Heat-induced seizures, premature mortality, and hyperactivity in a novel Scn1a nonsense model for Dravet syndrome.

Dravet syndrome (Dravet) is a severe congenital developmental genetic epilepsy caused by de novo mutations in the SCN1A gene. Nonsense mutations are found in ∼20% of the patients, and the R613X mutation was identified in multiple patients. Here we characterized the epileptic and non-epileptic phenotypes of a novel preclinical Dravet mouse model harboring the R613X nonsense Scn1a mutation. Scn1aWT/R613X mice, on a mixed C57BL/6J:129S1/SvImJ background, exhibited spontaneous seizures, susceptibility to heat-induced seizures, and premature mortality, recapitulating the core epileptic phenotypes of Dravet. In addition, these mice, available as an open-access model, demonstrated increased locomotor activity in the open-field test, modeling some non-epileptic Dravet-associated phenotypes. Conversely, Scn1aWT/R613X mice, on the pure 129S1/SvImJ background, had a normal life span and were easy to breed. Homozygous Scn1aR613X/R613X mice (pure 129S1/SvImJ background) died before P16. Our molecular analyses of hippocampal and cortical expression demonstrated that the premature stop codon induced by the R613X mutation reduced Scn1a mRNA and NaV1.1 protein levels to ∼50% in heterozygous Scn1aWT/R613X mice (on either genetic background), with marginal expression in homozygous Scn1aR613X/R613X mice. Together, we introduce a novel Dravet model carrying the R613X Scn1a nonsense mutation that can be used to study the molecular and neuronal basis of Dravet, as well as the development of new therapies associated with SCN1A nonsense mutations in Dravet.

Complications of Cochleostomy Versus Round Window Surgical Approaches: A Systematic Review and Meta-Analysis.

We conducted a systematic review and meta-analysis to compare round window (RW) and cochleostomy (C) surgical approaches for the placement of cochlear implants (CIs). After obtaining the Institutional Review Board (IRB) approval, 213 peer-reviewed articles published between January 1, 2000, and August 1, 2021, comparing RW and C approaches were identified via a search on Google Scholar, Cochrane, and PubMed. The inclusion criteria were articles having an English version and involving only human subjects (cadaveric or alive). Statistical analysis of compiled electrode-to-modiolus distances was performed with two-sample independent t-tests. Live patients were categorized as having complete hearing preservation (<10 dB threshold shift), partial hearing preservation (10-20 dB shift), or minimal hearing preservation (>20 dB shift). Chi-squared testing was used to compare the distribution of hearing preservation categories between surgical approaches. Due to the heterogeneous nature of the data, only summative information was provided on the effects of approaches on trauma, electrical impedance, speech perception, vestibular dysfunction, ease of scala tympani insertion, and scalar shift. A total of 3,797 CI patients were evaluated. The RW approach resulted in a smaller (0.15 mm smaller on average, p<0.05) electrode-to-modiolus distance when compared to the C approach. The RW approach (93.0%) led to statistically better hearing preservation than the C approach (84.3%) (p<0.05). The RW approach was also associated with better outcomes in terms of speech perception, ease of scala tympani insertion, and reduced scalar shift. No difference between approaches was found with regard to trauma, electrical impedance, and vestibular dysfunction. Based on our findings, the RW approach appears to have several benefits compared to the C approach.

Effectiveness of a Tele-Ophthalmology Vision Center in Treating Corneal Disorders and Its Associated Economic Benefits.

PURPOSE: The aim of this study was to study the clinical and economic impact of a teleophthalmology-based vision center (VC) in the management of corneal diseases in rural south India. METHODS: Data of patients with corneal disorders who visited the VC in the year 2019 were analyzed for the total number of outpatient visits, the proportion of corneal cases that were treated at the VC, those referred to the base hospital (BH), and the costs of treatment. RESULTS: In 2019, 1131 (10%) of 10,850 patients who visited the VC were diagnosed with corneal disorders. Of these, 950 (84%) patients were treated at the VC itself and did not require to be referred. The remaining 181 (16%) were referred to the BH. Of these 1131 patients, 836 (74%) patients presented with a painful acute corneal disorder. The most common painful acute corneal disorders included corneal foreign body (376, 33%), epithelial abrasions (205, 18%), and infectious keratitis (124, 11%). A patient can save approximately a minimum of INR 1200 (USD 16) by attending the VC rather than the BH. In 2019, by treating 950 patients, the VC saved approximately INR 114,0000 (USD 15,200) for the community. A similar calculation extrapolated to a 10-year period (2009-2019) revealed that by providing care for these corneal disorders at the community level, the VC saved approximately INR 705,8400 (USD 94,112) for the community. CONCLUSIONS: VC reduces the barriers to care by increasing the accessibility and affordability of treatment for patients with corneal disorders, resulting in a significant cost saving to the community.

