The Future of Atopic Dermatitis Treatment.

This chapter thoroughly examines recent breakthroughs in atopic dermatitis (AD) treatment, with a primary focus on the medications in the development pipeline. Biologics agents targeting new interleukin receptors like interleukin-31, interleukin-22, and interleukin-2 are discussed along with the novel pathway looking at the OX40-OX40L interaction. Oral agents and small molecule therapies like Janus kinase inhibitors, sphingosine-1-phosphate modulators, and Bruton's tyrosine kinase inhibitors are also discussed along with the various new topical medications. Newly approved topicals like phosphodiesterase-4 and JAK inhibitors are highlighted while also discussing the potential of tapinarof and emerging microbiome-targeted therapies. Beyond conventional approaches, the chapter touches upon unconventional therapies currently being studied. The goal of this chapter is to discuss new advances in AD treatment from medications in the initial stages of development to those nearing FDA approval.

Investigation on the insecticidal activities of cyanobacterial extracts as an alternative source for the management of fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae).

The Spodoptera frugiperda is a notorious pest with a broad host range. It severely damages crops, mainly in areas of the globewhere maize and sorghum are grown. The pest is difficult to control due to its adaptive nature and resistance to several insecticides available in the market. So, an identification of the alternative strategy is the prime important in the present context. Insecticidal activities of cyanobacterial extracts were evaluated in the laboratory as a biocomponent against S. frugiperda. The crude extracts of Nostoc muscorum and Spirulina sp. were prepared by using ethanol, methanol and petroleum ether solvents. Soxhlet apparatus was used for extraction. S. frugiperda larvae in their second instar were given access to fragments of maize leaf that had been treated with various cyanobacterial extracts. The findings displayed that the petroleum ether extract of N. muscorum had the lowest LC50 value of 155.22 ppm, followed by petroleum ether extracts of Spirulina, ethanol extract of N. Muscorum, methanol extract of N. muscorum, ethanol and methanol extract of Spirulina with an LC50 values of 456.02, 710, 780, 1050 and 1070 ppm respectively. Later, the effect of LC50 values on many biological parameters like the larval duration and pupal stages, the percentage of pupation, the weight of the pupal stage, the malformation of the pupal and adult stages, adult emergence percentage, fertility and the longevity of the male and female adult stages of S. frugiperda was examined. The gas chromatography-mass spectrometry (GC-MS) was used to analyse the crude extract to identify the bioactive components that were responsible for the insecticidal properties. The major compounds detected were diethyl phthalate (19.87 %), tetradecane (5.03%), hexadecanoic acid, ethyl ester (4.10 %), dodecane (4.03%), octadecane (3.72%), octadecanoic acid, methyl ester (3.40 %), ethyl oleate (3.11 %), methyl ester. octadecenoic acid (3.04 %), heptadecane (3.04 %) and phytol (3.02 %). The presence of several bioactive chemicals in the cyanobacterial extracts may be the reason for their insecticidal actions, thus it can be used as an alternative and new source to combat fall armyworm and other crop pests.

Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase.

Numerous expression systems, engineered strains, and cultivation systems have been developed globally but producing recombinant proteins in the soluble form continues to remain a challenge.  Escherichia coli, a preferred host for the recombinant production of biopharmaceuticals and other proteins. Up to 75% of human proteins expressed in E. coli have only 25% in an active soluble form. The proteolytic activity of Lon encoded protease triggers the inclusion bodies leading to heterogenous secreted proteins thereby hampering downstream processing and isolation. Putrescine monooxygenases are versatile with applications in iron acquisition, pathogen control, biotransformation, bio-remediation and redox reaction are still isolated from plant and microbial sources at low yields. As a prerequisite to developing protease knockout E. coli strains, using the Cre-loxP recombination strategy we have built a full-length Lon disruption cassette (5'lon-lox66-cre-KanR-lox71-3'lon) (3368 bp) consisting of upstream and downstream regions of Lon, loxP sites, and Cre gene driven by T7 promoter to the expression of Cre recombinase and a selectable kanamycin resistance gene. Here, after the integration of the knock-out cassette into the host genome, we show the production of homogeneous protein species of recombinant Putrescine monooxygenase by using an E. coli platform strain in which Lon gene is deleted. This Lon knock-out strain secreted more homogeneous protein at a volumetric yield of 60% of the wild-type strain. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01056-x.

