Chromatinization modulates topoisomerase II processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin's predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

Chromatinization Modulates Topoisomerase II Processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress in vivo. While cellular processes constantly create different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II). We observed that topo II was at least ~ 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a ~100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive (~ 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.

Catch bonds in sickle cell disease: Shear-enhanced adhesion of red blood cells to laminin.

Could the phenomenon of catch bonding-force-strengthened cellular adhesion-play a role in sickle cell disease, where abnormal red blood cell (RBC) adhesion obstructs blood flow? Here, we investigate the dynamics of sickle RBCs adhering to a surface functionalized with the protein laminin (a component of the extracellular matrix around blood vessels) under physiologically relevant microscale flow. First, using total internal reflectance microscopy we characterize the spatial fluctuations of the RBC membrane above the laminin surface before detachment. The complex dynamics we observe suggest the possibility of catch bonding, where the mean detachment time of the cell from the surface initially increases to a maximum and then decreases as a function of shear force. We next conduct a series of shear-induced detachment experiments on blood samples from 25 sickle cell disease patients, quantifying the number and duration of adhered cells under both sudden force jumps and linear force ramps. The experiments reveal that a subset of patients does indeed exhibit catch bonding. By fitting the data to a theoretical model of the bond dynamics, we can extract the mean bond lifetime versus force for each patient. The results show a striking heterogeneity among patients, both in terms of the qualitative behavior (whether or not there is catch bonding) and in the magnitudes of the lifetimes. Patients with large bond lifetimes at physiological forces are more likely to have certain adverse clinical features, like a diagnosis of pulmonary arterial hypertension and intracardiac shunts. By introducing an in vitro platform for fully characterizing RBC-laminin adhesion dynamics, our approach could contribute to the development of patient-specific antiadhesive therapies for sickle cell disease. The experimental setup is also easily generalizable to studying adhesion dynamics in other cell types, for example, leukocytes or cancer cells, and can incorporate disease-relevant environmental conditions like oxygen deprivation.

Integrating deep learning with microfluidics for biophysical classification of sickle red blood cells adhered to laminin.

Sickle cell disease, a genetic disorder affecting a sizeable global demographic, manifests in sickle red blood cells (sRBCs) with altered shape and biomechanics. sRBCs show heightened adhesive interactions with inflamed endothelium, triggering painful vascular occlusion events. Numerous studies employ microfluidic-assay-based monitoring tools to quantify characteristics of adhered sRBCs from high resolution channel images. The current image analysis workflow relies on detailed morphological characterization and cell counting by a specially trained worker. This is time and labor intensive, and prone to user bias artifacts. Here we establish a morphology based classification scheme to identify two naturally arising sRBC subpopulations-deformable and non-deformable sRBCs-utilizing novel visual markers that link to underlying cell biomechanical properties and hold promise for clinically relevant insights. We then set up a standardized, reproducible, and fully automated image analysis workflow designed to carry out this classification. This relies on a two part deep neural network architecture that works in tandem for segmentation of channel images and classification of adhered cells into subtypes. Network training utilized an extensive data set of images generated by the SCD BioChip, a microfluidic assay which injects clinical whole blood samples into protein-functionalized microchannels, mimicking physiological conditions in the microvasculature. Here we carried out the assay with the sub-endothelial protein laminin. The machine learning approach segmented the resulting channel images with 99.1±0.3% mean IoU on the validation set across 5 k-folds, classified detected sRBCs with 96.0±0.3% mean accuracy on the validation set across 5 k-folds, and matched trained personnel in overall characterization of whole channel images with R2 = 0.992, 0.987 and 0.834 for total, deformable and non-deformable sRBC counts respectively. Average analysis time per channel image was also improved by two orders of magnitude (∼ 2 minutes vs ∼ 2-3 hours) over manual characterization. Finally, the network results show an order of magnitude less variance in counts on repeat trials than humans. This kind of standardization is a prerequisite for the viability of any diagnostic technology, making our system suitable for affordable and high throughput disease monitoring.