Screening first-degree relatives of glaucoma patients reveals barriers to participation.

PURPOSE: To report the results of a glaucoma screening campaign targeting first-degree relatives of glaucoma patients in South India. METHODS: 1598 glaucoma patients were contacted via letter or letter and phone call and asked to bring their siblings and children to a glaucoma screening. Participants underwent standardised eye examinations and completed questionnaires that assessed barriers to participation and awareness of glaucoma risk. Two-proportion z-tests were used to compare categorical data. Costs associated with the screening were recorded. RESULTS: 206 probands (12.9%) attended the screening along with 50 siblings and children. Probands were nearly twice as likely to attend if they had been contacted via both letter and phone call rather than letter only. Over half of probands reported that their relatives could not participate because they did not live in the region, and one-fifth reported that their relatives had other commitments. Fifty-eight per cent of the siblings and children who attended did not know that they were at increased risk for glaucoma due to their family history, and 32.0% did not know that the relative who had invited them to the screening had glaucoma. Thirteen siblings and children (26.0% of those who attended) were found to have findings concerning for glaucoma. The average cost per first-degree relative who was screened was INR2422 (£26). CONCLUSION: Participation in this glaucoma screening campaign was poor. The major barrier to participation was distance from the screening site and associated indirect costs. Better strategies for bringing first-degree relatives in for examinations are needed.

Use of predictive models to identify patients who are likely to benefit from refraction at a follow-up visit after cataract surgery.

PURPOSE: To develop predictive models to identify cataract surgery patients who are more likely to benefit from refraction at a four-week postoperative exam. METHODS: In this retrospective study, we used data of all 86,776 cataract surgeries performed in 2015 at a large tertiary-care eye hospital in India. The outcome variable was a binary indicator of whether the difference between corrected distance visual acuity and uncorrected visual acuity at the four-week postoperative exam was at least two lines on the Snellen chart. We examined the following statistical models: logistic regression, decision tree, pruned decision tree, random forest, weighted k-nearest neighbor, and a neural network. Predictor variables included in each model were patient sex and age, source eye (left or right), preoperative visual acuity, first-day postoperative visual acuity, intraoperative and immediate postoperative complications, and combined surgeries. We compared the predictive performance of models and assessed their clinical impact in test samples. RESULTS: All models demonstrated predictive accuracy better than chance based on area under the receiver operating characteristic curve. In a targeting exercise with a fixed intervention budget, we found that gains from predictive models in identifying patients who would benefit from refraction ranged from 7.8% (increase from 1500 to 1617 patients) to 74% (increase from 250 to 435 patients). CONCLUSION: The use of predictive statistical models to identify patients who are likely to benefit from refraction at follow-up can improve the economic efficiency of interventions. Simpler models like logistic regression perform almost as well as more complex machine-learning models, but are easier to implement.

Long-Term Surgical Outcomes of Primary Congenital Glaucoma in a South Indian Population.

PURPOSE: To evaluate the long-term surgical outcomes of children with primary congenital glaucoma (PCG) in a South Indian population. DESIGN: Retrospective cohort study. PARTICIPANTS: Children with PCG who underwent primary surgery from 1997 through 2000 at a tertiary eye center in India with minimum of 5 years of follow-up. METHODS: This retrospective cohort study included children with PCG who underwent trabeculotomy, trabeculectomy, or combined trabeculotomy and trabeculectomy (CTT) as primary surgery from 1997 through 2000 at a tertiary eye center in India with minimum of 5 years of follow-up. Survival analyses were performed to determine cumulative probability of complete and qualified success. MAIN OUTCOME MEASURES: Intraocular pressure (IOP) control, glaucoma medication use, surgical success rates, and complications. RESULTS: The study included 50 eyes of 31 patients. Mean age at initial surgery was 5 months (range, 5 days-48 months) and 19 patients (61.3%) showed bilateral disease. Mean duration of follow-up was 10.9 ± 3.10 years (range, 6-18 years). Mean IOP was reduced from 28.58 ± 9.78 mmHg (range, 10-59 mmHg) before surgery to 17.13 ± 7.62 mmHg after surgery (range, 5-42 mmHg; P < 0.001) at final follow-up. Survival analysis showed that the probability of surgical success with CTT was 77.8% at 1 year, 66.2% at 2 years, 53.0% at 5 years, and 16.6% at 15 years. Visual acuity at last available follow-up correlated with surgical success (P = 0.042). CONCLUSIONS: Surgical success after long-term follow-up of children with PCG is low. The probability of surgical success declines over time. Children with PCG require life-long follow-up and management, even after initial surgical success, to prevent visual impairment and blindness.

