India, a global leader in agriculture, faces sustainability challenges in feeding its population. Although primarily a vegetarian population, the consumption of animal derived proteins has tremendously increased in recent years. Excessive dependency on animal proteins is not environmentally sustainable, necessitating the identification of alternative smart proteins. Smart proteins are environmentally benign and mimic the properties of animal proteins (dairy, egg and meat) and are derived from plant proteins, microbial fermentation, insects and cell culture meat (CCM) processes. This review critically evaluates the technological, safety, and sustainability challenges involved in production of smart proteins and their consumer acceptance from Indian context. Under current circumstances, plant-based proteins are most favorable; however, limited land availability and impending climate change makes them unsustainable in the long run. CCM is unaffordable with high input costs limiting its commercialization in near future. Microbial-derived proteins could be the most sustainable option for future owing to higher productivity and ability to grow on low-cost substrates. A circular economy approach integrating agri-horti waste valorization and C1 substrate synthesis with microbial biomass production offer economic viability. Considering the use of novel additives and processing techniques, evaluation of safety, allergenicity, and bioavailability of smart protein products is necessary before large-scale adoption.

Residues and byproducts generated after the extraction of essential oil (EO) from German Chamomile (Matricaria chamomilla L.) are often discarded or burnt causing severe resource wastage and pollution. Valorization of these residues and byproducts through a biorefinery approach generates myriad products of high value, leading to resource recovery and enhancement in the sustainability of Chamomile cultivation. Process residues such as Chamomile flower hydrodistillation filtrates (CFL), Chamomile flower residue (CR), and unutilized plant parts (aerial parts and roots) were characterized for their nutritional attributes, phytochemical composition and bioactive properties viz., antioxidant and α-glucosidase inhibition. A functional soup was developed utilizing the polyphenol rich CFL extracts and compared with commercial soups for their phytochemical and functional properties. The fixed oil of the Chamomile flower (CF) contained high concentrations of linoleic acid. The CFL extracts were abundant in polyphenols, specifically p-Coumaric acid constituting 50

Clustering Epilepsy (CE) is an epileptic disorder with neurological comorbidities caused by heterozygous variants of the X chromosome gene Protocadherin 19 (PCDH19). Recent studies have implicated dysregulation of the Nuclear Hormone Receptor (NHR) pathway in CE pathogenesis. To obtain a comprehensive overview of the impact and mechanisms of loss of PCDH19 function in CE pathogenesis, we have performed epigenomic, transcriptomic and proteomic analysis of CE relevant models. Our studies identified differential regulation and expression of Androgen Receptor (AR) and its targets in CE patient skin fibroblasts. Furthermore, our cell culture assays revealed the repression of PCDH19 expression mediated through ERα and the co-regulator FOXA1. We also identified a protein-protein interaction between PCDH19 and AR, expanding upon the intrinsic link between PCDH19 and the NHR pathway. Together, these results point to a novel mechanism of NHR signaling in the pathogenesis of CE that can be explored for potential therapeutic options.

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX ( Tr anscription- Ex port) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37–38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37–38 deletion male ( Thoc2 Δ/Y ) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2 Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.

Non-clustered protocadherins (ncPcdhs) are adhesive molecules with spatio-temporally regulated overlapping expression in the developing nervous system. Although their unique role in neurogenesis has been widely studied, their combinatorial role in brain physiology and pathology is poorly understood. Using probabilistic cell typing by in situ sequencing, we demonstrate combinatorial inter- and intra-familial expression of ncPcdhs in the developing mouse cortex and hippocampus, at single-cell resolution. We discovered the combinatorial expression of Protocadherin-19 (Pcdh19), a protein involved in PCDH19-clustering epilepsy, with Pcdh1, Pcdh9 or Cadherin 13 (Cdh13) in excitatory neurons. Using aggregation assays, we demonstrate a code-specific adhesion function of PCDH19; mosaic PCDH19 absence in PCDH19+9 and PCDH19 + CDH13, but not in PCDH19+1 codes, alters cell–cell interaction. Interestingly, we found that PCDH19 as a dominant protein in two heterophilic adhesion codes could promote trans-interaction between them. In addition, we discovered increased CDH13-mediated cell adhesion in the presence of PCDH19, suggesting a potential role of PCDH19 as an adhesion mediator of CDH13. Finally, we demonstrated novel cis-interactions between PCDH19 and PCDH1, PCDH9 and CDH13. These observations suggest that there is a unique combinatorial code with a cell- and region-specific characteristic where a single molecule defines the heterophilic cell–cell adhesion properties of each code.

