Gamma-Tubulin 1 (TUBG1) Mutation-Associated Lissencephaly and Microcephaly in an Indian Child: A Rare Case.

Malformations of cortical development (MCD) are a group of disorders affecting the normal development of the human cortex and are significant causes of delay in psychomotor development and epilepsy in children. Lissencephaly (smooth brain) forms a major group of brain malformations. Microtubules help in the migration of neuronal cells. Defect in tubulin gene alpha-tubulin (TUBA), beta-tubulin (TUBB), and gamma-tubulin (TUBG) leads to defective neuronal migration. This group of disorders is termed as "tubulinopathies." The important genes implicated in causing lissencephaly are LIS1, XLIS, and TUBA1A gene. Recently, a mutation in the TUBG1 gene is associated with it. Here, we report a one-and-a-half-year-old girl with global developmental delay, microcephaly, infantile-onset epilepsy, epileptic spasms, dysmorphism, and motor signs. There was no significant birth history. Neuroimaging (MRI) showed a broad thick gyri and a decreased number of sulci suggestive of lissencephaly/pachygyria spectrum. There was dilatation of the ventricles, and no grey matter heterotopia was noted. Sleep EEG showed multifocal epileptiform discharges. The child was treated with multiple anti-seizure medicines (ASMs). A genetic test, whole exome sequencing, was done to determine the etiology of MCD. A heterozygous missense variation in exon 6 of the TUBG1 gene was identified and reported as a "variant of unknown significance." Still, because the genotype matched with the clinical phenotype of the patient, it was considered clinically significant. Therefore, a complete diagnosis of TUBG1 mutation-associated cortical malformation (lissencephaly/pachygyria) with microcephaly and early-onset epilepsy was established. TUBG1 mutation is de novo in most cases, but parental testing is recommended. The parents of such patients need to be counseled about the need for prenatal testing and the risk of the disease to siblings. The overall prognosis in such cases is poor because of refractory seizures, physical limitations, and intellectual disability.

Calvarial Tuberculosis With Skin Tuberculosis in a Child: A Rare Case Report.

Calvarial tuberculosis (TB) is an uncommon form of skeletal TB. Early diagnosis can be challenging as they may exhibit diagnostic dilemmas. Another rare kind of skin TB is called TB verrucosa cutis. In this case, both of these uncommon forms were observed simultaneously and were effectively treated with first-line antitubercular therapy.

Reduction in the incidence of infusion-related phlebitis in a pediatric critical care unit of Eastern India: A quality improvement initiative.

Background: Phlebitis is one of the most common complications of the peripheral venous catheter (PVC) and adversely impacts future venous access, and bacterial phlebitis may lead to bloodstream infection. The objective of the study was to reduce the to reduce the incidence of infusion-related phlebitis in children admitted to the pediatric critical care unit. Methods: This Quality Initiative was implemented in the pediatric critical care unit of a tertiary care hospital between November 2019 and April 2020. Five interventions were identified (hand hygiene, use of transparent dressing, use of extension lines with PVCs, use of hard cardboard splints for joint immobilization, use of heparinized flush after medication administration) and were introduced sequentially. Over the next five weeks, a new intervention was introduced weekly while continuing the previous ones, if found to be working well as per improvement parameter, the phlebitis rate. From the sixth week onwards, all five interventions were applied together as a bundle. Results: Total seven hundred eighteen PVCs were sited in 284 (Male: female 1.58:1) patients during study period and a total of 56 incidences of phlebitis were observed. Mean baseline phlebitis rate was 48.5%. In the next 5 weeks when interventions were implemented as planned, phlebitis rate was 35.7% (n = 10), 16.6% (n = 03), 21.6% (n = 8), 10% (n = 05), and 13.3% (n = 2) respectively. Implementation of all five interventions together as a bundle led to reduction in phlebitis rate below 5 % consistently over the next 18 weeks (n = 8). Conclusion: A consistent reduction in PVC-related phlebitis can be achieved by the implementation of evidence-based interventions for the prevention of phlebitis, as a bundle.

