CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

MMS22L-TONSL functions in sister chromatid cohesion in a pathway parallel to DSCC1-RFC.

The leading strand-oriented alternative PCNA clamp loader DSCC1-RFC functions in DNA replication, repair, and sister chromatid cohesion (SCC), but how it facilitates these processes is incompletely understood. Here, we confirm that loss of human DSCC1 results in reduced fork speed, increased DNA damage, and defective SCC. Genome-wide CRISPR screens in DSCC1-KO cells reveal multiple synthetically lethal interactions, enriched for DNA replication and cell cycle regulation. We show that DSCC1-KO cells require POLE3 for survival. Co-depletion of DSCC1 and POLE3, which both interact with the catalytic polymerase ε subunit, additively impair DNA replication, suggesting that these factors contribute to leading-strand DNA replication in parallel ways. An additional hit is MMS22L, which in humans forms a heterodimer with TONSL. Synthetic lethality of DSCC1 and MMS22L-TONSL likely results from detrimental SCC loss. We show that MMS22L-TONSL, like DDX11, functions in a SCC establishment pathway parallel to DSCC1-RFC. Because both DSCC1-RFC and MMS22L facilitate ESCO2 recruitment to replication forks, we suggest that distinct ESCO2 recruitment pathways promote SCC establishment following either cohesin conversion or de novo cohesin loading.

The Yin and Yang of Immunity in Stem Cell Decision Guidance in Tissue Ecologies: An Infection Independent Perspective.

The impact of immune system and inflammation on organ homeostasis and tissue stem cell niches in the absence of pathogen invasion has long remained a conundrum in the field of regenerative medicine. The paradoxical role of immune components in promoting tissue injury as well as resolving tissue damage has complicated therapeutic targeting of inflammation as a means to attain tissue homeostasis in degenerative disease contexts. This confound could be resolved by an integrated intricate assessment of cross-talk between inflammatory components and micro- and macro-environmental factors existing in tissues during health and disease. Prudent fate choice decisions of stem cells and their differentiated progeny are key to maintain tissue integrity and function. Stem cells have to exercise this fate choice in consultation with other tissue components. With this respect tissue immune components, danger/damage sensing molecules driving sterile inflammatory signaling cascades and barrier cells having immune-surveillance functions play pivotal roles in supervising stem cell decisions in their niches. Stem cells learn from their previous damage encounters, either endogenous or exogenous, or adapt to persistent micro-environmental changes to orchestrate their decisions. Thus understanding the communication networks between stem cells and immune system components is essential to comprehend stem cell decisions in endogenous tissue niches. Further the systemic interactions between tissue niches integrated through immune networks serve as patrolling systems to establish communication links and orchestrate micro-immune ecologies to better organismal response to injury and promote regeneration. Understanding these communication links is key to devise immune-centric regenerative therapies. Thus the present review is an integrated attempt to provide a unified purview of how inflammation and immune cells provide guidance to stem cells for tissue sculpting during development, organismal aging and tissue crisis based on the current knowledge in the field.

Percutaneous Coronary Intervention in Anomalously arising Coronary Arteries.

OBJECTIVE: Anomalous origin of coronary arteries (ACA) is rare. The objective of this communication is to describe the difficulties in angiographic recognition and challenges in percutaneous management. METHODS: The material for this retrospective study was provided by contributing interventional cardiologists practicing in tertiary care centers. RESULTS: From 2010 to 2019, 27 patients underwent percutaneous coronary intervention (PCI) for ACA. Four groups were encountered including anomalous origin from opposite sinus [left anomalous coronary artery from opposite sinus (L-ACAOS, n = 5), right anomalous coronary artery from opposite sinus (R-ACAOS n = 4)], origin of left circumflex from right sinus (n = 12), and origin of right coronary artery from posterior sinus (n = 6). The selection of a guiding catheter is the crucial step and a wide range of accessories was required to achieve an excellent outcome. Radial access may have an advantage in R-ACAOS, although the majority had a successful procedure from the femoral approach. CONCLUSION: Percutaneous management of patients with anomalous coronary arteries is challenging but can be accomplished with an excellent immediate outcome.

Information theoretic perspective on genome clustering.

