Bilateral Iridochorioretinal Coloboma Managed with Low Vision Rehabilitation: A Case Report.

Ocular coloboma is a rare congenital disability. If involving the macula, it affects the patient's vision and subsequently affects childhood development and quality of life in the future. Appropriate low vision aid and timely rehabilitation can provide the best possible quality of life for visually impaired children. We report a 9-year-old boy who presented with a diminution of vision in both eyes, and who was just enrolled in pre-school. He was diagnosed with bilateral iridochorioretinal coloboma associated with nystagmus and unilateral cataract. After all the necessary evaluation, a telescope was prescribed for distance and a dome magnifier for near. Furthermore, a peaked cap and photo grey lens were given for outdoor activities. This case highlights the importance of low vision intervention in a visually impaired child. Appropriate low vision aid and rehabilitation can improve patients' lifestyle and academic performance who are diagnosed with iridochorioretinal coloboma. Keywords: case reports; coloboma; ocular; rehabilitation; training.

Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase.

F1Fo ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To further investigate the role of these residues and the chemical requirements for activity at these positions, we generated 13 substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and passive H+ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge or removal of polarity caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with cardiolipin.

An Infant with Congenital Diaphragmatic Eventration with Dextrocardia: A Case Report.

Diaphragmatic eventration is a rare condition, and its association with dextrocardia is even a rarer clinical entity. Patients are usually asymptomatic, but the typical features include rapid breathing and recurrent respiratory infections. Here we present a rare case of a seven months old infant, who presented with cough, noisy breathing and chest retraction. The patient was diagnosed to have dextrocardia with diaphragmatic eventration with pneumonia by chest imaging and was treated in coordination with the medical team for underlying pneumonia. Afterwards, plication of the diaphragm was done through the trans-abdominal approach and the symptoms gradually improved postoperatively. For dextrocardia, since there were no structural abnormalities, the patient was kept in regular follow-up in the pediatric cardiology unit. Though most patients are asymptomatic, diaphragmatic eventration increases the risk of recurrent chest infection and hampers the quality of life of the patient, so timely diagnosis and intervention will greatly improve their quality of life. Keywords: dextrocardia; diaphragm; diaphragmatic eventration.

Perception Regarding Early Clinical Exposure among Second Year Medical Students after Educating Pregnant Women on Physiological Changes during Pregnancy: A Mixed Methods Study.

INTRODUCTION: The traditional teaching-learning process should reform to improve the academic performance and understanding of the students. This study aimed to determine the perceptions of second-year medical students towards early clinical exposure about their approach to educating pregnant women on the physiology of pregnancy. METHODS: This was a descriptive cross-sectional study with a mixed-method design comprising both quantitative and qualitative components among second-year medical students of a medical college in Nepal from September 2019 to September 2020. After ethical approval from the Institutional Review Committee (Reference number: 207), 40 students included through the convenience sampling method. These students were subjected to early clinical exposure in the form of educating pregnant women on physiological changes during pregnancy. Data was entered and analyzed using Microsoft Excel 2016. Point estimate at 95% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: Among 34 responses, majority of the students 29 (85.28%) (73.36-97.20 at 95% Confidence Interval) were motivated to learn the physiology of pregnancy after the activity; 15 (44.11%) strongly agreed and 14 (41.17%) agreed to this statement. Thirty-two students (94.11%) claimed that the activity improved their understanding of the physiology of pregnancy. The majority of the students expressed that this approach is pragmatic which ignited more curiosity regarding the subject matter. CONCLUSIONS: The majority of the students had satisfactory perceptions regarding their early clinical exposure which was similar to standard data and they expressed that they would like to have similar activities in the future.

Amblyopia among Patients Attending the Outpatient Department of Ophthalmology of a Tertiary Care Centre: A Descriptive Cross-sectional Study.

Introduction: Amblyopia is defined as a reduction in visual acuity unilaterally or bilaterally without any detectable cause. It is a major public health issue in developing and underdeveloped countries. Its prevalence is usually underestimated because of proper study and lack of awareness. The aim of the study was to find out the prevalence of amblyopia among patients attending the Outpatient Department of Ophthalmology of a tertiary care centre. Methods: This descriptive cross-sectional study was conducted among outpatients visiting a tertiary care centre in the Outpatient Department of Ophthalmology between 1 January 2017 to 31 December 2019. Ethical approval was obtained from the Institutional Review Board (Registration number: 407/2020 P). All patients had gone through a comprehensive eye examination. Convenience sampling was used. Point estimate and 99% Confidence Interval were calculated. Results: Among 82972 patients, prevalence of amblyopia was 344 (0.41%) (0.37-0.46, 99% Confidence Interval). Amblyopia was more common in anisometropia 263 (63.50%). A total of 117 (34%) patients had no history of eye examination and were newly diagnosed with amblyopia. Astigmatism was the most common type of refractive error among 224 (56.70%) amblyopic patients. Conclusions: The prevalence of amblyopia was found to be lower than in previous studies conducted in similar settings. Early detection and diagnosis of amblyopia can help to design more effective plans and treatments to reduce amblyopia through optical correction and amblyopia therapy. Keywords: amblyopia; anisometropia; astigmatism; refractive errors.

