Activity Guided Azide-methyllysine Photo-trapping for Substrate Profiling of Lysine Demethylases.

Reversible post-translational modifications (PTMs) are key to establishing protein-protein and protein-nucleic acid interactions that govern a majority of the signaling pathways in cells. Sequence-specific PTMs are catalyzed by transferases, and their removal is carried out by a class of reverse-acting enzymes termed "detransferases". Currently available chemoproteomic approaches have been valuable in characterizing substrates of transferases. However, proteome-wide cataloging of the substrates of detransferases is challenging, mostly due to the loss of the epitope, rendering immunoprecipitation and activity-based methods ineffective. Herein, we develop a general chemoproteomic strategy called crosslinking-assisted substrate identification (CASI) for systematic characterization of cellular targets of detransferases and successfully apply it to lysine demethylases (KDMs) which catalyze the removal of methyl groups from lysine sidechain in histones to modulate gene transcription. By setting up a targeted azido-methylamino photo-reaction deep inside the active site of KDM4, engineered to carry p-azido phenylalanine, we reveal a novel "demethylome" that has escaped the traditional methods. The proteomic survey led to the identification of a battery of nonhistone substrates of KDM4, extending the biological footprint of KDM4 beyond its canonical functions in gene transcription. A notable finding of KDM4A-mediated demethylation of an evolutionarily conserved lysine residue in eukaryotic translational initiation factor argues for a much broader role of KDM4A in ribosomal processes. CASI, representing a substantive departure from earlier approaches by shifting focus from simple peptide-based probes to employing full-length photo-activatable demethylases, is poised to be applied to >400 human detransferases, many of which have remained poorly understood due to the lack of knowledge about their cellular targets.

The cancer journey through the lens of a sibling: a photovoice intervention for teen siblings of children with cancer.

Photovoice was employed as a clinical intervention to engage siblings of children with cancer, provide opportunity for sibling support, and elicit information about their lived experiences. Sibling support groups have been effective, however, none have utilized this intervention. Four teenagers who had a sibling diagnosed with cancer participated in a seven-week intervention group. Themes were identified to inform future clinical practice. Four main themes included: (i) support, (ii) changes, (iii) feelings, and (iv) Photovoice group experience. Photovoice, used as a clinical intervention, elicited valuable information and generated fruitful conversations, enabling siblings to relate to and learn from one another.

Engineering Methyllysine Writers and Readers for Allele-Specific Regulation of Protein-Protein Interactions.

Protein-protein interactions mediated by methyllysine are ubiquitous in biological systems. Specific perturbation of such interactions has remained a challenging endeavor. Herein, we describe an allele-specific strategy toward an engineered protein-protein interface orthogonal to the human proteome. We develop a methyltransferase (writer) variant that installs aryllysine moiety on histones that can only be recognized by an engineered chromodomain (reader). We establish biochemical integrity of the engineered interface, provide structural evidence for orthogonality and validate its applicability to identify transcriptional regulators. Our approach provides an unprecedented strategy for specific manipulation of the methyllysine interactome.

Allele-Specific Inhibition of Histone Demethylases.

Histone demethylases play a critical role in mammalian gene expression by removing methyl groups from lysine residues in degree- and site-specific manner. To specifically interrogate members and isoforms of this class of enzymes, we have developed demethylase variants with an expanded active site. The mutant enzymes are capable of performing lysine demethylation with wild-type proficiency, but are sensitive to inhibition by cofactor-competitive molecules embellished with a complementary steric "bump". The selected inhibitors show more than 20-fold selectivity over the wild-type demethylase, thus overcoming issues typical to pharmacological and genetic approaches. The mutant-inhibitor pairs are shown to act on a physiologically relevant full-length substrate. By engineering a conserved amino acid to achieve member-specific perturbation, this study provides a general approach for studying histone demethylases in diverse cellular processes.

Process optimization and characterization of 'sev' (traditional Indian extruded snack) with the incorporation of mushroom powder.