Shannon's information theoretic perspective of communication helps one to understand the storage and processing of information in one-dimensional sequences. An information theoretic analysis of 937 available completely sequenced prokaryotic genomes and 238 eukaryotic chromosomes is presented. Information content (Id) values were used to cluster these chromosomes. Chargaff's second parity rule i.e compositional self-complementarity, an empirical fact is observed in all the genomes, except for the proteobacteria Candidatus Hodgkinia cicadicola. High information content, arising out of biased base composition in all the 14 chromosomes of Plasmodium falciparum is found among two other genomes of prokaryotes viz. Buchnera aphidicola str. Cc (Cinara cedri) and Candidatus Carsonella ruddii PV. Despite size and compositional variations, both prokaryotic and eukaryotic genomes do not deviate significantly from an equiprobable and random situation. Eukaryotic chromosomes of an organism tend to have similar informational restraints as seen when a simple distance based method is used to cluster them. In eukaryotes, in certain cases, Id values are also similar for the two arms (p and q arm) of the chromosomes. The results of this current study confirm that the information content can provide insights into the clustering of genomes and the evolution of messaging strategies of the genomes. An efficient and robust Perl CGI standalone tool is created based on this information theory algorithm for the analysis of the whole genomes and is made available at https://github.com/AlagurajVeluchamy/InformationTheory.

Antibiotic use and resistance: Knowledge, attitudes and perceptions among primary care prescribers in South Africa.

BACKGROUND: Antibiotic resistance (ABR) is a major threat to global health, driven in part by inappropriate prescription of antibiotics in primary care. OBJECTIVES: To describe South African (SA) prescribers' knowledge of, attitudes to and perceptions of ABR. METHODS: We conducted a cross-sectional survey of knowledge of, attitudes to and perceptions of ABR among a convenience sample of primary healthcare providers in SA, the majority from the private sector. We used logistic regression to examine associations between knowledge and prescribing behaviours. RESULTS: Of 264 prescriber respondents, 95.8% (230/240) believed that ABR is a significant problem in SA and 66.5% (157/236) felt pressure from patients to prescribe antibiotics. The median knowledge score was 5/7, and scores were highest in respondents aged <55 years (p=0.0001). Prescribers with higher knowledge scores were more likely than those with lower scores to believe that to decrease ABR, narrow-spectrum antibiotics should be used (adjusted odds ratio (aOR) 1.29, 95% confidence interval (CI) 1 - 1.65) and more likely to report that explaining disease features that should prompt follow-up was a useful alternative to prescribing (aOR 1.47, 95% CI 1.058 - 2.04), and were less likely to report that antibiotics cannot harm the patient if they are not needed, so they prescribe when not necessary (aOR 0.57, 95% CI 0.38 - 0.84). CONCLUSIONS: Prescribers of antibiotics in the private sector in SA were aware of the problem of ABR, but felt pressure from patients to prescribe. Those with higher knowledge scores reported positive prescribing behaviours, suggesting that more education is needed to tackle the problem of ABR.

TSC loss distorts DNA replication programme and sensitises cells to genotoxic stress.

Tuberous Sclerosis (TSC) is characterized by exorbitant mTORC1 signalling and manifests as non-malignant, apoptosis-prone neoplasia. Previous reports have shown that TSC-/- cells are highly susceptible to mild, innocuous doses of genotoxic stress, which drive TSC-/- cells into apoptotic death. It has been argued that this hypersensitivity to stress derives from a metabolic/energetic shortfall in TSC-/- cells, but how metabolic dysregulation affects the DNA damage response and cell cycle alterations in TSC-/- cells exposed to genotoxic stress is not understood. We report here the occurrence of futile checkpoint responses and an unusual type of replicative stress (RS) in TSC1-/- fibroblasts exposed to low-dose genotoxins. This RS is characterized by elevated nucleotide incorporation rates despite only modest origin over-firing. Strikingly, an increased propensity for asymmetric fork progression and profuse chromosomal aberrations upon mild DNA damage confirmed that TSC loss indeed proved detrimental to stress adaptation. We conclude that low stress tolerance of TSC-/- cells manifests at the level of DNA replication control, imposing strong negative selection on genomic instability that could in turn detain TSC-mutant tumours benign.

