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Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings incurring large processing costs for industrial-scale biofuels or biochemicals production. Manipulating surface charge interactions to minimize non-productive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability, but with limited demonstrated success to date. Here we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe Thermobifida fusca . A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of -52 to 37 was computationally designed using Rosetta macromolecular modelling software. Activity for all mutants was rapidly characterized as soluble cell lysates and promising mutants (containing mutations either on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2-5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM containing endocellulases exhibited a 5-10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity towards complex lignocellulosic biomass derived substrates.
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PURPOSE: To evaluate the learning curve of continuous curvilinear capsulorhexis (CCC) and to assess the number of surgeries required to master it among residents in a postgraduate teaching institute. METHODS: The present prospective observational study was based on the completion time and complication rates related to CCC performed using various techniques by 10 students in the 2nd (JR2) and 3rd-year (JR3) of residency. CCC was performed either by a cystotome or capsulorhexis forceps or by a combined method in 253 eyes, of which 160 eyes (63.2%) were operated by JR3 and 93 (36.8%) by JR2. The complication rates were studied with respect to the number of capsular extensions, posterior capsular rent (PCR), zonular dehiscence, need for senior surgical assistance, and nucleus drop. RESULTS: The average time required for the completion of CCC was 412 ± 90.5 s. The average number of times residents required to fill the anterior chamber with viscoelastic was 6.9 ± 1.4. The average size of CCC was 7 ± 0.66 mm. Extended CCC was the most common complication. JR2 required assistance from a senior surgeon in 47 eyes (50.5%), whereas JR3 required assistance in 39 eyes (24.4%) (P = 0.0001). The rate of PCR was not significantly different in JR2 (7.5%) and JR3 (8.8%). CONCLUSION: CCC is a difficult step to master in the trainee. Focusing and practicing on this step will help to reduce the complications and maximize proficiency. Approximately 6-eight surgeries are required to master CCC.
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Surfactants with their intrinsic ability to solubilize lipid membranes are widely used as antibacterial agents, and their interactions with the bacterial cell envelope are complicated by their differential aggregation tendencies. We present a combined experimental and molecular dynamics investigation to unravel the molecular basis for the superior antimicrobial activity and faster kill kinetics of shorter-chain fatty acid surfactant, laurate, when compared with the longer-chain surfactants studied in contact time assays with live Escherichia coli (E. coli). From all-atom molecular dynamics simulations, translocation events across peptidoglycan were the highest for laurate followed by sodium dodecyl sulfate, myristate, palmitate, oleate, and stearate. The translocation kinetics were positively correlated with the critical micellar concentration, which determined the free monomer surfactant concentration available for translocation across peptidoglycan. Interestingly, aggregates showed a lower propensity to translocate across the peptidoglycan layer and longer translocation times were observed for oleate, thereby revealing an intrinsic sieving property of the bacterial cell wall. Molecular dynamics simulations with surfactant-incorporated bacterial inner membranes revealed the greatest hydrophobic mismatch and membrane thinning in the presence of laurate when compared with the other surfactants. The enhanced antimicrobial efficacy of laurate over oleate was further verified by experiments with giant unilamellar vesicles, and electroporation molecular dynamics simulations revealed greater inner membrane poration tendency in the presence of laurate when compared with the longer-chain surfactants. Our study provides molecular insights into surfactant translocation across peptidoglycan and chain length-induced structural disruption of the inner membrane, which correlate with contact time kill efficacies observed as a function of chain length with E. coli. The insights gained from our study uncover unexplored barrier properties of the bacterial cell envelope to rationalize the development of antimicrobial formulations and therapeutics.
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Antiinfecciosos , Tensoactivos , Tensoactivos/química , Escherichia coli , Ácido Oléico , Peptidoglicano/metabolismo , Lauratos , Pared CelularRESUMEN
AIM: To assess the incidence of vitreous loss and associated risk factors in residents performing manual small-incision cataract surgery (MSICS). METHODS: The present retrospective record review study was performed on 490 patients who underwent MSICS performed between November 2018 and December 2019 by 7 third-year postgraduate residents. The study group comprised of patients having intraoperative vitreous prolapse. All the surgeries were performed under supervision of a trained assistant. RESULTS: The mean age of the participants at the time of surgery was 68.42±2.05y. Of the 490 patients, 250 patients were male, and 240 patients were female (P=0.23). A total of 215 (43.9%) eyes had mature white cataract, 185 (37.8%) eyes had brown cataract, and 90 (18.3%) eyes had immature senile cataract. The incidence of intraoperative vitreous loss among residents was 2% (10/490). Vitreous loss occurred during hydrodissection [1/10 (10%)], nucleus delivery [3/10 (30%)], irrigation and aspiration [5/10 (50%)], and intraocular lens insertion [1/10 (10%)]. Multivariate stepwise Logistic regression analysis confirmed immature senile cataract [odds ratio (OR)=3.99; P=0.02], irrigation and aspiration of cortical material (OR=3.07; P=0.03), and anterior capsular extension (OR=3.22, P=0.03) as independent risk factors for vitreous loss. CONCLUSION: Immature senile cataract, irrigation and aspiration of cortical material, and anterior capsular extension are independent risk factors for vitreous loss. Our findings may serve as a guide for future trainers or residents learning MSICS.
