Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Bilateral spontaneous vertebral artery dissection complicated by bilateral posterior cerebral artery occlusion in a migraine patient: a case report with systematic review.

Migraines being a possible risk factor for spontaneous multivessel cervical artery dissection has been previously introduced but rarely discussed in literature. We present the case of a 32-year-old man with a history of migraines and a 2-week history of bilateral neck pain who was found to have bilateral Vertebral Artery dissection by CT angiography. The patient's stroke's etiology was spontaneous dissection followed by thromboembolism caused by bilateral Posterior Cerebral Artery (P1) occlusion. Due to an inability to protect his airway, he was scheduled to have a tracheostomy and percutaneous endoscopic gastrostomy (PEG). Over the following weeks, the patient continued to be unresponsive to stimuli, unable to follow commands, and unable to exhibit active/purposeful movement. As a result, the patient was transitioned to inpatient palliative care with total parenteral nutrition. We conducted a systematic literature review querying four databases: MEDLINE, Embase, CINAHL, and Academic Search Complete. Eligibility criteria were applied based on article type, title, abstract, and full text screening. Four case reports and three case-control studies discussing patients with a past medical history of migraines presenting with unilateral or bilateral vertebral artery dissection were identified and included in this review. We describe the possibility of the patient's migraine history and potentially associated vasculopathy as a predisposing factor in the development of Vertebral Artery Dissection. Further research is needed to fully understand the exact mechanism occurring that predisposes migraine patients to spontaneous arterial wall injury.

Human papillomavirus 16 promotes microhomology-mediated end-joining.

Squamous cell carcinomas (SCCs) arising from aerodigestive or anogenital epithelium that are associated with the human papillomavirus (HPV) are far more readily cured with radiation therapy than HPV-negative SCCs. The mechanism behind this increased radiosensitivity has been proposed to be secondary to defects in DNA repair, although the specific repair pathways that are disrupted have not been elucidated. To gain insight into this important biomarker of radiosensitivity, we first examined genomic patterns reflective of defects in DNA double-strand break repair, comparing HPV-associated and HPV-negative head and neck cancers (HNSCC). Compared to HPV-negative HNSCC genomes, HPV+ cases demonstrated a marked increase in the proportion of deletions with flanking microhomology, a signature associated with a backup, error-prone double-strand break repair pathway known as microhomology-mediated end-joining (MMEJ). Then, using 3 different methodologies to comprehensively profile double-strand break repair pathways in isogenic paired cell lines, we demonstrate that the HPV16 E7 oncoprotein suppresses canonical nonhomologous end-joining (NHEJ) and promotes error-prone MMEJ, providing a mechanistic rationale for the clinical radiosensitivity of these cancers.

Risk factors for pneumonia mortality in under-five children in a tertiary care hospital of Darjeeling district of West Bengal: A prospective case-control study.

BACKGROUND: Among children admitted with pneumonia, several modifiable predictors have been identified for deaths in children in hospitals. Despite the presence of a several national programs designed to address most of the risk factors directly or indirectly it is surprising that they continue to be common in children with pneumonia. OBJECTIVES: The objective is to determine the risk factors for pneumonia mortality in under-five children in a tertiary care hospital of Darjeeling district of West Bengal. METHODS: An analytical study with case-control design was conducted between May 2016 and October 2017. Children aged 2-59 months admitted with the diagnosis of pneumonia were followed up after admission and who died were recruited as cases and two consecutive age- and sex-matched controls were recruited among children who were declared cured and discharged. A total of 95 cases and 190 controls were studied and the risk factors were compared in the pair matched groups by the conditional logistic regression. RESULTS: Factors influencing childhood pneumonia mortality were severely underweight (adjusted odds ratio [AOR]: 3.66 [1.28,10.46]) unimmunized child (AOR 4.18 [1.53, 11.41]), lack of exclusive breast feeding (AOR: 3.12 [1.23, 7.91]), past history of diarrhea or acute respiratory infection in the last 3 months (AOR: 7.27 [3.68, 14.36]), hypoxemia on admission (AOR: 2.53 [1.14, 5.61]), sub-center as the first contact health facility (AOR: 6.49 [2.15, 19.67]), and antibiotic not received at first contact (AOR: 3.18 [1.36, 7.43]). CONCLUSIONS: Most of the risk factors for death in children between 6 and 59 months of age are directly or indirectly related to health service delivery and can be ameliorated through proper structural and administrative measures.