Construction and characterization of a temperature-sensitive pRC4 replicon for Rhodococcus and Gordonia.

Temperature-sensitive plasmids are useful for genome engineering and several synthetic biology applications. There are only limited reports on temperature-sensitive plasmids for Rhodococcus and none for Gordonia. Here, we report the construction of a temperature-sensitive pRC4 replicon that is functional in Rhodococcus and Gordonia. The amino acid residues were predicted for the temperature-sensitive phenotype in the pRC4 replicon using in silico methods and molecular simulation of the DNA-binding replication protein with the origin of replication. The amino acid residues were mutated, and the temperature-sensitive phenotype was validated in Gordonia sp. IITR100. Similar results were also observed in Rhodococcus erythropolis, suggesting that the temperature-sensitive phenotype was exhibited across genera.

Detoxification and decolorization of complex textile effluent in an enzyme membrane reactor: batch and continuous studies.

There is an urgent need to look for bio-based technologies to address the pollution related to textile dyes in waterbodies. The aim of this study was to evaluate an engineered laccase variant, LCC1-62 of Cyathus bulleri, expressed in recombinant Pichia pastoris, for the decolorization and detoxification of real textile effluent. The partially purified laccase effectively (~60-100%) decolorized combined effluent from different dyeing units at a laccase concentration of 500 U/L at a 50-mL level. Decolorization and detoxification of the combined effluents, from a local textile mill, were evaluated at 0.3 L volumetric level in a ray-flow membrane reactor in batch and continuous modes of operation. In batch studies, maximum decolorization of 97% and detoxification of 96% occurred at a hydraulic retention time (HRT) of 6 h without any additional laccase requirement. In continuous studies, the reactor was operated at an HRT of 6 h with a lower enzyme dosage (~120 U/L of the effluent). Decolorization was accompanied by a loss in laccase activity which was restored to ~120 U/L by the addition of laccase in two regimes. The addition of laccase, when the residual laccase activity decreased to 40% (~50 U/L), resulted in high decolorization (~5 ppm residual dye concentration) and low variance (σ2) of 2.77, while laccase addition, when the residual dye concentration decreased to ~8% (~10 U/L), resulted in an average dye concentration of 13 ppm with a high variance of 62.08. The first regime was implemented, and the continuous reactor was operated for over 80 h at an HRT of 3 and 6 h, with the latter resulting in ~95% decolorization and 96% reduction in the mutagenicity of the effluent. Less than 10% membrane fouling was observed over long operations of the reactor. The findings strongly suggest the feasibility of using LCC1-62 in an enzyme membrane reactor for large-scale treatment of textile effluents.

Human Adenovirus Infection Causing Hyperinflammatory Syndrome Mimicking Multisystem Inflammatory Syndrome in Children (MIS-C): A Case Report.

Transmission of human adenovirus (HAdV) infection and the associated clinical disease can be sporadic or epidemic and manifestations may range from mild infection to severe disease. HAdV has been seen to behave as a proinflammatory virus that can trigger the release of high levels of inflammatory cytokines and chemokines in children. Here, we report an unusual case of an infant with HAdV infection who presented with respiratory illness, with a protracted course, complicated with hyperinflammation and multi-system involvement with clinical characteristics mimicking multisystem inflammatory syndrome in children (MIS-C) and Kawasaki disease. The patient was an 11-month-old male infant with a background of infantile epilepsy, epileptic encephalopathy, hemimegaloencephaly, and global developmental delay, diagnosed as Ohtahara syndrome. He was admitted with a three-day history of cough, cold, fever, and respiratory distress. Management was initiated with a heated humidified high-flow nasal cannula and given ceftriaxone and hypertonic saline nebulization. Additionally, he developed loose motion on the fifth day of admission. The reverse transcriptase polymerase chain reaction (RT-PCR) of the nasopharyngeal swab was positive for HAdV. Due to persistent fever, elevated inflammatory markers, multisystem involvement (diarrhea, coagulopathy), an absence of a clear microbial etiology, and an epidemiologic link to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, MIS-C was diagnosed. The first dose of intravenous immunoglobulins (IVIG) was administered over the course of 48 hours and the baby required a second dose of IVIG as the fever failed to settle after the first dose. Within 24 hours of the second IVIG dose, defervescence occurred. His platelet count started to rise, and the baby developed thrombocytosis in the third week of illness. Echocardiography was suggestive of dilatation of mild left main coronary artery. He was weaned off oxygen support by day 14 and discharged on day 17. To our knowledge, this is the first reported case of HAdV infection with hyperinflammatory syndrome and vasculitis akin to MIS-C and Kawasaki disease.

Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

Mechanically robust and highly bactericidal macroporous polymeric gels based on quaternized N,N-(dimethylamino)ethyl methacrylate possessing varying alkyl chain lengths.

In this paper, macroporous antimicrobial polymeric gels (MAPGs) functionalized with active quaternary ammonium cations attached to varying hydrocarbon chain lengths have been fabricated. Apart from the change in the alkyl chain length attached to the quaternary ammonium cation, the amount of crosslinker was also varied during the fabrication of the macroporous gels. The prepared gels were characterized using Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, field emission scanning electron microscopy (FE-SEM) and swelling studies. In addition, the mechanical properties of the fabricated macroporous gels were studied using compression and tensile testing. The antimicrobial activity of the gels has been determined for Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) as well as Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus). Antimicrobial activity, as well as the mechanical properties of the macroporous gels, was found to be influenced by the alkyl chain length attached to the quaternary ammonium cations as well as by the amount of crosslinker used for the fabrication of the gel. In addition, on increasing the alkyl chain length from C4 (butyl) to C8 (octyl), the effectiveness of the polymeric gels increased. It was observed that the gels derived using a tertiary amine (NMe2) containing monomer showed relatively low antimicrobial activity as compared to the gels obtained using quaternized monomers (C4 (butyl), C6 (hexyl), and C8 (octyl)). The gels based on the quaternized C8 monomer displayed the highest antimicrobial activity and mechanical stability as compared to the gels based on the C4 and C6 monomers.

Employing mutants to study the role of a three-membered bacterial consortium in growth promotion of Cajanus cajan.

Bacterial consortia, comprising plant growth promoting (PGP) rhizobacteria, are known to outcompete their impacts on plant attributes compared to their individual application. However, tracking of individual bacterial strains post application as consortium, remains challenging. The primary goal of this study was to develop an efficient method of tracking bioinoculants by generating spontaneous mutants of three different bacterial strains in an established consortium, using antibiotic-based screening, followed by their enumeration after application in Cajanus cajan. Mutants were generated for consortium members, viz. Azotobacter chroococcum (A), Priestia megaterium (formerly Bacillus megaterium) (B), and Pseudomonas sp. (P), against streptomycin, kanamycin and rifampicin, respectively. Those mutants having similar growth rates and PGP properties as compared to wild type bacterial strains were selected to test their efficacy in plant growth promotion. Selected mutant strains were applied as mono, dual and triple cultures to C. cajan grown hydroponically. Enumeration of mutant bacterial strains was carried out to check their viability. Bacterial colonization on roots was also analyzed. The application of triple (mutant) inoculants improved plant growth attributes significantly in comparison to mono and dual culture treatments and control. Cell enumeration revealed that the abundance of each bacterial strain increased till the 5th day of treatment. No significant change was observed later in their abundance for any treatment. The triple culture treatment showed greater abundance of bacterial mutant strains in comparison to mono- or dual cultures. To the best of our knowledge, this is the first mutant-based study to have reported the successful tracking and enumeration of bacterial consortium members, post their application in C. cajan.

