Cysteine-independent CRISPR-Associated Protein Labeling for Presentation and Co-delivery of Molecules Toward Genetic and Epigenetic Regulations.

Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for in vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to rapidly detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.

Effect of substrate salinity and pH on life history traits of the bluetongue virus vector Culicoides peregrinus.

Habitat selection of Culicoides spp. (Diptera: Ceratopogonidae) is influenced by the physicochemical factors such as temperature, pH, salinity, moisture, conductivity, organic and inorganic compounds of substrates. These factors determine the life history traits of the vectors. We studied the influence of substrate salinity (0-40 parts per thousand, ppt) and pH (pH 1-13) on oviposition, egg hatching, larval survivability, and adult emergence of Culicoides peregrinus Kieffer under laboratory conditions. Most eggs (80.74%) were laid in 0 ppt and 95% in pH 7 but lowered with increased salinity and pH levels. It was observed that the females did not lay eggs in 30 ppt to 40 ppt salinity; pH 1 and pH 13 but interestingly up to 95% of the eggs were retained within the abdomen. Little effect of salinity and pH on egg hatching was observed up to 5 ppt and 10 ppt except at the extreme values of 40 ppt and pH 1, pH 13. Pupation did not occur in rearing plates with high salinities, 30 ppt and 40 ppt, although the few eggs hatched when exposed to such salinity. In low salinity (0 to 2 ppt), occurrence of adult emergence was more and then decreased with increasing salinity. Maximum emergence was seen when the rearing media was alkaline. This study deals with the suitability of breeding substrate of C. peregrinus when exposed to salinity and pH ranges. Our study suggests the ambient salinity and pH ranges to be maintained during laboratory rearing of this vector species.

N-Terminal Pro-Brain Natriuretic Peptide Levels in Kawasaki Disease, Sepsis and Other Febrile Illnesses.

OBJECTIVE: To compare the values of N-Terminal Pro-Brain Natriuretic Peptide (NT-proBNP) in acute phase of Kawasaki disease (KD), sepsis and other acute febrile illnesses. METHODS: We conducted a cross-sectional study on 40 KD patients and 40 age- and sex-matched controls with sepsis and other febrile illnesses between January, 2019 and June, 2020. Complete blood count, C-reactive protein (CRP), liver and renal function tests, serum electrolytes, chest X-ray, NT-proBNP level, Bactec blood culture, urine microscopy and culture along with detailed physical examination was done for all cases and control. RESULTS: Mean (SD) values of NT-proBNP levels in acute KD was higher than sepsis/other febrile illnesses (914.9 vs 219 pg/mL; P=0.001). Also, the number of KD patients whose NT-proBNP was elevated were significantly higher compared to controls (70% vs 32.5%; P<0.001). CONCLUSION: NT-proBNP may be useful as a biomarker in the diagnosis of acute phase KD.

A taxonomic revision of the Indian species of the 'Aterinervis' group of Culicoides Latreille Subgenus Hoffmania Fox (Diptera: Ceratopogonidae).

The seven species of Culicoides spp. belonging to the Aterinervis Group of subgenus Hoffmania Fox reported from India are revised. The study is based on type material and fresh specimens trapped during the Annual Biodiversity Assessment (2nd & 4th) of Neora Valley National Park (NVNP) in the Darjeeling-Sikkim Himalaya of India. Comparative redescriptions of adult male and female of Culicoides isoregalis, C. neoregalis, C. pararegalis, C. pseudoregalis, C. quasiregalis, C. regalis and C. subregalis are provided along with the formal transfer of the nominate species, Culicoides aterinervis from subgenus Culicoides Latreille to Hoffmania. A key to the Indian species belonging to the Aterinervis group is provided along with a list of the Culicoides species present in the Darjeeling-Sikkim Himalayas.

In vitro biochemical characterization and identification of hemolytic bacteria associated with life history of Culicoides peregrinus (Diptera: Ceratopogonidae), a vector of bluetongue virus.

