Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

Repeat expansions confer WRN dependence in microsatellite-unstable cancers.

The RecQ DNA helicase WRN is a synthetic lethal target for cancer cells with microsatellite instability (MSI), a form of genetic hypermutability that arises from impaired mismatch repair1-4. Depletion of WRN induces widespread DNA double-strand breaks in MSI cells, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which WRN protects MSI-associated cancers from double-strand breaks remains unclear. Here we show that TA-dinucleotide repeats are highly unstable in MSI cells and undergo large-scale expansions, distinct from previously described insertion or deletion mutations of a few nucleotides5. Expanded TA repeats form non-B DNA secondary structures that stall replication forks, activate the ATR checkpoint kinase, and require unwinding by the WRN helicase. In the absence of WRN, the expanded TA-dinucleotide repeats are susceptible to cleavage by the MUS81 nuclease, leading to massive chromosome shattering. These findings identify a distinct biomarker that underlies the synthetic lethal dependence on WRN, and support the development of therapeutic agents that target WRN for MSI-associated cancers.

ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways.

Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.

DNA Breaks and End Resection Measured Genome-wide by End Sequencing.

DNA double-strand breaks (DSBs) arise during physiological transcription, DNA replication, and antigen receptor diversification. Mistargeting or misprocessing of DSBs can result in pathological structural variation and mutation. Here we describe a sensitive method (END-seq) to monitor DNA end resection and DSBs genome-wide at base-pair resolution in vivo. We utilized END-seq to determine the frequency and spectrum of restriction-enzyme-, zinc-finger-nuclease-, and RAG-induced DSBs. Beyond sequence preference, chromatin features dictate the repertoire of these genome-modifying enzymes. END-seq can detect at least one DSB per cell among 10,000 cells not harboring DSBs, and we estimate that up to one out of 60 cells contains off-target RAG cleavage. In addition to site-specific cleavage, we detect DSBs distributed over extended regions during immunoglobulin class-switch recombination. Thus, END-seq provides a snapshot of DNA ends genome-wide, which can be utilized for understanding genome-editing specificities and the influence of chromatin on DSB pathway choice.

Clinical relevance of PD-1 positive CD8 T-cells in gastric cancer.

BACKGROUND: We evaluated the relevance of PD-1+CD8+ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME). METHODS: Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the "3G" chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs. RESULTS: In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B+] (r = 0.714, p < 0.001) and proliferative [Ki-67+] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1high compared to CD8PD-1low tumors. CONCLUSION: This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1+CD8+ T-cells being associated with improved OS. CD8PD-1high tumors have distinct features of an immunologically active, T-cell inflamed TME.

Artificially inserted strong promoter containing multiple G-quadruplexes induces long-range chromatin modification.

Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained.

BBD optimized antioxidants of Crotalaria candicans and its nanoconjugates, exert potent in vivo anti-biofilm effects against MRSA.

Crotalaria genus is extensively dispersed in tropical and subtropical provinces, and it is found to harbor antioxidant flavonoids. Response surface methodology-based optimization was carried out for the purpose of efficient extraction involving a suitable solvent which can maximize the yield along with higher total phenolic content and total flavonoid content (TFC). Optimization conditions for extraction of C.candicans flavonoids (CCF) based on variables such as solvent, solid-solvent ratio and extraction temperature were evaluated. The optimized conditions were found as Solvent i.e., Aqueous-ethanol (53.42%), Solid-solvent ratio (1:15.83 w/v) and temperature (44.42 °C) and resulted to obtain the TFC as 176.23 mg QRET/g C. candicans extract with the yield 27.42 mg CCF/g (C. candicans dry weight). LC-MS analysis of CCF, revealed the presence of seven major flavonoids. The antioxidant flavonoids were further used to functionalize the zero-valent silver (ZVAgF) and copper (ZVCuF) nanoparticles. The ZVAgF and ZVCuF were investigated using UV-Vis spectrophotometry, FT-IR spectroscopy and X-ray diffractometry to confirm the presence of the zero valent metals and possible functional groups which capped the elemental metal. Further transmission electron microscopy, dynamic light scattering method and zeta-potential studies were done to understand their respective structural and morphological properties. The efficacy of the as-prepared ZVAgF/ZVCuF as antibiofilm agents on Methicillin-resistant Staphylococcus aureus (MRSA) with the mechanism studies have been explored. The MRSA-colony count from the infection zebrafish (in vivo) model, portrayed a reduction of > 1.9 fold for ZVCuF and > twofold for ZVAgF, with no alteration in liver morphology when treated with ZVAgF, implying that the nanoparticles were safe and biocompatible.

Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability.

Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here we discover a checkpoint-regulated cascade of chromatin signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favoured by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.

Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for proper lariat debranching and ectodermal differentiation.

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.

Boolean modeling and fault diagnosis in oxidative stress response.