Determination of chlorantraniliprole 18.5% SC in the paddy ecosystem and its risk assessment.

Chlorantraniliprole belongsto theanthranilic diamide group is widely used against broad range of lepidopteron pests in a variety of vegetable and rice pests includingyellow rice stem borer and leaf folder. Supervised field trials were conducted duringRabi (2018-2019) and Kharif (2019) to evaluate the dissipation pattern and risk assessment of chlorantraniliprole 18.5% SC in paddy ecosystem following foliar application at 30 and 60 g a.i. ha-1 in two different cropping seasons.Modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) technique was used for the extraction of CAP residues with acetonitrile and determined by LC-MS/MS (ESI +).The limit of quantification (LOQ) was 0.01 µg g-1 for paddy leaf, straw, husk, and brown rice, respectively and 0.005 µg g-1 for soil. The average recoveries obtained were 84.30-88.92% from paddy leaf, 94.25-97.81% from straw, 90.21-93.38% from husk, 93.57-96.40% from brown rice and 89.93-91.14% from soil. The residues in paddy leaf dissipated within 35-40 days with a half-life of 4.33-5.07 days in Rabi and 3.92-4.86 days in Kharif at 30 and 60 g a.i. ha-1, respectively. The residues in soil dissipated within 15-21 days with a half-life of 14.44-15.75 days in Rabi and 13.33-14.44 days in Kharif at respective doses. At harvest chlorantraniliprole residues were not detected in straw, husk, and brown rice. The dietary risk of paddy leaf (green fodder) for cattle was found safe for consumption as the hazard index is less than one. Soil ecological risk assessment was found to be less than one (RQ < 0.1) for earthworms (Eisenia foetida) and arthropods (Aphidiusrhopalosiphi). The presentmethod could be useful inthe analysis ofchlorantraniliproleresidues in different cereals and vegetable crop ecosystems and application at recommended dose is safe for the final produce at harvest.

In silico structural, phylogenetic and drug target analysis of putrescine monooxygenase from Shewanella putrefaciens-95.

BACKGROUND: The enormous and irresponsible use of antibiotics has led to the emergence of resistant strains of bacteria globally. A new approach to combat this crisis has been nutritional immunity limiting the availability of nutrients to pathogens. Targeting the siderophore biosynthetic pathway that helps in iron acquisition, an essential microelement in the bacterial system has been the topic of interest in recent days that backs the concept of nutritional immunity. Supporting this view, we have chosen to study a key enzyme in the biosynthetic pathway of putrebactin called putrescine monooxygenase (SpPMO) from Shewanella putrefaciens. In our previous study, we co-expressed putrescine monooxygenase recombinantly in Escherichia coli BL21 Star (DE3). The bioinformatic analysis and screening of inhibitors will broaden the scope of SpPMO as a drug target. RESULTS: In the present study, we have analysed the physicochemical properties of the target enzyme and other N-hydroxylating monooxygenases (NMOs) using ExPASy server. The target enzyme SpPMO and most of the selected NMOs have a slightly acidic isoelectric point and are medially thermostable and generally insoluble. The multiple sequence alignment identified the GXGXX(N/A), DXXXFATGYXXXXP motives and conserved amino acids involved in FAD binding, NADP binding, secondary structure formation and substrate binding. The phylogenetic analysis indicated the distribution of the monooxygenases into different clades according to their substrate specificity. Further, a 3D model of SpPMO was predicted using I-TASSER online tool with DfoA from Erwinia amylovora as a template. The model was validated using the SAVES server and deposited to the Protein Model Database with the accession number PM0082222. The molecular docking analysis with different substrates revealed the presence of a putrescine binding pocket made of conserved amino acids and another binding pocket present on the surface of the protein wherein all other ligands interact with high binding affinity. The molecular docking of naturally occurring inhibitor molecules with SpPMO 3D model identified curcumin and niazirin with 1.83 and 2.81 µM inhibition constants as two promising inhibitors. Further studies on kinetic parameters of curcumin and niazirin inhibitors in vitro determined the Ki to be 2.6±0.0036 µM and 18.38±0.008 µM respectively. CONCLUSION: This analysis will help us understand the structural, phylogenetic and drug target aspects of putrescine monooxygenase from Shewanella putrefaciens-95 in detail. It sheds light on the precautionary measures that can be developed to inhibit the enzyme and thereby the secondary infections caused by them.