Clustering Epilepsy (CE) is a neurological disorder caused by pathogenic variants of the Protocadherin 19 (PCDH19) gene. PCDH19 encodes a protein involved in cell adhesion and Estrogen Receptor α mediated-gene regulation. To gain further insights into the molecular role of PCDH19 in the brain, we investigated the PCDH19 interactome in the developing mouse hippocampus and cortex. Combined with a meta-analysis of all reported PCDH19 interacting proteins, our results show that PCDH19 interacts with proteins involved in actin, microtubule, and gene regulation. We report CAPZA1, αN-catenin and, importantly, β-catenin as novel PCDH19 interacting proteins. Furthermore, we show that PCDH19 is a regulator of β-catenin transcriptional activity, and that this pathway is disrupted in CE individuals. Overall, our results support the involvement of PCDH19 in the cytoskeletal network and point to signalling pathways where PCDH19 plays critical roles.

Chhurpe is a naturally fermented traditional dairy food of high altitude Western Himalayan region. They are generally prepared from cow or yak milk and are consumed during harsh winters. The present study was conducted to characterize the different Chhurpe samples traditionally prepared by the ethnic groups utilizing milk from different animal breeds such as cow, yak, Zomo (cow × yak) , and Germo (Zomo × yak). Nutritional characterization revealed that 100 g of Chhurpe could completely meet the dietary protein requirements of children and adults with high concentrations of methionine and lysine. Tryptophan and valine were the limiting amino acids among all the Chhurpe samples. Palmitic, stearic, and oleic acids were the predominant fatty acids. The Chhurpe samples were a rich source of micronutrients such as calcium, iron, and zinc meeting above 70% of recommended dietary allowances (RDA) among children (3–10 years) and up to 20% RDA for adults. Culture-independent metagenomic analysis revealed that lactic acid bacteria were the predominant group, consisting of genera such as Lactobacillus , Leuconostoc , Lactococcus , and Streptococcus followed by acetic acid bacteria, mainly Acetobacter. At the species level, Lactobacillus delbrueckii was the abundant strain among all the Chhurpe samples. Species diversity was significantly higher in Chhurpe prepared from Zomo milk. Probiotic bacterial strains such as Lactobacillus helveticus, L. delbrueckii, L. brevis , and Leuconostoc mesenteroides were identified in the Zomo Chhurpe indicating their superior quality. The present study was an attempt to popularize Chhurpe and promote its wider consumption by highlighting its nutritional properties.

Aicardi Syndrome (AIC) is a rare neurodevelopmental disorder recognized by the classical triad of agenesis of the corpus callosum, chorioretinal lacunae and infantile epileptic spasms syndrome. The diagnostic criteria of AIC were revised in 2005 to include additional phenotypes that are frequently observed in this patient group. AIC has been traditionally considered as X-linked and male lethal because it almost exclusively affects females. Despite numerous genetic and genomic investigations on AIC, a unifying X-linked cause has not been identified. Here, we performed exome and genome sequencing of 10 females with AIC or suspected AIC based on current criteria. We identified a unique de novo variant, each in different genes: KMT2B, SLF1, SMARCB1, SZT2 and WNT8B, in five of these females. Notably, genomic analyses of coding and non-coding single nucleotide variants, short tandem repeats and structural variation highlighted a distinct lack of X-linked candidate genes. We assessed the likely pathogenicity of our candidate autosomal variants using the TOPflash assay for WNT8B and morpholino knockdown in zebrafish (Danio rerio) embryos for other candidates. We show expression of Wnt8b and Slf1 are restricted to clinically relevant cortical tissues during mouse development. Our findings suggest that AIC is genetically heterogeneous with implicated genes converging on molecular pathways central to cortical development.