BACKGROUND: Oxidative stress is a consequence of normal and abnormal cellular metabolism and is linked to the development of human diseases. The effective functioning of the pathway responding to oxidative stress protects the cellular DNA against oxidative damage; conversely the failure of the oxidative stress response mechanism can induce aberrant cellular behavior leading to diseases such as neurodegenerative disorders and cancer. Thus, understanding the normal signaling present in oxidative stress response pathways and determining possible signaling alterations leading to disease could provide us with useful pointers for therapeutic purposes. Using knowledge of oxidative stress response pathways from the literature, we developed a Boolean network model whose simulated behavior is consistent with earlier experimental observations from the literature. Concatenating the oxidative stress response pathways with the PI3-Kinase-Akt pathway, the oxidative stress is linked to the phenotype of apoptosis, once again through a Boolean network model. Furthermore, we present an approach for pinpointing possible fault locations by using temporal variations in the oxidative stress input and observing the resulting deviations in the apoptotic signature from the normally predicted pathway. Such an approach could potentially form the basis for designing more effective combination therapies against complex diseases such as cancer. RESULTS: In this paper, we have developed a Boolean network model for the oxidative stress response. This model was developed based on pathway information from the current literature pertaining to oxidative stress. Where applicable, the behaviour predicted by the model is in agreement with experimental observations from the published literature. We have also linked the oxidative stress response to the phenomenon of apoptosis via the PI3k/Akt pathway. CONCLUSIONS: It is our hope that some of the additional predictions here, such as those pertaining to the oscillatory behaviour of certain genes in the presence of oxidative stress, will be experimentally validated in the near future. Of course, it should be pointed out that the theoretical procedure presented here for pinpointing fault locations in a biological network with feedback will need to be further simplified before it can be even considered for practical biological validation.

When Knowledge Is Not Enough: Applying a Behavioral Design Approach to Improve Fever Case Management in Nigeria.

BACKGROUND: We sought to encourage health care providers to adhere to national malaria case management guidelines. This requires them to conduct malaria parasite tests for every patient presenting with a fever and provide malaria treatment only to those who test positive for malaria. Our goal was to make it easier for providers to follow guidelines by addressing drivers of nonadherence uncovered through facility observations and interviews with staff and clients. IMPLEMENTATION AND MONITORING: The case management interventions were piloted in 12 public health facilities in Akwa Ibom, Kebbi, and Nasarawa states in Nigeria between October and December 2019. Participating facilities included 1 hospital and 3 primary health centers in each state. Relevant changes included the following: (1) providers at each facility participated in facilitated discussions to correct misconceptions about the reliability of malaria test kits; (2) testing procedures were integrated into existing triage systems; (3) treatment algorithms were integrated into medical record forms; (4) providers were issued pictorial brochures outlining danger signs to share with clients, together with instructions for when to seek further care; and (5) a process was created for facilities to monitor their own adherence to guidelines. LESSONS LEARNED: The lessons learned include: (1) disentangling the drivers of behavior allows for more targeted solutions, (2) solutions that streamline processes for overburdened providers allow them to redirect their attention and efforts where they can be most impactful, and (3) changing staff perceptions of workplace norms can support a holistic and sustained approach to behavior change.

Disrupted control of origin activation compromises genome integrity upon destabilization of Polε and dysfunction of the TRP53-CDKN1A/P21 axis.

The maintenance of genome stability relies on coordinated control of origin activation and replication fork progression. How the interplay between these processes influences human genetic disease and cancer remains incompletely characterized. Here we show that mouse cells featuring Polε instability exhibit impaired genome-wide activation of DNA replication origins, in an origin-location-independent manner. Strikingly, Trp53 ablation in primary Polε hypomorphic cells increased Polε levels and origin activation and reduced DNA damage in a transcription-dependent manner. Transcriptome analysis of primary Trp53 knockout cells revealed that the TRP53-CDKN1A/P21 axis maintains appropriate levels of replication factors and CDK activity during unchallenged S phase. Loss of this control mechanism deregulates origin activation and perturbs genome-wide replication fork progression. Thus, while our data support an impaired origin activation model for genetic diseases affecting CMG formation, we propose that loss of the TRP53-CDKN1A/P21 tumor suppressor axis induces inappropriate origin activation and deregulates genome-wide fork progression.

Joule heating effects on electroosmotic flow in insulator-based dielectrophoresis.

Insulator-based dielectrophoresis (iDEP) is an emerging technology that has been successfully used to manipulate a variety of particles in microfluidic devices. However, due to the locally amplified electric field around the in-channel insulator, Joule heating often becomes an unavoidable issue that may disturb the electroosmotic flow and affect the particle motion. This work presents the first experimental study of Joule heating effects on electroosmotic flow in a typical iDEP device, e.g., a constriction microchannel, under DC-biased AC voltages. A numerical model is also developed to simulate the observed flow pattern by solving the coupled electric, energy, and fluid equations in a simplified two-dimensional geometry. It is observed that depending on the magnitude of the DC voltage, a pair of counter-rotating fluid circulations can occur at either the downstream end alone or each end of the channel constriction. Moreover, the pair at the downstream end appears larger in size than that at the upstream end due to DC electroosmotic flow. These fluid circulations, which are reasonably simulated by the numerical model, form as a result of the action of the electric field on Joule heating-induced fluid inhomogeneities in the constriction region.