Spoken Language Identification Using Deep Learning.

The process of detecting language from an audio clip by an unknown speaker, regardless of gender, manner of speaking, and distinct age speaker, is defined as spoken language identification (SLID). The considerable task is to recognize the features that can distinguish between languages clearly and efficiently. The model uses audio files and converts those files into spectrogram images. It applies the convolutional neural network (CNN) to bring out main attributes or features to detect output easily. The main objective is to detect languages out of English, French, Spanish, and German, Estonian, Tamil, Mandarin, Turkish, Chinese, Arabic, Hindi, Indonesian, Portuguese, Japanese, Latin, Dutch, Portuguese, Pushto, Romanian, Korean, Russian, Swedish, Tamil, Thai, and Urdu. An experiment was conducted on different audio files using the Kaggle dataset named spoken language identification. These audio files are comprised of utterances, each of them spanning over a fixed duration of 10 seconds. The whole dataset is split into training and test sets. Preparatory results give an overall accuracy of 98%. Extensive and accurate testing show an overall accuracy of 88%.

Characterization of a butyrate kinase from Desulfovibrio vulgaris str. Hildenborough.

Short and branched chain fatty acid kinases participate in both bacterial anabolic and catabolic processes, including fermentation, through the reversible, ATP-dependent synthesis of acyl phosphates. This study reports biochemical properties of a predicted butyrate kinase from Desulfovibrio vulgaris str. Hildenborough (DvBuk) expressed heterologously and purified from Escherichia coli. Gel filtration chromatography indicates purified DvBuk is active as a dimer. The optimum temperature and pH for DvBuk activity is 44°C and 7.5, respectively. The enzyme displays enhanced thermal stability in the presence of substrates as observed for similar enzymes. Measurement of kcat and KM for various substrates reveals DvBuk exhibits the highest catalytic efficiencies for butyrate, valerate and isobutyrate. In particular, these measurements reveal this enzyme's apparent high affinity for C4 fatty acids relative to other butyrate kinases. These results have implications on structure and function relationships within the ASKHA superfamily of phosphotransferases, particularly regarding the acyl binding pocket, as well as potential physiological roles for this enzyme in Desulfovibrio vulgaris str. Hildenborough.

Knowledge regarding avulsion, reimplantation and mouthguards in high school children: Organised sports-related orodental injuries.

PURPOSE: To assess knowledge, awareness and attitude of high school children involved in various sports activities regarding avulsion, reimplantation and mouthguard use. MATERIALS AND METHODS: An interview-based study was conducted among high school children in the age group of 8-16 years (n = 1105). A questionnaire was given to children and parents which included questions based on knowledge of children regarding avulsion, reimplantation, consequences of losing teeth, mouthgards and their usage and preferred media for tooth transportation. The research proposal was submitted to the ethics committee and the study design was approved. Data analysis included descriptive statistics and Statistical Package for the Social Sciences (SPSS) software. RESULTS: Only 37.6% of the children knew that their teeth can be completely knocked out with injury and 20.1% knew that a knocked-out tooth can be replanted. The preferred media for tooth transportation were gauze/cotton (27.2%), handkerchief (17.5%), paper (14%), water (13.8%) and cellophane (1.3%), i.e. 81.3% of the children preferred dry transport media. Among the children playing contact sport, 53.8% did not know about mouthguards and the rest 46.2% knew about it, but most of them never used it. CONCLUSION: Considering the lack of awareness among children, as well as the people around them, there appears to be a crucial need for education of public and to determine dental manpower and dentists to increase awareness.

Pathogenesis, Clinical Features, Diagnosis, and Management of Radiation Hazards in Dentistry.