The residue at position 5 of the N-terminal region of Src and Fyn modulates their myristoylation, palmitoylation, and membrane interactions.

The interactions of Src family kinases (SFKs) with the plasma membrane are crucial for their activity. They depend on their fatty-acylated N-termini, containing N-myristate and either a polybasic cluster (in Src) or palmitoylation sites (e.g., Fyn). To investigate the roles of these moieties in SFK membrane association, we used fluorescence recovery after photobleaching beam-size analysis to study the membrane interactions of c-Src-GFP (green fluorescent protein) or Fyn-GFP fatty-acylation mutants. Our studies showed for the first time that the membrane association of Fyn is more stable than that of Src, an effect lost in a Fyn mutant lacking the palmitoylation sites. Unexpectedly, Src-S3C/S6C (containing cysteines at positions 3/6, which are palmitoylated in Fyn) exhibited fast cytoplasmic diffusion insensitive to palmitoylation inhibitors, suggesting defective fatty acylation. Further replacement of the charged Lys-5 by neutral Gln to resemble Fyn (Src-S3C/S6C/K5Q) restored Fyn-like membrane interactions, indicating that Lys-5 in the context of Src-S3C/S6C interferes with its myristoylation/palmitoylation. This was validated by direct myristoylation and palmitoylation studies, which indicated that the residue at position 5 regulates the membrane interactions of Src versus Fyn. Moreover, the palmitoylation levels correlated with targeting to detergent-resistant membranes (rafts) and to caveolin-1. Palmitoylation-dependent preferential containment of Fyn in rafts may contribute to its lower transformation potential.

Neurobehavioral toxicity in progeny of rat mothers exposed to methylmercury during gestation.

INTRODUCTION: Methylmercury (MeHg) is recognized as one of the most hazardous environmental pollutants. This may be a concern to long-term consumption of contaminated fish and seafood for health risk to pregnant women and their children. AIM: An animal study was conducted to assess the effect of MeHg exposure on rodent offspring following in utero exposure. METHODS: Pregnant Wister rats were treated by gavage with MeHg at dose levels of 0.5, 1.0 and 2.0 mg/kg/day from gestation day (GD) 5 till parturition, and then were allowed to deliver. RESULTS: Dams treated with 2.0 mg/kg/day MeHg group showed signs of toxicity such as gait alterations and hyperactivity resulting in the failure to deliver sustainable viable pups. MeHg had significant effects on body weight gain of dams during GD 5 till parturition. MeHg had no significant effects on the ages of physical developments such as pinna detachment, incisor eruptions or eye opening as well as alter cliff avoidance, surface righting, swimming ontogeny, startle reflex, pivoting, negative geotaxis, or forelimb and hindlimb grip strength in either sex. Exposure to 1.0 mg/kg/day MeHg treatment group prolonged gestation period, retard mid-air righting in male pups, shortened forelimb grip strength measured on rotating rod in either sex and enhanced open field behaviour in male pups. Data obtained from Functional Observation Battery (FOB) also revealed impairment of neuromotor performance in male pups. The male pups appeared to be more susceptible than the female pups. CONCLUSION: Overall, the dose level of MeHg in the present study produced a few adverse effects on the neurobehavioral parameters, and it may alter neuromotor performance of the male pups.

Influence of gestational exposure on the effects of prenatal exposure to methyl mercury on postnatal development in rats.

Fish and other aquatic organisms are important source of dietary proteins for the human population. Fish meat, however, is contaminated with methyl mercury (MeHg), a potent neurotoxin. The well known Minamata and Niigata epidemic outcomes in Japan have raised the awareness of the health risk resulting from consumption of fish (and shellfish) from water basins polluted with industrial wastes containing mercury. In the present study, pregnant rat dams were exposed to environmental toxic elements--methyl mercury, 1000-1200 h, daily from the fifth gestation day (GD5) till parturition. Three groups of animals were given, by gavages, MeHg (0.5, 1.0 and 2.0 mg/kg/day) and control group received 0.9% saline at the same time. All animals were allowed to deliver and wean their offspring. Pups were evaluated for early development effects. There was a significant effect of treatment on somatic growth such as reduction in percentage of maternal weight gain (20.62%) at higher dose level whereas there was no change in percentage of live birth (100.00%) with 0.5 and 1.0 mg/kg dose treatment groups. There was a significant increase in the percentage of resorption (100.00%) per litter with 2.0 mg/kg/day MeHg dose. Average gestation length (days) and percentage resorption per litter or percentage foetuses/malformations per litter were not affected at 0.5 and 1.0 mg/kg/day dose level. The results of the study confirmed the high-teratogenic potential of MeHg and the need of payng increased attention to MeHg concerning its exogenous use during pregnancy.

