Genome-wide investigations on regulatory functions of RECQ1 helicase.

DNA helicase RECQ1 (also known as RECQL or RECQL1) is a candidate breast cancer susceptibility gene significantly correlated with clinical outcomes of sporadic breast cancer patients. Prior studies have suggested that RECQ1 maintains genomic stability by regulating a wide variety of core cellular functions including DNA replication, DNA damage response, and transcription. However, it is unclear which, if any, of these are the primary functions of RECQ1 as related to its role in suppressing breast cancer. We describe here an unbiased integrative genomics approach that enabled us to discover a previously unknown regulatory role of RECQ1 in promoting Estrogen Receptor alpha (ERα) expression and the expression of specific ERα target genes in ER positive breast cancer cells. We discuss potential future applications of similar experimental strategies in advancing the mechanistic understanding and elucidating specific new details of genome-wide functions of RECQ1 and other RecQ helicases in maintaining genomic stability and preventing cancer.

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.

Mn(III) Porphyrin, MnTnBuOE-2-PyP5+, Commonly Known as a Mimic of Superoxide Dismutase Enzyme, Protects Cardiomyocytes from Hypoxia/Reoxygenation Induced Injury via Reducing Oxidative Stress.

Myocardial ischemia-reperfusion injury (I/R) causes damage to cardiomyocytes through oxidative stress and apoptosis. We investigated the cardioprotective effects of MnTnBuOE-2-PyP5+ (BMX-001), a superoxide dismutase mimic, in an in vitro model of I/R injury in H9c2 cardiomyocytes. We found that BMX-001 protected against hypoxia/reoxygenation (H/R)-induced oxidative stress, as evident by a significant reduction in intracellular and mitochondrial superoxide levels. BMX-001 pre-treatment also reduced H/R-induced cardiomyocyte apoptosis, as marked by a reduction in TUNEL-positive cells. We further demonstrated that BMX-001 pre-treatment significantly improved mitochondrial function, particularly O2 consumption, in mouse adult cardiomyocytes subjected to H/R. BMX-001 treatment also attenuated cardiolipin peroxidation, 4-hydroxynonenal (4-HNE) level, and 4-HNE adducted proteins following H/R injury. Finally, the pre-treatment with BMX-001 improved cell viability and lactate dehydrogenase (LDH) activity in H9c2 cells following H/R injury. Our findings suggest that BMX-001 has therapeutic potential as a cardioprotective agent against oxidative stress-induced H/R damage in H9c2 cardiomyocytes.

The ATX-LPA Axis Regulates Vascular Permeability during Cerebral Ischemic-Reperfusion.

Endothelial permeability is a major complication that must be addressed during stroke treatment. Study of the mechanisms underlying blood−brain barrier (BBB) disruption and management of the hypoxic stress-induced permeability of the endothelium following reperfusion are both urgently needed for stroke management. Lysophosphatidic acid (LPA), a bioactive lipid essential for basic cellular functions, causes unfavorable outcomes during stroke progression. LPA-producing enzyme autotaxin (ATX) is regulated in ischemic stroke. We used an electrical cell-substrate impedance sensor (ECIS) to measure endothelial permeability. Mitochondrial bioenergetics were obtained using a Seahorse analyzer. AR-2 probe fluorescence assay was used to measure ATX activity. LPA increased endothelial permeability and reduced junctional protein expression in mouse brain microvascular endothelial cells (MBMEC). LPA receptor inhibitors Ki16425 and AM095 attenuated the LPA-induced changes in the endothelial permeability and junctional proteins. LPA significantly diminished mitochondrial function in MBMEC. ATX was upregulated (p < 0.05) in brain microvascular endothelial cells under hypoxic reperfusion. ATX activity and permeability were attenuated with the use of an ATX inhibitor in a mouse stroke model. The upregulation of ATX with hypoxic reperfusion leads to LPA production in brain endothelial cells favoring permeability. Inhibition of the ATX−LPA−LPAR axis could be therapeutically targeted in stroke to achieve better outcomes.

Rotenone-Induced 4-HNE Aggresome Formation and Degradation in HL-1 Cardiomyocytes: Role of Autophagy Flux.