Mushroom is a nutritious and palatable food with various health attributes. The mushroom powder is a rich source of vegetable protein. In the present study, efforts were made to optimize the process for making 'sev', a traditional Indian extruded snack, with the incorporation of mushroom powder (Agaricus bisporus) to the Bengal gram (Cicer arietinum L.) flour ('besan'). Response surface methodology with central composite rotary design was used to determine the optimum level of various ingredients for 'sev' which was then analyzed for various physicochemical and sensory characteristics. Storage studies of mushroom 'sev' (with and without rice starch/mashed potato) were done at 37 ± 1 °C. The product containing rice starch was found to be highly acceptable over that with mashed potato. The optimized mushroom 'sev' contains 7.93% mushroom powder, 8.91% rice flour, 75.95% Bengal gram flour and 7.2% other ingredients with high scores for different sensory attributes viz., colour (8.4), appearance (8.3), flavour (8.3), texture (8.5) and overall acceptability (8.4). The mushroom powder based 'sev' had 2.79% higher protein content than the control sample (without mushroom powder) with a storage life of at least 7 days at 37 ± 1 °C.

In vivo effect of two different dietary fiber blends on the milk calcium bioavailability.

Milk is a valuable source of dietary calcium and it becomes important to establish whether incorporation of dietary fiber (DF), a health promoting food constituent, would lead to any undesirable impact on the bioavailability of milk calcium or not. The DF fortified spray dried partly skimmed milk powder with prestandardized fiber Blend-I (psyllium husk, oat fiber, MCC, inulin) and fiber Blend-II (psyllium husk, oat bran, wheat fiber and inulin) was subjected to rat-feeding studies to examine the possible effects on the bioavailability of milk calcium. The differences for calcium absorption and retention among diets containing DF Blend-I, DF Blend-II and cellulose (control) were found to be non-significant. It was evident that the milk calcium bioavailability of the diets containing two fiber formulations tested (at the levels studied) was at par with that of control standard diet containing only cellulose as DF. Therefore, it is reasonable to incorporate these DF blends into dairy products, and thereby add value.

Complementary Steric Engineering at the Protein-Ligand Interface for Analogue-Sensitive TET Oxygenases.

Ten-eleven translocation (TET) enzymes employ O2, earth-abundant iron, and 2-ketoglutarate (2KG) to perform iterative C-H oxidation of 5-methylcytosine in DNA to control expression of the mammalian genome. Given that more than 60 such C-H oxygenases are present in humans, determining context-dependent functions of each of these enzymes is a pivotal challenge. In an effort to tackle the problem, we developed analogue-sensitive TET enzymes to perturb the activity of a specific member. We rationally engineered the TET2-2KG interface to develop TET2 variants with an expanded active site that can be specifically inhibited by the N-oxalylglycine (NOG) derivatives carrying a complementary steric "bump". Herein, we describe the identification and engineering of a bulky gatekeeper residue for TET proteins, characterize the orthogonal mutant-inhibitor pairs, and show generality of the approach. Employing cell-permeable NOG analogues, we show that the TET2 mutant can be specifically inhibited to conditionally modulate cytosine methylation in chromosomal DNA in intact human cells. Finally, we demonstrate application of the orthogonal mutant-inhibitor pair to probe transcriptional activity of a specific TET member in cells. Our work provides a general platform for developing analogue-sensitive 2KG-dependent oxygenases to unravel their functions in diverse signaling processes.

miRNet - dissecting miRNA-target interactions and functional associations through network-based visual analysis.

MicroRNAs (miRNAs) can regulate nearly all biological processes and their dysregulation is implicated in various complex diseases and pathological conditions. Recent years have seen a growing number of functional studies of miRNAs using high-throughput experimental technologies, which have produced a large amount of high-quality data regarding miRNA target genes and their interactions with small molecules, long non-coding RNAs, epigenetic modifiers, disease associations, etc These rich sets of information have enabled the creation of comprehensive networks linking miRNAs with various biologically important entities to shed light on their collective functions and regulatory mechanisms. Here, we introduce miRNet, an easy-to-use web-based tool that offers statistical, visual and network-based approaches to help researchers understand miRNAs functions and regulatory mechanisms. The key features of miRNet include: (i) a comprehensive knowledge base integrating high-quality miRNA-target interaction data from 11 databases; (ii) support for differential expression analysis of data from microarray, RNA-seq and quantitative PCR; (iii) implementation of a flexible interface for data filtering, refinement and customization during network creation; (iv) a powerful fully featured network visualization system coupled with enrichment analysis. miRNet offers a comprehensive tool suite to enable statistical analysis and functional interpretation of various data generated from current miRNA studies. miRNet is freely available at http://www.mirnet.ca.

