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.

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.

Microarray profiling and identification of core promoter sequence in Gordonia.

Gordonia are Gram-positive bacteria which have immense biotechnological potential. Genomes of several Gordonia spp. have been sequenced but a detailed analysis of the differentially expressed genes during growth, the promoters which drive their expression and the information on the core promoter sequence is lacking. Here, we report the identification of core promoter sequence in Gordonia sp. IITR100. The GC content of the promoters was found to be within a range of 62-65%. The 5'-UTR length in the genes was also analysed and about 56% promoters were found to have long 5'-UTR. The functionality of the promoters was validated by microarray profiling. Based on the differential expression of genes, two growth phase dependent promoters PdsbA and Pglx were isolated and analysed. They add to the existing repertoire of the promoters functional in both Gram-negative and Gram-positive bacteria. Our results suggest that the core promoter sequence identified is conserved in members of Gordonia spp. and is similar to that of other members of Actinobacteria.

Genomic deletions in Rhodococcus based on transformation of linear heterologous DNA.

Several genome engineering methods have been developed for Rhodococcus. However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in Rhodococcus spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of rodA and orphan parA. None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan parA and rodA resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of rodA and orphan parA genes by electroporation of heterologous linear DNA in Rhodococcus spp.

Functional characterization of the transcription regulator WhiB1 from Gordonia sp. IITR100.

WhiB is a transcription regulator which has been reported to be involved in the regulation of cell morphogenesis, cell division, antibiotic resistance, stress, etc., in several members of the family Actinomycetes. The present study describes functional characterization of a WhiB family protein, WhiB1 (protein ID: WP_065632651.1), from Gordonia sp. IITR100. We demonstrate that WhiB1 affects chromosome segregation and cell morphology in recombinant Escherichia coli, Gordonia sp. IITR100 as well as in Rhodococcus erythropolis. Multiple sequence alignment suggests that WhiB1 is a conserved protein among members of the family Actinomycetes. It has been reported that overexpression of WhiB1 leads to repression of the biodesulfurization operon in recombinant E. coli, Gordonia sp. IITR100 and R. erythropolis. A WhiB1-mut containing a point mutation Q116A in the DNA binding domain of WhiB1 led to partial alleviation of repression of the biodesulfurization operon. We show for the first time that the WhiB family protein WhiB1 is also involved in repression of the biodesulfurization operon by directly binding to the dsz promoter DNA.

Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli.

The Gram-negative bacterium Escherichia coli has been the work horse for recombinant protein production since the past several years. However, most of the gene expression systems used either require expensive inducers or exhibit low strength. In the present study, we have generated a strong promoter by repeated rounds of random mutagenesis in a stationary phase promoter isolated from Gordonia sp. IITR100. The promoter activity increased 16-fold as compared to the wild-type promoter. The resultant synthetic promoter showed ß-galactosidase activities of ~16,000 Miller units which is comparable to the strong T7 promoter ~13,000 Miller units. The amount of LacZ produced by the synthetic promoter was found to be active for several days in stationary phase. The advantage of this synthetic promoter over T7 promoter includes its stationary phase auto-inducibility thereby saving the cost of addition of inducers. Expression of GFPuv was observed in all the cells of E. coli due to the absence of requirement of inducer. A general-purpose vector containing the synthetic promoter with an MCS ready for use has been developed in the study. It has also been used to demonstrate the production of two heterologous proteins.

Rhodoccoccus erythropolis Is Different from Other Members of Actinobacteria: Monoploidy, Overlapping Replication Cycle, and Unique Segregation Pattern.

Among actinomycetes, chromosome organization and segregation studies have been limited to Streptomyces coelicolor, Corynebacterium glutamicum, and Mycobacterium spp. There are differences with respect to ploidy and chromosome organization pattern in these bacteria. Here, we report on chromosome replication, organization, and segregation in Rhodococcus erythropolis PR4, which has a circular genome of 6.5 Mbp. The origin of replication of R. erythropolis PR4 was identified, and the DNA content in the cell under different growth conditions was determined. Our results suggest that the number of origins increases as the growth medium becomes rich, suggesting an overlapping replication cell cycle in this bacterium. Subcellular localization of the origin region revealed polar positioning in minimal and rich media. The terminus, which is the last region to be replicated and segregated, was found to be localized at the cell center in large cells. The middle markers corresponding to the 1.5-Mb and 4.7-Mb loci did not overlap, suggesting discontinuity in the segregation of the two arms of the chromosome. Chromosome segregation was not affected by inhibiting cell division. Deletion of parA or parB affected chromosome segregation. Unlike in C. glutamicum and Streptomyces spp., diploidy or polyploidy was not observed in R. erythropolis PR4. Our results suggest that R. erythropolis is different from other members of Actinobacteria; it is monoploid and has a unique chromosome segregation pattern. This is the first report on chromosome organization, replication, and segregation in R. erythropolis PR4.IMPORTANCE Rhodococci are highly versatile Gram-positive bacteria with high bioremediation potential. Some rhodococci are pathogenic and have been suggested as emerging threats. No studies on the replication, segregation, and cell cycle of these bacteria have been reported. Here, we demonstrate that the genus Rhodococcus is different from other actinomycetes, such as members of the genera Corynebacterium, Mycobacterium, and Streptomyces, with respect to ploidy and chromosome organization and segregation. Such studies will be useful not only in designing better therapeutics pathogenic strains in the future but also for studying genome maintenance in strains used for bioremediation.