Generalized woolly hair with diventricular arrythmogenic cardiomyopathy: a rare variant of Naxos disease.

Woolly hair may occur as an isolated problem of cosmetic concern or can be a part of a systemic disease (woolly hair syndrome) with underlying fatal cardiomyopathy. Two characteristic associations of woolly hair syndrome are Naxos disease and Carvajal syndrome. Naxos disease is characterized by woolly hair, palmoplantar keratoderma, and arrythmogenic right ventricular cardiomyopathy.In this report we describe a case of a young girl who presented with heart failure and was subsequently diagnosed as a case of generalized woolly hair with biventricular arrythmogenic cardiomyopathy.Our case represented a rare variant of Naxos disease in the advanced stage of arrythmogenic right ventricular cardiomyopathy; biventricular failure may occur with involvement of the interventricular septum and left ventricle causing congestive heart failure.

High Specificity of HPV Cell-Free DNA Tests in Persons With HIV for the Detection of HPV-Related Cancer.

INTRODUCTION: Persons with HIV (PWH) experience high rates of human papillomavirus (HPV)-associated cancers compared with the general population. Plasma HPV cell-free DNA (cfDNA) tests are sensitive in patients with known HPV-associated cancers. It is not known whether these tests can screen for invasive cancers in populations with high burdens of nonmalignant HPV disease such as PWH. It was not known whether HPV infection and/or noninvasive anal high-grade squamous intraepithelial lesions (HSIL) alone in this population would result in detectable HPV cfDNA, which would result in a high number of false positives if HPV cfDNA is used to screen for invasive cancers. METHODS: We conducted a prospective study of PWH in 2 cohorts: 20 without anal HSIL and 20 with anal HSIL. We tested anal and vaginal swabs for HPV infection, and HPV genotyped the biopsies of anal HSIL. Finally, we performed HPV cfDNA droplet digital polymerase chain reaction to test for HPV16/18/33 from plasma samples. RESULTS: In the combined cohorts, the median age was 56 years, 12.5% were cisgender women, and none had detectable HIV. In total, 84.6% had prevalent anovaginal HPV infection, including 10 participants with HPV16, 13 with HPV18, and 2 with HPV33 infections. Five and 2 participants had HPV16 and HPV33 detected in anal HSIL, respectively. Despite the high prevalence of HPV infection and anal HSIL, no participant had HPV16/18/33 detectable cfDNA by droplet digital polymerase chain reaction. CONCLUSIONS: These results provide a strong rationale for investigating the use of HPV cfDNA in a screening setting for suspected HPV-related invasive cancers in PWH.

Newborn joint mechanics.

INTRODUCTION: We aimed to evaluate joint mechanics in newborn by goniometric assessment of major joints in healthy babies born at different gestational ages (GAs). MATERIALS AND METHODS: An institution based observational study was carried out on healthy newborn babies within two days of birth. Study subjects were born at 28-41 completed weeks of gestation. The major joints of upper and lower limbs were assessed with manual goniometer for joint angles in relation to specific passive movements and range of motion (ROM) calculated where applicable. All measurements were made by a single observer with careful consideration of plane of movement and axes involved. Strength of association between joint angles and GA was quantified by Pearson's r coefficient. RESULTS: Six major joints (shoulder, elbow, wrist, hip, knee, and ankle) were evaluated on either side in 433 babies. No significant differences were found between male and female babies and left or right side of the body. For most joints, a secular declining trend of joint angle or ROM was noted with good to strong inverse correlation with GA. The strongest associations were for flexion-extension ROM and adduction-abduction ROM at shoulder, palmar flexion at wrist and dorsiflexion at ankle joint with r values of -0.76, -0.75, -0.75, and -0.75, respectively. CONCLUSIONS: The reading of a specific joint angle in the newborn infants was found to be dependent on GA. Precise calibration of gestation appropriate joint angles had laid down the foundation for functional assessment of multimodal joint mechanics.HighlightsEvaluation of newborn joint angles require stringent attention toward the plane and axis of the particular joint movement being assessed.Major joint angles and range of motion in newborn infants were observed to follow a secular declining trend according to the gestational age.Precise estimation of gestation appropriate joint angle will be helpful to understand the mechanics of musculoskeletal medicine in newborn.