Sustainable agriculture practices involve the application of environment-friendly plant growth promoters and additives that do not negatively impact the health of the ecosystem. Stringent regulatory frameworks restricting the use of synthetic agrochemicals and the increase in demand for organically grown crops have paved the way for the development of novel bio-based plant growth promoters. In this context, microalgae biomass and derived agrochemicals offer novel sources of plant growth promotors that enhance crop productivity and impart disease resistance. These beneficial effects could be attributed to the presence of wide range of biomolecules such as soluble amino acid (AA), micronutrients, polysaccharides, phytohormones and other signaling molecules in microalgae biomass. In addition, their phototrophic nature, high photosynthetic efficiency, and wide environmental adaptability make them an attractive source of biostimulants, biofertilizers and biopesticides. The present review aims to describe the various plant growth promoting metabolites produced by microalgae and their effects on plant growth and productivity. Further, the effects elicited by microalgae biostimulants with respect to different modes of applications such as seed treatments, foliar spray and soil/root drenching is reviewed in detail. In addition, the ability of microalgae metabolites to impart tolerance against various abiotic and biotic stressors along with the mechanism of action is discussed in this paper. Although the use of microalgae based biofertilizers and biostimulants is gaining popularity, the high nutrient and water requirements and energy intensive downstream processes makes microalgae based technology commercially unsustainable. Addressing this challenge, we propose a circular economy model of microalgae mediated bioremediation coupled with biorefinery approaches of generating high value metabolites along with biofertilizer applications. We discuss and review new trends in enhancing the sustainability of microalgae biomass production by co-cultivation of algae with hydroponics and utilization of agriculture effluents.

The present study was aimed at developing Arthrospira platensis (Spirulina) fortified traditional foods of the Indian subcontinent, namely sattu (multigrain beverage mix) and chikki (peanut bar) and evaluating their ability to promote recovery from protein and iron deficiency anaemia (IDA) using albino Wistar rats. Addition of Spirulina (at 4% w/w Spirulina inclusion levels) enriched the protein content by 20.33% in sattu and 15.65% in chikki while the iron content was enhanced by 45% in sattu and 29.6% in chikki. In addition, the total carotenoid and polyphenol content and antioxidant capacity of the food products improved after Spirulina incorporation. Supplementation of 100 g of Spirulina fortified food products meets more than 50% of recommended dietary allowances (RDA) of protein, dietary fiber, iron and zinc for the age group 3 to 10 years of children. Spirulina contributed between 11% and 22% of RDA for protein and iron, respectively; however it contributed very negligibly to RDA of dietary fibre with respect to the nutrient requirements for the target age group. Supplementation of Spirulina fortified foods individually promoted bodyweight gain in malnourished rats and restored haemoglobin, serum protein, albumin, serum iron, and hepcidin levels and reduced the iron binding capacity indicating recovery from IDA. Spirulina supplementation ameliorated malnutrition induced oxidative stress in the liver, spleen and kidneys by reducing the lipid peroxidation and enhancing superoxide dismutase and glutathione activities. Histopathological analysis revealed that supplementation of Spirulina fortified foods reversed pathological changes such as fatty changes in the liver cells, thinning of cardiac muscle fibers and degeneration of intestinal villi. Fe-protein deficiency significantly altered the gut microflora by reducing the abundance of beneficial microbes. However, supplementation of Spirulina fortified foods improved the levels of beneficial gut microbes such as Lactobacillus reuteri and Akkermansia muciniphila while reducing the abundance of Helicobacteraceae, Enterobacteria and Clostridia. In summary, supplementation of Spirulina fortified foods promoted recovery from protein and iron deficiency indicating the bioavailability of nutrients (iron and protein) from Spirulina at par with casein and ferrous ascorbate.

