Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs.

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. We used inverse PCR of non-B microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures integrated at a common ectopic chromosomal site to show that these non-B DNAs generate highly mutagenized eccDNAs by replication-dependent mechanisms. Mutagenesis occurs within the non-B DNAs and extends several kilobases bidirectionally into flanking and nonallelic DNA. Each non-B DNA exhibits a different pattern of mutagenesis, while sister clones containing the same non-B DNA also display distinct patterns of recombination, microhomology-mediated template switching and base substitutions. Mutations include mismatches, short duplications, long nontemplated insertions, large deletions and template switches to sister chromatids and nonallelic chromosomes. Drug-induced replication stress or the depletion of DNA repair factors Rad51, the COPS2 signalosome subunit or POLη change the pattern of template switching and alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA double strand breaks to account for the generation and circularization of mutagenized eccDNAs and the appearance of genomic homologous recombination deficiency (HRD) scars. These results may help to explain the appearance of tumor eccDNAS and their roles in neoantigen production, oncogenesis and resistance to chemotherapy.

Microsatellite break-induced replication generates highly mutagenized extrachromosomal circular DNAs.

Extrachromosomal circular DNAs (eccDNAs) are produced from all regions of the eucaryotic genome. In tumors, highly transcribed eccDNAs have been implicated in oncogenesis, neoantigen production and resistance to chemotherapy. Here we show that unstable microsatellites capable of forming hairpin, triplex, quadruplex and AT-rich structures generate eccDNAs when integrated at a common ectopic site in human cells. These non-B DNA prone microsatellites form eccDNAs by replication-dependent mechanisms. The microsatellite-based eccDNAs are highly mutagenized and display template switches to sister chromatids and to nonallelic chromosomal sites. High frequency mutagenesis occurs within the eccDNA microsatellites and extends bidirectionally for several kilobases into flanking DNA and nonallelic DNA. Mutations include mismatches, short duplications, longer nontemplated insertions and large deletions. Template switching leads to recurrent deletions and recombination domains within the eccDNAs. Template switching events are microhomology-mediated, but do not occur at all potential sites of complementarity. Each microsatellite exhibits a distinct pattern of recombination, microhomology choice and base substitution signature. Depletion of Rad51, the COPS2 signalosome subunit or POLη alter the eccDNA mutagenic profiles. We propose an asynchronous capture model based on break-induced replication from microsatellite-induced DNA breaks for the generation and circularization of mutagenized eccDNAs and genomic homologous recombination deficiency (HRD) scars.

Intra thecal baclofen pump as an ad hoc measure for a case of severe tardive dystonia refractory to multiple lesioning.

Tardive dystonia is an infrequent ailment in patient reliant with chronic antipsychotic medication. The front-line envoy in the treatment of this illness is set into motion with oral agents including baclofen, benzodiazepines, and other antispasmodics. Regardless of an extensive therapy, the patients are not able to control of their spasticity/ dystonia. The authors reported a case of severe tardive dystonia treated with baclofen therapy in a patient frigid to medical therapy and multiple lesioning. Case Report: A 31-year-old female, diagnosed as a case of depressive illness and being managed with neuroleptic medications, who went onto develop tardive dystonia progressively worsening over a 4-year duration. After a comprehensive and meticulous evaluation of her neurological and psychological stratum, globus pallidus interna lesioning was reputed as the best course of action. As intended, staged lesioning was executed bilaterally with a trivial resolution eventually succumbing into recurrence, compelling a repeat lesioning. It was inaptly discouraging to see her crippled with the plight. Determined, not to give upon her, a way out with a baclofen therapy was proposed. A test dose with a 100 mcg of baclofen with an increment up to 150 mcg over a 3-day period demonstrated a promising prospect. On that account, the insertion of the baclofen pump was performed with an outstanding aftermath in her neurological endeavor. Clinical Discussion: Tardive dystonia is believed to be caused by striatal dopamine receptor super-sensitivity persuaded by the dopamine-antagonizing action of antipsychotic drugs. The first line of treatment being oral agents including oral baclofen, benzodiazepines, and antispasmodics. If the patient suffers from an early-onset primary generalized dystonia, then treatment with deep brain stimulation of the globus pallidus interna is the approved and preferred treatment approach. Recurrence of the symptoms despite of multiple lesioning can be overcome by intrathecal baclofen pump infusion as stated by many research. It is not uncommon to face complications in such a procedure, but the benefits outreach the risk, which makes it a choice of treatment. Conclusion: The use of a continuous intrathecal baclofen pump for cases with tardive dystonia refractory to conventional therapy, it has been approved as one of the safest and capable procedures.