Effect of fiber blends, total solids, heat treatment, whey protein concentrate and stage of sugar incorporation on dietary fiber-fortified Kheer.

Owing to the proven beneficial role of dietary fiber (DF) on human health, feasibility of incorporating commercially available soluble and insoluble DF preparations into rice-milk-pudding (kheer, a popular Indian delicacy) was studied through process modification. The novel approach of preparing reduced fat DF-fortified-kheer (DFFK) by developing liquid/cream phase and particulate/rice phase separately, and subsequently blending the two was developed. The major processing variables studied were total solids (TS) in the liquid phase, type of fiber blend, flavor-simulation through heat treatment or added whey protein, and the presence of sugar in water for pre-cooking of rice. Reduced fat DFFK made from three different pre-standardized fiber blends was quite acceptable to the sensory panel (overall rating 7.5). With increasing TS in milk up to 16.5%, sensory acceptability of DFFK increased. There was a small but perceivable improvement in the flavour of DFFK when precooking of rice was carried out in sweetened water. Reduced fat DFFK from different fiber blends was found to be reasonably close to conventional kheer. DFFK prepared from Blend-I provided 3.31 g dietary fiber/100 kcal (suitable for the claim "High-in-Fiber") with 38.71% Reduced-Fat. With the developed process of fortification of kheer with DF, it is possible to reduce fat (and thus calories) to cater to the needs of consumers seeking good health.

Determination of relationship between sensory viscosity rating and instrumental flow behaviour of soluble dietary fibers.

The shear-thinning low, medium and high-viscosity fiber preparations (0.15-1.05 % psyllium husk, 0.07-0.6 % guar gum, 0.15-1.20 % gum tragacanth, 0.1-0.8 % gum karaya, 0.15-1.05 % high-viscosity Carboxy Methyl Cellulose and 0.1-0.7 % xanthan gum) showed that the consistency coefficient (k) was a function of concentration, the relationship being exponential (R(2), 0.87-0.96; P < 0.01). The flow behaviour index (n) (except for gum karaya and CMC) was exponentially related to concentration (R(2), 0.61-0.98). The relationship between k and sensory viscosity rating (SVR) was essentially linear in nearly all cases. The SVR could be predicted from the consistency coefficient using the regression equations developed. Also, the relationship of k with fiber concentration would make it possible to identify the concentration of a particular gum required to have desired consistency in terms of SVR.

Psychometric validation of the Clubhouse climate questionnaire as an autonomy support measure for people with severe mental illness.

BACKGROUND: Self-determination represents a paradigm shift from "shoulds" to "decisions and behaviors" as the best options for helping people with mental illness achieving recovery goals. Autonomy support plays an important role in self-determination. AIMS: The purpose of this study was to validate the Clubhouse climate questionnaire (CCQ) as an autonomy support measure. METHOD: One hundred and twenty-four participants were recruited from eight Clubhouse programs in Hawaii. Measurement structure of the CCQ was evaluated using exploratory and confirmatory factor analysis. FINDINGS: CCQ is a unidimensional scale with good reliability and acceptable levels of divergent and convergent validity. CONCLUSIONS: CCQ is a brief, reliable and valid instrument for assessing autonomy support and contributes to the use of self-determination as a paradigm for improving recovery outcomes in psychiatric rehabilitation.

Effect of male parental gamma irradiation on host suitability of its F1 progeny of a lepidopteran tropical pest, Spodoptera litura (Fabr.) towards development and virulence of entomopathogenic nematodes, Steinernema thermophilum.

The suitability of F1 progeny insect larvae of the irradiated male parent, Spodoptera litura (Fabr.) for infective juveniles (IJs) of entomopathogenic nematodes (EPN), Steinernema thermophilum was assessed to comprehend the feasibility of combining EPNs with nuclear pest control tactic. As compared to the control, the IJs induced faster host mortality with reduced proliferation in F1 host larvae. IJs derived from F1 host larvae exhibited almost similar proliferation capacity on normal hosts as in control. Further, the molecular basis of EPNs induced mortality in F1 host larvae was evaluated. Dual stress of EPN infection and irradiation induced downregulation of the relative mRNA expression of antimicrobial genes and upregulated expression of antioxidative genes. A pronounced effect of EPNs in association with irradiation stress was apparent on host mortality. Radiation induced sterile F1 insect larvae of S. litura acted as a reasonably suitable host for EPNs and also provided the environment for developing viable EPNs for their potential use as biocontrol agents.