Crystal structure of the Thr316Ala mutant of a yeast JAMM deubiquitinase: implication of active-site loop dynamics in catalysis.

AMSH, an endosome-associated deubiquitinase (DUB) with a high specificity for Lys63-linked polyubiquitin chains, plays an important role in endosomal-lysosomal sorting and down-regulation of cell-surface receptors. AMSH belongs to the JAMM family of DUBs that contain two insertion segments, Ins-1 and Ins-2, in the catalytic domain relative to the JAMM core found in the archaebacterial AfJAMM. Structural analyses of the AMSH homologs human AMSH-LP and fission yeast Sst2 reveal a flap-like structure formed by Ins-2 near the active site that appears to open and close during its catalytic cycle. A conserved phenylalanine residue of the flap interacts with a conserved aspartate residue of the Ins-1 ß-turn to form a closed `lid' over the active site in the substrate-bound state. Analyses of these two residues (Phe403 and Asp315) in Sst2 showed that their interaction plays an important role in controlling the flexibility of Ins-2. The Lys63-linked diubiquitin substrate-bound form of Sst2 showed that the conserved phenylalanine also interacts with Thr316 of Ins-1, which is substituted by tyrosine in other AMSH orthologs. Although Thr316 makes no direct interaction with the substrate, its mutation to alanine resulted in a significant loss of activity. In order to understand the contribution of Thr316 to catalysis, the crystal structure of this mutant was determined. In spite of the effect of the mutation on catalytic activity, the structure of the Sst2 Thr316Ala mutant did not reveal significant changes in either the overall structure or the active-site arrangement relative to the wild type. The Phe403-Thr316 van der Waals interaction is impaired by the Thr316Ala mutation, abrogating the adoption of the closed active-site conformation required for catalysis. Since van der Waals interactions with phenylalanine are conserved across substrate-bound forms of AMSH-LP and Sst2, these interactions may be critical for loop immobilization and the positioning of the isopeptide bond of Lys63-linked polyubiquitin-chain substrates.

Prevalence of Kawasaki Disease in a Tertiary Care Hospital: A Descriptive Cross-sectional Study.

INTRODUCTION: Kawasaki disease is an acute vasculitis of unknown etiology. The epidemiological data available for Nepal remains insufficient. In Nepal, Kawasaki disease has only been reported in cases of brief reports, leaving the true disease burden unknown. Many cases go undiagnosed and untreated due to a lack of knowledge regarding this entity. The objective of this study was to find the prevalence of Kawasaki disease in a tertiary care hospital. METHODS: This descriptive cross-sectional study was carried out in a tertiary care hospital of Nepal from 2013 to 2018 after taking ethical approval from the Institutional Review Committee. The sample size was calculated and the consecutive sampling method was done. Data collection and entry was done in Microsoft Excel, point estimate at 99% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: The overall prevalence of Kawasaki disease was found to be 0.10% at 95% Confidence Interval (0.07-0.13%) among 11,416 patients under the age of 5 years admitted in pediatrics ward. There were 4 (33.33%) cases of complete Kawasaki and 8 (66.67%) cases of incomplete Kawasaki. There were 9 (75%) males and 3 (25%) females and the male to female ratio was 3:1. There was a male preponderance. The age at diagnosis ranged between 4 and 60 months. The median age at diagnosis was 10.5 months. The most common presentation was fever, conjunctivitis, rash, and oral changes. CONCLUSIONS: Prevalence of Kawasaki disease was found to be lesser compared to other studies done in other countries. Knowledge of Kawasaki disease among Nepalese pediatricians should be enhanced to guarantee the appropriate diagnosis and treatment of this disease.

Strains of the toxic and bloom-forming Nodularia spumigena (cyanobacteria) can degrade methylphosphonate and release methane.

Nodularia spumigena is a nitrogen-fixing cyanobacterium that forms toxic blooms in the Baltic Sea each summer and the availability of phosphorous is an important factor limiting the formation of these blooms. Bioinformatic analysis identified a phosphonate degrading (phn) gene cluster in the genome of N. spumigena suggesting that this bacterium may use phosphonates as a phosphorus source. Our results show that strains of N. spumigena could grow in medium containing methylphosphonic acid (MPn) as the sole source of phosphorous and released methane when growing in medium containing MPn. We analyzed the total transcriptomes of N. spumigena UHCC 0039 grown using MPn and compared them with cultures growing in Pi-replete medium. The phnJ, phosphonate lyase gene, was upregulated when MPn was the sole source of phosphorus, suggesting that the expression of this gene could be used to indicate the presence of bioavailable phosphonates. Otherwise, growth on MPn resulted in only a minor reconstruction of the transcriptome and enabled good growth. However, N. spumigena strains were not able to utilize any of the anthropogenic phosphonates tested. The phosphonate utilizing pathway may offer N. spumigena a competitive advantage in the Pi-limited cyanobacterial blooms of the Baltic Sea.

