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Reassembling Traditional Dance reveals traditional dance choreography in contemporary Singapore and Southeast Asia to be a zone of intense interaction between two interwoven but distinct sets of postcolonial and globalized elements. The first is the colonial assemblage of governance practices formed in the era of British colonialism in Malaya, which continues to define the discourse of tradition in Singapore. I identify how racial and temporal logic rooted in colonial epistemologies replicates itself as grant frameworks, self-Orientalizing marketing strategies and festival themes, rendering non-western dance within the familiar frame of the “traditional”. The entrepreneurial assemblage of contemporary, neoliberal Singapore on the other hand is constituted by neoliberal policy instruments that now transform community-based performance cultures like Indian, Malay and other “traditional” dance, into sites of creative economic regulation, adding market values to the mix of factors that shape urban Indian identity performance in Singapore today.I offer choreographic reassembly as an organizing conceptual framework to analyze choreographers emerging in relation to both the colonial and entrepreneurial assemblages. Choreographic reassembly entails deliberate and conscious experimentation with dance forms and aesthetics to meet immediate concerns of contemporary professional life as an artist in a complex, postcolonial, globalized, multiracial and multicultural art ecology. Choreographic reassembly is therefore the deliberate reshuffling and play with conventional and unconventional elements in a given traditional dance form, as a response to transformations in the context of cultural production in different sites. A range of examples ranging from early experiments by Singaporean choreographers K.P. and Santha Bhaskar in the 1950s, to more recent works by Singaporean, Indian, Malaysian, Thai and Indonesian choreographers are explored as cases of choreographic reassembly. Methodologically, this dissertation employs performance and dance analysis, historiography and ethnography. The concept of choreographic reassembly is indebted to Collier and Ong’s conceptual model of “global assemblages” drawn from contemporary, urban anthropological studies of globalization. Notions of cultural mutation and cross-generic experimentation draw from Donna Haraway’s philosophical interventions into anthropocentric epistemologies by “staying with the trouble” of colonial and capitalist inheritances.

Objectives: Acute repetitive seizures (ARSs) are one of the few commonly encountered neurological emergencies in children. There is a need for an appropriate timeline-based treatment protocol, which will be shown to be safe and efficacious in a clinical study. Materials and Methods: This was a retrospective chart review to determine the efficacy of a pre-specified treatment protocol for the management of ARSs in children aged 1–18 years. The treatment protocol was specifically applied in children with a diagnosis of epilepsy and not critically ill, who met the criteria for ARSs, with the exemption of new onset of ARSs. The first tier of treatment protocol focused on intravenous lorazepam, optimization of dose of existing anti-seizure medications (ASMs), and control of triggers like acute febrile illness, while second-tier focused on adding one or two additional ASMs, commonly used in cases with seizure clusters or status epilepticus. Results: We included the first 100 consecutive patients (7.6 ± 3.2 years, 63% boys). Our treatment protocol was successful in 89 patients (58 and 31 required first-tier and second-tier treatment). The absence of pre-existing drug-resistant epilepsy and the presence of acute febrile illness as a triggering factor (P = 0.02 and 0.03) were associated with the success of the first tier of the treatment protocol. Excessive sedation (n = 29), incoordination (n = 14), transient gait instability (n = 11), and excessive irritability (n = 5) were the most common adverse effects observed during the initial 1 week. Conclusion: This pre-specified treatment protocol is safe and efficacious in controlling ARSs in cases with established epilepsy who are not critically sick. External validation from other parts of the world/centers and a more diverse epilepsy population are required before generalizing the protocol into clinical practice.

Older children and adults with spinal muscular atrophy (SMA) have been shown to have more anxiety, depression, and other behavioral problems in a few studies. But no similar studies have been performed in infants and young children with SMA.Behavioral co-morbidities of young children with SMA were compared with healthy and children with chronic non-neurological illness control group. Infant Behavior Questionnaire-Revised (IBQ-R) and parent-report version of chid behavior checklist (CBCL) were used for infants and preschool-age children respectively.A total of 35 SMA children (age at symptom onset-5.9 ± 2.8 months, at enrolment-21.4 ± 7.1 months, 65% boys, 11, 19 and 5 were SMA 1, 2 and 3 respectively) and 24 siblings (38.6 ± 11.2 months, 71% boys) were enrolled. We also enrolled 15 children with nephrotic syndrome as age and gender matched control to SMA children in age-group 2-5 years (27.7 ± 9.1 months, 67% boys). In infants with SMA, the scale scores of IBQ-R were significantly higher for distress to limitation, fear, sadness, and falling reactivity/rate of recovery from distress (p = 0.005; 0.03; 0.001, and 0.04) and lower for soothability as compared to healthy control group (p = 0.04). Similarly, for the three dimensions of temperament computed from these 13 domains, the mean scale score for surgency/extraversion was lower and negative affectivity was higher (p = 0.04 and 0.03), in infants with SMA as compared to healthy controls. For preschool age group, the internalizing problem scores (p = 0.009 and 0.03) and stress problem scores (p = 0.002 and 0.04) were higher in the SMA group, as compared to both the healthy control group and diseased control group. While assessing the syndrome scale scores, the score was higher for emotionally reactive (p = 0.0002 and 0.01) and anxious domains (p 0.0001 and p = 0.0002) compared to both healthy and diseased control groups.Infants and young children with SMA suffer from increased internalizing problems like anxiety, depression and probably their healthy siblings are also at increased levels of stress, depicted by increased somatic complaints.