Spatiotemporal imaging and analysis of mouse and human liver bud morphogenesis.

BACKGROUND: The process of liver organogenesis has served as a paradigm for organ formation. However, there remains a lack of understanding regarding early mouse and human liver bud morphogenesis and early liver volumetric growth. Elucidating dynamic changes in liver volumes is critical for understanding organ development, implementing toxicological studies, and for modeling hPSC-derived liver organoid growth. New visualization, analysis, and experimental techniques are desperately needed. RESULTS: Here, we combine observational data with digital resources, new 3D imaging approaches, retrospective analysis of liver volume data, mathematical modeling, and experiments with hPSC-derived liver organoids. Mouse and human liver organogenesis, characterized by exponential growth, demonstrate distinct spatial features and growth curves over time, which we mathematically modeled using Gompertz models. Visualization of liver-epithelial and septum transversum mesenchyme (STM) interactions suggests extended interactions, which together with new spatial features may be responsible for extensive exponential growth. These STM interactions are modeled with a novel in vitro human pluripotent stem cell (hPSC)-derived hepatic organoid system that exhibits cell migration. CONCLUSIONS: Our methods enhance our understanding of liver organogenesis, with new 3D visualization, analysis, mathematical modeling, and in vitro models with hPSCs. Our approach highlights mouse and human differences and provides potential hypothesis for further investigation in vitro and in vivo.

Rising Mental Health Incidence Among Adolescents in Westchester, NY.

CONTEXT: Many governments have publicly released healthcare data, which can be mined for insights about disease conditions, and their impact on society. METHODS: We present a big-data analytics approach to investigate data in the New York Statewide Planning and Research Cooperative System (SPARCS) consisting of 20 million patient records. FINDINGS: Whereas the age group 30-48 years exhibited an 18% decline in mental health (MH) disorders from 2009 to 2016, the age group 0-17 years showed a 5.4% increase. MH issues amongst the age group 0-17 years comprise a significant expenditure in New York State. Within this age group, we find a higher prevalence of MH disorders in females and minority populations. Westchester County has seen a 32% increase in incidences and a 41% increase in costs. CONCLUSIONS: Our approach is scalable to data from multiple government agencies and provides an independent perspective on health care issues, which can prove valuable to policy and decision-makers.

Determination of 77 Multiclass Pesticides and Their Metabolitesin Capsicum and Tomato Using GC-MS/MS and LC-MS/MS.

A quick, sensitive, and reproducible analytical method for the determination of 77 multiclass pesticides and their metabolites in Capsicum and tomato by gas and liquid chromatography tandem mass spectrometry was standardized and validated. The limit of detection of 0.19 to 10.91 and limit of quantification of 0.63 to 36.34 µg·kg-1 for Capsicum and 0.10 to 9.55 µg·kg-1 (LOD) and 0.35 to 33.43 µg·kg-1 (LOQ) for tomato. The method involves extraction of sample with acetonitrile, purification by dispersive solid phase extraction using primary secondary amine and graphitized carbon black. The recoveries of all pesticides were in the range of 75 to 110% with a relative standard deviation of less than 20%. Similarly, the method precision was evaluated interms of repeatability (RSDr) and reproducibility (RSDwR) by spiking of mixed pesticides standards at 100 µg·kg-1 recorded anRSD of less than 20%. The matrix effect was acceptable and no significant variation was observed in both the matrices except for few pesticides. The estimated measurement uncertainty found acceptable for all the pesticides. This method found suitable for analysis of vegetable samples drawn from market and farm gates.