A Protocol for the Detection of Fusion Transcripts Using RNA-Sequencing Data.

Fusion transcripts are formed when two genes or their mRNAs fuse to produce a novel gene or chimeric transcript. Fusion genes are well-known cancer biomarkers used for cancer diagnosis and as therapeutic targets. Gene fusions are also found in normal physiology and lead to the evolution of novel genes that contribute to better survival and adaptation for an organism. Various in vitro approaches, such as FISH, PCR, RT-PCR, and chromosome banding techniques, have been used to detect gene fusion. However, all these approaches have low resolution and throughput. Due to the development of high-throughput next-generation sequencing technologies, the detection of fusion transcript becomes feasible using whole genome sequencing, RNA-Seq data, and bioinformatics tools. This chapter will overview the general computational protocol for fusion transcript detection from RNA-sequencing datasets.

Challenges, Barriers, and Facilitators in Telemedicine Implementation in India: A Scoping Review.

Telemedicine has revolutionized healthcare access by breaking geographical barriers and facilitating remote consultations. The eSanjeevani platform has been pivotal in India, especially during the COVID-19 pandemic. Despite its benefits, implementing telemedicine faces various challenges and barriers. This scoping review aims to identify these challenges, barriers, and facilitators in the Indian context. This review follows the preferred reporting items for systematic reviews and meta-analyses extension for scoping reviews (PRISMA-ScR) guidelines. A comprehensive literature search was conducted using PubMed and Google Scholar to identify studies published between January 2019 and January 2024. Studies on telemedicine adoption, barriers, and facilitators in India were included. Data were extracted and synthesized from 26 quantitative, qualitative, and mixed-methods research studies. Individual-level challenges include limited digital literacy, particularly among older adults and those in rural areas, and health literacy issues. Barriers such as limited smartphone access, unreliable internet connectivity, and socio-cultural preferences for in-person consultations were identified. Facilitators at this level include family involvement, training programs, and community outreach. Provider-level challenges involve a lack of training and concerns about care quality, while barriers include insufficient infrastructure and medico-legal concerns. Facilitators include ongoing training, clear guidelines, and user-friendly telehealth systems. System-level challenges include integrating telemedicine into existing infrastructure and ensuring data security. Barriers include inadequate funding and resistance to change, while facilitators include policy support, investment in infrastructure, and collaborative efforts. Telemedicine holds great potential to enhance remote healthcare access in India, but its successful implementation requires addressing significant challenges and barriers. Strengthening digital infrastructure, enhancing digital literacy, standardizing protocols, and developing clear regulatory frameworks are essential. Collaborative efforts and tailored approaches that respect local cultures can further facilitate telemedicine adoption. Continuous research and public awareness campaigns are necessary to ensure telemedicine's sustainable and effective use in India.

In Silico Identification of tRNA Fragments, Novel Candidates for Cancer Biomarkers, and Therapeutic Targets.

The identification of a wide variety of RNA molecules using high-throughput sequencing techniques in the transcriptome pool of living organisms has revealed hidden regulatory insights in the cell. The class of non-coding RNA fragments produced from transfer RNA, or tRFs, is one such example. They are heterogeneously sized molecules with lengths ranging between 15 and 50 nt. They have a history of being dysregulated in human malignancies and other illnesses. The detection of these molecules has been made easier by a variety of bioinformatics techniques. The various types of tRFs and how they relate to cancer are covered in this chapter. It also provides a summary of the biological significance of tRFs reported in human cancer. Additionally, it emphasizes the utilities of databases and computational tools that have been created by different research teams for the investigation of tRFs. This will further aid the exploration and analysis of tRFs in cancer research and will support future advancement and a better comprehension of these molecules.

Role of vesicular monoamine transporter-2 for treating attention deficit hyperactivity disorder: a review.