Objectives: This study aims to compare the cost-effectiveness of oral prednisolone and adrenocorticotropic hormone injection in West syndrome patients, the two most common hormonal therapies used for this condition. Materials and Methods: In this prospective and observational study, we documented sociodemographic, epilepsy, and development-related variables at baseline and up to 6 months after starting hormonal therapy, in all consecutive eligible patients of WS between August 2019 and June 2021, apart from the direct medical and non-medical costs and indirect health-care costs. We selected cost per quality-adjusted life-year (QALY) gained, per one patient with spasm freedom, one positive responder (>50% reduction in spasms), one relapse-free patient, and one patient with development gain. We determined whether incremental cost-effectiveness ratio for these parameters crossed the threshold value in base-case analysis and alternate scenario analysis. Results: Out of 52 patients screened, 38 and 13 patients enrolled in ACTH and prednisolone group. On D28, 76% and 71% achieved spasm cessation (P = 0.78) and the total cost of treatment was INR 19783 and 8956 (P = 0.01), in ACTH and prednisolone group respectively. For all pre-specified parameters, the cost/effectiveness ratios including cost/QALY gain were higher in ACTH group and the corresponding ICER values for all these parameters crossed the threshold cost value of INR 148,777 in base-case analysis and also in alternative scenario analysis. Conclusion: Treatment with oral prednisolone is more cost-effective as compared to ACTH injection for children with WS.

Objectives: The primary objective of the study was to compare the number of patients with febrile seizure recurrence within 1 year of presenting to our institute, among patients who received and didn’t receive oral iron supplementation. Materials and Methods: This prospective intervention study with historical controls was conducted to compare the number of patients with febrile seizure recurrence within 1 year, among patients who received and did not receive oral iron supplementation. The intervention group additionally received prophylactic iron supplementation of 20 mg biweekly for 1 year. Results: A total of 53 patients each were enrolled in both the groups, with comparable baseline characteristics. Although there was a trend toward a lower rate of recurrence of febrile seizures in the interventional group, as compared to the control group, it did not reach the point of statistical significance (P = 0.35). Both in the worst-case scenario and best-case scenario, there was a trend toward less risk of recurrence of febrile seizure in the intervention group, but it did not reach the point of statistical significance (P = 0.43 and 0.52). For the original scenario, worst-case scenario, and best-case scenario, the absolute risk reduction was 6.5%, 7%, and 6%, respectively, with corresponding number needed to treat (NNT) being 15, 14, and 16, respectively. The trend for absolute risk reduction was more pronounced in those with complex febrile seizures with an NNT of 6.5, but it still did not reach the point of statistical significance (P = 0.16). Moderate/severe IDA was also found to be an independent risk factor for recurrence of febrile seizure in the intervention group (P = 0.03). Conclusion: Oral serum iron supplementation does not significantly reduce the recurrence rate of febrile seizures in children aged 6–60 months. However, there is a trend toward reduction in the frequency of recurrence of febrile seizures, which is more pronounced in the subset with complex febrile seizures.

Inflammatory granulomas (neurocysticercosis [NCC] and tuberculomas) are important causes of seizures in children and adults in the developing world. Although several studies have explored seizure characteristics individually in patients with either NCC or tuberculoma, none has compared the seizure recurrence rate between them. This study included patients aged 5 to 18 years with viable parenchymal NCC or tuberculomas who had completed regular follow-up of at least 12 months at a tertiary institute in India. Their baseline seizure and electroencephalographic characteristics, antiseizure medications (ASMs), and breakthrough seizure rates at 12, 24, and 52 weeks were noted. A total of 103 and 54 patients with active NCC and tuberculomas respectively were included. The number of patients who had at least one breakthrough seizure at 12, 24, and 52 weeks in both groups was comparable (P = 0.32, 0.27, and 0.13), and the vast majority were controlled on monotherapy (98% cases in each group). The proportion of patients who required an increase in the dose or change of ASMs or polytherapy, the proportion of children who had status epilepticus at or before 12 months, abnormal electroencephalogram at 12 months, and calcified and active granuloma in neuroimaging at 12 months were also comparable between the two groups (P > 0.05 for all). The number of patients who had ASM-related adverse events and discontinued ASM due to serious adverse events was comparable between both groups, except for hepatotoxicity in the tuberculoma group. The predictors for breakthrough seizures that were found to be statistically significant in the NCC group were the presence of perilesional edema in the baseline magnetic resonance imaging (P = 0.02) and more than five active granulomas (P = 0.01); predictors in the tuberculoma group were the presence of severe perilesional edema causing midline shift in the baseline magnetic resonance imaging (P = 0.01) and more than five active granulomas (P = 0.04). The recurrence rates of breakthrough seizures over the next 12 months in newly detected cases of active NCC and tuberculomas were comparable.