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic properties of the flap in the Trp mutant, compared to the wild-type enzyme, manifest as a defect in interactions that facilitate the rate-limiting step. Consistent with this notion, the Trp mutant was able to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme, indicating altered mobility and hence reduced selectivity.

Intercalating dyes for enhanced contrast in second-harmonic generation imaging of protein crystals.

The second-harmonic generation (SHG) activity of protein crystals was found to be enhanced by up to ∼1000-fold by the intercalation of SHG phores within the crystal lattice. Unlike the intercalation of fluorophores, the SHG phores produced no significant background SHG from solvated dye or from dye intercalated into amorphous aggregates. The polarization-dependent SHG is consistent with the chromophores adopting the symmetry of the crystal lattice. In addition, the degree of enhancement for different symmetries of dyes is consistent with theoretical predictions based on the molecular nonlinear optical response. Kinetics studies indicate that intercalation arises over a timeframe of several minutes in lysozyme, with detectable enhancements within seconds. These results provide a potential means to increase the overall diversity of protein crystals and crystal sizes amenable to characterization by SHG microscopy.

Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal-lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.45, 1.7, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn(2+). One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn(2+) and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 ± 0.6 µM for the binding of ubiquitin to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (~20 µM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (~20 µM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect.

Effect of urogenital cleaning with paper soap on bacterial contamination rate while collecting midstream urine specimens.

CONTEXT: Urinary tract infection (UTI) is one of the major health problems. Urine culture is considered as a gold standard method for the diagnosis of UTI. But, improper sample collection can lead to contamination with normal urogenital flora. Use of any portable disinfectant that can reduce contamination rate would be the significant help in urine culture interpretation. AIMS: To observe the effect of urogenital cleaning with paper soap on bacterial contamination rate while collecting specimens. MATERIALS AND METHODS: A cross-sectional comparative study was done in 600 patients aged 15-45 years, equally divided into three groups. The first group was given sterile container and instructed to collect midstream clean catch urine (MSU) after urogenital cleaning with provided piece of paper soap. The second group was given sterile container and strictly instructed to collect the MSU sample after urogenital cleansing by tap water only. The third group was given the sterile container and asked for midstream urine. Collected specimens were inoculated in CLED media, incubated aerobically for overnight at 37°C. Reporting of culture was done according to the guideline of American Society of Microbiology. RESULTS: The contamination rate in the three groups were 6.0%, 13.0%, and 27.5%, respectively (P value < 0.05), which was statistically significant. CONCLUSIONS: Contamination rate was significantly lower in group who provided urine specimen after urogenital cleaning with paper soap. Thus, cleaning the urogenital area may reduce the need of the repeat sample to rule out actual contamination and prevent from the unnecessary antibiotic treatment.

Analysis of the composition of lipid in human meibum from normal infants, children, adolescents, adults, and adults with meibomian gland dysfunction using ¹H-NMR spectroscopy.

PURPOSE: This study represents a first step toward the evaluation of possible compositional differences in meibum from normal donors (Mn) and donors with meibomian gland dysfunction (Md) by (1)H-NMR spectroscopy. The results highlight the applicability of (1)H-NMR spectroscopy for the quantitative analysis of waxes, cholesteryl esters, and glycerides in meibum lipid (ML). METHODS: Meibum was obtained from 41 normal donors and 51 donors with meibomian gland dysfunction (MGD). (1)H-NMR spectroscopy was used to quantify the amount of waxes, glycerides, and cholesteryl esters in human meibum. RESULTS: The relative amount of cholesteryl esters in Mn increased with age and was 40% (P < 0.05) lower in Md. Interestingly, the relative levels of cholesteryl esters in infant meibum were comparable to those in Md. The relative amounts of glycerides were not affected significantly by age or MGD. CONCLUSIONS: The changes in cholesteryl ester could be used as a molecular marker for MGD and could potentially be applied to follow the efficacy of drug therapy in the treatment of MGD. The similarity of the levels of cholesteryl esters in infant meibum and Md suggests that the relative amounts of these meibum components alone are unlikely to be responsible for the increased stability of the infant tear film and decreased stability of the tear film with MGD. This study reveals the complexity of human MLs and the changes that occur with age and disease. Understanding the factors that lead to such variations is of utmost relevance in the design of effective therapies.