Basics of high throughput sequencing Summary

Sequencing can provide genomic characterisation of a specific organism, as well as of a whole environmental or clinical sample. High Throughput Sequencing (HTS) makes it possible to generate an enormous amount of genomic data at gradually decreasing costs and almost in real-time. HTS is used, among others, in medicine, veterinary medicine, microbiology, virology and epidemiology. The paper presents practical aspects of the HTS technology. It describes generations of sequencing, which vary in throughput, read length, accuracy and costs ̶ and thus are used for different applications. The stages of HTS, as well as their purposes and pitfalls, are presented: extraction of the genetic material, library preparation, sequencing and data processing. For success of the whole process, all stages need to follow strict quality control measurements. Choosing the right sequencing platform, proper sample and library preparation procedures, as well as adequate bioinformatic tools are crucial for high quality results.

Detection of Polioviruses in Sewage Using Cell Culture and Molecular Methods

The work presented here demonstrates the utility of a two-step algorithm for environmental poliovirus surveillance based on: preselection of sewage samples tested for the presence of enteroviral genetic material-RT-PCR assay and detection of infectious viruses by cell culture technique (L20B for polioviruses and RD for polio and other non-polio enteroviruses). RD and L20B cell lines were tested to determine their sensitivity for isolation of viruses from environmental samples (sewage). Finally, we wanted to determine if sewage concentration affects the results obtained for RT-PCR and cell cultures.

Effect of Mycoplasma synoviae strains isolated from the respiratory and reproductive tracts of chicken on SPF chicken embryos

Mycoplasma synoviae (MS) infections in poultry are an important epidemiological and economic problem in poultry production all over the world. The differences between M. synoviae strains are related to the pathogenicity and the course of the disease. In recent years, the pathogenicity of M. synoviae strains has increased, and some of them are capable of causing serious infections. Both horizontal and vertical transmission routes play an important role in MS infection in flocks. The aim of the study was to determine the impact of infection with selected MS strains obtained from chickens showing a clinical form of MS infection on SPF chicken embryos. Ten strains of M. synoviae were used for this purpose. The strains were isolated from the respiratory tract and the oviduct of chickens with symptoms typical of infection with this pathogen. Genetic material isolated from liquid cultures of these strains was confirmed by molecular (PCR and LAMP) and microbiological methods. The selected M. synoviae strains belonged to six different genotypes. Significant differences in virulence between the strains were demonstrated. In nine infected groups of embryos, M. synoviae strains caused weight loss, and in seven groups they produced anatomopathological changes characteristic of mycoplasma infections. The most pathogenic for SPF chicken embryos turned out to be strains characterized as genotype F isolated from the chicken oviduct and strains of genotype C isolated from the respiratory tract. One strain of genotype H isolated from the respiratory tract showed no pathogenic effect on SPF chicken embryos. The study showed that infections with M. synoviae can have a significant impact on the production of chicken chicks in commercial hatcheries and the economy of the poultry industry.

Nutrient variability of rice landraces (Oryza sativa L.) from Manipur, Northeast India and its nutrients supply potential in rice-based diets

Purpose Rice landraces are essential for supplying beneficial traits for developing improved rice varieties with better nutritional quality. Nevertheless, in a yield-driven environment, grain nutritional quality has been ignored especially that of rice landraces. Given this, the purpose of this study is to evaluate the content and nutritional variability of rice landraces from Manipur. Design/methodology/approach Thirty-three most popular rice landraces were collected as dry paddy samples from Manipur and transported to the National Institute of Nutrition, Hyderabad, by air. All the paddy samples were processed and analyzed for 35 nutrient parameters using standard methodologies. Findings The mean nutrient content of Nagaland brown rice was: protein 7.5 ± 0.8, fat 3.0 ± 0.3, TDF 5.5 ± 0.4 and ash 1.2 ± 0.2 g/100g. The range of water soluble-vitamin content in mg/100g, was 0.1–0.43 for Thiamine and for Niacin 2.1–3.5, while the content in µg/100g was 40–64 for Riboflavin, 0.5–3.9 for Pantothenic acid and 20–118 for Pyridoxine. A relatively large coefficient of variation was observed for iron (25%), manganese (28%), copper (32%), calcium (13%) and phosphorus (11%). Manipur rice landraces have significantly higher total dietary fiber and lower phytate contents than modern varieties. Milling led to steep losses of nutrients, and limiting to 5% milling substantially improves nutrient retention in milled rice. Research limitations/implications Future nutrition interventions should use rice with superior nutrient quality to improve nutrient intakes. Manipur rice landraces conserved over generations can broaden the genetic base of breeding stocks especially in the face of climate change. Originality/value The paper presents comprehensive nutritional data of 33 rice landraces from the state of Manipur, India. The results indicate large nutrient variability even within these 33 rice landraces with important traits such as high total dietary fiber and low phytate contents. The study highlights the importance of conserving the existing rich genetic material of Manipur rice landraces to develop varieties that combine higher yields with stress tolerance and superior grain nutritional value to improve the food and nutrient security.