Gut bacterial communities in insects provide several beneficial roles like nutrition, digestion, fecundity, and survival of the host. The microbial communities of Culicoides spp. (Diptera: Ceratopogonidae) vary with parity, developmental stages, and environmental factors. Previous studies have revealed the presence of hemolytic bacteria in adult Culicoides peregrinus Kieffer (Diptera: Ceratopogonidae), an important vector of bluetongue virus (BTV). Our objectives were (i) to identify bacterial communities with hemolytic activities associated with all life stages and (ii) to compare between reared and field-collected adults including age graded females. Bacterial identification followed Sanger sequencing of 16S rRNA. In vitro biochemical characterizations including antibiotic sensitivity tests were also done. The majority of bacterial species were beta hemolytic with one, Alcaligenes faecalis, showing alpha hemolysis. Most bacterial species were observed in field-collected adults except Proteus spp. Throughout the life history of the vector, Bacillus cereus (CU6A, CU1E) and Paenibacillus sp. (CU9G) were detected indicating their possible role in blood digestion within the gut of this vector species. In vivo hemolytic activities of these culturable bacterial communities within this vector may be addressed in future. These hemolytic bacterial communities may be targeted to develop novel and effective strategies for vector control.

A Metagenomic Based Approach on Abundance and Diversity of Bacterial Communities Across the Life Stages of Culicoides peregrinus (Diptera: Ceratopogonidae) a Vector of Bluetongue Virus.

During larval rearing of Culicoides peregrinus Kieffer (Diptera: Ceratopogonidae) it was obligatory to add a small quantity of mud from larval habitat to nutrient broth in culture plates. This initiated microbial growth in rearing plates which facilitated growth and development of immature. The primary aim was to enumerate gut microbial communities across the different life stages of C. peregrinus. Amplicon sequencing of the V3-V4 hypervariable region (16S rDNA) was done on Illumina Miseq platform to detect gut bacterial communities at different life stages, while ITS regions (18S rRNA) were targeted for fungal communities of the 4th instar larvae. The major findings were: 1) Phylum Proteobacteria and Firmicutes were the most abundant throughout the life stages, along with the highest bacterial alpha diversity in the egg, 2) bacterial compositions were similar to laboratory reared and field collected adults, and 3) abundant fungal phyla associated with the larval gut were Ascomycota and Basidiomycota. Furthermore, analyses of the gut microbiome with METAGENassist might be indicative of their likely function in the natural habitat. Abundant gut-associated bacteria and/or fungal genera detected in the present study could be used as dietary supplements to establish laboratory colonies for further vectorial research. While, individual roles of the bacteria or fungi in paratransgenesis are warned for their possible utilization to frame the management strategy in upcoming works.

Risk factors in IVIG-resistant Kawasaki disease and correlation with Japanese scoring systems - a study from Eastern India.

OBJECTIVES: To assess the risk factors of intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD) and to evaluate the performance of the three Japanese risk-scoring systems, namely the Kobayashi, Egami, and Sano scores in predicting IVIG resistance among the Indian patients. METHODS: Prospective observational study on children admitted with KD at Institute of Child Health, Kolkata, over a period of 16 months, from January 2019 to April 2020. The study included 70 KD patients all of whom were treated with IVIG. Clinical parameters, laboratory variables, and risk scores were compared between the IVIG-responsive and the IVIG-resistant groups. RESULTS: A total of 31.4% were IVIG non-responders. Skin rash was found to be significantly associated with IVIG-resistant KD. The IVIG-resistant group had higher total bilirubin, lower albumin, higher CRP levels, and higher ALT and AST levels. High Kobayashi score, high Egami score, and high Sano score were significantly associated with IVIG resistance, individually. Sano score had the highest sensitivity (81.8%) and Kobayashi score had the highest specificity (77.1%) in our cohort. CONCLUSION: The presence of skin rash, high total bilirubin, high CRP, high AST, high ALT, and low albumin were important predictors of IVIG resistance in our population. Among the three scores, Sano score is the most reliable in identifying potential non-responders to IVIG. But Sano score lacked good specificity. Therefore, Indian KD patients may need an exclusive scoring system to predict non-responsiveness to IVIG so that a more aggressive therapy can be instituted at the earliest. Key points • Early prediction of IVIG-resistant KD is necessary to limit cardiac injuries. • Sano score has high sensitivity to predict IVIG resistance in Indian population.

