to improvements in caregiver QOL and mental health. These data support the routine provision of interventions to improve well-being in caregivers of patients with advanced cancer.

Association of diabetes, hypertension, and their combination with basal symptoms and treatment responses in overactive bladder patients.

Introduction: Pelvic hypoperfusion caused by atherosclerosis has been proposed as a cause of lower urinary tract dysfunction including overactive bladder syndrome (OAB). Limited data indicate that OAB patients with concomitant diabetes or hypertension, known risk factors of atherosclerosis, may exhibit greater baseline OAB symptoms and slightly smaller therapeutic responses to treatment, but the impact of a combined presence of diabetes and hypertension has not been reported. Therefore, we have explored whether the combined presence of both comorbidities is associated with greater baseline OAB symptoms than that of either comorbidity alone. Secondary questions were exploration of the impact of either comorbidity on baseline symptoms, and of the impact of either comorbidity alone and their combination on therapeutic responses. Methods: Data from two non-interventional studies applying treatment with propiverine ER 30 or 45 mg/d for 12 weeks were analyzed. Results: Number of urgency episodes in the combination group was greater than with each comorbidity alone. The impact of comorbidities on baseline intensity of incontinence, frequency or nocturia or Patient Perception of Bladder Condition was less consistent or absent. Either comorbidity alone was associated with a smaller % improvement of symptoms, and their combination had a greater effect than either alone. However, all attenuations associated with comorbidity were small relative to the overall improvement. Conclusions: We conclude that comorbidities of diabetes and hypertension have detectable effects on OAB symptoms and treatment responses, but the small magnitude of these alterations does not justify changing existing paradigms for the clinical management of OAB.

Alcohol Use, HIV Stigma and Quality of Life Among Alcohol Consuming Men Living with HIV in India: A Mediation Analysis.

This study examined the hypothesis that HIV-related stigma mediates the effect of alcohol use on health-related quality of life (HRQoL) among alcohol consuming Indian men living with HIV (PLWH). The study used baseline data from a randomized controlled clinical trial entitled 'Alcohol and ART adherence: Assessment, Intervention, and Modeling in India. Participants completed surveys assessing demographic characteristics, alcohol use, HIV-related stigma, HRQoL. Mediation analysis was conducted to establish the mediation effect of HIV-related stigma on the relationship between alcohol use and HRQoL. The final mediation model showed that the effect of alcohol use on HRQoL were partially mediated by overall HIV-related stigma. Specially, 27.1% of the effects of alcohol use on HRQoL was mediated through overall HIV stigma. In the HIV stigma subdomain analyses, negative self-image mediated 14% and concerns with public attitudes (anticipated stigma) mediated 17.3% of the effect of alcohol use on HRQoL respectively. The findings suggest that efforts to reduce the negative impact of alcohol use on HRQoL and improve HRQoL among PLWH should include interventions addressing both alcohol use and specific forms of HIV-related stigma.

RESUMEN: Este estudio examinó la hipótesis de que el estigma relacionado con el VIH mediaría el efecto del consumo de alcohol en la calidad de vida relacionada con la salud entre hombres indios que consumen alcohol y viven con VIH. El estudio utilizó datos de línea base de un ensayo clínico aleatorizado controlado titulado "Consumo de alcohol y adherencia al TAR: evaluación, intervención y modelización en India". Los participantes completaron encuestas que evaluaron características demográficas, consumo de alcohol, estigma relacionado con el VIH y calidad de vida relacionada con la salud (CVRS). Se realizó un análisis de mediación para establecer el efecto de la mediación del estigma relacionado con el VIH en la relación entre el consumo de alcohol y la CVRS. El modelo final de mediación mostró que el efecto del consumo de alcohol en la CVRS fue parcialmente mediado por el estigma general relacionado con el VIH. Específicamente, el 27,1% de los efectos del consumo de alcohol en la CVRS se medió a través del estigma general relacionado con el VIH. En los análisis de subdominios del estigma del VIH, la imagen negativa de sí mismo medió el 14% y las preocupaciones sobre las actitudes públicas (estigma anticipado) mediaron el 17,3% del efecto del consumo de alcohol en la CVRS, respectivamente. Los resultados sugieren que los esfuerzos para reducir el impacto negativo del consumo de alcohol en la CVRS y mejorar la CVRS entre las personas que viven con VIH deberían incluir intervenciones que aborden tanto el consumo de alcohol como formas específicas de estigma relacionado con el VIH.

DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells.

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.

PI3Kß controls immune evasion in PTEN-deficient breast tumours.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kß isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kß activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kß led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kß inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kß inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kß controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kß inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic.

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.