This systematic review explored how virtual reality (VR) has been used to rehabilitate aphasia.Empirical studies were included where VR was used to target language, well-being, or quality of life in adults with acquired language impairment. Degenerative communication disabilities were excluded. Seven health databases were searched in October 2021. Risk of Bias was assessed using published checklists and completeness of intervention reporting evaluated. Narrative synthesis described forms of VR, rationales given, outcome measures, communication functions targeted, characteristics of interventions, and outcomes achieved within the framework of impairment, activity, and participation.Fourteen studies, involving 229 participants, met the criteria. The studies employed four forms of VR with various rationales given. Interventions used published and novel protocols. Primary outcomes targeted language impairment (12/14), activity (1/14), and well-being (1/14) and achieved positive outcomes in impairment and activity. All studies were exploratory. Risk of bias was high. Findings are discussed in the context of gains achieved by VR in other health contexts and the multi-user gaming literature.Uses of VR in aphasia rehabilitation described in the literature are limited. Most applications target the remediation of language impairments. Opportunities to address activity, participation, and wider aspects of well-being are rare.IMPLICATIONS FOR REHABILITATIONResearch documenting the use of virtual reality (VR) to rehabilitate aphasia is limited and exploratory, so does not yet offer clear guidance for clinicians.Many of the identified studies have used known published protocols (e.g., naming therapy or scripts therapy) delivered through the novel VR format and focus on language impairment outcomes.VR offers clinicians a unique opportunity to address communication activity and participation through the use of multi-user virtual worlds, but this has only been explored by only two research teams.

This systematic review explored coping with voice problems in professional voice users. The objectives were to: 1) evaluate how voice-related coping is assessed in professional voice users 2) investigate how they cope with voice problems, and 3) identify factors associated with voice-related coping.Systematic review.A systematic literature search of ten electronic databases using both EBSCOhost and OVID online platforms was conducted following the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. Only peer-reviewed articles which assessed coping in the context of voice problems in professional voice users were included. Methodological quality was assessed using Johanna-Briggs Institute Critical Appraisal checklists. Data analysis was conducted using narrative synthesis.Following deduplication, abstract and full-text screening, seven articles were included in the review. All participants (n=2484) were teachers; no other professional voice users were covered. 98% of the cases studied were females. The tools used to assess voice-related coping were Utrecht Coping List (UCL) and Voice Disability Coping Questionnaire (VDCQ). Studies which used UCL reported a passive coping pattern in teachers with high vocal handicap whereas VDCQ showed increased use of social support. Factors associated with coping were not examined by any of the studies.Seeking social support was highlighted as a frequently used coping strategy across studies and measures. Teachers with high vocal handicap used a passive coping pattern and active coping styles were not significantly used. Current evidence does not sufficiently specify factors affecting coping in professional voice users. More research on voice-related coping involving all professional voice users is warranted to identify associated factors and further ascertain its influence on vocal health.

Introduction: Cluster headache is rare in children and only a few clinical studies have systematically evaluated cluster headache in children. Methods: This study was conducted between August 2019 and December 2021 with the primary aim to evaluate the feasibility and utility of the Cluster Headache Severity Scale in determining the severity of cluster headache in patients aged less than 18 years and monitoring response to prescribed treatment. Secondary objectives were to evaluate the feasibility and utility of Cluster Headache Quality of Life, Cluster Headache Index, and 6-item Headache Impact Test in pediatric cluster headache patients to assess the quality of life, severity, and impact of cluster headache. Results: A total of 32 children (age of onset 11.9 ± 2.3 years, age of diagnosis 13.7 ± 2.4 years, 68% boys) were enrolled. Although 30 cases had their headache episodes occurring during nighttime, only 16 children had a Children’s Sleep Habits Questionnaire (CSHQ) score >41 at baseline. All children responded to prednisolone as bridging therapy and 23 of 32 showed adequate pain relief after sumatriptan nasal spray for an acute attack. The average time taken for completion of Cluster Headache Index, Cluster Headache Severity Scale, Cluster Headache Quality of Life, and Headache Impact Test–6 scores were 5.2 ± 0.7, 5.1 ± 0.8, 27.4 ± 3.5, and 6.2 ± 0.8 minutes, respectively. The interrater reliability was good for Cluster Headache Severity Scale, Cluster Headache Quality of Life, and Headache Impact Test–6 (Cronbach α 0.93, 0.81, and 0.89, respectively). There was a strong positive correlation between the Cluster Headache Severity Scale score with Headache Impact Test–6 score and Cluster Headache Quality of Life score (correlation coefficient r = 0.90 and 0.98). Conclusion: Majority of pediatric cluster headache patients are likely to respond to prednisolone and sumatriptan. Cluster Headache Severity Scale, Cluster Headache Quality of Life, and Headache Impact Test–6 can be used for pediatric cluster headache patients for treatment monitoring.

