Application of Quality by Design in the Development of Hydrogen Sulfide Donor Loaded Polymeric Microparticles.

Hydrogen sulfide (H2S) is a multifaceted gasotransmitter molecule which has potential applications in many pathological conditions including in lowering intraocular pressure and providing retinal neuroprotection. However, its unique physicochemical properties pose several challenges for developing its efficient and safe delivery method system. This study aims to overcome challenges related to H2S toxicity, gaseous nature, and narrow therapeutic concentrations range by developing polymeric microparticles to sustain the release of H2S for an extended period. Various formulation parameters and their interactions are quantitatively identified using Quality-by-Design (QbD) approach to optimize the microparticle-based H2S donor (HSD) delivery system. Microparticles were prepared using a solvent-evaporation coacervation process by using polycaprolactone (PCL), soy lecithin, dichloromethane, Na2S.9H2O, and silicone oil as polymer, surfactant, solvent, HSD, and dispersion medium, respectively. The microparticles were characterized for size, size distribution, entrapment efficiency, and H2S release profile. A Main Effects Screening (MES) and a Response Surface Design (RSD) model-based Box-Behnken Design (BBD) was developed to establish the relationship between critical process parameters (CPPs) and critical quality attributes (CQAs) qualitatively and quantitatively. The MES model identified polymer to drug ratio and dispersion medium quantity as significant CPPs among others, while the RSD model established their quantitative relationship. Finally, the target product performance was validated by comparing predicted and experimental outcomes. The QbD approach helped in achieving overall desired microparticle characteristics with fewer trials and provided a mathematical relationship between the CPPs and the CQAs useful for further manipulation and optimization of release profile up to at least 30 days.

Liver transplant for primary biliary tract neuroendocrine tumor in a nine-year-old girl.

BACKGROUND: Neuroendocrine tumors (NETs) are rare epithelial neoplasms that arise most commonly from the gastrointestinal tract. In pediatrics, the most common site of origin is in the appendix, with the liver being the most common site of metastasis. Neuroendocrine tumors arising from the biliary tract are extremely rare. METHODS: We describe a case of a nine-year-old girl who presented with obstructive cholestasis and was found to have multiple liver masses identified on biopsy as well-differentiated neuroendocrine tumor with an unknown primary tumor site. RESULT: The patient underwent extensive investigation to identify a primary tumor site, including endoscopy, endoscopic ultrasound, and capsule endoscopy. The patient ultimately underwent definitive management with liver transplant, and on explant was discovered to have multiple well-differentiated neuroendocrine tumors, WHO Grade 1, with extensive infiltration into the submucosa of bile duct, consistent with primary biliary tract neuroendocrine tumor. CONCLUSION: Identifying the site of the primary tumor in NETs found within the liver can be challenging. To determine if an extrahepatic primary tumor exists, workup should include endoscopy, EUS, and capsule endoscopy. Children with well-differentiated hepatic NETs, with no identifiable primary tumor, and an unresectable tumor, are considered favorable candidates for liver transplantation.

Chronic Liver Disease in Patients with Prolidase Deficiency: A Case Series.

Introduction: Prolidase deficiency is a rare autosomal recessive disorder caused by variants in the PEPD gene. Patients usually have multi-organ involvement and a wide range of clinical features including recurrent skin ulcers, dysmorphic facial features, recurrent infections, intellectual disability, and splenomegaly. Studies have shown that patients with prolidase deficiency may have hepatic manifestations including hepatomegaly and abnormal liver enzymes. However, there is no detailed description of liver disease in this patient population. Case Presentation: Here, we present 3 patients with prolidase deficiency with varying extents of hepatic involvement. Conclusion: Prolidase deficiency patients with liver disease should be followed up long term to understand more about the pathophysiology and the impact of liver disease on long-term outcomes.

Comparison of colorimetric, spectroscopic and electrochemical techniques for quantification of hydrogen sulfide.

Background: Hydrogen sulfide (H2S), an endogenous gasotransmitter, has potential applications in several conditions. However, its quantification in simulated physiological solutions is a major challenge due to its gaseous nature and other physicochemical properties. Aim: This study was designed to compare four commonly used H2S detection and quantification methods in aqueous solutions. Methods: The four techniques compared were one colorimetric, one chromatographic and two electrochemical methods. Results: Colorimetric and chromatographic methods quantified H2S in millimolar and micromole ranges, respectively. The electrochemical methods quantified H2S in the nanomole and picomole ranges and were less time-consuming. Conclusion: The H2S quantification method should be selected based on the specific requirements of a research project in terms of sensitivity, response time and cost-effectiveness.

SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein histidines.

Inteins are auto-processing domains that implement a multistep biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis (Mtu). Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wildtype (WT) precursor protein. Structural analysis and molecular dynamics (MD) simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen-specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.

Nodular regenerative hyperplasia in X-linked agammaglobulinemia: An underestimated and severe complication.

BACKGROUND: Late-onset complications in X-linked agammaglobulinemia (XLA) are increasingly recognized. Nodular regenerative hyperplasia (NRH) has been reported in primary immunodeficiency but data in XLA are limited. OBJECTIVES: This study sought to describe NRH prevalence, associated features, and impact in patients with XLA. METHODS: Medical records of all patients with XLA referred to the National Institutes of Health between October 1994 and June 2019 were reviewed. Liver biopsies were performed when clinically indicated. Patients were stratified into NRH+ or NRH- groups, according to their NRH biopsy status. Fisher exact test and Mann-Whitney test were used for statistical comparisons. RESULTS: Records of 21 patients with XLA were reviewed, with a cumulative follow-up of 129 patient-years. Eight patients underwent ≥1 liver biopsy of whom 6 (29% of the National Institutes of Health XLA cohort) were NRH+. The median age at NRH diagnosis was 20 years (range, 17-31). Among patients who had liver biopsies, alkaline phosphatase levels were only increased in patients who were NRH+ (P = .04). Persistently low platelet count (<100,000 per µL for >6 months), mildly to highly elevated hepatic venous pressure gradient and either hepatomegaly and/or splenomegaly were present in all patients who were NRH+. In opposition, persistently low platelet counts were not seen in patients who were NRH-, and hepatosplenomegaly was observed in only 1 patient who was NRH-. Hepatic venous pressure gradient was normal in the only patient tested who was NRH-. All-cause mortality was higher among patients who were NRH+ (5 of 6, 83%) than in the rest of the cohort (1 of 15, 7% among patients who were NRH- and who were classified as unknown; P = .002). CONCLUSIONS: NRH is an underreported, frequent, and severe complication in XLA, which is associated with increased morbidity and mortality.

Identification of marker genes in Alzheimer's disease using a machine-learning model.

Alzheimer's Disease (AD) is one of the most common causes of dementia, mostly affecting the elderly population. Currently, there is no proper diagnostic tool or method available for the detection of AD. The present study used two distinct data sets of AD genes, which could be potential biomarkers in the diagnosis. The differentially expressed genes (DEGs) curated from both datasets were used for machine learning classification, tissue expression annotation and co-expression analysis. Further, CNPY3, GPR84, HIST1H2AB, HIST1H2AE, IFNAR1, LMO3, MYO18A, N4BP2L1, PML, SLC4A4, ST8SIA4, TLE1 and N4BP2L1 were identified as highly significant DEGs and exhibited co-expression with other query genes. Moreover, a tissue expression study found that these genes are also expressed in the brain tissue. In addition to the earlier studies for marker gene identification, we have considered a different set of machine learning classifiers to improve the accuracy rate from the analysis. Amongst all the six classification algorithms, J48 emerged as the best classifier, which could be used for differentiating healthy and diseased samples. SMO/SVM and Logit Boost further followed J48 to achieve the classification accuracy.

DNA barcode, multiplex PCR and qPCR assay for diagnosis of pathogens infecting pulse crops to facilitate safe exchange and healthy conservation of germplasm.