RATIONALE: The Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) classifies attention deficit hyperactivity disorder (ADHD) as a neurodevelopmental disorder that interferes with human functioning and development. As the clinical presentation of ADHD involves a deficiency in executive function, neurocognitive deficits involving distinctive neuropathological changes must be present for clinical diagnosis. OBJECTIVES: The vesicular monoamine transporter (VMAT), specifically VMAT-2, plays a role in ADHD pathogenesis. In addition, experimental data show that the stimulants (amphetamines and methylphenidate) are first-line treatments for the condition because of their extensive interaction with VMAT-2. The interactions of peptides, bupropion, and nutritional supplements with VMAT-2 receptors have been researched, but more evidence is needed to elucidate their pharmacodynamic properties. Therefore, this literature review evaluated the current pharmacological treatment modalities, peptides, and nutritional supplements for ADHD that target the VMAT-2 system. METHODS, RESULTS, AND CONCLUSIONS: We obtained relevant studies from several platforms, including the National Center for Biotechnology, Clinical Key, Access Medicine, and PubMed. From the results of these studies, we observed that stimulants interact highly with the VMAT-2 transporter, with omega-3 fatty acids, peptides, and bupropion exerting some modulatory activity on VMAT-2. These agents should be considered for the future treatment of ADHD, although clinical-level research involving human participants is necessary.

Phase Separation and Aggregation of a Globular Folded Protein Small Ubiquitin-like Modifier 1 (SUMO1).

Liquid-liquid phase separation (LLPS) plays a crucial role in cellular organization, primarily driven by intrinsically disordered proteins (IDPs) leading to the formation of biomolecular condensates. A folded protein SUMO that post-translationally modifies cellular proteins has recently emerged as a regulator of LLPS. Given its compact structure and limited flexibility, the precise role of SUMO in condensate formation remains to be investigated. Here, we show the rapid phase separation of SUMO1 into micrometer-sized liquid-like condensates in inert crowders under physiological conditions. Subsequent time-dependent conformational changes and aggregation are probed by label-free methods (tryptophan fluorescence and Raman spectroscopy). Remarkably, experiments on a SUMO1 variant lacking the N-terminal disordered region further corroborate the role of its structured part in phase transitions. Our findings highlight the potential of folded proteins to engage in LLPS and emphasize further investigation into the influence of the SUMO tag on IDPs associated with membrane-less assemblies in cells.

Lymphoepithelial Carcinoma of the Breast.

Lymphoepithelial malignancy is an extremely rare carcinoma of the breast characterized by a confusing histopathological picture resembling medullary carcinomas, lymphoma, etc. It has also been reported in other regions of the body like salivary glands, nasopharyngeal area and sometimes the lung. Due to its rare presence and difficult diagnosis, the treatment is often prolonged and delayed. Here we present a case report of a 56-year-old lady who was eventually diagnosed as lymphoepithelial carcinoma of the breast. Her journey of evaluation and treatment was fraught with pathological nuances and an elimination drill of multiple differentials before concluding this rare diagnosis. Although lymphoepithelial-like carcinoma is a rare entity, multiple cases have been reported in the literature and their review is mandated to further our clinical knowledge about the oncological treatment and expected prognosis of such cases in the future. Our patient underwent a simple mastectomy, followed by chemotherapy, radiotherapy, and is completely asymptomatic now. She has been cancer-free for the last seven years so far.

Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes.

Pneumocystis jirovecii is an important etiological agent of pneumonia that is underdiagnosed due to the inability to culture the organism. The 2019 PERCH study identified Pneumocystis as the top fungal cause of pneumonia in HIV-negative children using a PCR cutoff of 104 copies of Pneumocystis per mL of sample in nasopharyngeal/oropharyngeal (NP/OP) specimens. Given that Pneumocystis consists of an environmental ascus form and a trophic from (the latter is the form that attaches to the lung epithelium), it is possible that life-form-specific molecular assays may be useful for diagnosis. However, to accomplish this goal, these assays require genotypic information, as the current fungal genomic data are largely from the US and Europe. To genotype Pneumocystis across the globe, we developed an NGS-based genotyping assay focused on genes expressed in asci as well as trophs using PERCH throat swabs from Africa, Bangladesh, and Thailand, as well as North American samples. The NGS panel reliably detected 21 fungal targets in these samples and revealed unique genotypes in genes expressed in trophs, including Meu10, an ascospore assembly gene; two in mitochondrial gene ATP8, and the intergenic region between COX1 and ATP8. This assay can be used for enhanced Pneumocystis epidemiology to study outbreaks but also permits more accurate RT-CPR- or CRISPR-based assays to be performed to improve the non-bronchoscopic diagnosis of this under-reported fungal pathogen.