The clinical assessment scale for autoimmune encephalitis (CASE) is a recently developed and validated scale to rate the severity of autoimmune encephalitis (AE) in adults. But it is yet to be validated in pediatric AE cases.In a prospective observational study, we determined the validity and prognostic utility of CASE in the pediatric population with a diagnosis of probable or definite AE. We also determined clinical, neuroimaging, or laboratory-based prognostic factors for favorable clinical outcomes at 3 months after presentation. We used weighted kappa statistics and the intra-class correlation coefficient of individual item scores and total scores for determining inter-observer and intra-observer reliability respectively.We enrolled a total of 54 patients (28 girls, probable [45%] or definite [55%] AE). Functional status score (FSS), CASE score, and other scores showed significant improvement at the time of discharge and 3-months, as compared to baseline (p 0.0001). The intra-observer and interobserver reliability of the total scores was excellent (k = 0.94 and 0.95 respectively). CASE was also found to have good internal consistency (Cronbach-α = 0.83). The corrected item-total correlations of all items were0.40. The correlation between the total CASE score and FSS score at admission, at discharge, and at 3 months was strong (r = 0.90, 0.92, and 0.94, p 0.001). In multivariate analysis, only seropositivity or definite AE and CASE score at baseline was found to be significant predictive factors for functional status at 3 months (p = 0.03, 0.01).CASE score can be used for monitoring the severity of pediatric AE patients. It also has prognostic usefulness for predicting functional independence on follow-up.

Only a few studies have explored prognostic factors for tuberculous meningitis (TBM) in children, and an easily applicable bedside prognostic score for TBM has not been developed yet. We compared the sociodemographic, clinical, radiological, and cerebrospinal fluid parameters in the cohort of 94 TBM cases aged 1 to 18 years, with at least 6 months of completed follow-up and determined the prognostic factors associated with poor functional outcome. We assessed our proposed prognostic model using both discrimination and calibration and subsequently used the bootstrap method to validate the model internally. We finally derived an easily applicable bedside prognostic score by rounding off the regression coefficients to the nearest integers. A total of 39 (41%) and 55 (59%) patients had poor and good functional outcomes, respectively, at the end of 6 months (12 died, 13%). In multivariate analysis, a high baseline Pediatric Cerebral Performance Category (PCPC) score, brain infarction in neuroimaging, tonic motor posturing, younger age, and stage III TBM were independent predictors of poor functional outcomes. The final model showed good discrimination (area under the curve = 88.2%, P < 0.001) and good calibration (Hosmer–Lemeshow test, P = 0.53). Bootstrapping also confirmed the internal validity of this model. The PITAS (PCPC score [P], brain infarction in neuroimaging [I], tonic motor posturing [T], age [A], and stage of TBM [S]) score developed from this model has a score ranging from 0 to 12, with a higher score predicting a higher risk of poor functional outcome. The PITAS score performed better than medical research council staging alone in predicting poor functional outcomes (area under the curve = 87.1% versus 82.3%). Our study’s PITAS score, developed and internally validated, has good sensitivity and specificity in predicting poor functional outcomes in pediatric TBM cases at 6 months.

Vinpocetine as a neuroprotective agent is effective in acute ischemic stroke in some randomized controlled trials (RCTs). Since the last systematic review has been published in 2008, which didn't find conclusive evidence favoring its use, two more RCTs have also been completed.Relevant electronic databases were searched with a suitable combination of Medical Subject Headings terms to detect publications describing RCTs exploring the safety and efficacy of vinpocetine in patients with acute ischemic stroke. The risk of bias was determined by using the Cochrane Collaboration's tool for assessing the risk of bias in RCTs after full-text review and relevant data extraction. Higgins and Thompson's IFour placebo-controlled RCTs enrolling a total of 601 and 236 patients in vinpocetine and placebo groups, respectively, were included. The number of patients with death or significant disability was lower in the vinpocetine group than that in the placebo group at both 1 and 3 months (relative risk 0.80, 95% confidence interval [CI] 0.65-0.99 and relative risk 0.67, CI 0.48-0.92, p = 0.04 and 0.02, respectively). The degree of disability in participants at 1 month and 3 months was also lower in vinpocetine group than that in the placebo group (standardized mean difference (SMD) 0.49, 95% CI 0.03-0.95 and SMD 1.22, CI 0.23-2.24, p = 0.001 and 0.04, respectively). Change in mini-mental state examination score compared with baseline at trial enrolment was also better in the vinpocetine group than in the placebo group (pooled weighted mean difference 0.92, 95% CI 0.02-1.82, p = 0.04).Vinpocetine has some promising efficacy in patients with ischemic stroke when used in the acute stage in reducing the disability, but presently there is not enough evidence to suggest that it also reduces case fatality. More double-blind, placebo-controlled RCTs of adequate sample size are needed before making recommendations for the routine administration of vinpocetine for all patients with acute ischemic stroke.