Suppressors of Break-Induced Replication in Human Cells.

Short tandem DNA repeats are drivers of genome instability. To identify suppressors of break-induced mutagenesis human cells, unbiased genetic screens were conducted using a lentiviral shRNA library. The recipient cells possessed fragile non-B DNA that could induce DNA double-strand breaks (DSBs), integrated at an ectopic chromosomal site adjacent to a thymidine kinase marker gene. Mutagenesis of the thymidine kinase gene rendered cells resistant to the nucleoside analog ganciclovir (GCV). The screen identified genes that have established roles in DNA replication and repair, chromatin modification, responses to ionizing radiation, and genes encoding proteins enriched at replication forks. Novel loci implicated in BIR included olfactory receptors, the G0S2 oncogene/tumor suppressor axis, the EIF3H-METTL3 translational regulator, and the SUDS3 subunit of the Sin3A corepressor. Consistent with a role in suppressing BIR, siRNA knockdown of selected candidates increased the frequency of the GCVr phenotype and increased DNA rearrangements near the ectopic non-B DNA. Inverse PCR and DNA sequence analyses showed that hits identified in the screen increased genome instability. Further analysis quantitated repeat-induced hypermutagenesis at the ectopic site and showed that knockdown of a primary hit, COPS2, induced mutagenic hotspots, remodeled the replication fork, and increased nonallelic chromosome template switches.

Venture of a Tertiary Care Neurosurgical Center in Course of COVID-19 Lockdown without RT-PCR.

"I will not permit considerations of age, disease or disability, creed, ethnic origin, gender, nationality, political affiliation, race, sexual orientation, social standing or any other factor to intervene between my duty and my patient." Obliged by the aforementioned oath, no medical practitioner shall sit in a moral judgment on any patient but will treat their illness to the best of their ability whatever the circumstances. A clear concord was yet to be authorized after the World Health Organization (WHO) declared the global pandemic of severe acute respiratory syndrome coronavirus 2infection. As a diagnostic modality, WHO recommended real-time reverse transcription-polymerase chain reaction (RT-PCR) as a reliable test; however, its availability in a deprived nation like ours became a major restraining factor. Despite an asset of having high specificity, RT-PCR for coronavirus disease 2019has its own liability of having low sensitivity. Henceforth, as time passed by, the validity of the rapid diagnostic tests was put into question. In later months, a few centers around our periphery started conducting RT-PCR, but the time taken to obtain the result was long-drawn-out process and the patient who needed urgent neurosurgical intervention at Annapurna Neurological Institute and Allied Sciences had to wait. We would like to share our expedition through peaks and valleys of managing 215 patients during the vicious circle of lockdown and global pandemic.

Stable G-quadruplex DNA structures promote replication-dependent genome instability.

G-quadruplex (G4)-prone structures are abundant in mammalian genomes, where they have been shown to influence DNA replication, transcription, and genome stability. In this article, we constructed cells with a single ectopic homopurine/homopyrimidine repeat tract derived from the polycystic kidney disease type 1 (PKD1) locus, which is capable of forming triplex (H3) and G4 DNA structures. We show that ligand stabilization of these G4 structures results in deletions of the G4 consensus sequence, as well as kilobase deletions spanning the G4 and ectopic sites. Furthermore, we show that DNA double-strand breaks at the ectopic site are dependent on the nuclease Mus81. Hypermutagenesis during sister chromatid repair extends several kilobases from the G4 site and breaks at the G4 site resulting in microhomology-mediated translocations. To determine whether H3 or G4 structures are responsible for homopurine/homopyrimidine tract instability, we derived constructs and cell lines from the PKD1 repeat, which can only form H3 or G4 structures. Under normal growth conditions, we found that G4 cell lines lost the G4 consensus sequence early during clonal outgrowth, whereas H3 cells showed DNA instability early during outgrowth but only lost reporter gene expression after prolonged growth. Thus, both the H3 and G4 non-B conformation DNAs exhibit genomic instability, but they respond differently to endogenous replication stress. Our results show that the outcomes of replication-dependent double-strand breaks at non-B-DNAs model the instability observed in microhomology-mediated break-induced replication (BIR). Marked variability in the frequency of mutagenesis during BIR suggests possible dynamic heterogeneity in the BIR replisome.