INTRODUCTION: With the advent of newer radiographic diagnostic procedures of the maxillofacial region, there is a drastic increase in the use of Ionizing radiation which further leads to increased chance of radiation hazards among the patients and the health care workers. In addition to the diagnostic information extracted, the radiation exposure carries the potential to induce carcinogenesis in the exposed individual. However, the amount of Radiation exposure in dentistry is significantly low but it is still harmful owing to the requirement of repeated radiographic examination during the dental treatment. Therefore, to ensure minimum and inevitable exposure during dental treatment, it is necessary to follow principles of radiation protection and safety. RECOMMENDATIONS: Several studies in the literature have revealed that the attitude and knowledge of the dental professionals regarding radiation safety is not up to the mark. Henceforth, there is a necessity of implementing certain basic guidelines regarding radiation safety and protection. Further state dental councils must advocate new and interesting methods of education regarding the same and should introduce strict rules and penalties for this spectrum of field. CONCLUSION: This present short commentary is to familiarize the dental practitioner regarding the methods to minimize the risk of the radiation hazards. Further this article will also educate the dental practitioners regarding the pathogenesis of Radiation effects during Radiation therapy of head and neck region along with pertinent management protocols.

A study of sports related occurrence of traumatic orodental injuries and associated risk factors in high school students in north India.

BACKGROUND: Oral and dental injuries contribute to a major part of sports related injuries in children. Trauma occurring in developing years disrupts normal social functioning and brings about a major impact on quality of life due to their cumulative effect. OBJECTIVES: To assess the prevalence and causes of various sports related traumatic orodental injuries among 8 to 16 year school students along with identifying the associated risk factors in North India. PATIENTS AND METHODS: A cross sectional study consisting of high school students of different organized sports teams aged 8-16 years was carried out in geographical area of north India. The students were selected by multistage cluster sampling methodology. 1105 students from 19 school teams (sports teams) and sports academies participated in study through structured interview and clinical examination in different sports situations. RESULTS: 30.3% (n = 335) of students suffered from orodental injuries. A higher number of girls had injury (32%) than boys (29%), though the difference was not significant. Most of the students suffered from soft tissue injuries (48%) followed by tooth fractures (43%). Maximum numbers of injuries were reported in high velocity (44.1%) and medium intensity sports (46.6%) (P < 0.001) Maximum injuries occurred in basketball (50%) and lowest in the field of badminton (6.1%) (P < 0.05). Amateurs (52%) suffered the most from injuries as per level of coaching. Only 6% of boys and 2% of girls used mouthguards. CONCLUSIONS: The result of the present study confirmed that students participating in different organized sports at high school level are at a very high risk of getting orodental injury. Hence knowledge and education regarding prevention of traumatic injuries is of paramount importance.

The prevalence, radiographic appearance, and characteristics of zygomatic air cell defects (ZACDs) in symptomatic temporomandibular joint disorder patients in North Indian population.

PURPOSE: Although the incidence of zygomatic air cell defects (ZACDs) is significantly low in general population, still they pose as a risk factor during surgical procedures like eminectomies and eminoplasties because there is a risk of spread of infection intracranially. Furthermore, such procedures are more common in TMD patients. With this fact in mind, this study was designed to find out the prevalence, radiographic appearance, and characteristics of zygomatic air cell defects in diagnosed symptomatic temporomandibular joint disorder patients. METHOD: The study comprised of evaluation of panoramic radiographs of 70 temporomandibular joint disorder (TMD) patients in the age range of 18-30 years selected on the basis of Research Diagnostic Criteria (RDC/TMD). The radiographs were evaluated regarding the presence, variations, and characteristics of ZACDs. Groups were compared by χ (2) analysis. RESULTS: ZACDs were identified in 21 TMD subjects out of 70, giving an overall prevalence of 30 %. Out of 21 ZACDs, nine were in males (42.8 %) and 12 were in females (52.38 %). ZACDs were unilateral in ten TMD patients (47.61 %) and were bilateral in 11 patients (52.38 %). CONCLUSION: It can be concluded that the number of ZACDs is surprisingly more in diagnosed TMD patients. This strengthens the need for thorough preoperative imaging evaluation of ZACDs in such patients. Further longitudinal studies are required to find out the long-term effect of ZACDs on symptomatic as well as non-symptomatic TMD subjects. This may prove helpful to appreciate that whether ZACDs have any role in the development TMDs and vice versa. As with many TMD studies, caution should be exercised in interpreting these results until further studies have been carried out on this topic. Ideally, some prospective randomised evaluations with "hard" evidence of the diagnosis with MRI support.