Expression and purification of codon-optimized cre recombinase in E. coli.

The presence of antibiotic resistance genes in genetically modified bacteria raises a regulatory concern in the production of therapeutic proteins and additionally reduces the number of plasmids available for propagation in a cell. Cre recombinase from bacteriophage P1, involved in Cre/loxP mechanism is one of the widely used systems for selectable marker gene removal. We have overexpressed codon-optimized cre gene in pColdIV and pET28a(+) vector systems and purified His6-Cre recombinase by immobilized metal affinity chromatography. N-terminal His6 tagged Cre recombinase obtained was approximately 26 fold purified and promoted the site-specific recombination of two loxP sites of linearized pLox2+ vector allowing the excision of a re-circularized plasmid and a short stretch of DNA containing the recombined loxP site. The results of the expression using two vectors, purification and activity assessment of His6 tagged Cre recombinase is presented here.

Advances in cloning, structural and bioremediation aspects of nitrile hydratases.

Nitrile hydratase (NHase) is a prominent enzyme in many microorganisms for its nitrile metabolism. The potentiality in the bioconversion of nitriles to its high-value amides has been extensively used in industries for the production of acrylamide and nicotinamide which are essential chemicals. Enzymologists are still considering NHases for its potential biotechnological applications including biotransformations and bioremediations. But most of the nitrile hydratases have limitations like the low expression, low thermostability and enantioselectivity. Though considerable data has been generated in the area of gene configuration, crystal structure, kinetic mechanism and photoreactivity of NHases, there is a need for constant improvement to develop a robust biocatalyst for bioremediation of toxic nitriles. With these considerations, in the present review, we report advances with the main focus to structure, catalytic mechanism, cloning strategy, gene expression, bioinformatic tools, metagenomics, thermostability and current bioremediation applications of NHases.

Endophyte fungi, Cladosporium species-mediated synthesis of silver nanoparticles possessing in vitro antioxidant, anti-diabetic and anti-Alzheimer activity.

This research study is to develop an easy and eco-friendly method for the synthesis of AgNPs using aqueous extract of endophytic fungi, Cladosporium species (CsAgNPs) and investigated the effects of antioxidant, anti-diabetic and anti-acetylcholinesterase (AChE), anti-butyrylcholinesterase (BChE) activity. Cladosporium species-mediated silver nanoparticles were characterized by UV-Vis spectrophotometer, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX). The aqueous extract of Cladosporium species has shown the presence of carbohydrates, tannin, phenolic glycosides, terpenoids, alkaloids, phenol and anthraquinones. At 438 nm conformed the absorbance of AgNPs. The SEM result confirms that size, morphology and high density of the synthesized nanoparticles with huge disparity in the particle size distribution. The FTIR analysis confirmed the important biological compounds responsible for reduction of silver. Strong absorption property of AgNPs was studied by EDX. In antioxidant activity, CsAgNPs showed the involvement of NADPH-dependent reductase in the formation of AgNPs. The AgNPs has reduced the activity of α-amylase, α-glucosidase and dipeptidyl peptidase IV in vitro antidiabetic activity. The CsAgNPs showed significant glucose uptake in 3T3L1 cell line. The AgNPs have shown excellent inhibitory activity against AChE and BChE. To our best knowledge, this is the first on the synthesis of silver nanoparticles using endophytic fungi, Cladosporium species isolated from healthy leaf of Loranthus micranthus. Hence, to validate our results the in vivo animal studies at molecular level are needed to develop an antioxidant, anti-diabetic and anti-cholinesterase agent.

Treatment outcome and mortality at one and half year follow-up of HIV infected TB patients under TB control programme in a district of South India.