Reactive oxygen species (ROS) cause oxidative stress by generating reactive aldehydes known as 4-hydroxynonenal (4-HNE). 4-HNE modifies protein via covalent adduction; however, little is known about the degradation mechanism of 4-HNE-adducted proteins. Autophagy is a dynamic process that maintains cellular homeostasis by removing damaged organelles and proteins. In this study, we determined the role of a superoxide dismutase (SOD) mimetic MnTnBuOE-2-PyP5+ (MnP, BMX-001) on rotenone-induced 4-HNE aggresome degradation in HL-1 cardiomyocytes. A rotenone treatment (500 nM) given for 24 h demonstrated both increased ROS and 4-HNE aggresome accumulation in HL-1 cardiomyocytes. In addition, cardiomyocytes treated with rotenone displayed an increase in the autophagy marker LC3-II, as shown by immunoblotting and immunofluorescence. A pre-treatment with MnP (20 µM) for 24 h attenuated rotenone-induced ROS formation. An MnP pre-treatment showed decreased 4-HNE aggresomes and LC3-II formation. A rotenone-induced increase in autophagosomes was attenuated by a pre-treatment with MnP, as shown by fluorescent-tagged LC3 (tfLC3). Rotenone increased tubulin hyperacetylation through the ROS-mediated pathway, which was attenuated by MnP. The disruption of autophagy caused HL-1 cell death because a 3-methyladenine inhibitor of autophagosomes caused reduced cell death. Yet, rapamycin, an inducer of autophagy, increased cell death. These results indicated that a pre-treatment with MnP decreased rotenone-induced 4-HNE aggresomes by enhancing the degradation process.

A new sub-pathway of long-patch base excision repair involving 5' gap formation.

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.

The DNA repair helicase RECQ1 has a checkpoint-dependent role in mediating DNA damage responses induced by gemcitabine.

The response of cancer cells to therapeutic drugs that cause DNA damage depends on genes playing a role in DNA repair. RecQ-like helicase 1 (RECQ1), a DNA repair helicase, is critical for genome stability, and loss-of-function mutations in the RECQ1 gene are associated with increased susceptibility to breast cancer. In this study, using a CRISPR/Cas9-edited cell-based model, we show that the genetic or functional loss of RECQ1 sensitizes MDA-MB-231 breast cancer cells to gemcitabine, a nucleoside analog used in chemotherapy for triple-negative breast cancer. RECQ1 loss led to defective ATR Ser/Thr kinase (ATR)/checkpoint kinase 1 (ChK1) activation and greater DNA damage accumulation in response to gemcitabine treatment. Dual deficiency of MUS81 structure-specific endonuclease subunit (MUS81) and RECQ1 increased gemcitabine-induced, replication-associated DNA double-stranded breaks. Consistent with defective checkpoint activation, a ChK1 inhibitor further sensitized RECQ1-deficient cells to gemcitabine and increased cell death. Our results reveal an important role for RECQ1 in controlling cell cycle checkpoint activation in response to gemcitabine-induced replication stress.

Stress Marks on the Genome: Use or Lose?

Oxidative stress and the resulting damage to DNA are inevitable consequence of endogenous physiological processes further amplified by cellular responses to environmental exposures. If left unrepaired, oxidative DNA lesions can block essential processes such as transcription and replication or can induce mutations. Emerging data also indicate that oxidative base modifications such as 8-oxoG in gene promoters may serve as epigenetic marks, and/or provide a platform for coordination of the initial steps of DNA repair and the assembly of the transcriptional machinery to launch adequate gene expression alterations. Here, we briefly review the current understanding of oxidative lesions in genome stability maintenance and regulation of basal and inducible transcription.

Transcriptome guided identification of novel functions of RECQ1 helicase.

Gene expression changes in the functional absence of a specific RecQ protein, and how that relates to disease outcomes including cancer predisposition and premature aging in RecQ helicase associated syndromes, are poorly understood. Here we describe detailed experimental strategy for identification of RECQ1-regulated transcriptome that led us to uncover a novel association of RECQ1 in regulation of cancer cell migration and invasion. We initiated a focused study to determine whether RECQ1, the most abundant RecQ protein in humans, alters gene expression and also investigated whether RECQ1 binds with G4 motifs predicted to form G-quadruplex structures in the target gene promoters. Rescue of mRNA expression of select RECQ1-downregulated genes harboring G4 motifs required wild-type RECQ1 helicase. However, some RECQ1-regulated genes are also regulated by BLM and WRN proteins regardless of the presence or absence of G4 motifs. The approach described here is applicable for systematic comparison of gene expression signatures of individual RecQ proteins in isogenic background, and to elucidate their participation in transcription regulation through G-quadruplex recognition and/or resolution. Such strategies might also reveal molecular pathways that drive the pathogenesis of cancer and other diseases in specific RecQ deficiency.

Menstrual pattern among adolescent girls of Pokhara Valley: a cross sectional study.