Surgical Management among Patients with Spontaneous Supratentorial Intracerebral Haemorrhage Admitted in a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Spontaneous intracerebral haemorrhage is the second most common form of stroke and the most deadly one. An understanding of changing trends in the epidemiology of intracerebral haemorrhage prevalence, its risk factors, current practice in management, case fatality, and long-term outcome is essential to measure the effectiveness of stroke prevention and various treatment efforts. The objective of this study was to find out the prevalence of surgical management among patients with spontaneous supratentorial intracerebral haemorrhage in a tertiary centre. Methods: A descriptive cross-sectional study was conducted in the Department of Neurosurgery from January 2017 to December 2019. Ethical approval was obtained from the Institutional Review Committee (Reference number: 06/2020/IRC-ANIAS). A convenience sampling method was used. Data of the patients were retrieved from online medical records. Point estimate and 95% Confidence Interval were calculated. Results: Among 221 patients with spontaneous supratentorial intracerebral haemorrhage, 115 (52.04%) (45.45-58.63, 95% Confidence Interval) underwent surgical management. In-hospital mortality was seen in 23 (20%) and survivors at 3 months were 78 (67.82%) patients. Conclusions: The prevalence of surgical management among spontaneous supratentorial intracerebral haemorrhages was higher than in other studies done in a similar setting. Keywords: intracerebral haemorrhage; mortality; surgical procedure.

Early internal capsule infarction following globus pallidus internus lesioning for cervical dystonia.

INTRODUCTION AND IMPORTANCE: Ischemia associated with lesioning for the treatment of cervical dystonia is a very rare phenomenon. We reported a case of early internal capsule infarction following GPi lesioning for cervical dystonia. CASE PRESENTATION: A 56-year-old man with one year history of progressive onset of cervical dystonia which was unresponsive to medical therapy. He was planned for bilateral globus pallidus internus pallidotomy. After completion of the procedure on one side and the successful completion of stimulation and test lesion on another side, the patient suddenly developed dysarthria and one sided weakness. Post-operative magnetic resonance imaging revealed acute infarction in the posterior end of the internal capsule. The patient was managed conservatively and underwent physiotherapy and rehabilitation care. He recovered gradually with modified ranking scale 3 on discharge. CLINICAL DISCUSSION: Globus pallidus internus lesioning or ablation is one of the surgical treatment for dystonia and movement disorder. Though considered as a safe technique, various risk are associated with the procedure. Ischemia is one of the associated risk but is a very rare phenomenon. Though the patient did not have any signs of pre-operative ischemia, the occlusion of one of the perforators to internal capsule during ablation may be the cause of intra-operative ischemia. CONCLUSION: Ischemia associated with lesioning in the treatment of cervical dystonia is a very rare phenomenon. Though, ischemia related with radiofrequency ablation is very rare and sporadic, one has to be very cautious during GPi pallidotomy to prevent occlusion of perforators to internal capsule.

Customized Cost-Effective Polymethyl-Methacrylate Cranioplasty Implant Using Three-Dimensional Printer.

There is no doubt that many synthetic materials used in cranioplasty have given good result regarding patient's calvarial shape. However, the use of these materials is costly to the patient and requires complex intraoperative process. There has been a long history regarding the use of acrylic bone cement called as polymethyl-methacrylate (PMMA) as an implant due to its desirable properties. Here, we present three cases of simple, cost effective manually sculpted calvarial defect using three-dimensional (3D) printer. Sharing the achievement and challenges, we want to focus that the 3D customized implant of PMMA can be used as bone substitute.

Progress in neurosurgery: Contributions of women neurosurgeons in Asia and Australasia.