Assessment of the relationship of yield and its contributing traits in wheat

Wheat is an essential source of nutrition for humans and an important cereal crop of Pakistan due to its widespread use as food. To fulfill the nutrition needs of boosted population, crop yield must be improved. In this perspective, we conducted the experiment reported in this paper to achieve correlation and path coefficient attributes given by yield-related traits. Our study enables to understand the association and contribution of nine traits toward grain production. The genetic material was comprised of seven parents and twelve F1 hybrids. Correlation analysis showed that grain yield per spike, thousand grain weight, spike length, and number of tillers per plant have positive and significant correlation with grain yield per plant, at both genotypic and phenotypic level. Path coefficient analysis revealed that direct effects of number of tillers per plant, grain yield per spike, and spike length were positive. This data persuaded that selection based on these traits could be useful for breeding purposes to higher grain yield.

Epigenetic Signatures in Antidepressant Treatment Response: a Methylome-wide Association Study in the EMC Trial

Although the currently available antidepressants are well established in the treatment of major depressive disorder (MDD), there is strong variability in the response of individual patients. Reliable predictors to guide treatment decisions before or in an early stage of treatment are needed. DNA-methylation has been proven a useful biomarker in different clinical conditions, but its importance for mechanisms of antidepressant response has not yet been determined. 80 MDD patients were selected out of >500 participants from the Early Medication Change (EMC) cohort with available genetic material based on their antidepressant response after four weeks and stratified into clear responders and age- and sex-matched non-responders (N=40, each). Early improvement after two weeks was analyzed as a secondary outcome. DNA-methylation was determined using the Illumina EPIC BeadChip. Epigenome-wide association studies were performed and differentially methylated regions (DMRs) identified using the comb-p algorithm. Enrichment was tested for hallmark gene-sets and in genome-wide association studies of depression and antidepressant response. No epigenome-wide significant differentially methylated positions were found for treatment response or early improvement. Twenty DMRs were associated with response; the strongest in an enhancer region in SORBS2, which has been related to cardiovascular diseases and type II diabetes. Another DMR was located in CYP2C18, a gene previously linked to antidepressant response. Results pointed towards differential methylation in genes associated with cardiac function, neuroticism, and depression. Linking differential methylation to antidepressant treatment response is an emerging topic and represents a step towards personalized medicine, potentially facilitating the prediction of patients’ response before treatment.

Organic dry pea (Pisum sativum L.) biofortification for better human health

A primary criticism of organic agriculture is its lower yield and nutritional quality compared to conventional systems. Nutritionally, dry pea (Pisum sativum L.) is a rich source of low digestible carbohydrates, protein, and micronutrients. This study aimed to evaluate dry pea cultivars and advanced breeding lines using on-farm field selections to inform the development of biofortified organic cultivars with increased yield and nutritional quality. A total of 44 dry pea entries were grown in two USDA-certified organic on-farm locations in South Carolina (SC), United States of America (USA) for two years. Seed yield and protein for dry pea ranged from 61 to 3833 kg ha-1 and 12.6 to 34.2 g/100 g, respectively, with low heritability estimates. Total prebiotic carbohydrate concentration ranged from 14.7 to 26.6 g/100 g. A 100-g serving of organic dry pea provides 73.5 to 133% of the recommended daily allowance (%RDA) of prebiotic carbohydrates. Heritability estimates for individual prebiotic carbohydrates ranged from 0.27 to 0.82. Organic dry peas are rich in minerals [iron (Fe): 1.9–26.2 mg/100 g; zinc (Zn): 1.1–7.5 mg/100 g] and have low to moderate concentrations of phytic acid (PA:18.8–516 mg/100 g). The significant cultivar, location, and year effects were evident for grain yield, thousand seed weight (1000-seed weight), and protein, but results for other nutritional traits varied with genotype, environment, and interactions. “AAC Carver,” “Jetset,” and “Mystique” were the best-adapted cultivars with high yield, and “CDC Striker,” “Fiddle,” and “Hampton” had the highest protein concentration. These cultivars are the best performing cultivars that should be incorporated into organic dry pea breeding programs to develop cultivars suitable for organic production. In conclusion, organic dry pea has potential as a winter cash crop in southern climates. Still, it will require selecting diverse genetic material and location sourcing to develop improved cultivars with a higher yield, disease resistance, and nutritional quality.