Genome Sequence of a Potent Biosurfactant-Producing Bacterium, Franconibacter sp. Strain IITDAS19.

Here, we report the whole-genome sequence of Franconibacter sp. strain IITDAS19, a potent biosurfactant-producing bacterium that was isolated from oil-contaminated soil. The sequence provided information on the genes and enzymes responsible for the biosynthesis of the biosurfactant.

Characterization of divergent promoters PmaiA and Phyd from Gordonia: Co-expression and regulation by CRP.

Divergent promoters are often responsible for controlling gene expression of related genes of the same pathway or for coordinating regulation at different time points. There are relatively few reports on characterization of divergent promoters in bacteria. In the present study, microarray profiling was carried out to analyze gene expression during growth of Gordonia sp. IITR100, which led to the identification of 35 % of adjacent gene candidates that are divergently transcribed. We focus here on the in-depth characterization of one such pair of genes. Two divergent promoters, PmaiA and Phyd, drive the expression of genes encoding maleate cis-trans isomerase (maiA) and hydantoinase (hyd), respectively. Our findings reveal asymmetric promoter activity with higher activity in the reverse orientation (Phyd) as compared to the forward orientation (PmaiA). Minimal promoter region for each orientation was identified by deletion mapping. Deletion of a 5'-untranslated region of each gene resulted in an increase in promoter activity. A putative binding site for CRP (Catabolite Repressor Protein) transcription regulator was also identified in the 80 bp common regulatory region between the -35 hexamers of the two promoters. The results of this study suggest that CRP-mediated repression of PmaiA occurs only in the cells grown in glucose. Phyd, on the other hand, is not repressed by CRP. However, deletion of the CRP binding site located between -95 to -110 upstream to the transcription start site of the maiA gene resulted in increased activity of PmaiA and decreased activity of Phyd. A single CRP binding site, therefore, affects the two promoters differently.

Enhanced Oil Recovery using a Combination of Biosurfactants.

Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been emerging as promising alternatives to chemical surfactants due to less toxicity, high biodegradability, environmental compatibility and tolerance to extreme environmental conditions. Here, we illustrate the methods used for screening of microbes capable of producing biosurfactants. The biosurfactant producing microbes were identified using drop collapse, oil spreading, and emulsion index assays. Biosurfactant production was validated by determining the reduction in surface tension of the media due to growth of the microbial members. We also describe the methods involved in characterization and identification of biosurfactants. Thin layer chromatography of the extracted biosurfactant followed by differential staining of the plates was performed to determine the nature of the biosurfactant. LCMS, 1H NMR, and FT-IR were used to chemically identify the biosurfactant. We further illustrate the methods to evaluate the application of the combination of produced biosurfactants for enhancing residual oil recovery in a simulated sand pack column.

Expression of Escherichia coli malic enzyme gene in Zymomonas mobilis for production of malic acid.

Malic acid is one of the organic acids which is used in various industries including food and pharmaceuticals. Biotechnological production of malic acid by an efficient microorganism is highly desirable as the process will be eco-friendly and cost-effective. In this study, malic acid synthesis by Zymomonas mobilis was studied by expressing Escherichia coli malic enzyme gene under Pchap, Ptac and Ppdc promoters. The mae+ recombinants were obtained by recombineering-based genomic integration of Pchap-mae, Ptac-mae and Ppdc-mae sequences. The Ppdc promoter showed the highest expression of malic enzyme and the Pchap the lowest. However, cell growth was limited in mae+ recombinant containing Ppdc promoter. The metabolic analysis showed the highest level of malic acid in Ppdc-mae recombinant (2.84 g/L), which was about eight times higher than that in the wild type strain. The study showed that these three promoters can be used to produce organic acids in Z. mobilis.

Isolation and chemical characterization of the biosurfactant produced by Gordonia sp. IITR100.