BACKGROUND: Menstruation is a normal physiological process occurring every month throughout the reproductive age of the females. However, significant variation in menstrual pattern is observed among adolescents. The objective of this study was to determine menstrual pattern among adolescent girls of Pokhara Valley. METHOD: A cross sectional study was conducted in seven schools of Pokhara Valley. A total of 260 adolescent girls were included in the study. Girls were requested to complete questionnaire containing 19 items. Selected girls voluntarily agreed to answer questionnaire under the supervision of the researcher and then the data was analyzed. RESULTS: Mean age of the girls was 14.0 ± 1.3 years. Mean age at menarche was 12.2 ± 0.9 years. The mean cycle length of the subjects was 34.8 ± 11.8 days. It was observed that as many as 167(64.2%) girls had irregular menstrual cycle and significant association was found between regularity of menstruation and ethnicity. Seven (2.7%) girls had a menstrual cycle length shorter than 21 days, 60(23.1%) had cycle longer than 35 days and 193(74.2%) had a normal cycle length between 21 and 35 days. Majority 231(88.8%) had normal duration of menstruation. Dysmenorrhoea was reported by more than half of the girls and significant association was found between severity of dysmenorrhoea with school absenteeism and treatment needed. CONCLUSION: Dysmenorrhoea was the most common menstrual problem among adolescent girls. Girls' school attendance was affected due to menstrual pain. Girls with severe dysmenorrhea needed medical treatment. Irregular menstruation was reported by more than half of the girls and significant association was found with the ethnicity.

RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin.

RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologues, have remained poorly understood. We show that RECQ1 associates with FEN-1 (flap endonuclease-1) in nuclear extracts and exhibits direct protein interaction in vitro. Recombinant RECQ1 significantly stimulated FEN-1 endonucleolytic cleavage of 5'-flap DNA substrates containing non-telomeric or telomeric repeat sequence. RECQ1 and FEN-1 were constitutively present at telomeres and their binding to the telomeric chromatin was enhanced following DNA damage. Telomere residence of FEN-1 was dependent on RECQ1 since depletion of RECQ1 reduced FEN-1 binding to telomeres in unperturbed cycling cells. Our results confirm a conserved collaboration of human RecQ helicases with FEN-1 and suggest both overlapping and specialized roles of RECQ1 in the processing of DNA structure intermediates proposed to arise during replication, repair and recombination.

Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome.

Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.

Cellular deficiency of Werner syndrome protein or RECQ1 promotes genotoxic potential of hydroquinone and benzo[a]pyrene exposure.

The 5 known RecQ helicases in humans (RECQ1, BLM, WRN, RECQL4, and RECQ5) have demonstrated roles in diverse genome maintenance mechanisms but their functions in safeguarding the genome from environmental toxicants are poorly understood. Here, we have evaluated a potential role of WRN (mutated in Werner syndrome) and RECQ1 (the most abundant homolog of WRN) in hydroquinone (HQ)- and benzo[a]pyrene (BaP)-induced genotoxicity. Silencing of WRN or RECQ1 expression in HeLa cells increased their sensitivity to HQ and BaP but elicited distinct DNA damage response. The RECQ1-depleted cells exhibited increased replication protein A phosphorylation, Chk1 activation, and DNA double-strand breaks (DSBs) as compared to control or WRN-depleted cells following exposure to BaP treatment. The BaP-induced DSBs in RECQ1-depleted cells were dependent on DNA-dependent protein kinase activity. Notably, loss of WRN in RECQ1-depleted cells ameliorated BaP toxicity. Collectively, our results provide first indication of nonredundant participation of WRN and RECQ1 in protection from the potentially carcinogenic effects of BaP and HQ.

Hyper-IgE syndrome with a novel STAT3 mutation-a single center study from India.

BACKGROUND: Hyper IgE syndrome (HIES) is a rare primary immunodeficiency disorder characterized by the triad of elevated IgE and eosinophilia, eczema and recurrent skin and pulmonary infections. Mutation in the STAT3 gene accounts for majority of the autosomal dominant and sporadic forms of HIES. OBJECTIVE: To report clinical and molecular analyses of patients with Hyper IgE syndrome from a single tertiary care center in India. METHODS: Four patients with suspected HIES were studied. Flowcytometry for T(H)17 cell numbers and phosphoSTAT3, and STAT3 gene sequencing were performed. RESULTS: T(H)17 cells were significantly reduced. Mutations were found in the DNA-binding domain in three and a mutation in the transactivation domain in one patient. One of the mutations detected was a novel mutation (g54792 c.1018A> C p.K340Q) in the DNA binding domain. Mycobacterial infection, which is usually not commonly associated with HIES was found in two of our cases, one with a cutaneous abscess in the shoulder, and the other with BCG site reactivation. CONCLUSIONS: A novel mutation in the STAT3 is reported. Mycobacterial infections can be seen in the spectrum of HIES related infections.