A comparative study of IL-6, CRP and NT-proBNP levels in post-COVID multisystem inflammatory syndrome in children (MISC) and Kawasaki disease patients.

BACKGROUND: Post-COVID multisystem hyperinflammatory syndrome in children (MISC) has clinical and laboratory similarities with Kawasaki disease (KD). Inflammatory markers like C-reactive protein (CRP), interleukin 6 (IL6) as well as N-terminal probrain natriuretic peptide (NT-proBNP) are elevated in both. This study attempts a comparative analysis of the 3 markers in an attempt at early differentiation for planning appropriate management. METHODOLOGY: This analytical study conducted at the Institute of Child Health, Kolkata, India compared the levels of the above 3 markers at admission between 72 patients with KD, 30% of whom had coronary artery lesions (CALs) collected over a period of 18 months (Jan 2017-June 2018), with 71 MISC patients over a period of 6 months (July 2020-December 2020). The non-parametric Mann-Whitney U test was used to test for similarity in distributions of the samples of CRP, NT-proBNP and IL6 in KD and MISC patients using correction factor for similar ranks. The 3 parameters were compared using receiver operating characteristic (ROC) curve analysis. RESULTS: Mean IL6 value in KD was 83.22 pg/mL and in MISC 199.91 pg/mL, which was not found to be statistically significant (P = .322 > .05).However mean NT-proBNP (914.91 pg/mL) with CRP level (96.32 mg/L) in KD was significantly lower (P < .05 for both cases) than that in MISC (9141.16 pg/mL and 145.66 mg/L respectively). ROC analysis showed NT-proBNP has the best sensitivity and specificity in predicting MISC. CONCLUSION: NT-proBNP and CRP are significantly higher among MISC patients; ROC analysis shows levels >935.7 pg/mL and >99.55 mg/L respectively might act as a guide to differentiate between them.

Computational Insight into TM-N x Embedded Graphene Bifunctional Electrocatalysts for Oxygen Evolution and Reduction Reactions.

Due to the energy crisis, development of bifunctional electrocatalysts for both oxygen evolution and reduction reactions is highly demanding. In this study, we have systematically investigated the bifunctional activity of metal (Co/Rh/Ir) and N co-doped graphene systems with varying N-dopant concentrations (TM-N x @G, x = 0, 2, 4) using first-principles calculations. Charge transfer from the metal sites to the adsorbed intermediates and the adsorption free energy of the intermediates play important roles to help understand the potential-determining step and overpotential values for oxygen evolution reaction (OER)/oxygen reduction reaction (ORR). A dual volcano plot for all the systems using a common descriptor ΔG OH* has been constructed. We find that the systems having ΔG OH* values in the range of 0.40-0.70 eV can act as bifunctional electrocatalysts. Our study not only highlights the importance of metal and non-metal co-doped graphene as bifunctional catalysts but also can serve as a promising strategy for the design of efficient OER/ORR electrocatalysts.

Electronic structures and second hyperpolarizabilities of alkaline earth metal complexes end-capped with NA2 (A = H, Li, Na).

The ground state structures and NLO properties of a number of alkaline earth metal complexes end-capped with NA2 groups (A = H, Li, Na) are calculated by employing the CAM-B3LYP, wB97XD and B2PLYP functionals along with MP2 and CCSD(T) for 6-311++G(d,p), 6-311++G(3df,3pd), aug-cc-pVTZ, aug-pc-2 and Hypol basis sets. The complexes are found to be significantly stable. The magnitude of second hyperpolarizability enhances appreciably with increase in the number of magnesium and calcium atoms in the chain, which has been indicated by the power law dependence γ = a + bn(c) with c values ranging from 2.4-4.3 for Mg and 2.4-3.7 for Ca complexes, respectively. The largest second-hyperpolarizability (10(9) au) is obtained for the complex Ca7(NNa2)2 at the CAM-B3LYP level. The two state model has been used to explain the variation of hyperpolarizabilities.