Development of modified CTAB and Trizol protocols to isolate high molecular weight (HMW) RNA from polyphenol and polysaccharides rich pigeonpea (Cajanuscajan (L.) Millsp.

Pigeonpea (Cajanuscajan L.) is a legume crop that contains high levels of polyphenolic compounds and polysaccharides that become a hindrance in extracting good-quality and enough amount of RNA from its tissues. With the existing methods of RNA isolation, the phenolic compounds may co-precipitate or bind to the RNA giving false results. Therefore, in the present study, we have modified conventional CTAB and Trizol-based methods which resulted in good quality with the absorbance A260/A280 ratios in the range of 1.83 to 1.98 and A260/230 ratios in the range of 2.0-2.23, revealed RNA to be of high purity and free of contaminants. Both of the proposed protocols yielded a good quantity of RNA ranging from 289 to 422µg per gram of tissue. Distinctly visible bands of 28S and 18S rRNA were observed without degradation or smear, which indicated the presence of intact RNA. RT-PCR analysis showed that isolated RNA was quantitatively sufficient and compliant for the subsequent gene expression analysis.

Identification, characterization, and comprehensive expression profiling of floral master regulators in pigeon pea (Cajanus cajan [L.] Millspaugh).

Pigeon pea is an important protein-rich pulse crop. Identification of flowering master regulators in pigeon pea is highly imperative as indeterminacy and late flowering are impediments towards yield improvement. A genome-wide analysis was performed to explore flowering orthologous groups in pigeon pea. Among the 412 floral orthologs identified in pigeon pea, 148 genes belong to the meristem identity, photoperiod-responsive, and circadian clock-associated ortholog groups. Our comparative genomics study revealed purifying selection pressures (ka/ks) on floral orthologs, and duplication patterns and evolution through synteny with other model species. Phylogenetic analysis of floral genes substantiated a connection between pigeon pea plant architecture and flowering time as all the PEBP domain-containing genes belong to meristem identity floral networks of pigeon pea. Expression profiling of eleven major orthologs in contrasting determinate and indeterminate genotypes indicated that these orthologs might be involved in flowering regulation. Expression of floral inducer, FT, and floral repressor, TFL1, was non-comparable in indeterminate genotypes across all the developmental stages of pigeon pea. However, dynamic FT/TFL1 expression ratio detected in all tissues of both the genotypes suggested their role in floral transition. One TFL1 ortholog having high sequence conserveness across pigeon pea genotypes showed differential expression indicating genotype-dependent regulation of this ortholog. Presence of conserved 6mA-methylation patterns in light-responsive elements and in other cis-regulatory elements of FT and TFL1 across different plant genotypes indicated possible involvement of epigenetic regulation in flowering.

First report of milkweed (Euphorbia geniculata) as an alternative host for Colletotrichum truncatum in soybean fields in India.

Soybean (Glycine max, L.), a major oilseed crop of India faces anthracnose disease caused by Colletotrichum truncatum (Nataraj et al. 2021). Several weeds serve as alternative hosts for Colletotrichum spp. (Hartman et al. 1986). Around 24.67% of soybean fields in the study area were infested with Euphorbia geniculata (Kutariye et al. 2021). In September 2021, milkweed plants died in the field, showing irregular circular lesions with wavy margins on the stem, change in color of veins and veinlets from brown to black and leaves exhibiting a twisted appearance at ICAR-Indian Institute of Soybean Research, India. Later on plants completely died and acervuli of average size 284 µm were visualized under stereo microscopy. Twenty milkweed samples were collected, rinsed, and surface sterilized with NaOCl (1%). Fungus isolation was done from leaf and stem and transferred to sterilized Petri plates with Potato dextrose agar (PDA). The plates were incubated at 25 ± 2°C for 48 h with dark/light (10h/14h) cycle. The fungi produced circular, raised, black to light grey colonies. Sickle shaped aseptate conidia, measuring 23.14 µm length, 3.18 µm width and hyphal width 5.49 µm were confirmed using a compound microscope with 20X magnification. The fungus was purified via hyphal tip method and pure culture was maintained on PDA at (26 ± 2°C). Milkweed seedlings in clay pots were inoculated with a conidial suspension of the fungus (106 conidia/mL) prepared from ten days old culture using serial dilution technique. Soybean variety JS 95-60 was inoculated by atomizing 20 ml of the same suspension on each plant. The negative controls for both milkweed and soybean were inoculated with sterile distilled water. Veinal necrosis and acervuli formation were observed on both milkweed and soybean, but no signs or symptoms of disease were observed in the controls. The re-isolated fungus from both the diseased hosts resembled original culture as they produced black to light grey colonies, sickle shaped aseptate conidia and ITS sequence (OR124845) exhibiting 100% resemblance to C. truncatum isolate C-17 (MN736513), thus confirming Koch's postulates. The pathogen was classified as Colletotrichum spp. based on morphological and cultural characters and the pathogenicity test (Rajput et al. 2021). To confirm identity of the pathogen infecting milkweed, DNA was extracted from the reisolated fungus using the HiPurA Fungal DNA Purification Kit (HiMedia, India). The internal transcribed spacer (ITS) region, beta-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified (Kumar et al. 2021). The GAPDH gene was amplified under similar reaction conditions except for annealing temp 59°C. For species level identification, the ITS, TUB2 and GAPDH gene sequences were submitted to GenBank with accession numbers OR004468, OQ869780 and OQ869781, respectively. The BLAST analysis of TUB2 and GAPDH gene showed sequence homology of 100% and 98.43% respectively with C. truncatum culture-collection CBS:151.35 (GU228156, GU228254). The isolate was identified as C. truncatum on the basis of molecular analysis, corroborating the above morphological identification. This is the first report of C. truncatum infecting milkweed in India, indicating milkweed as an alternative host in soybean fields, potentially raising inoculum levels and carryover between crops.