Background & objectives: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder and is caused mainly by deletion, duplication and point mutations in the DMD gene. Diagnosis of DMD has been a challenge as the mutations in the DMD gene are heterogeneous and require more than one diagnostic strategy for the validation of the mutation. This study was planned to evaluate the targeted next-generation sequencing (NGS) as a single platform to detect all types of mutations in the DMD gene, thereby reducing the time and costs compared to conventional sequential testing and also provide precise genetic information for emerging gene therapies. Methods: The study included 20 unrelated families and 22 patients from an Indian population who were screened for DMD based on phenotypes such as scoliosis, toe walking and loss of ambulation. Peripheral blood DNA was isolated and subjected to multiplex ligation-dependent probe amplification (MLPA) and targeted NGS of the DMD gene to identify the nature of the mutation. Results: In the study patients, 77 per cent of large deletion mutations and 23 per cent single-nucleotide variations (SNVs) were identified. Novel mutations were also identified along with reported deletions, point mutations and partial deletions within the exon of the DMD gene. Interpretation & conclusions: Our findings showed the importance of NGS in the routine diagnostic practice in the identification of DMD mutations over sequential testing. It may be used as a single-point diagnostic strategy irrespective of the mutation type, thereby reducing the turnaround time and cost for multiple diagnostic tests such as MLPA and Sanger sequencing. Though MLPA is a sensitive technique and is the first line of a diagnostic test, the targeted NGS of the DMD gene may have an advantage of having a single diagnostic test. A study on a larger number of patients is needed to highlight NGS as a single, comprehensive platform for the diagnosis of DMD.

Background: Schizophrenia, a chronic severe psychiatric illness of unknown aetiology, has been shown to be associated with HLA alleles but at varied degree in different population. The present study has focussed on analysing the frequency of HLA class I and class II alleles in persons with schizophrenia from South India. Methods: Ninety seven individuals with schizophrenia and 103 age- and gender-matched controls were typed for HLA- A, B, C, DRB1 and DQB1 loci by next-generation sequencing in Illumina MiniSeq using MIA FORA NGS FLEX HLA typing kit. Results: The results showed that HLA-A*01:01:01, B*37:01:01 and C*01:02:01 were positively associated with schizophrenia while HLA-B*35:03:01 and DRB1*04:03:01 were negatively associated. Gender-specific associations revealed that DRB1*10:01:01 and DQB1*05:01:01 were positively associated while DQB1*03:02:01 was negatively associated with female subjects with schizophrenia. A*24:02:01~B*37:01:01~C*06:02:01~DRB1*10:01:01~DQB1*05:01:01 is the predominant haplotype in schizophrenia population when compared to healthy controls. Amino acid association in susceptible and protective alleles has shown that the presence of peptide in the peptide-binding groves of mature HLA-A protein (K, M, V, R and V at 44th, 67th, 150th, 156th and 158th position), HLA-B protein (D and S at 77th and 99th position) and HLA-C protein (M at 99th position) confer susceptibility to the disease, only in the absence of E (Glutamic acid) at 74th position in mature HLA-DRB1 protein. Interaction of amino acids in protective alleles namely B*35:01:01 and DRB1*04:03:01 has revealed that aspartic acid at 114th (D) position in mature HLA-B protein and glutamic acid (E) at 74th position of mature HLA-DRB1 protein have a combined effect in protecting against the disease. Conclusion: The study has revealed the HLA association with schizophrenia in south Indian population. The amino acid interaction with the disease needs to be confirmed in a larger population.

Aim: To compare the effects of pretreatment with doxycycline and chlorhexidine on microtensile bond strength to dentin in class I cavities restored with composite resin. Materials and Methods: Class I cavities were prepared on the occlusal surface of 30 freshly-extracted caries-free molars. In group 1, 2% chlorhexidine was applied to the acid etched surface, placed in contact for 20 seconds, and gently air dried without rinsing. In group 2, 2% doxycycline was applied to the acid etched surface, placed in contact for 20 seconds, and gently air dried without rinsing. In group 3, the control group, just the conventional bonding procedure was used without any pre-treatment. The cavities were restored with resin composite using a bulk method. After thermocycling, the specimens were serially sectioned to obtain resin dentin sticks with a rectangular cross-sectional area of approximately 1mm2 using microtome. The microtensile bond strength values were tested using Universal testing machine at crosshead speed of 0.5 mm/min until failure. The results obtained were analyzed by One-way ANOVA and Tukey's HSD tests for statistical significance. Results: The pre-treatment with chlorhexidine and doxycycline showed a significant effect in preserving the in vitro bond strength, when compared to the control group (p=0.000). Among the test groups, the chlorhexidine treatment resulted in a higher bond strength value than doxycycline group (p=0.000). Conclusion: Pre-treatment with MMP inhibitors resulted in higher bond strength values. Chlorhexidine group showed a significantly higher bond strength value compared to the Doxycycline group. Keywords: Chlorhexidine, Dentin, Doxycycline, MMP inhibitors, Microtensile bond strength

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10–12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3′-TCC, TTC, and TTT, and the consensus sequence is 3′-Pu(Py)2–3. Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10−6), yeast mitochondrial Rpo41 (6 × 10−6), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10−5) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10−4. The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine–purine mismatches were relatively frequent (10−5–10−6), whereas C→G, U→A, G→C, and C→A errors from purine–purine and pyrimidine–pyrimidine mismatches were rare (10−7–10−10). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10−4). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of in vitro transcribed RNAs.