The DNA barcodes were developed from ITS region for the identification of fungal plant pathogens namely, Alternaria alternata and A. tenuissima both causing leaf spots, Ascochyta rabiei causing Ascochyta blight, Fusarium oxysporum f. sp. ciceris causing wilt, Macrophomina phaseolina causing dry root rot, Rhizoctonia solani causing web blight and wet root rot, Sclerotium (Athelia) rolfsii causing collar rot, Sclerotinia sclerotiorum causing stem rot and Cercospora canescens and Pseudocercospora cruenta both causing leaf spots in pulse crops. Barcode compliance for A. alternata (DBTPQ001-18), A. tenuissima (DBTPQ002-18), A. rabiei (DBTPQ003-18), F. oxysporum f. sp. ciceris (DBTPQ004-18), M. phaseolina (DBTPQ005-18), R. solani (DBTPQ006-18), S. rolfsii (DBTPQ007-18), S. sclerotiorum (DBTPQ008-18), C. canescens (DBTPQ009-18) and P. cruenta (DBTPQ029-20) have been generated based on the Barcode of Life Data System (BOLD) system. In addition to ITS, other genomic regions were also explored and on the basis of sequence variation they were ranked as TEF-α > SSU > LSU > ß-tubulin. These genes could be considered for secondary barcode and phylogenetic relatedness. ITS-based markers for the detection of A. alternata (BAA2aF and BAA2aR) and R. solani (BRS17cF and BRS17cR) were developed which provided 400 bp and 220 bp amplicons, respectively. While, for F. oxysporum f. sp. ciceris, COX1-based marker (FOCox1F and FOCox3R) was developed which amplified 150 bp. The markers proved highly specific and sensitive with detection limit of 0.0001 ng of template DNA using qPCR and simultaneously detected these three pathogens. The DNA barcodes and diagnostics developed are suitable for quick and reliable detection of these pathogens during quarantine processing and field diagnostics.

Quality evaluation of near-isogenic line of the wheat variety HD2733 carrying the Lr24/Sr24 genomic region.

A near-isogenic line (NIL) of the Indian wheat variety HD2733, carrying an introgressed Lr24/Sr24 genomic region was used for studying the effect of this introgression on quality traits. Data on the grain yield and 21 quality traits were recorded in this NIL and its recurrent parent (RP), both of which were grown in a randomized block design for two consecutive years. The statistical analysis revealed that grain yield was on par between the NIL and the RP. The NIL and its RP were both hard grained but the NIL showed a grain hardness index reduced by 9.7%. However, quality traits such as grain weight, protein content, sedimentation value, gluten traits, and solvent retention capacity were significantly higher in the NIL. The NIL also showed an increase in dough stability, a lower degree of softening and a higher farinograph quality number. These results indicated that the NIL could be utilized for hard grain, high protein and strong gluten-based products. An overall improvement in the quality of the NIL over its recurrent parent and without any yield penalty suggests that the Lr24/Sr24 genomic region could be gainfully utilized in wheat breeding for improving the industrial quality of wheat without jeopardising grain yield. The authors suggest that the improved quality of the NIL may be due to the genomic segment carried along with the Lr24/Sr24 genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02679-x.

A single centre experience of treatment outcomes for Helicobacter pylori infection among inner-city children and adolescents.

AIM: This study assessed treatment outcomes of Helicobacter pylori (H pylori) infection among inner-city children. METHODS: This was a retrospective study of patients aged 1-21 years who underwent initial treatment for H pylori infection from 2011 to 2015. We included patients who completed 2 weeks of treatment with documented adequate compliance after H pylori infection was diagnosed. Treatment outcomes were measured based on stool H pylori antigen and/or histology. RESULTS: Of the total 261 patients diagnosed with H pylori, 239 completed the first-line treatment. The regimens used included amoxicillin/clarithromycin/proton pump inhibitor (PPI) in 207/239 patients (86.6%), amoxicillin/metronidazole/PPI in 14/239 patients (5.8%) and other regimens in 18/239 patients (7.5%). H pylori eradication status was tested in 111/207 (53.6%) patients treated with amoxicillin/clarithromycin/PPI, and the eradication was achieved in 84/111(75.7%) patients. The treatment success rates for amoxicillin/metronidazole/PPI and other regimens were 71.4% (5/7) and 63.6% (7/11), respectively. There was no statistical significance of post-treatment stool H pylori antigen results between PPI (n = 31) and no PPI (n = 43) users. CONCLUSION: The study showed an eradication rate of 75.7% with the regimen amoxicillin/clarithromycin/PPI suggesting significant antibiotic resistance in our population. The use of PPI did not influence post-treatment stool H pylori antigen results.

Marker-assisted transfer of PinaD1a gene to develop soft grain wheat cultivars.