Spinal muscular dystrophy - a revisit of the diagnosis and treatment modalities.

Spinal Muscular Atrophy (SMA) is a pan-ethnic disorder and generally characterized as prevalent lethal genetic disease of infants. It is an autosomal recessive neuromuscular disease caused by degeneration of alpha motor neurons in the spinal cord, resulting in progressive proximal muscle weakness and paralysis. Due to the high carrier frequency (1:50), the burden of this genetic disorder is very heavy in developing countries. Till date no absolute cure or effective treatment of the disease is available in clinical practice, whereas minor enhancement of SMN protein levels can be beneficial. It can be achieved by augmenting SMN2 transcription, stimulating exon 7 splicing and protein stabilization. Due to its low prevalence among population, costly screening and diagnosis, the disease is still lacking proper management. SMN is expressed almost in all tissues of body, still the reason why only lower motor neurons are affected in SMA is unknown. Research is still going on and with advancement of innovative therapies and gene modification, improved outcome may come in near future. Presently, supportive care including respiratory, nutritional, psychiatric and orthopaedic management can ameliorate clinical symptoms and improve survival rates if SMA is diagnosed early in life. Routine prenatal and new-born screening can help with potential benefits and timely management. In this review, the concept of newer methodological system and recent advances for molecular diagnosis of SMA with the variability in the clinical features is stressed. The public health community should remain alert to the rapidly changing developments in early detection and treatment of SMA.

Fibrous Dysplasia of Paranasal Sinuses - An Atypical Presentation.

Fibrous Dysplasia is a benign bone disease of unknown etiology. The involvement of the craniofacial skeleton is not uncommon but rarely involves the paranasal sinus. We report a case of fibrous dysplasia of paranasal sinuses, describing its unusual clinical presentation, radiological features, histopathological appearance and surgical management.

In vitro synthesis of 9,10-dihydroxyhexadecanoic acid using recombinant Escherichia coli.

BACKGROUND: Hydroxy fatty acids are widely used in food, chemical and cosmetic industries. A variety of dihydroxy fatty acids have been synthesized so far; however, no studies have been done on the synthesis of 9,10-dihydroxyhexadecanoic acid. In the present study recombinant E. coli has been used for the heterologous expression of fatty acid hydroxylating enzymes and the whole cell lysate of the induced culture was used for in vitro production of 9,10-dihydroxyhexadecanoic acid. RESULTS: A first of its kind proof of principle has been successfully demonstrated for the production of 9,10-dihydroxyhexadecanoic acid using three different enzymes viz. fatty acid desaturase (FAD) from Saccharomyces cerevisiae, epoxide hydrolase (EH) from Caenorhabditis elegance and epoxygenase (EPOX) from Stokasia laevis. The genes for these proteins were codon-optimised, synthesised and cloned in pET 28a (+) vector. The culture conditions for induction of these three proteins in E. coli were optimised in shake flask. The induced cell lysates were used both singly and in combination along with the trans-supply of hexadecanoic acid and 9-hexadecenoic acid, followed by product profiling by GC-MS. Formation of 9,10-dihydroxyhexadecanoic acid was successfully achieved when combination of induced cell lysates of recombinant E. coli containing FAD, EH, and EPOX were incubated with 9-hexadecenoic acid. CONCLUSIONS: The in vitro production of 9,10-dihydroxyhexadecanoic acid synthesis using three fatty acid modification genes from different sources has been successfully demonstrated. The strategy adopted can be used for the production of similar compounds.

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.

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.

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.

Discovering the Untouched Perspective of Endangered North American Herb Actaea racemosa.

Actaea racemosa (AR), sometimes also known as black cohosh, is a perennial herb that grows in the Ranunculaceae family that effloresces in the middle of summer. This herb is currently present throughout south and west North America despite being endangered in the eastern section of the continent. Certain information about the photochemistry and biological potential of this herb is available. In accordance with the scant available ethno-medical reports, this herb possesses antioxidant, antidiabetic, anti-inflammatory, antiosteoporosis, and anticancer properties. As per the available literature, caffeic acid, isoferulic acid, actein, 23-epi-26 deoxycatein, cimicifugoside, and ferukinolic acid are the key components found in different parts of AR. To date, no thorough research or systematic review has been done to highlight the traditional, biological, and phytochemical benefits of this herb. Consequently, further research is needed to gain a deeper understanding of this therapeutic herb, particularly about its separation and pharmacological screening of its insulating portion for a range of biological functions. The goal of this review was to compile the most recent data on the phytochemical presence of AR. in relation to its ethnomedical applications, methods of extraction, pharmacological applications, and future potential.

Novel expression system for Corynebacterium acetoacidophilum and Escherichia coli based on the T7 RNA polymerase-dependent promoter.

The industrially important species of corynebacteria viz. Corynebacterium acetoacidophilum appear to be alternative hosts for recombinant protein production; despite many efforts, a strong promoter-based system in corynebacteria has not been established so far. Described here is a T7 promoter-based expression system which was functional in both gram-positive C. acetoacidophilum and gram-negative Escherichia coli in an external inducer independent manner. This is the very first report of a T7 expression system for Corynebacterium sp. Also, it is a useful addition in the existing T7 expression systems of E. coli.

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.

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.

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.

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.

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.