Functional Difficulties of Patients Seeking Low Vision Services in South India.

PURPOSE: The aim of the study was to determine the functional concerns of patients with different clinical and demographic characteristics seeking low vision care in South India. DESIGN: Cross-sectional clinic-based survey. METHODS: Consecutive new patients evaluated by the low vision service at Aravind Eye Care System (AECS), Madurai, India, India from September 2016 to March 2017 were recruited. Clinical and sociodemographic data were collected and participants underwent a semistructured survey to determine vision-related functional concerns. Analyses were conducted to determine associations with functional concerns. This study was approved by the AECS Institutional Review Board and all participants provided informed consent. RESULTS: The study included 419 participants (mean age 42.0 years, 65.2% male). Retinal dystrophy (35.8%) and acquired retinal disease (22.0%) were the most common diagnoses. The most frequently cited functional concerns were reading (37.7%), mobility (19.9%), and facial identification (13.8%). The number of functional concerns did not vary by diagnosis, age, sex, education, occupation, or presenting visual acuity (P > 0.05). Participants with retinal dystrophy were more likely to cite problems with night vision (P < .001). Age was significantly associated with greater difficulty recognizing faces [odds ratio (OR) = 1.20, 95% confidence interval (CI) = 1.01-1.43] and less night vision difficulty (OR = 0.75, 95% CI = 0.60-1.00). Worse presenting visual acuity was significantly associated with reporting a mobility problem (OR = 2.87, 95% CI = 2.09-3.93). CONCLUSIONS: This study supports the expansion of low vision services in India targeted to common functional concerns including reading, mobility, and facial identification. However, results do not support the use of ocular diagnosis for this purpose.

Air within surgical arterial graft on computed tomography-An alarming finding of a rare complication.

We present a case of a 61 years old lady operated 2 years back for severe superior mesenteric artery stenosis with a surgical vascular graft and presenting as acute severe abdominal pain and vomiting. Her CT angiography showed occlusion of the surgical vascular graft with graft migration into small bowel. Both the findings of graft occlusion and bowel perforation were optimally demonstrated on the CT angiography study. The alarm of bowel perforation in addition to graft infection was raised by the presence of air pockets within the graft and its communication with bowel lumen. Coexistent graft infection was evident on graft culture.

Residents' Learning Curve for Manual Small-Incision Cataract Surgery at Aravind Eye Hospital, India.

PURPOSE: The goal of this study was to document the resident learning curve for manual small-incision cataract surgery (MSICS) and to identify implications for the design of ophthalmology residency programs aimed to train surgeons for developing countries. DESIGN: Hospital-based retrospective cohort study. PARTICIPANTS: All 38 residents entering 2 postgraduate residency programs at Aravind Eye Hospital, Madurai, in 2012 and 2013. METHODS: Surgical complications and reoperations for all MSICSs performed by residents during the residency training period were evaluated using a computerized patient database. Multivariate logistic regression models were used to estimate the effect of the cumulative number of surgeries performed on incidence of intraoperative complications, postoperative complications, and reoperations, controlling for covariates. MAIN OUTCOME MEASURES: Incidence of intraoperative and first-day postoperative complications of Oxford Cataract Treatment and Evaluation Team (OCTET) grades II and III and the incidence of reoperations. Analyses controlled for patient-, resident-, and residency program-level covariates. RESULTS: The study evaluated 13 159 surgeries performed by the 38 residents between October 15, 2012, and August 24, 2016. The mean number of surgeries performed by a resident was 346.3 (standard deviation, 269.4). Three hundred forty-two eyes (2.60%) with at least 1 intraoperative complication, 234 eyes (1.78%) with at least 1 first-day postoperative complication, and 154 reoperations (1.17%) were observed. After controlling for baseline covariates, increasing surgical experience was associated with reduced risk of intraoperative and postoperative complications, as well as reoperations. The odds decreased by 17% (intraoperative complications), 12% (postoperative complications measured 1 day after surgery), and 7% (reoperations) per 100 additional surgeries performed. Patient-level factors such as older age, left eye surgery, and lower preoperative uncorrected visual acuity were found to be associated with higher risk of intraoperative complications (P < 0.01 for all). CONCLUSIONS: The risk of surgical complications and reoperations in MSICS decreased steadily with surgical experience gained by resident surgeons. We recommend that ophthalmology residency programs in developing nations teaching MSICS provide opportunities to perform 300 surgeries or more by residents so as to achieve rates of intraoperative and postoperative complications of less than 2%.