A case of a bloated face: SVC syndrome relieved by an endovascular approach.

Superior vena cava (SVC) syndrome, or obstruction of blood returning from the head and upper extremities, is a syndrome that is rapidly increasing in the cardiovascular patient population due to the increasing use of transvenous devices such as permanent pacemakers, implantable cardioverter defibrillators (ICDs), and indwelling venous access devices for hemodialysis. This syndrome in the past has been seen predominately in the cancer population with malignancy being the most common reason for SVC syndrome. The management of this syndrome has largely been with a medical/supportive care approach or with surgical bypass. Given the advancement in the field of endovascular interventions and the increasing expertise in performing these procedures, an endovascular approach to relieving the SVC obstruction is rapidly becoming the treatment of choice for these patients. We describe a case of a patient who had a chronic indwelling port-a-cath who developed SVC syndrome, which was treated with an endovascular approach with stenting of the SVC/brachiocephalic vein junction.

Multidisciplinary approach to the rehabilitation of a tooth with two trauma episodes: systematic review and report of a case.

Studies have shown that some children and adolescents are affected only once with dental trauma, while others seem to be accident-prone and suffer from multiple dental trauma episodes. Less is known about treatment consequences related to repeated traumatic dental injuries to the same tooth and treatment guidelines are not well established. Complicated crown fractures and crown-root fractures pose difficulties for dentists to establish adequate treatment plans because these fractures require multidisciplinary knowledge and approach for a correct case planning and prognosis. The objective of this paper was to present and discuss a case of a child who sustained a second trauma to the same tooth following treatment of an earlier sustained crown fracture. The research-based background for establishment of the treatment plan is discussed. Reattachment of tooth fragment is a minimal invasive and esthetic method. Essential advantage of the reattached teeth is the fact that all the alternative methods as direct adhesive resin reconstruction, veneers and crowns can be performed in case of failure or a refracture. As a consequence of initial trauma, the tooth suffered a complicated crown fracture which was resolved by endodontic therapy and fragment reattachment. During follow-up, the child suffered a second trauma resulting in dislodgement and fracture of the reattached fragment and a crown-root fracture extending subgingivally with involvement of the biologic width. A conservative restorative option is described. After 1 year of follow-up, the clinical and radiographic findings demonstrated that the adopted clinical protocol was successful and yielded healthy periodontal tissues with no signs of periradicular pathosis. The occurrence of repeated traumatic dental injuries to teeth involving conservative management of both crown-root fracture and complicated crown fracture on the same tooth is extremely rare and a challenge for dental professionals to treat.

Trauma-induced dentigerous cyst involving an inverted impacted mesiodens: case report.

There have been only a small number of studies on the association of dentigerous cysts with supernumerary teeth. The purpose of this article was to report the case of a dentigerous cyst associated with an impacted inverted mesiodens that developed secondary to trauma to its predecessor, a non-vital permanent maxillary central incisor. As a consequence of trauma, the central incisor's root development was prematurely arrested and the open apex lay close to the follicle of the underlying inverted mesiodens. The negligent attitude of both the child and parent in seeking dental treatment was a contributing factor. The case was further complicated by impaction of the adjacent permanent central incisor due to the presence of another unerupted but normally oriented mesiodens. Occlusal and Intraoral periapical radiographs revealed a well-defined radiolucent area surrounding the inverted mesiodens. Microscopic examination revealed a cystic cavity that was lined by 2-3 cell thick non-keratinized stratified squamous epithelium resembling reduced enamel epithelium. Dentigerous cysts associated with impacted permanent teeth are not uncommon but the cysts which are induced by trauma are uncommon. Development of trauma-induced dentigerous cyst around an inverted impacted mesiodens associated with the periapical area of a traumatized, non-vital, immature permanent central incisor is a rare occurrence.