Microarray analysis of genes from animals treated with a traditional formulation ChandraprabhaVati reveals its therapeutic targets.

BACKGROUND: Traditional medicinal preparations have not received global acceptance, and their therapeutic benefits remain disputed due to lack of scientific evidence on their mechanism of action. Microarray analysis has emerged as a powerful technique that can aid in understanding the complex signaling networks activated by these formulations and thereby assess their beneficial as well as adverse effects. AIM: The present work aims to investigate the differential influence of ChandraprabhaVati, Ayurvedic formulation used in the treatment of diabetes, anemia, urinary, respiratory, skin and liver disorders. MATERIALS AND METHODS: The RNA from the liver of rats treated with different doses of ChandraprabhaVati for 28 days was isolated and studied for the genome-wide changes in the expression. RESULTS: The results revealed several molecular targets that could contribute to the therapeutic effects of ChandraprabhaVati. Several genes have been differentially expressed, among those miRNAs miR-434, miR877, and miRlet7e contribute to the anti-diabetic, anti-fibrotic and anti-inflammatory of CPV. The rejuvenative activity of CPV may be due to the MeOX1 and Upf3b genes. Up-regulation of Hbaa2 gene facilitates the anti-anemic effect. Interestingly gender-specific differential expressions of genes were also observed. Rab3d were found to be altered in female when compared to male animals. CONCLUSION: Thus the microarray data for the CPV treated animals has revealed molecular targets that may be responsible for the various known therapeutic effects and also identified new beneficial effects of CPV.

The clinician crowdsourcing challenge: using participatory design to seed implementation strategies.

BACKGROUND: In healthcare settings, system and organization leaders often control the selection and design of implementation strategies even though frontline workers may have the most intimate understanding of the care delivery process, and factors that optimize and constrain evidence-based practice implementation within the local system. Innovation tournaments, a structured participatory design strategy to crowdsource ideas, are a promising approach to participatory design that may increase the effectiveness of implementation strategies by involving end users (i.e., clinicians). We utilized a system-wide innovation tournament to garner ideas from clinicians about how to enhance the use of evidence-based practices (EBPs) within a large public behavioral health system. METHODS: Our innovation tournament occurred in three phases. First, we invited over 500 clinicians to share, through a web-based platform, their ideas regarding how their organizations could best support use of EBPs. Clinicians could rate and comment on ideas submitted by others. Second, submissions were judged by an expert panel (including behavioral scientists, system leaders, and payers) based on their rated enthusiasm for the idea. Third, we held a community-facing event during which the six clinicians who submitted winning ideas presented their strategies to 85 attendees representing a cross-section of clinicians and system and organizational leaders. RESULTS: We had a high rate of participation (12.3%), more than double the average rate of previous tournaments conducted in other settings (5%). A total of 65 ideas were submitted by 55 participants representing 38 organizations. The most common categories of ideas pertained to training (42%), financing and compensation (26%), clinician support and preparation tools (22%), and EBP-focused supervision (17%). The expert panel and clinicians differed on their ratings of the ideas, highlighting value of seeking input from multiple stakeholder groups when developing implementation strategies. CONCLUSIONS: Innovation tournaments are a useful and feasible methodology for engaging end users, system leaders, and behavioral scientists through a structured approach to developing implementation strategies. The process and resultant strategies engendered significant enthusiasm and engagement from participants at all levels of a healthcare system. Research is needed to compare the effectiveness of strategies developed through innovation tournaments to strategies developed through design approaches.

A unique mutator phenotype reveals complementary oncogenic lesions leading to acute leukemia.

Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4-32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2cre/cre NHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.

Hypoxia Stress Response Pathways: Modeling and Targeted Therapy.

Hypoxia is a consequence of the decrease in the oxygen reaching the tissues of the body. It is a prominent feature of most solid tumors and is known to promote malignant progression, metastatic capacity, resistance to chemotherapy, and leads to poor patient prognosis. When a cell is under hypoxic stress, a cascade of cell signals is initiated through a family of transcription factors named as hypoxia inducible factors (HIFs). During hypoxia, HIF stabilizes and enters the nucleus and binds to the DNA via the hypoxia response element (HRE) and leads to the translation of downstream genes. The decision of adaptation or cell death depends on the extent of hypoxic stress faced by the cells. Proper understanding of hypoxic stress response is critical for understanding the mechanism of tumor cell adaptation to hypoxia and to develop efficient therapeutic interventions. In this paper, we develop a Boolean network model with targeted drug intervention in a cell that mimics persistent hypoxia. This hypoxic pathway is combined with pathways that help the cell adapt to the situation or undergo cell death. It is linked to apoptosis, cell survival, and energy production via the p53/Mdm2, PI3k/Akt/mTOR, and Glycolysis/TCA cycle pathways, respectively. In this model, we have incorporated eight known anticancer drugs that target these pathways. Through simulations, we have identified drug combinations that provided overall benefits to the cell in comparison to the no intervention case. Where applicable, the behavior predicted by this model is in agreement with experimental observations from the published literature.