Host-Delivered RNA Interference for Durable Pest Resistance in Plants: Advanced Methods, Challenges, and Applications.

Insect-pests infestation greatly affects global agricultural production and is projected to become more severe in upcoming years. There is concern about pesticide application being ineffective due to insect resistance and environmental toxicity. Reduced effectiveness of Bt toxins also made the scientific community shift toward alternative strategies to control devastating agricultural pests. With the advent of host-delivered RNA interference, also known as host-induced gene silencing, targeted insect genes have been suppressed through genetic engineering tools to deliver a novel insect-pest resistance strategy for combating a number of agricultural pests. This review recapitulates the possible mechanism of host-delivered RNA interference (HD-RNAi), in particular, the silencing of target genes of insect-pests. We emphasize the development of the latest strategies against evolving insect targets including designing of artificial microRNAs, vector constructs, and the benefit of using plastid transformation to transform target RNA-interfering genes. Advantages of using HD-RNAi over other small RNA delivery modes and also the supremacy of HD-RNAi over the CRISPR-Cas system particularly for insect resistance have been described. However, the broader application of this technology is restricted due to its several limitations. Using artificial miRNA designs, the host-delivered RNAi + Bt combinatorial approach and chloroplast transformation can overcome limitations of RNAi. With careful design and delivery approaches, RNAi promises to be extremely valuable and effective plant protection strategy to attain durable insect-pest resistance in crops. Development of transgenic plant using novel strategies to achieve durable resistance against the target insect.

Amelioration of cold-induced sweetening in potato by RNAi mediated silencing of StUGPase encoding UDP-glucose pyrophosphorylase.

Cold-induced sweetening (CIS) is an unwanted physiological phenomenon in which reducing sugars (RS) get accumulated in potato (Solanum tuberosum) upon cold storage. High RS content makes potato commercially unsuitable for processing due to the unacceptable brown color in processed products like chips, fries, etc., and the production of a potential carcinogen, acrylamide. UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose towards the synthesis of sucrose and is also involved in the regulation of CIS in potato. The objective of the present work was RNAi-mediated downregulation of the StUGPase expression level in potato for the development of CIS tolerant potato. Hairpin RNA (hpRNA) gene construct was developed by placing UGPase cDNA fragment in sense and antisense orientation intervened by GBSS intron. Internodal stem explants (cv. Kufri Chipsona-4) were transformed with hpRNA gene construct, and 22 transgenic lines were obtained by PCR screening of putative transformants. Four transgenic lines showed the highest level of RS content reduction following 30 days of cold storage, with reductions in sucrose and RS (glucose & fructose) levels of up to 46% and 57.5%, respectively. Cold stored transgenic potato of these four lines produced acceptable chip colour upon processing. The selected transgenic lines carried two to five copies of the transgene. Northern hybridization revealed an accumulation of siRNA with a concomitant decrease in the StUGPase transcript level in these selected transgenic lines. The present work demonstrates the efficacy of StUGPase silencing in controlling CIS in potato, and the strategy can be employed for the development of CIS tolerant potato varieties.

Probiotic and Functional Characterization of Pediococcus acidilactici Isolated from Bhaati jaanr, Traditional Fermented Rice Porridge.