An unusual presentation of hypothyroidism.

We describe a case of a 42-year-old male, with a 5-year history of recurrent gum bleeding, easy bruising, and chronic fatigue who presented for management of possible bleeding complications prior to a planned dental procedure. After extensive work-up, the patient was diagnosed with acquired von Willebrand Disease and underlying hypothyroidism with a thyrotropin (TSH) of 321 mIU/L (0.35-5.5) and total thyroxine (T(4)) of less than 1 microg/dL (4.5-12.5). He was started on levothyroxine and therapy and when compliant with treatment, the mucosal bleeding and symptoms of hypothyroidism were resolved. A subsequent TSH and total T(4) samples were drawn and found to be 6.3 mIU/L and 4.1 microg/dL, respectively.

Silyl-heparin bonding improves the patency and in vivo thromboresistance of carbon-coated polytetrafluoroethylene vascular grafts.

OBJECTIVES: Our purpose was to improve the performance of carbon-coated expanded polytetrafluoroethylene vascular grafts by bonding the grafts with silyl-heparin, a biologically active heparin analog, using polyethylene glycol as a cross-linking agent. Material and method Silyl-heparin-bonded carbon-coated expanded polytetrafluoroethylene vascular grafts (Bard Peripheral Vascular, Tempe, Ariz), were evaluated for patency and platelet deposition 2 hours, 7 days, and 30 days after graft implantation in a canine bilateral aortoiliac artery model. Platelet deposition was determined by injection of autologous, (111)Indium-radiolabeled platelets, followed by a 2-hour circulation period prior to graft explantation. Histologic studies were performed on a 2-mm longitudinal strip of each graft (7-day and 30-day groups). Heparin activity of the explanted silyl-heparin grafts was determined by using an antithrombin-III based thrombin binding assay. RESULTS: Overall chronic graft patency (7-day and 30-day groups) was 100% for the silyl-heparin bonded (16/16) grafts versus 68.75% for control (11/16) grafts (P =.043). Acute 2-hour graft patency was 100% for the silyl-heparin bonded (6/6) grafts versus 83.3% for control (5/6) grafts. Radiolabeled platelet deposition studies revealed a significantly lower amount of platelets deposited on the silyl-heparin grafts as compared with control grafts in the 30-day group (13.8 +/- 7.18 vs 28.4 +/- 9.73, CPM per cm2 per million platelets, mean +/- SD, P =.0451, Wilcoxon rank sum test). In the 2-hour group of dogs, a trend towards a lower deposition of platelets on the silyl-heparin grafts was observed. There was no significant difference in platelet deposition between the two grafts in the 7-day group. Histologic studies revealed a significant reduction in intraluminal graft thrombus present on the silyl-heparin grafts as compared with control grafts in the 30-day group of animals. In contrast, there was no difference in amount of graft thrombus present on both graft types in the 7-day group of dogs. Pre-implant heparin activity on the silyl-heparin bonded grafts was 2.0 IU/cm(2) (international units[IU]/cm(2)). Heparin activity remained present on the silyl-heparin grafts after explantation at all 3 time points (2 hours: above upper limit of assay, upper limit = 0.57, n = 6; 7 days: 0.106 +/- 0.015, n = 5; 30 days: 0.007 +/- 0.001, n = 5; mean +/- SD, IU/cm(2)). CONCLUSION: Silyl-heparin bonding onto carbon-coated expanded polytetrafluoroethylene vascular grafts resulted in (1) improved graft patency, (2) increased in vivo graft thromboresistance, and (3) a significant reduction in intraluminal graft thrombus. This graft may prove to be useful in the clinical setting.