Chromatin condensation dynamics and implications of induced premature chromosome condensation.

Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing 'prophasing' of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.

The impact of coinfection with human immunodeficiency virus and pulmonary tuberculosis on the success of bronchial artery embolisation.

OBJECTIVE: Pulmonary tuberculosis (PTB) is often associated with human immunodeficiency virus (HIV) in South Africa. Bronchial artery embolisation (BAE) is a specialised, expensive and risky procedure. The aim of this study was to investigate the impact of coinfection with HIV and PTB on the success of BAE. METHODS: A retrospective cross-sectional study of sequential BAE procedures during 2006 and 2007 was performed. Rates of procedural and clinical outcome, reasons for failures and the impact of cluster of differentiation cell type 4 (CD4) level on failure were investigated. Patients were included if they presented with massive or life-threatening haemoptysis with a diagnosis of previous or active PTB and their HIV status was known, for the first two attempts at BAE only. RESULTS: The study population consisted of 74 patients who were HIV positive and 33 who were HIV negative. Statistically, procedural success did not imply a clinically successful outcome, and HIV status and CD4 level did not correlate significantly with procedural success. Statistically, no technical reason had an impact on the success of the procedure when correlated with HIV status. The detection of lymphadenopathy was noted in 19.1% of patients who were HIV positive and in 42.4% of patients who were HIV negative, and was the only feature of significance. CONCLUSION: Coinfection with HIV does not have an impact on the success of BAE in patients with active PTB or with the sequelae of PTB who present with massive or life-threatening haemoptysis. Technical success does not imply clinical success, regardless of HIV status. Improvement in technique locally may improve outcome. ADVANCES IN KNOWLEDGE: PTB coinfection with HIV should not affect the decision to consider BAE.

Possible cross-sensitivity between sertraline and paroxetine in a panic disorder patient.

Cross-sensitivity due to paroxetine and sertraline, the SSRIs, is rarely reported in the literature. We report an adverse drug reaction to paroxetine and sertraline in a patient of panic disorder, who initially developed a maculopapular, erythematous, pruritic rash in the third week with sertraline 50 mg/day. The rash resolved within 2 days of its discontinuation and oral supplementation of diphenhydramine and betamethasone. 10 days following discontinuation of sertraline, the patient was shifted on sustain release paroxetine 12.5 mg/day when another skin reaction with the same appearance and distribution appeared on day 4 of it, suggesting a possibility of cross-sensitivity, a drug class effect. This case report intends to improve the awareness among clinicians to use caution when choosing an alternative SSRIs.

Differential gene expression analysis in antimony-unresponsive Indian kala azar (visceral leishmaniasis) clinical isolates by DNA microarray.

In this study, cDNA microarray analysis of a closely related species, Leishmania major, was used as a screening tool to compare antimonial-resistant and susceptible clinical isolates of Leishmania donovani in order to to identify candidate genes on the basis of antimony resistance. Clinically confirmed resistant isolate 39 and sensitive isolate 2001 were used in this study. Many differentially regulated genes were identified whose expression levels differ in sodium antimony gluconate (SAG)-treated patients. Interestingly, genes on the array, showing changes in expression of over 2-fold revealed the identity of ABC transporters, which are known determinants of drug resistance in laboratory mutants. The functionality of the transporters was validated by flow cytometry which, being biologically informative, provides direct clues to gene function. The results suggest that isolate 39 could have developed resistance by an increased multidrug resistance protein (MRP)-like pump. This study provides preliminary clues to the role of a thiol-dependent efflux system in antimonial resistant clinical isolates of Leishmania donovani.