At the end of the first 100 years of neurosurgery as a specialty, it is appropriate to look back and then imagine the future. As neurosurgery celebrates its first century, the increasing role of women neurosurgeons is a major theme. This article documents the early women pioneers in neurosurgery in Asia and Australasia. The contributions of these trailblazers to the origins, academics, and professional organizations of neurosurgery are highlighted. The first woman neurosurgeon of the region, Dr. T.S. Kanaka of India, completed her training in 1968, not long after the trailblazers in Europe and North America. She heralded the vibrant communities of neurosurgical women that have developed in the vast and diverse nations of the region, and the many formal and informal groups of women in neurosurgery that have introduced and promoted talented women in the profession. Contributions of women neurosurgeons to academic medicine and society as a whole are briefly highlighted, as are their challenges in this male-dominated specialty. The region is home to many deeply conservative societies; in fact, some nations in the region have not yet trained their first woman neurosurgeon. The fortitude of these individuals to achieve at the highest levels of neurosurgery indicates great potential for future growth of women in the profession, but also demonstrates the need for initiatives and advocacy to reach the full potential of gender equity.

Study of Anterior Commissure-Posterior Commissure Distance among Nepalese Cohort.

INTRODUCTION: The main aim of this study is to determine the anterior commissure-posterior commissure (AC-PC) distance in Nepalese cohort and has comparison of intercommissural distance of Nepalese cohort with some other races. MATERIALS AND METHODS: The 47 patients, with mean age of 50 years, included in the study had undergone DBS or lesioning (Pallidotomy). Data were collected through the magnetic resonance imaging under DBS protocol and the manipulation, marking of AC and PC was done in Inomed Planning Software (IPS). RESULTS: The data revealed average AC-PC distance of Nepalese cohort to be 24.86 ± 2.08 mm, ranging from 16 mm to 30 mm. The study among 29 males and 18 females illustrated male to have longer AC-PC as compared to female (25.38 mm male and 24.02 mm female). The results also confirmed age related changes in AC-PC distance which was linearly increasing with the age. CONCLUSION: Comparison of data from other studies revealed Nepalese cohort to have almost similar AC-PC distance with Asian population, while that was longer in Caucasian and shorter in Hispanic Population.

A Simplified Risk Stratification Method for Women With Stage I Endometrial Carcinoma.

OBJECTIVES: Available risk stratification methods for women with endometrial carcinoma are controversially defined. We sought to develop a simplified and an individualized prognostic index for cancer recurrence in women with International Federation of Gynecology and Obstetrics (FIGO) stage I endometrial carcinoma, solely of endometrioid histology. MATERIALS AND METHODS: We identified 976 women who underwent a hysterectomy and did not receive any adjuvant therapy. Cox proportional hazards model was used to identify independent predictors of recurrence. Prognostic groups were created based on the number of independent predictors of recurrence (0, 1, or 2 or 3 risk factors). These groups were then validated using a separate cohort of 611 women treated at another academic institution. The model's performance for predicting cancer recurrence was measured by the concordance probability estimate along with a 95% confidence interval. RESULTS: Median follow-up was 65 months. The final recurrence model included 3 risk groups based on 3 independent predictors of recurrence (tumor grade 2 or 3, the presence of lymphovascular space invasion and stage IB). Five-year recurrence rates were 4%, 16%, and 44% for groups 0, 1, and 2 or 3, respectively. The performance of the model was very good with a concordance probability estimate of 0.72 and 0.80 for the development and validation cohorts, respectively. CONCLUSIONS: On the basis of 3 well-known prognostic factors, we have developed and externally validated a simplified prognostic model that accurately predicts cancer recurrence in women with stage I endometrial carcinoma. This simplified predictive tool may be helpful in estimating individualized risk of recurrence and guide counseling with regard to adjuvant treatment.

Breast fibroadenomas are not associated with increased breast cancer risk in an African American contemporary cohort of women with benign breast disease.