Traditional fermented foods are the ideal source of novel probiotic isolates which are known to have significant therapeutic benefits and play a vital role as bioprotective agents. Bhaati jaanr is an ethnic fermented rice beverage popularly consumed in sub-Himalayan regions. The strain UAMS was isolated from Bhaati jaanr based on high butyrate production and evaluated for the potential probiotic characteristics. MALDI-TOF MS and 16 s rRNA gene sequencing revealed the identity of strains as Pediococcus acidilactici. The isolated strain exhibited high tolerance to gastric and bile stress, autoaggregation, hydrophobicity, and adherence to colon cells. Antibiotic susceptibility testing results showed the resistance of the isolated strain toward tested common antibiotics and the pathogenic determinants were absent in PCR-based detection. Moreover, the organism was able to inhibit the growth of Listeria, Salmonella, Staphylococcus, and Enterococcus species. The isolate was found to be a high butyrate producer along with other short-chain fatty acids and exhibited an anti-proliferative effect against colon cancer cells HT29 and SW480. Therefore, our study represents Pediococcus acidilactici UAMS as a potent putative probiotic with bioprotective abilities.

A comprehensive survey on the biomedical signal processing methods for the detection of COVID-19.

The novel coronavirus, renamed SARS-CoV-2 and most commonly referred to as COVID-19, has infected nearly 44.83 million people in 224 countries and has been designated SARS-CoV-2. In this study, we used 'web of Science', 'Scopus' and 'goggle scholar' with the keywords of "SARS-CoV-2 detection" or "coronavirus 2019 detection" or "COVID 2019 detection" or "COVID 19 detection" "corona virus techniques for detection of COVID-19", "audio techniques for detection of COVID-19", "speech techniques for detection of COVID-19", for period of 2019-2021. Some COVID-19 instances have an impact on speech production, which suggests that researchers should look for signs of disease detection in speech utilising audio and speech recognition signals from humans to better understand the condition. It is presented in this review that an overview of human audio signals is presented using an AI (Artificial Intelligence) model to diagnose, spread awareness, and monitor COVID-19, employing bio and non-obtrusive signals that communicated human speech and non-speech audio information is presented. Development of accurate and rapid screening techniques that permit testing at a reasonable cost is critical in the current COVID-19 pandemic crisis, according to the World Health Organization. In this context, certain existing investigations have shown potential in the detection of COVID 19 diagnostic signals from relevant auditory noises, which is a promising development. According to authors, it is not a single "perfect" COVID-19 test that is required, but rather a combination of rapid and affordable tests, non-clinic pre-screening tools, and tools from a variety of supply chains and technologies that will allow us to safely return to our normal lives while we await the completion of the hassle free COVID-19 vaccination process for all ages. This review was able to gather information on biomedical signal processing in the detection of speech, coughing sounds, and breathing signals for the purpose of diagnosing and screening the COVID-19 virus.

Elucidation of immunological response and its regulatory network by P-TUFT-ALT-2: a promising fusion protein vaccine for human lymphatic filariasis.

Human lymphatic filariasis, a mosquito-borne neglected tropical parasitic disease, needs an early development of prophylactic agents such as a vaccine for its successful elimination. Our earlier study suggested the enhanced immunological response by fusion protein (P-TUFT-ALT-2) of Tuftsin and ALT-2 in a mice model. We cultured human peripheral blood mononuclear cells (PBMCs) and treated cells with Escherichia coli-expressed ALT-2 (E-ALT-2) and P-TUFT-ALT-2. Real-time polymerase chain reaction was performed to identify the mRNA copy number of various cytokine and transcription factor genes. The recombinant vaccine candidate was also validated for humans by immunoreactivity with human sera samples of natural infection. In this study, P-TUFT-ALT-2 stimulated 12% higher PBMC proliferation in endemic normal (EN) individuals than E-ALT-2 alone. There was enhanced production of IFN γ, IL-2, IL-5 and IL-12, indicating a balanced Th1/Th2 response. However, higher expression of IL-5 and lower IL-4 validate the humoral response through an IL-5-dependent manner. Also, high level of IL-17 indicates a strong Th/Treg regulation over T-cell activation. The upregulated T-bet might have enhanced IFN-γ production, whereas GATA-3 was supposed to enhance IL-5 expression. The fusion protein also exhibited 15-16% higher reactivity with EN clinical sera, exposing the upregulation of IgG1 and IgM in natural infection. The higher reactivity of P-TUFT-ALT-2 with sera of natural infection (EN) was validated indirectly by B-cell activation through various cytokines and regulatory genes produced from different T cells. Thus, these findings endorse P-TUFT-ALT-2 as a potential vaccine candidate for human lymphatic filariasis.