The global demand for protein ingredients is continuously increasing owing to the growing population, rising incomes, increased urbanization, and aging population. Conventionally, animal-derived products (dairy, egg, and meat) satisfy the major dietary protein requirements of humans. With the global population set to reach 9.6 billion by 2050, there would be a huge deficit in meeting dietary protein requirements. Therefore, it is necessary to identify sustainable alternative protein sources that could complement high-quality animal proteins. In recent years, microalgae have been advocated as a potential industrial source of edible proteins owing to their wide and excellent ecological adaptation. Microalgae can grow in marginal areas utilizing non-potable wastewaters with high photosynthetic efficiency. Previously microalgae species such as Arthospira, and Chlorella have been used as single-cell proteins (SCP) with limited application in pharmaceutical industries. In recent years, the demand for innovative and sustainable functional ingredients for food applications has renewed the interest worldwide in microalgae proteins. The present review aims to provide a holistic view of various aspects related to the production and processing of edible proteins from microalgae biomass. A critical review of available literature on the nutritional quality, techno-functional properties, applications in food and feed sectors, and biological activities is presented. Further, challenges associated with each stage of processing are discussed. From the literature review, it can be summarized that microalgae proteins are comparable to reference proteins both in terms of amino acid (AA) quality and techno-functional properties. However recalcitrant cell wall poses a challenge in digestibility and effective utilization of the microalgae proteins. Further, poor sensory scores and palatability of microalgae biomass limit its applications in the food and feed sector. Novel applications of microalgae proteins include meat analogues, emulsifying agents, and bioactive peptides. Development of low-cost cultivation strategies, wet biomass-based downstream processing along with the bio-refinery approach of complete biomass volarization would enhance the sustainability quotient for human food applications.

Immunoprecipitation (IP) of endogenously expressed proteins is one of the most biologically relevant techniques to identify protein-protein interactions. We describe an adaptable IP protocol reliant on a specific antibody to the target protein. We detail a quantitative proteomics workflow for the unbiased identification of co-immunoprecipitating proteins, known collectively as an interactome. This includes protocols for the tryptic digestion, Tandem Mass Tag labeling and fractionation of peptides, and their identification and quantification using liquid chromatography-mass spectrometry including computational and statistical analysis. For complete details on the use and execution of this protocol, please refer to Johnson et al. (2020).

Naturally occurring forage species in the high-altitude pasturelands of Western Himalaya are traditionally being utilized for feeding of livestock. However, the nutritional quality evaluation and validation of their potential as feed for livestock needs to be undertaken. Here, we evaluated proximate compositions, minerals and amino acids (AAs) of five high-altitude forage species, namely, Festuca kashmiriana L., Medicago sativa L., Trifolium pratense L., Medicago falcate L. and Melilotus indica L. The results revealed that proximate compositions, mineral and AAs varied significantly among the forage species. The carbohydrate, crude protein, moisture content, crude fat, crude fiber, crude ash, total phenol, oil absorption capacity and water absorption capacity were found in the range of 17.44-37.27 mg/100 mg, 3.34-14.71 mg/100 mg, 88.73%-90.72%, 0.98-2.32 mg/100 mg, 11.16-24.16%, 7.71-34.49%, 292.50-488.12 mu g/100 mg, 3.91-4.67 g/g and 2.64-3.41 g/g, respectively. Elemental composition showed that calcium was the predominant element among the minerals (13.91-132.05 mg/kg DM) followed by magnesium (4.60-12.92 mg/kg DM), iron (2.04-76.13 mg/kg DM) and zinc (1.07-2.17 mg/kg DM). Furthermore, we found that these high-altitude forage species are rich in essential AAs like histidine, tryptophan, valine, leucine, phenylalanine and to some extent isoleucine and tyrosine. In addition, these five species showed distinct proteomes but shared a similar functional group. The proteome profiling of these forage species will help to understand the molecular basis of nutritional enrichment and stress tolerance potential against harsh environmental conditions of high altitudes. Overall, we conclude that traditionally used high-altitude forage species are nutritionally rich and can be recommended as part of the daily nutritive feed for livestock animals.