BACKGROUND: There is paucity of data from India on the impact of HIV related immunosuppression in response to TB treatment and mortality among HIV infected TB patients. We assessed the TB treatment outcome and mortality in a cohort of HIV infected TB patients treated with intermittent short course chemotherapy under TB control programme in a high HIV prevalent district of south India. METHODOLOGY/ FINDINGS: Among 3798 TB patients registered for treatment in Mysore district from July 2007 to June 2008, 281 HIV infected patients formed the study group. The socio-demographic and treatment related data of these patients was obtained from TB and HIV programme records and patient interviews 19 months after TB treatment initiation by field investigators. Treatment success rate of 281 patients was 75% while in smear positive pulmonary tuberculosis cases it was 62%, attributable to defaults (16%) and deaths (19%). Only 2 patients had treatment failure. Overall, 83 (30%) patients were reported dead; 26 while on treatment and 57 after TB treatment. Association of treatment related factors with treatment outcome and survival status was studied through logistic regression analysis. Factors significantly associated with 'unfavourable outcome' were disease classification as Pulmonary [aOR-1.96, CI (1.02-3.77)], type of patient as retreatment [aOR-4.78, CI (2.12-10.76)], and non initiation of ART [aOR-4.90, CI (1.85-12.96)]. Factors associated with 'Death' were non initiation of ART [aOR-2.80, CI (1.15-6.81)] and CPT [aOR-3.46, CI (1.47-8.14)]. CONCLUSION: Despite the treatment success of 75% the high mortality (30%) in the study group is a matter of concern and needs immediate intervention. Non initiation of ART has emerged as a high risk factor for unfavourable treatment outcome and mortality. These findings underscore the importance of expanding and improving delivery of ART services as a priority and reconsideration of the programme guidelines for ART initiation in HIV infected TB patients.

Analyzing the catalytic mechanism of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.

In order to gain insight into the catalytic mechanism of Fe-type nitrile hydratases (NHase), the pH and temperature dependence of the kinetic parameters k cat, K m, and k cat/ K m along with the solvent isotope effect were examined for the Fe-type NHase from Comamonas testosteroni Ni1 ( CtNHase). CtNHase was found to exhibit a bell-shaped curve for plots of relative activity vs pH over pH values 4-10 for the hydration of acrylonitrile and was found to display maximal activity at pH approximately 7.2. Fits of these data provided a p K ES1 value of 6.1 +/- 0.1, a p K ES2 value of 9.1 +/- 0.2 ( k' cat = 10.1 +/- 0.3 s (-1)), a p K E1 value of 6.2 +/- 0.1, and a p K E2 value of 9.2 +/- 0.1 ( k' cat/ K' m of 2.0 +/- 0.2 s (-1) mM (-1)). Proton inventory studies indicate that two protons are transferred in the rate-limiting step of the reaction at pH 7.2. Since CtNHase is stable to 25 degrees C, an Arrhenius plot was constructed by plotting ln( k cat) vs 1/ T, providing an E a of 33.3 +/- 1.5 kJ/mol. Delta H degrees of ionization values were also determined, thus helping to identify the ionizing groups exhibiting the p K ES1 and p K ES2 values. Based on Delta H degrees ion data, p K ES1 is assigned to betaTyr68 while p K ES2 is assigned to betaArg52, betaArg157, or alphaSer116 (NHases are alpha 2beta 2 heterotetramers). Given the strong similarities in the kinetic data obtained for both Co- and Fe-type NHase enzymes, both types of NHase enzymes likely hydrate nitriles in a similar fashion.

Chaperone-assisted expression, purification, and characterization of recombinant nitrile hydratase NI1 from Comamonas testosteroni.

Nitrile hydratases (NHases) are industrially important iron- and cobalt-containing enzymes that are used in the large-scale synthesis of acrylamide. Heterologous expression of NHases has been complicated by the fact that other proteins (activators or metallochaperones) appear to be required to produce NHases in their catalytically active form. We report a novel heterologous system for the expression of catalytically active iron-containing NI1 NHase in Escherichia coli, involving coexpression with the E. coli GroES and GroEL chaperones. The purified recombinant enzyme was found to be highly similar to the enzyme purified from Comamonas testosteroni according to its spectroscopic features, catalytic properties with various substrates, and post-translational modifications. In addition, we report a rapid and convenient spectrophotometric method to monitor the activity of NI1 NHase during purification.