Practical consensus recommendations regarding the management of sentinel lymph node issues in early breast cancer.

Axillary lymph node involvement is a very important poor prognostic factor in the clinical staging and management of breast cancer patients. Traditionally, axillary lymph node dissection (ALND) has been used for determining the status of the axillary lymph nodes. More recently the sentinel lymph node biopsy (SLNB) procedure has gained wider acceptance as the standard of care, having the advantage of being less invasivewhile providing good accuracy. This expert group used data from published literature, practical experience and opinion of a large group of academic oncologists to arrive at these practical consensus recommendations in regards with the use of the two different procedures and other issues in patients with early breast cancer for the benefit of community oncologists.

Association of Pseudoexfoliation With Systemic Vascular Diseases in a South Indian Population.

IMPORTANCE: Ocular pseudoexfoliation (PEX) syndrome may be associated with systemic vascular diseases, which might suggest a broader health significance of PEX, although previous reports are conflicting. OBJECTIVE: To determine whether prespecified vascular risk factors and cardiac abnormalities are more common among patients with PEX than among control individuals without PEX. DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional analysis of patients recruited into the Aravind Pseudoexfoliation study included South Indian patients older than 40 years with or without PEX who required cataract surgery. Surgical procedures were performed at 4 tertiary Aravind eye hospitals in Tamil Nadu, India, from December 2, 2010, through March 26, 2012. Nine hundred thirty patients with PEX and 476 non-PEX controls underwent detailed ocular examinations, including specific ocular features reflecting PEX. Patients also underwent evaluation for multiple systemic potential cardiovascular diseases and their risk factors. The data collection for this analysis on systemic vascular diseases started on December 2, 2010, and ended on April 30, 2014. This study analysis was specified in the study protocol. MAIN OUTCOMES AND MEASURES: Five cardiovascular outcomes compared between patients with and without PEX included blood glucose, cholesterol, and homocysteine levels; blood pressure; and cardiac morbidity (defined by electrocardiographic [ECG] abnormalities). RESULTS: The study analysis included 930 patients in the PEX group and 476 in the non-PEX group. The mean (SD) ages of patients in the PEX and non-PEX groups were 64.8 (6.8) and 59.9 (7.3) years (P < .001), respectively. More patients in the PEX group were men (470 [50.5%] vs 460 women [49.5%]) than in the non-PEX group (201 [42.2%] vs 275 women [57.8%]; P < .001). In multivariable analyses adjusting for age and sex, higher systolic blood pressure values were noted for the PEX group (difference [Δ], 4.0 mm Hg; 95% CI, 1.7-6.2 mm Hg; P = .001). Also, patients in the PEX group were more likely to demonstrate an ECG abnormality than in the non-PEX group (odds ratio, 1.64; 95% CI, 1.04-2.60; P = .03). Pseudoexfoliation was not observed to be associated with a higher level of blood glucose (Δ, 6.2 mg/dL; 95% CI, -2.0 to 14.3 mg/dL; P = .14), serum cholesterol (Δ, -0.6 mg/dL; 95% CI, -5.1 to 4.0 mg/dL; P = .81), or serum homocysteine level (Δ, 0.004 mg/L; 95% CI, -0.12 to 0.14; P = .96). CONCLUSIONS AND RELEVANCE: Of the 5 cardiovascular outcomes examined in South Indian patients requiring cataract surgery, PEX was associated with higher systolic blood pressure and more frequent ECG abnormalities but not with higher blood glucose, serum cholesterol, or serum homocysteine levels. Patients with PEX are more likely to require attention to blood pressure and cardiac morbidity.

Direct lineage conversion of adult mouse liver cells and B lymphocytes to neural stem cells.

Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors.

Cell-type-specific repression by methyl-CpG-binding protein 2 is biased toward long genes.