Protein ubiquitination is a widespread, multifunctional, posttranslational protein modification, best known for its ability to direct protein degradation via the ubiquitin proteasome system (UPS). Ubiquitination is also reversible, and the human genome encodes over 90 deubiquitinating enzymes (DUBs), many of which appear to target specific subsets of ubiquitinated proteins. This review focuses on the roles of DUBs in neurodevelopmental disorders (NDDs). We present the current genetic evidence connecting 12 DUBs to a range of NDDs and the functional studies implicating at least 19 additional DUBs as candidate NDD genes. We highlight how the study of DUBs in NDDs offers critical insights into the role of protein degradation during brain development. Because one of the major known functions of a DUB is to antagonize the UPS, loss of function of DUB genes has been shown to culminate in loss of abundance of its protein substrates. The identification and study of NDD DUB substrates in the developing brain is revealing that they regulate networks of proteins that themselves are encoded by NDD genes. We describe the new technologies that are enabling the full resolution of DUB protein networks in the developing brain, with the view that this knowledge can direct the development of new therapeutic paradigms. The fact that the abundance of many NDD proteins is regulated by the UPS presents an exciting opportunity to combat NDDs caused by haploinsufficiency, because the loss of abundance of NDD proteins can be potentially rectified by antagonizing their UPS-based degradation.

TIMMDC1 encodes the Translocase of Inner Mitochondrial Membrane Domain-Containing protein 1 (TIMMDC1) subunit of complex I of the electron transport chain responsible for ATP production. We studied a consanguineous family with two affected children, now deceased, who presented with failure to thrive in the early postnatal period, poor feeding, hypotonia, peripheral neuropathy and drug-resistant epilepsy. Genome sequencing data revealed a known, deep intronic pathogenic variant TIMMDC1 c.597-1340A>G, also present in gnomAD (~1/5000 frequency), that enhances aberrant splicing. Using RNA and protein analysis we show almost complete loss of TIMMDC1 protein and compromised mitochondrial complex I function. We have designed and applied two different splice-switching antisense oligonucleotides (SSO) to restore normal TIMMDC1 mRNA processing and protein levels in patients' cells. Quantitative proteomics and real-time metabolic analysis of mitochondrial function on patient fibroblasts treated with SSOs showed restoration of complex I subunit abundance and function. SSO-mediated therapy of this inevitably fatal TIMMDC1 neurologic disorder is an attractive possibility.

Steroids yield great influence on neurological development through nuclear hormone receptor (NHR)-mediated gene regulation. We recently reported that cell adhesion molecule protocadherin 19 (encoded by the PCDH19 gene) is involved in the coregulation of steroid receptor activity on gene expression. PCDH19 variants cause early-onset developmental epileptic encephalopathy clustering epilepsy (CE), with altered steroidogenesis and NHR-related gene expression being identified in these individuals. The implication of hormonal pathways in CE pathogenesis has led to the investigation of various steroid-based antiepileptic drugs in the treatment of this disorder, with mixed results so far. Therefore, there are many unmet challenges in assessing the antiseizure targets and efficiency of steroid-based therapeutics for CE. We review and assess the evidence for and against the implication of neurosteroids in the pathogenesis of CE and in view of their possible clinical benefit.

PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.

The pioneering discovery research of X-linked intellectual disability (XLID) genes has benefitted thousands of individuals worldwide; however, approximately 30% of XLID families still remain unresolved. We postulated that noncoding variants that affect gene regulation or splicing may account for the lack of a genetic diagnosis in some cases. Detecting pathogenic, gene-regulatory variants with the same sensitivity and specificity as structural and coding variants is a major challenge for Mendelian disorders. Here, we describe three pedigrees with suggestive XLID where distinctive phenotypes associated with known genes guided the identification of three different noncoding variants. We used comprehensive structural, single-nucleotide, and repeat expansion analyses of genome sequencing. RNA-Seq from patient-derived cell lines, reverse-transcription polymerase chain reactions, Western blots, and reporter gene assays were used to confirm the functional effect of three fundamentally different classes of pathogenic noncoding variants: a retrotransposon insertion, a novel intronic splice donor, and a canonical splice variant of an untranslated exon. In one family, we excluded a rare coding variant in ARX, a known XLID gene, in favor of a regulatory noncoding variant in OFD1 that correlated with the clinical phenotype. Our results underscore the value of genomic research on unresolved XLID families to aid novel, pathogenic noncoding variant discovery.