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Multisystem inflammatory syndrome in children (MIS-C) is a post-infectious immune-mediated condition, seen 3-5 weeks after COVID-19. Maternal SARS-CoV-2 may potentially cause a similar hyperinflammatory syndrome in neonates due to transplacental transfer of antibodies. We reviewed the perinatal history, clinical features, and outcomes of 20 neonates with features consistent with MIS-C related to maternal SARS-CoV-2 in Kolhapur, India, from 1 September 2020 to 30 April 2021. Anti-SARS-CoV-2 IgG and IgM antibodies were tested in all neonates. Fifteen singletons and five twins born to eighteen mothers with a history of COVID-19 disease or exposure during pregnancy presented with features consistent with MIS-C during the first 5 days after birth. Nineteen were positive for anti-SARS-CoV-2 IgG and all were negative for IgM antibodies. All mothers were asymptomatic and therefore not tested by RTPCR-SARS-CoV-2 at delivery. Eighteen neonates (90%) had cardiac involvement with prolonged QTc, 2:1 AV block, cardiogenic shock, or coronary dilatation. Other findings included respiratory failure (40%), fever (10%), feeding intolerance (30%), melena (10%), and renal failure (5%). All infants had elevated inflammatory biomarkers and received steroids and IVIG. Two infants died. We speculate that maternal SARS-CoV-2 and transplacental antibodies cause multisystem inflammatory syndrome in neonates (MIS-N). Immunomodulation may be beneficial in some cases, but further studies are needed.
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Context Computerized tomography (CT) is an invaluable imaging investigation for evaluating COVID-19 disease. CT detects early changes of COVID-19 pneumonia and predicts the disease prognosis based on a semiquantitative 25-point CT severity score (CT-SS). India launched its vaccination drive in January 2021 with two different vaccines being approved by the government. These vaccines are believed to prevent the disease itself, in majority of the cases and at least decrease disease severity, in the rest. Aim This study aims to evaluate the CT-SS in vaccinated and non-vaccinated subjects who have been diagnosed with COVID-pneumonia or are COVID suspects. Subjects and Methods A total of 3,235 patients with typical COVID-19 related imaging findings on HRCT thorax were included in the study. These subjects were divided into three age categories, 18-44, 45-59 and ≥60 years. The CT severity scores were allotted by experienced radiologists. Medians of the scores in different age groups were compared amongst vaccinated and non-vaccinated individuals using the Kruskal-Wallis H test. A p- value < 0.05 was considered significant. All results were shown with 95% confidence interval. Results The difference in the medians amongst the vaccinated and non-vaccinated groups was significant, p -values being < 0.001 in all age categories. Conclusion The mean CT-SS was less in vaccinated subjects and the difference in median CT-SS amongst vaccinated and non-vaccinated individuals was statistically significant, thus sending an important message that it is mandatory for the population at large to get vaccinated to reduce infection rate/disease severity.
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The bacterial cell envelope is a complex multilayered structure evolved to protect bacteria in hostile environments. An understanding of the molecular basis for the interaction and transport of antibacterial therapeutics with the bacterial cell envelope will enable the development of drug molecules to combat bacterial infections and suppress the emergence of drug-resistant strains. Here we report the successful creation of an in vitro supported lipid bilayer (SLB) platform of the outer membrane (OM) of E. coli, an archetypical Gram-negative bacterium, containing the full smooth lipopolysaccharide (S-LPS) architecture of the membrane. Using this platform, we performed fluorescence correlation spectroscopy (FCS) in combination with molecular dynamics (MD) simulations to measure lipid diffusivities and provide molecular insights into the transport of natural antimicrobial agent thymol. Lipid diffusivities measured on symmetric supported lipid bilayers made up of inner membrane lipids show a distinct increase in the presence of thymol as also corroborated by MD simulations. However, lipid diffusivities in the asymmetric OM consisting of only S-LPS are invariant upon exposure to thymol. Increasing the phospholipid content in the LPS-containing outer leaflet improved the penetration toward thymol as reflected in slightly higher relative diffusivity changes in the inner leaflet when compared with the outer leaflet. Free-energy computations reveal the presence of a barrier (â¼6 kT) only in the core-saccharide region of the OM for the translocation of thymol while the external O-antigen part is easily traversed. In contrast, thymol spontaneously inserts into the inner membrane. In addition to providing leaflet-resolved penetration barriers in bacterial membranes, we also assess the ability of small molecules to penetrate various membrane components. With rising bacterial resistance, our study opens up the possibility of screening potential antimicrobial drug candidates using these realistic model platforms for Gram-negative bacteria.
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Escherichia coli , Timol , Antibacterianos , Bacterias , Membrana Celular , Membrana Dobles de Lípidos , LipopolisacáridosRESUMEN
OBJECTIVES: To evaluate the perinatal outcome with the abnormal umbilical cord coiling index. STUDY DESIGN: This prospective study was carried out in the department of OBG at Adichunchangiri Institute of Medical Sciences, B.G.Nagara, Mandya, Karnataka, India from January 2008 to August 2010. 200 patients who were in active labour with term gestations, irrespective of their parities, who had singleton pregnancies with live babies who were either delivered by vaginal or LSCS were included in the study. Umbilical cord coiling index was calculated and it was correlated with various perinatal parameters like birth weight, meconium stained liquor, Apgar score, ponderal index and foetal growth restriction. Chi square and Fisher exact tests were used to find the significance of study parameters. RESULTS: There was a significant correlation between the hypercoiled cords (UCI >90(th) percentile) and IUGR of the babies (p value of < 0.001) and low ponderal indices (a p value of 0.022) Hypocoiled cords ( UCI which was < 10(th) percentile) were significantly associated with meconium staining (p < 0.001), Apgar score at 1 min of <4 and at 5 min of <7 (p value 0.065), LSCS rates (p value of 0.008) and NICU admissions (p <0.001). CONCLUSION: Hypercoiled cords or UCI which was > 90(th) percentile was associated with IUGR and low ponderal indices. Hypocoiled cords or UCI which was <10th percentile was associated with meconium staining, Apgar score at 1 min of <4 and at 5 min of <7, more LSCS rates and more NICU admissions.