Grain softness has been a major trait of interest in wheat because of its role in producing flour suitable for making high-quality biscuits, cookies, cakes and some other products. In the present study, marker-assisted backcross breeding scheme was deployed to develop advanced wheat lines with soft grains. The Australian soft-grained variety Barham was used as the donor parent to transfer the puroindoline grain softness gene Pina-D1a to the Indian variety, DBW14, which is hard grained and has PinaD1bPinbD1a genes. Foreground selection with allele-specific PCR-based primer for Pina-D1a (positive selection) was used to identify heterozygous BC1F1 plants. Background selection with 173 polymorphic SSR primers covering all the 21 chromosomes was also carried out, in the foreground-selected BC1F1 plants. BC1F2 plants were selected by ascertaining the presence of Pina-D1a (positive selection) and absence of Pina-D1b (negative selection). Using the approach of positive, negative and background selection with molecular markers, 15 BC1F2 and 31 BC2F1 plants were finally selected. The 15 BC1F2 plants were selfed and the 31 BC2F1 plants were further backcrossed and selfed to raise BC3F1 and BC2F2 progenies, respectively. A part of the BC2F2 seed of each of the 31 plants was analyzed for grain hardness index (GHI) with single-kernel characterization system. The GHI varied from 12.1 to 37.1 in the seeds borne on the 31 BC2F1 plants. The reasons for this variation and further course of action are discussed.

Rheological evaluations and molecular marker analysis of cultivated bread wheat varieties of India.

The aim of this study was to screen Indian cultivated wheat varieties and list out the parameters/genes required to be improved for an end-product. Therefore, 30 Indian wheat varieties under cultivation by farmers were screened for 14 physico-chemical and rheological parameters, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for high molecular weight glutenin subunits (HMW-GS), DNA based molecular markers for low molecular weight glutenin subunits (LMW-GS) and puroindolines (Pin) genes. Based on grain texture, sedimentation value, farinographic, alveographic, HMW-GS and LMW-GS and biscuit making parameters, HS490 was found to be a highly suited for biscuit and soft wheat products. HI1563 and DBW14 were also found to possess characteristics such as low protein, low to medium SDS-sedimentation value and combination of 2*, 7+8 and 2+12 (HMW-GS). DBW14 also had LMW alleles desirable for biscuit quality. DBW14 needs to be improved for grain softness to make it suitable for biscuit quality while both grain softness and LMW alleles need to be improved for HI1563 to improve its biscuit spread factor and alveographic indices for extensible gluten. Rest varieties showed moderate to very strong gluten but the gluten lacked extensibility. Only four varieties K307, DBW39, NI5439 and DBW17 possessed high flour protein and moderately strong gluten. They had more balanced deformation energy (W) and configuration ratio (P/L) combination suggestive of strong and extensible gluten needed for raised bread making. Marker assisted backcross breeding is suggested as solution to produce end-use specific varieties where appropriate alleles at only a few loci need to be incorporated.

Impact of Parental Presence at Infants' Bedside on Neonatal Abstinence Syndrome.

BACKGROUND: Despite increased incidence of neonatal abstinence syndrome (NAS) over the past decade, minimal data exist on benefits of parental presence at the bedside on NAS outcomes. OBJECTIVE: To examine the association between rates of parental presence and NAS outcomes. METHODS: This was a retrospective, single-center cohort study of infants treated pharmacologically for NAS using a rooming-in model of care. Parental presence was documented every 4 hours with nursing cares. We obtained demographic data for mothers and infants and assessed covariates confounding NAS severity and time spent at the bedside. Outcomes included length of stay (LOS) at the hospital, extent of pharmacotherapy, and mean Finnegan withdrawal score. Multiple linear regression modeling assessed the association of parental presence with outcomes. RESULTS: For the 86 mother-infant dyads, the mean parental presence during scoring was on average 54.4% (95% confidence interval [CI], 48.8%-60.7%) of the infant's hospitalization. Maximum (100%) parental presence was associated with a 9 day shorter LOS (r = -0.31; 95% CI, -0.48 to -0.10; P < .01), 8 fewer days of infant opioid therapy (r = -0.34; 95% CI, -0.52 to -0.15; P < .001), and 1 point lower mean Finnegan score (r = -0.35; 95% CI, -0.52 to -0.15; P < .01). After adjusting for breastfeeding, parental presence remained significantly associated with reduced NAS score and opioid treatment days. CONCLUSIONS: More parental time spent at the infant's bedside was associated with decreased NAS severity. This has important implications for clinical practice guidelines for NAS.

Tumor necrosis factor-α-mediated suppression of dual-specificity phosphatase 4: crosstalk between NFκB and MAPK regulates endothelial cell survival.

We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70-84% and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.