BACKGROUND: Fibroadenomas are common benign breast lesions, and studies of European American women indicate a persistent, increased risk of breast cancer after diagnosing a fibroadenoma on biopsy. This association has not been independently assessed in African American women, despite reports that these women are more likely to present with fibroadenomas. METHODS: The study cohort included 3853 African American women with a breast biopsy completed between 1997 and 2010 in metropolitan Detroit. Biopsies were microscopically reviewed for benign breast lesions, including fibroadenoma, proliferative disease, and atypia. Risk of breast cancer within the cohort was estimated using relative risk ratios and 95% CIs calculated using multivariable log-binomial regression. Relative risk of breast cancer in this cohort compared with African American women in the broader metropolitan Detroit population was estimated using standardized incidence ratios (SIRs). RESULTS: Fibroadenomas occurred more frequently in biopsies of younger women, and other types of benign breast lesions were less likely to occur when a fibroadenoma was present (p = 0.008 for lobular hyperplasia; all other p values < 0.01). Unlike women with other benign lesions (SIR, 1.41; 95% CI, 1.20, 1.66), women with fibroadenomas did not have an increased risk of developing breast cancer compared with the general population (SIR, 0.94; 95% CI, 0.75, 1.18). Biopsies that indicated a fibroadenoma were associated with a reduced risk of breast cancer after adjusting for age at biopsy, proliferation, and atypia (relative risk, 0.67; 95% CI, 0.48, 0.93) compared with biopsies without a fibroadenoma. CONCLUSIONS: These findings have important implications for breast cancer risk models and clinical assessment, particularly among African American women, in whom fibroadenomas are common.

Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis.

Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M. tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M. tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.

Prospective study of transsphenoidal pituitary surgery: is tumor volume a predictor for the residual tumor?

BACKGROUND: The presence of residual tumor after surgery for pituitary adenoma may necessitate further treatment. The suprasellar and parasellar extension of the tumor have been widely considered as the predictors for residual tumor. However there is scarcity of studies regarding the preoperative tumor volume and residual tumor. This study was conducted to evaluate if tumor volume could predict the outcome of transsphenoidal pituitary surgery. METHODS: A prospective study was designed and 48 patients who underwent transsphenoidal pituitary surgery within 1 year in the First Affiliated Hospital of Xi'an Jiaotong University were included in this study. The preoperative tumor volume and immediate postoperative tumor volume (within 4 - 7 days) were calculated in the contrast magnetic resonance imaging by using the formula of ellipsoid. All these volumes were divided into three subgroups, i.e. group 1, group 2 and group 3 with preoperative volume of less than 4 cm(3), 4 - 8 cm(3), and more than 8 cm(3) respectively. The parasellar and suprasellar extension of the tumor were also classified by Knosp and modified Hardy's classifications. RESULTS: Baseline characteristics were comparable. The preoperative tumor volume of more than 8 cm(3) (group 3, (12.1 ± 1.1) cm(3)) had increased risk on postoperative tumor residue (P < 0.01) than the other two groups ((2.1 ± 0.3) cm(3) and (6.1 ± 0.3) cm(3) in groups 1 and 2). The mean postoperative volume in group 3 patients ((2.2 ± 0.1) cm(3)) was significantly higher than the other two groups (P < 0.01). CONCLUSION: Preoperative volume of more than 8 cm(3) can be considered as a predictor for postoperative residual volume.

Risk factors analysis on traumatic brain injury prognosis.

OBJECTIVE: To investigate the independent risk factors of traumatic brain injury (TBI) prognosis. METHODS: A retrospective analysis was performed in 885 hospitalized TBI patients from January 1, 2003 to January 1,2010 in the First Affiliated Hospital of Medical College of Xi'an Jiaotong University .Single-factor and logisticregression analysis were conducted to evaluate the association of different variables with TBI outcome. RESULTS: The single-factor analysis revealed significant association between several variables and TBI outcome, including age ( P=0.044 for the age group 40-60, Pü0.001 for the age group ≥60), complications ( P<0.001), cerebrospinal fluid leakage( P<0.001), Glasgow Coma Scale(GCS) ( P<0.001), pupillary light reflex ( P<0.001), shock ( P<0.001), associated extra-craniallesions ( P=0.01), subdural hematoma ( P<0.001), cerebral contusion ( P<0.001), diffuse axonal injury ( P<0.001), and subarachnoid hemorrhage( P<0.001), suggesting theinfluence of those factors on the prognosis of TBI. Furthermore, logistic regression analysis identified age, GCS score, pupillary light reflex, subdural hematoma,and subarachnoid hemorrhage as independent risk factors of TBI prognosis. CONCLUSION: Age, GCS score, papillary lightreflex, subdural hematoma, and subarachnoid hemorrhage may be risk factors influencing the prognosis of TBI. Paying attention to those factors might improve the outcome of TBI in clinical treatment.