Mutations in methyl-CpG-binding protein 2 (MeCP2) cause Rett syndrome and related autism spectrum disorders (Amir et al., 1999). MeCP2 is believed to be required for proper regulation of brain gene expression, but prior microarray studies in Mecp2 knock-out mice using brain tissue homogenates have revealed only subtle changes in gene expression (Tudor et al., 2002; Nuber et al., 2005; Jordan et al., 2007; Chahrour et al., 2008). Here, by profiling discrete subtypes of neurons we uncovered more dramatic effects of MeCP2 on gene expression, overcoming the "dilution problem" associated with assaying homogenates of complex tissues. The results reveal misregulation of genes involved in neuronal connectivity and communication. Importantly, genes upregulated following loss of MeCP2 are biased toward longer genes but this is not true for downregulated genes, suggesting MeCP2 may selectively repress long genes. Because genes involved in neuronal connectivity and communication, such as cell adhesion and cell-cell signaling genes, are enriched among longer genes, their misregulation following loss of MeCP2 suggests a possible etiology for altered circuit function in Rett syndrome.

Actinomycetes diversity among rRNA gene clones and cellular isolates from Sambhar salt lake, India.

The vertical stratification of actinomycetes diversity in Sambhar salt lake (India's largest salt lake) was investigated by using cultivable and uncultivable approaches. The isolates from cultured approaches were clustered on the basis of cultural, morphological, biochemical, and cell wall characteristics, and results were further strengthened by 16S rDNA-RFLP into five major groups. 16S rDNA sequencing of the representative isolates from each clusters was identified as belonging to Streptomyces, Actinopolyspora, Microbispora, Saccharopolyspora, and Actinoplanes genera, while culture independent group was established as Streptomyces (130 clones, 20 OTUs), Micromonospora (96 clones, 7 OTUs), Streptosporangium (79 clones, 9 OTUs), Thermomonospora (46 clones, 8 OTUs), and Dactylosporangium (58 clones, 8 OTUs). The diversity assessment using Shannon and Wiener index was found to be 1.55, 1.52, 1.55, and 1.49 from surface lake water, at depth of 1.5 m, shallow layer of water with algal population, and finally at depth of 2.5 m, respectively. We observed diversity in terms of the species richness as Streptomyces is dominant genus in both culture dependent and culture independent techniques followed by Microbispora (culture dependent methods) and Micromonospora (culture independent method) genera, respectively.

Extensions of MADM (mosaic analysis with double markers) in mice.

Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM.

Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice.

Synapse assembly requires trans-synaptic signals between the pre- and postsynapse, but our understanding of the essential organizational molecules involved in this process remains incomplete. Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains. Here we show that two Drosophila Teneurins, Ten-m and Ten-a, are required for neuromuscular synapse organization and target selection. Ten-a is presynaptic whereas Ten-m is mostly postsynaptic; neuronal Ten-a and muscle Ten-m form a complex in vivo. Pre- or postsynaptic Teneurin perturbations cause severe synapse loss and impair many facets of organization trans-synaptically and cell autonomously. These include defects in active zone apposition, release sites, membrane and vesicle organization, and synaptic transmission. Moreover, the presynaptic microtubule and postsynaptic spectrin cytoskeletons are severely disrupted, suggesting a mechanism whereby Teneurins organize the cytoskeleton, which in turn affects other aspects of synapse development. Supporting this, Ten-m physically interacts with α-Spectrin. Genetic analyses of teneurin and neuroligin reveal that they have differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates target selection between specific motor neurons and muscles. Our study identifies the Teneurins as a key bi-directional trans-synaptic signal involved in general synapse organization, and demonstrates that proteins such as these can also regulate target selection.

Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species.

Restriction fragment length analysis of PCR amplified 16S rDNA with AluI revealed the presence of a 265 bp fragment in all species of Bacillus with the exception of B. cereus and B. thuringiensis, which contains two restriction sites within this fragment which results in three smaller fragments totalling to 265 bp. Some distant species of Bacillus with no evidence of this fragment could be delineated into other genera based on phenotypic and genotypic parameters. BLAST search for homologous sequences of individual species revealed that it is a highly conserved region. Multiple alignment of the fragment suggests that a region between 160 and 265 bp of the 265 bp fragment was a hypervariable region and were highly species-specific. A set of primers was designed for amplification of this hypervariable region. Partial sequencing of the hypervariable region within the 265 bp fragment seems an index for identification of Bacillus species.