Herbal Medicine for the Treatmentof Viral Infections: A Systemic Review

The term “Antiviral agents” has been defined in very wide terms as substances other than a virus or virus containing vaccine or specific antibody which can build either a protective or therapeutic effect to the direct measurable advantage of the virus infected host. Viruses are simple in form which are very tiny germs. They comprise of genetic material inside of a protein coating. Viruses cause amicable infectious diseases like common cold, flu and warts. They also cause severe diseases such as HIV/AIDS, Ebola, avian influenzas, dengue virus and COVID-19. Viral diseases are very complex and are easily spread. Herbs and herbal medicines were the foremost in treating infections from centuries over the world in every civilization. Modern science has narrowed the importance of herbal medicine in the past two centuries. But, the side effects and new varieties of diseases creating challenges to modern science. So, usage of herbal medicines is again attaining interests these days. Herbal products for different treatments have achieved a lot of popularity in the last couple of decades. Thus, discovering novel antiviral drugs is of extremely important and natural products are an excellent source for such discoveries. There are many herbs which are excellent sources for the antiviral properties to treat viral infections. This review provides the verified data on the herbal substances with antiviral activity, and some of the herbal marketed antiviral agents like CORONIL TABLETS from Patanjali and different companies had made an attempt to treat viral infections in this pandemic situation. Therefore, herbal plants proved to be a major resort for the treatment of diseases and sickness by traditional healers in many societies.

Role of F-box Protein Cdc4 in Fungal Virulence and Sexual Reproduction of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic yeast-like pathogen that mainly infects immunocompromised individuals and causes fatal meningitis. Sexual reproduction can promote the exchange of genetic material between different strains of C. neoformans, which is one of the reasons leading to the emergence of highly pathogenic and drug-resistant strains of C. neoformans. Although much research has been done on the regulation mechanism of Cryptococcus sexual reproduction, there are few studies on the sexual reproduction regulation of Cryptococcus by the ubiquitin-proteasome system. This study identified an F-box protein, Cdc4, which contains a putative F-box domain and eight WD40 domains. The expression pattern analysis showed that the CDC4 gene was expressed in various developmental stages of C. neoformans, and the Cdc4 protein was localized in the nucleus of cryptococcal cells. In vitro stress responses assays showed that the CDC4 overexpression strains are sensitive to SDS and MMS but not Congo red, implying that Cdc4 may regulate the cell membrane integrity and repair of DNA damage of C. neoformans. Fungal virulence assay showed that although the cdc4Δ mutant grows normally and can produce typical virulence factors such as capsule and melanin, the cdc4Δ mutant completely loses its pathogenicity in a mouse systemic-infection model. Fungal mating assays showed that Cdc4 is also essential for fungal sexual reproduction in C. neoformans. Although normal mating hyphae were observed during mating, the basidiospores’ production was blocked in bilateral mating between cdc4Δ mutants. Fungal nuclei development assay showed that the nuclei failed to undergo meiosis after fusion inside the basidia during the bilateral mating of cdc4Δ mutants, indicating that Cdc4 is critical to regulating meiosis during cryptococcal mating. In summary, our study revealed that the F-box protein Cdc4 is critical for fungal virulence and sexual reproduction in C. neoformans.

Putative host-derived insertions in the genome of circulating SARS-CoV-2 variants

Insertions in the SARS-CoV-2 genome have the potential to drive viral evolution, but the source of the insertions is often unknown. Recent proposals have suggested that human RNAs could be a source of some insertions, but the small size of many insertions makes this difficult to confirm. Through an analysis of available direct RNA sequencing data from SARS-CoV-2 infected cells, we show that viral-host chimeric RNAs are formed through what are likely stochastic RNA-dependent RNA polymerase template switching events. Through an analysis of the publicly available GISAID SARS-CoV-2 genome collection, we then identified two genomic insertions in circulating SARS-CoV-2 variants that are identical to regions of the human 18S and 28S rRNAs. These results provide direct evidence of the formation of viral-host chimeric sequences and the integration of host genetic material into the SARS-CoV-2 genome, highlighting the potential importance of host-derived insertions in viral evolution.