The 3.6 GHz SPARC T5 processor is Oracle's next generation CMT SoC processor implemented in TSMC's 28 nm process with 1.5 billion transistors. Significant performance improvements were made by doubling the previous generations number of cores to 16 and L3 cache size to 8 MB while increasing bandwidth by nearly 3×. Power efficiency was improved through features like DVFS, core-pair cycle skipping and SerDes power scaling. The SPARC T5 processor has been designed to fit in systems that can scale from 1 to 8 sockets, or 128 to 1024 threads, in glueless fashion. The diverse system-level bandwidth requirements of up to 5.65 TB/sec in these systems are met by advanced SERDES design that handles up to 30 dB loss in an area and power efficient manner. The different thermal envelopes of these systems are addressed by power management features that span software, system and chip design.

The V protein of the paramyxovirus subfamily Paramyxovirinae is an important virulence factor that can interfere with host innate immunity by inactivating the cytosolic pathogen recognition receptor MDA5. This interference is a result of a protein-protein interaction between the highly conserved carboxyl-terminal domain of the V protein and the helicase domain of MDA5. The V protein C-terminal domain (CTD) is an evolutionarily conserved 49- to 68-amino-acid region that coordinates two zinc atoms per protein chain. Site-directed mutagenesis of conserved residues in the V protein CTD has revealed both universal and virus-specific requirements for zinc coordination in MDA5 engagement and has also identified other conserved residues as critical for MDA5 interaction and interference. Mutation of these residues produces V proteins that are specifically defective for MDA5 interference and not impaired in targeting STAT1 for proteasomal degradation via the VDC ubiquitin ligase complex. Results demonstrate that mutation of conserved charged residues in the V proteins of Nipah virus, measles virus, and mumps virus also abolishes MDA5 interaction. These findings clearly define molecular determinants for MDA5 inhibition by the paramyxovirus V proteins.

Over the last few years, salivary diagnostics has become a topic of discussion for many researchers primarily due to several advantages that offer over other diagnostic methods. Diagnostic techniques that involve oral fluids are mainly of interest as they are relatively easy to use, lower in cost and non-invasive. This review provides a brief overview of saliva as a best diagnostic tool and how salivary fluid can play a significant role in the early and easy detection of some major viruses such as HIV, HPV, Influenza, Zika and Hepatitis.

Measles virus, a member of the Morbillivirus family, infects millions of people each year despite the availability of effective vaccines. The V protein of measles virus is an important virulence factor that can interfere with host innate immunity by inactivating alpha/beta interferon (IFN-α/β) and IFN-γ signaling through protein interactions with signal transducer and activator of transcription proteins STAT1 and STAT2. Here we demonstrate that although STAT1 interference results from protein interactions within a V protein N-terminal region encompassed by amino acids 110 to 130, detection of STAT1 interaction and IFN-γ signaling inhibition requires the presence of cellular STAT2. Cell-specific variability in STAT1 interference was observed to correlate with V protein expression level. A more direct target for measles virus V protein-mediated IFN-α/β evasion is STAT2. Results indicate that the widely conserved C-terminal zinc finger domain of measles virus V protein is both necessary and sufficient to bind STAT2 and disrupt IFN-α/β signal transduction. Mutagenesis and molecular modeling define a contact surface for STAT2 association that includes aspartic acid residue 248 as critical for STAT2 interference and IFN antiviral immune suppression. These findings clearly define the molecular determinants for measles virus IFN evasion and validate specific targets as candidates for therapeutic intervention.

The second in the Niagara series of processors (Niagara2) from Sun Microsystems is based on the power-efficient chip multi-threading (CMT) architecture optimized for Space, Watts (Power), and Performance (SWaP) [SWap Rating = Performance/(Space * Power) ]. It doubles the throughput performance and performance/watt, and provides >10times improvement in floating point throughput performance as compared to UltraSPARC T1 (Niagara1). There are two 10 Gb Ethernet ports on chip. Niagara2 has eight SPARC cores, each supporting concurrent execution of eight threads for 64 threads total. Each SPARC core has a floating point and graphics unit and an advanced cryptographic unit which provides high enough bandwidth to run the two 10 Gb Ethernet ports encrypted at wire speeds. There is a 4 MB Level2 cache on chip. Each of the four on-chip memory controllers controls two FBDIMM channels. Niagara2 has 503 million transistors on a 342 mm2 die packaged in a flip-chip glass ceramic package with 1831 pins. The chip is built in Texas Instruments' 65 nm 11LM triple-Vt CMOS process. It operates at 1.4 GHz at 1.1 V and consumes 84 W.