Biosurfactants are amphipathic molecules produced from microorganisms. There are relatively few species known where the detailed chemical characterization of biosurfactant has been reported. Here, we report isolation and chemical characterization of the biosurfactant produced by a biodesulfurizing bacterium Gordonia sp. IITR100. Biosurfactant production was determined by performing oil spreading, drop-collapse, Emulsion index (E24), and Bacterial adhesion to hydrocarbons (BATH) assay. The biosurfactant was identified as a glycolipid by LCMS and GCMS analysis. The chemical structure was further confirmed by performing FTIR and NMR of the extracted biosurfactant. The emulsion formed by the biosurfactant was found to be stable between temperatures of 4°C to 30°C, pH of 6 to 10 and salt concentrations up to 2%. It was successful in reducing the surface tension of the aqueous media from 61.06 mN/m to 36.82 mN/m. The biosurfactant produced can be used in petroleum, detergents, soaps, the food and beverage industry and the healthcare industry.

Characterization of a potent biosurfactant produced from Franconibacter sp. IITDAS19 and its application in enhanced oil recovery.

Biosurfactants are surface-active molecules produced from microorganisms either on the cell surface or secreted extracellularly. Several biosurfactant producing microorganisms have been isolated to date, but they differ in their efficacy towards different types of hydrocarbons. Here, we report the isolation and characterization of a biosurfactant producing bacterium Franconibacter sp. IITDAS19 from crude oil contaminated soil. The biosurfactant was isolated, purified and characterized. It was identified as a glycolipid. It was found to be very stable at wide range of temperatures, pH and salt concentrations. It could reduce the surface tension of the water from 71 mN/m to 31 mN/m. IITDAS19 showed very high efficacy towards both aliphatic and aromatic hydrocarbons. It resulted in about 63% recovery of residual oil in a sand pack column. Our results suggested that the produced biosurfactant can be used for enhanced oil recovery. To our knowledge, this is the first report demonstrating the detailed characterization of a biosurfactant from Franconibacter spp.

Evaluation and optimization of antibiotic usage in upper respiratory tract infections in children at a tertiary care outpatient department: A clinical audit.

INTRODUCTION: Inappropriate antibiotic (ab)use contributes to antimicrobial resistance. Upper respiratory tract infection (URTI) is the most common reason for antibiotic prescription in an outpatient department (OPD). Several factors influence the high and unjustified antibiotic use in a common ailment. MATERIALS AND METHODS: A clinical audit was performed to assess antibiotic prescription rate (APR) for URTI in the pediatric OPD against the available benchmark. The prescription pattern was assessed, and interventions were formulated to improve prescription behavior. Data of all children attending OPD and fulfilling the criteria for URTI group were collected from the online hospital management system and analyzed. Interventions, in the form of discussions, presentations, posters, and guidelines (Indian Ministry of Health Guidelines for URTI) regarding etiology of URTI, and indications for antibiotic prescription were implemented. Data were monitored and feedback to consultants was given. RESULTS: The baseline APR was 14.7%. There was wide variation in APR (4.1%-53.1%) among consultants. Three consultants had a rate of 53.1%, 29.7%, and 28.6%, which was very high. Postintervention, the average APR decreased to 8.7%, a reduction of 40.8%. There was a reduction in APR among consultants with high APR as well. There was reduction in the use of azithromycin, a drug recommended for patients with penicillin allergy, from 21.2% to 14.4% (32.1% reduction). Amoxycillin plus clavulanic acid combination and amoxicillin alone continued to be the most prescribed antibiotics. CONCLUSION: Interventions through clinical audit were useful in reducing APR. The APR of 8.7% achieved in this study postintervention can be used as a benchmark by other institutions to assess APR in children with URTI.

Characterization of Zymomonas mobilis promoters that are functional in Escherichia coli.