Evolving narratives on signal functions for monitoring maternal and newborn health services: A meta-narrative inspired review.

Emergency obstetric care (EmOC) signal functions are a shortlist of key clinical interventions capable of averting deaths from the five main direct causes of maternal mortality; they have been used since 1997 as a part of an EmOC monitoring framework to track the availability of EmOC services in low- and middle-income settings. Their widespread use and proposed adaptation to include other types of care, such as care for newborns, is testimony to their legacy as part of the measurement architecture within reproductive health. Yet, much has changed in the landscape of maternal and newborn health (MNH) since the initial introduction of EmOC signal functions. As part of a project to revise the EmOC monitoring framework, we carried out a meta-narrative inspired review to reflect on how signal functions have been developed and conceptualised over the past two decades, and how different narratives, which have emerged alongside the evolving MNH landscape, have played a role in the conceptualisation of the signal function measurement. We identified three overarching narrative traditions: 1) clinical 2) health systems and 3) human rights, that dominated the discourse and critique around the use of signal functions. Through an iterative synthesis process including 19 final articles selected for the review, we explored patterns of conciliation and areas of contradiction between the three narrative traditions. We summarised five meta-themes around the use of signal functions: i) framing the boundaries; ii) moving beyond clinical capability; iii) capturing the woods versus the trees; iv) grouping signal functions and v) measurement challenges. We intend for this review to contribute to a better understanding of the discourses around signal functions, and to provide insight for the future roles of this monitoring approach for emergency obstetric and newborn care.

Increased utilisation and quality: a focus on inequality and a learning health system approach-explaining Nepal's success in reducing maternal and newborn mortality 2000-2020.

INTRODUCTION: Maternal mortality in Nepal dropped from 553 to 186 per 100 000 live births during 2000-2017 (66% decline). Neonatal mortality dropped from 40 to 21 per 1000 live births during 2000-2018 (48% decline). Stillbirths dropped from 28 to 18 per 1000 births during 2000-2019 (34% decline). Nepal outperformed other countries in these mortality improvements when adjusted for economic growth, making Nepal a 'success'. Our study describes mechanisms which contributed to these achievements. METHODS: A mixed-method case study was used to identify drivers of mortality decline. Methods used included a literature review, key-informant interviews, focus-group discussions, secondary analysis of datasets, and validation workshops. RESULTS: Despite geographical challenges and periods of political instability, Nepal massively increased the percentage of women delivering in health facilities with skilled birth attendance between 2000 and 2019. Although challenges remain, there was also evidence in improved quality and equity-of-access to antenatal care and childbirth services. The study found policymaking and implementation processes were adaptive, evidence-informed, made use of data and research, and involved participants inside and outside government. There was a consistent focus on reducing inequalities. CONCLUSION: Policies and programmes Nepal implemented between 2000 and 2020 to improve maternal and newborn health outcomes were not unique. In this paper, we argue that Nepal was able to move rapidly from stage 2 to stage 3 in the mortality transition framework not because of what they did, but how they did it. Despite its achievements, Nepal still faces many challenges in ensuring equal access to quality-care for all women and newborns.

Immune infiltration, aggressive pathology, and poor survival outcomes in RECQL helicase deficient breast cancers.

RECQL is essential for genomic stability. Here, we evaluated RECQL in 449 pure ductal carcinomas in situ (DCIS), 152 DCIS components of mixed DCIS/invasive breast cancer (IBC) tumors, 157 IBC components of mixed DCIS/IBC and 50 normal epithelial terminal ductal lobular units (TDLUs). In 726 IBCs, CD8+, FOXP3+, IL17+, PDL1+, PD1+ T-cell infiltration (TILs) were investigated in RECQL deficient and proficient cancers. Tumor mutation burden (TMB) was evaluated in five RECQL germ-line mutation carriers with IBC by genome sequencing. Compared with normal epithelial cells, a striking reduction in nuclear RECQL in DCIS was evident with aggressive pathology and poor survival. In RECQL deficient IBCs, CD8+, FOXP3+, IL17+ or PDL1+ TILs were linked with aggressive pathology and shorter survival. In germline RECQL mutation carriers, increased TMB was observed in 4/5 tumors. We conclude that RECQL loss is an early event in breast cancer and promote immune cell infiltration.