Templated Insertions Are Associated Specifically with BRCA2 Deficiency and Overall Survival in Advanced Ovarian Cancer.

Cancer cells defective in homologous recombination (HR) are responsive to DNA-crosslinking chemotherapies, PARP inhibitors, and inhibitors of polymerase theta (Pol θ), a key mediator of the backup pathway alternative end-joining. Such cancers include those with pathogenic biallelic alterations in core HR genes and another cohort of cases that exhibit sensitivity to the same agents and harbor genomic hallmarks of HR deficiency (HRD). These HRD signatures include a single-base substitution pattern, large rearrangements, characteristic tandem duplications, and small deletions. Here, we used what is now known about the backup pathway alternative end-joining (Alt-EJ) through the key factor Pol θ to design and test novel signatures of polymerase theta-mediated (TMEJ) repair. We generated two novel signatures; a signature composed of small deletions with microhomology and another consisting of small, templated insertions (TINS). We find that TINS consistent with TMEJ repair are highly specific to tumors with pathogenic biallelic mutations in BRCA2 and that high TINS genomic signature content in advanced ovarian cancers associate with overall survival following treatment with platinum agents. In addition, the combination of TINS with other HRD metrics significantly improves the association of platinum sensitivity with survival compared with current state-of-the-art signatures. IMPLICATIONS: Small, templated insertions indicative of theta-mediated end-joining likely can be used in conjunction with other HRD mutational signatures as a prognostic tool for patient response to therapies targeting HR deficiency.

A multi-throughput mechanical loading system for mouse intervertebral disc.

Mechanical loading plays an important role in maintaining disc health and function, and in particular, excessive mechanical loading has been identified as one of major reasons of disc degeneration. Intervertebral disc organ culture serves as a valuable tool to study disc biology/pathology. In this study, we report the development and validation of a new mouse disc organ culture system by dynamically applying compression loading in a customized micro-culture device tailored for mouse lumbar discs. Precise axial compression force was delivered by a computer-controlled system consisting of a robust micromechanical linear actuator, a force sensitive resistor, and a precision micro-stepping machinery. Customized PDMS-based loading chambers allowed simultaneous loading of six discs per regimen, which streamlined the workflow to reach sufficient statistic power. The detrimental loading regimen of mouse lumbar discs (0.5 MPa of axial compression at 1Hz for 7 days) was demonstrated through live-dead assay, histology, and fluorescence probe based collagen staining. In addition, various mechanical compression profiles were simulated using different materials and geometry designs, potentiating for more sophisticated loading protocols. In summary, we developed a new mechanical loading system for dynamic axial compression of mouse discs, which created a unique avenue to study disc pathogenesis with enriched mouse species-related resources, and complemented the existing spectrum of bioreactor systems predominately for discs of human and large animals.

Molecular Detection and Assessment of Intervertebral Disc Degeneration via a Collagen Hybridizing Peptide.

During aging, wear, and tear of intervertebral discs, human discs undergo a series of morphological and biochemical changes. Degradation of extracellular matrix proteins, e.g., collagen, arises as an important contributor and accelerator in this process. Existing methods to detect collagen degradation at the tissue level include histology and immunohistochemistry. Unfortunately, most of these methods only depict overall collagen content without the ability to specifically discern degraded collagen and to assess the severity of degeneration. To fill this technological gap, we developed a robust and simple approach to detect and assess early disc degeneration with a collagen hybridizing peptide (CHP) that hybridizes with the flawed triple helix structure in degraded collagen. Intriguingly, the CHP signal in mouse lumbar discs exhibited a linear incremental pattern with age. This finding was corroborated with histological analysis based on established methods. When comparing this analysis, a positive linear correlation was found between CHP fluorescence intensity and the histological score with a regression value of r 2 = 0.9478. In degenerative mouse discs elicited by pro-inflammatory stimuli (IL-1ß and LPS) ex vivo, the newly developed approach empowered prediction of the severity of disc degeneration. We further demonstrated higher CHP signals in a degenerative human disc tissue when compared to a normal sample. These findings also resonated with histological analysis. This approach lays a solid foundation for specific detection and assessment of intervertebral disc degeneration at the molecular level and will promote development of future disc regeneration strategies.