Steroidogenic factor 1 (SF1) is a member of the NR5A subfamily of nuclear hormone receptors and is considered a master regulator of reproduction because it regulates a number of genes encoding reproductive hormones and enzymes involved in steroid hormone biosynthesis. Like other NR5A members, SF1 harbors a highly conserved approximately 30-residue segment called the FTZ-F1 box C-terminal to the core DNA binding domain (DBD) common to all nuclear receptors and binds to 9-bp DNA sequences as a monomer. Here we describe the solution structure of the SF1 DBD in complex with an atypical sequence in the proximal promoter region of the inhibin-α gene that encodes a subunit of a reproductive hormone. SF1 forms a specific complex with the DNA through a bipartite motif binding to the major and minor grooves through the core DBD and the N-terminal segment of the FTZ-F1 box, respectively, in a manner previously described for two other monomeric receptors, nerve growth factor-induced-B and estrogen-related receptor 2. However, unlike these receptors, SF1 harbors a helix in the C-terminal segment of the FTZ-F1 box that interacts with both the core DBD and DNA and serves as an important determinant of stability of the complex. We propose that the FTZ-F1 helix along with the core DBD serves as a platform for interactions with coactivators and other DNA-bound factors in the vicinity.

RNA viruses in the paramyxovirus family have evolved a number of strategies to escape host cell surveillance and antiviral responses. One mechanism exploited by a number of viruses in this family is direct targeting of cytokine-inducible transcription regulators in the STAT family. Diverse members of this large virus family effectively suppress STAT signaling by the actions of their V proteins, or the related proteins derived from alternate viral mRNAs. These viral proteins have distinct means of targeting STATs, resulting in a variety of negative effects on STATs and their signal transduction. Recent developments in understanding STAT targeting will be reviewed.

Detection of pathogens by the mammalian innate immune system is mediated by pattern recognition receptors. For viruses, nucleic acids are often the trigger for innate responses that culminate in antiviral gene expression, including the production of type I interferon (IFN-α/β). Foreign nucleic acids outside the cell can be recognized by transmembrane Toll-like receptors at the cell surface or in the lumen of endocytic vesicles (9). Intracellular nucleic acids are recognized by cytoplasmic receptor proteins (24). In both cases, receptor binding to the nucleic acid ligand triggers a signal transduction cascade that activates immediate transcriptional responses, including the induction of the antiviral cytokines in the IFN family. An important class of receptors for the detection of cytosolic nonself RNAs is represented by the proteins encoded by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (24). These proteins share functional domains, including an amino-terminal protein interaction motif that functions in signal transduction and is homologous to the caspase activation and recruitment domain (CARD) (8) and a C-terminal DECH-box family RNA helicase domain. The helicase domains share approximately 30 to 40% overall sequence identity. Experimental and structural studies have demonstrated a third functional region at the extreme C terminus of RIG-I that forms a zinc-mediated fold and functions as a regulatory domain for the recognition and discrimination of RNA 5′ ends (6, 20). Current evidence supports a model in which binding to RNA ligand induces a change in accessibility to the CARD, allowing it to interact with downstream signaling proteins, including the mitochondrial resident IPS-1 (also identified as MAVS, VISA, and Cardiff [12, 15, 21, 28]). This association coordinates a serine kinase-mediated cascade that activates latent transcription factors, including NF-κB and IFN regulatory factor 3, culminating in the expression of IFN-β and a number of other crucial antiviral effector genes (8). A third helicase protein, LGP2, resembles RIG-I and MDA5 in the helicase domain, also exhibiting 30 to 40% sequence identity. This overall level of similarity may underestimate the relatedness of these proteins, as they are more identical in their key helicase sequence motifs and encompassing domains (2). Significantly, LGP2 lacks the N-terminal CARD homology region. As a result, LGP2 expression is not typically associated with the ability to directly activate downstream signaling. Ectopic expression of LGP2 interferes with double-stranded RNA (dsRNA) and virus-induced antiviral signaling, while reduction of LGP2 expression by RNA interference results in enhanced IFN-β synthesis and antiviral responses. Expression of LGP2, like that of RIG-I and MDA5, is induced by virus infection, nucleic acid transfection, and IFN stimulation, suggesting it has the properties of a negative feedback inhibitor (14, 19, 20, 29). The importance of these RNA helicase proteins in antiviral responses is validated by the phenotypes of mice harboring targeted disruptions in these genes (9). Deficiency in RIG-I leads to a widespread enhancement in replication of many RNA virus types, due to suppressed IFN biosynthesis and antiviral responses (11). Cells deficient in RIG-I exhibit a general defect in the ability to respond to foreign dsRNAs or single-stranded RNAs (ssRNAs) bearing phosphorylated 5′ ends. MDA5 deficiency showed a more specific phenotype, resulting in increased susceptibility to picornavirus infection and insensitivity to the synthetic dsRNA analog poly(I:C) (7). LGP2 deficiency leads to a more complex phenotype (27). LGP2-deficient mice are more sensitive to IFN induction by cytosolic poly(I:C), an MDA5 ligand, and more resistant to vesicular stomatitis virus, a virus that triggers RIG-I signaling. Negative regulation of the IFN response remains intact overall, indicating that LGP2 may not be the primary negative regulator of type I IFN production. However, LGP2 deficiency results in a suppressed IFN response to infection with encephalomyocarditis virus, a picornavirus that has been demonstrated to trigger MDA5-mediated antiviral signaling. These data suggest that LGP2 executes both negative and positive regulatory functions related to RIG-I and MDA5 signaling. The disparate effects of LGP2 deficiency are difficult to interpret without a better mechanistic understanding of LGP2 functions in cellular innate antiviral immunity. Further affirmation of the antiviral role of the helicase proteins derives from the fact that individual viruses have evolved means to evade or disrupt their activity, either by direct interference or by antagonizing the signaling intermediates. The large Paramyxovirus family of negative-strand RNA viruses is well known to evade IFN antiviral responses. Most viruses within this family encode a protein, called V, that is essential for IFN signaling evasion. In addition to suppressing the activity of signal transducer and activator of transcription (STAT) proteins to compromise IFN signal transduction, paramyxovirus V proteins can also limit the induction of IFN gene expression by interfering with the MDA5 protein (1, 3). Associations between the V protein C-terminal domain (CTD), a 60- to 70-residue zinc binding fold that is the conserved hallmark of V proteins, and the MDA5 helicase domain prevents signal transduction downstream of poly(I:C), a synthetic RNA ligand that is a known MDA5 activator (1, 3). This inhibition was observed for all tested V proteins of the large Paramyxovirus family (3). Despite the potential importance of the V protein-MDA5 interface as an antiviral target, the mechanistic basis and consequences for MDA5 interference by V proteins are poorly understood and difficult to reconcile with the lack of phenotype in MDA5-deficient mice. Recent evidence indicates that MDA5 is involved in the detection of Sendai virus-defective interfering (DI) genomes, providing plausible biological relevance to the observed V protein interference that may not have been apparent from gene disruption studies (31). Identification of the MDA5 target recognized by paramyxovirus V proteins led to the discovery that LGP2 is also a target for V protein antagonism. Results demonstrate that the helicase C domain of both proteins functions as a V protein binding region common to both MDA5 and LGP2 but absent in RIG-I. We demonstrate that interaction with the V protein interferes with the catalytic activity of both target helicases, providing a biochemical basis for V protein helicase antagonism.