Zymomonas mobilis ZM4 is a gram-negative, facultative anaerobic, natural ethanologenic bacterium used in industrial production of bio-products. For expression of genes, promoters are required. However, most of the promoters reported from Z. mobilis poorly function in Escherichia coli. This makes the process of expression and screening labor-intensive. In the present study, we compared the strengths of two Z. mobilis promoters, Pchap and Ppap, which drive the expression of chaperonin and phosphatase PAP2 family protein, respectively, with Ptac promoter. In E. coli, the Ptac promoter was found to be the strongest followed by Ppap and Ppdc, while in Z. mobilis, Ppdc was found to be the strongest and Pchap the weakest promoter. Further characterization of the promoters was done by cloning the gfpuv gene which expresses the green fluorescent protein, under their control and measuring the fluorescence of the E. coli transformants. The activity of these promoters was also studied at different pH (pH 5, 7 and 9) and different temperatures (30°C, 37°C and 42°C) in exponential and stationary phases. Both Pchap and Ppap promoters showed higher activity in stationary phase than in exponential phase. Since the promoters were active at all temperatures and pH studied, they can be used for gene expression in E. coli under desired environmental conditions.

Structural and functional characterization of a novel biosurfactant from Bacillus sp. IITD106.

Plant saponins are attractive biosurfactants and have been used to enhance phytoremediation. There are only limited reports on saponins produced by bacteria. Here, we report structural and functional characterization of a novel saponin produced by Bacillus sp. IITD106. Biosurfactant production was determined by emulsion index, drop collapse, oil displacement and hemolytic assays. The biosurfactant was stable over a range of temperature (30 °C to 70 °C), salinity (0-150 g liter-1) and pH (4-10). The surface tension of the medium reduced from 58.89 mN/m to 27.29 mN/m using the isolated biosurfactant. Chromatographic analysis revealed the biosurfactant to be a glycolipid. LCMS, FT-IR and NMR analysis identified the biosurfactant to be a saponin containing two sugar groups and a 5 ringed triterpene sapogenin unit. Genome sequencing of the strain revealed the presence of genes responsible for biosynthesis of saponin. Statistical optimization of culture medium resulted in 9.3-fold increase in biosurfactant production. Kinetics study of biosurfactant production performed in a stirred tank batch bioreactor resulted in 6.04 g liter-1 and 6.9 g liter-1 biomass and biosurfactant concentration, respectively. The biosurfactant was found to solubilize polycyclic aromatic hydrocarbons. The potential of cell free biosurfactant containing broth to enhance oil recovery was tested in a sand pack column and recovery of 63% of residual oil was observed. To our knowledge this is the first report of saponin production by any of the strains of Bacillus.

Bending is required for activation of dsz operon by the TetR family protein (DszGR).

The dsz operon responsible for the biodesulfurization of organosulfurs is under the control of a 385 bp long promoter. Recently, a TetR family protein was identified which served as an activator of operon. Here we report that the TetR family protein (WP_058249973.1), named DszGR can specifically activate the dsz operon. Direct binding of the DszGR to DNA was observed at single molecule level by AFM. It was found that the binding of DszGR to the promoter DNA induces a bend by about âˆ¼40-50° degrees which may not be enough for the activation of the promoter. Thus, bendability in the promoter sequence was analyzed. The results show that the promoter has a curvature at around -235 and -200 bp with respect to dszA start codon. On mutating this region, a decrease in activity of the promoter was observed. Our results suggest that the DszGR protein binds to the upstream sequences and induces a bend, which is facilitated by further bending of the DNA which is required for dsz promoter activity. IHF binding site present in the promoter, and a significant reduction in desulphurization activity in the absence of either IHF subunits, suggested role of IHF in regulation of the dsz operon.

Draft Genome Sequence of Bacillus sp. Strain IITD106, Isolated from Oil-Contaminated Soil.

Here, we report the whole-genome sequence of Bacillus sp. strain IITD106. The bacterium has the unique ability to produce saponins. The complete nucleotide sequence will provide insights into the various genes and regulators involved in the biosynthesis of saponin.