Background: BRAF is one of the most frequently activated oncogenes in melanoma comprising 40–60% of all mutations. About ninety percent of activating BRAF mutations occur at the V600E position enabling specific therapeutic targeting with inhibitors such as vemurafenib. Detection of the BRAF V600E mutation is therefore important for guiding treatment; however, access to tissue for testing is limited. We have recently shown that microvesicles (exosomes) isolated from serum/plasma contain RNA (exoRNA) and that tumor-derived exoRNA can be extracted from microvesicles circulating in peripheral blood. Thus the detection of the BRAF V600E mutation in blood represents a potentially useful biomarker for non-invasive diagnosis and monitoring therapeutic response. Methods: ExoRNA was extracted from the serum and plasma of 18 patients with biopsy confirmed grade IV melanoma and 12 healthy controls. Patients were consented and samples were collected in accordance with protocols approved by the IRB at MGH and LMU. Some patients included in the study were undergoing chemotherapy or drug treatment during blood draw (13/18), while others did not receive any treatment (5/18). BRAF V600E mutation in the isolated exoRNA from serum/plasma was measured by two methods. qPCR based assay: This assay used Taqman MGB™ probes and ARMS primers. The assay was able to detect mutations as low as Ultra-deep Sequencing assay using Illumina HiSeq technology: Eight melanoma patient samples were used for this assay (3 from group I and 5 from group II). Using PCR, a 100 bp region of BRAF encompassing the V600E mutation locus was amplified. Illumina library preparation protocol and bioinformatics methodology was then optimized to identify BRAF V600E mutations from ultra-high coverage amplicon sequencing data. To establish the sensitivity of the sequencing platform, we also made admixtures of synthetic DNA oligos with known sequences diluting the V600E variant in up to 10,000 copies of the wild type. We found that the lower detection limit for the rare mutant variant is about 1 in 5000 wild type molecules. Results: qPCR results indicate that 66% patients had concordance with biopsy. The healthy controls tested were all negative for V600E (0/12), giving the assay a specificity of 100% Ultra deep sequencing of exoRNA indicated that 62% patients had concordance with biopsy. By this method 2 million sequencing reads were generated per sample. The fraction of V600E to wild type variant was estimated to range from 0.05% to 10% in the samples. Conclusions: Both qPCR and ultra-deep sequencing have identified the BRAF V600E mutation from microvesicles circulating in peripheral blood in patients with malignant melanoma. Ultra-deep sequencing further allows accurate estimation of the fraction of tumor-derived microvesicles which may be used as a surrogate marker for the actual tumor burden. ExoRNA from peripheral blood has the potential to act as a novel class of diagnostic tests for both mutation detection and therapy response monitoring without the need for invasive biopsy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C139.