In silico analysis of SARS-CoV-2 genomes: Insights from SARS encoded non-coding RNAs.

The recent pandemic caused by Severe Acute Respiratory Syndrome Coronavirus-2 has resulted in enormous deaths around the world. Clues from genomic sequences of parent and their mutants can be obtained to understand the evolving pathogenesis of this virus. Apart from the viral proteins, virus-encoded microRNAs (miRNAs) have been shown to play a vital role in regulating viral pathogenesis. Thus we sought to investigate the miRNAs encoded by SARS-CoV-2, its mutants, and the host. Here, we present the results obtained using a dual approach i.e (i) identifying host-encoded miRNAs that might regulate viral pathogenesis and (ii) identifying viral-encoded miRNAs that might regulate host cell signaling pathways and aid in viral pathogenesis. Analysis utilizing the first approach resulted in the identification of ten host-encoded miRNAs that could target the SARS, SARS-CoV-2, and its mutants. Interestingly our analysis revealed that there is a significantly higher number of host miRNAs that could target the SARS-CoV-2 genome as compared to the SARS reference genome. Results from the second approach resulted in the identification of a set of virus-encoded miRNAs which might regulate host signaling pathways. Our analysis further identified a similar "GA" rich motif in the SARS-CoV-2 and its mutant genomes that was shown to play a vital role in lung pathogenesis during severe SARS infections. In summary, we have identified human and virus-encoded miRNAs that might regulate the pathogenesis of SARS coronaviruses and describe similar non-coding RNA sequences in SARS-CoV-2 that were shown to regulate SARS-induced lung pathology in mice.

Integrated miRNA, target mRNA, and metabolome profiling of Tinospora cordifolia with reference to berberine biosynthesis.

MicroRNAs play a central role in gene regulation and emerge as novel targets for secondary metabolites improvement in plants. The crops thus can be improved through knowledge obtained by the study of miRNAs because of their conserved nature in gene regulation. The present study has been carried out on Tinospora cordifolia (T. cordifolia), because of its illimitable application for the treatment of various diseases. This plant has tremendous medicinal properties, yet unexplored at the molecular level, and has not received much recognition in the scientific field. Thus, here computational analysis was performed to identify T. cordifolia miRNAs using EST database. Using these miRNAs, we predicted their targets which were found to be associated with the regulation of diverse gene networks including 433 berberine biosynthesis genes in T. cordifolia. Further, selected miRNAs were validated and their expression was detected in different T. cordifolia tissues followed by expression analysis of their target mRNAs. These data were then compared with the metabolic profile of T. cordifolia with an emphasis on therapeutically important compound berberine. In this study, we did simultaneous miRNA/target gene expression and metabolome analysis which opens a new way for initiating new proposition and prioritization of miRNAs/genes/metabolites for targeted follow­up metabolic engineering experimentations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03342-9.

Pioneer Role of Extracellular Vesicles as Modulators of Cancer Initiation in Progression, Drug Therapy, and Vaccine Prospects.

Cancer is one of the leading diseases, causing deaths worldwide. Nearly 10 million deaths were reported in 2020 due to cancer alone. Several factors are involved in cancer progressions, such as lifestyle and genetic characteristics. According to a recent report, extracellular vesicles (EVs) are involved in cancer initiation, progression, and therapy failure. EVs can play a major role in intracellular communication, the maintenance of tissue homeostasis, and pathogenesis in several types of diseases. In a healthy person, EVs carry different cargoes, such as miRNA, lncRNA etc., to help other body functions. On the other hand, the same EV in a tumor microenvironment carries cargoes such as miRNA, lncRNA, etc., to initiate or help cancer progression at various stages. These stages may include the proliferation of cells and escape from apoptosis, angiogenesis, cell invasion, and metastasis, reprogramming energy metabolism, evasion of the immune response, and transfer of mutations. Tumor-derived EVs manipulate by altering normal functions of the body and affect the epigenetics of normal cells by limiting the genetic makeup through transferring mutations, histone modifications, etc. Tumor-derived EVs also pose therapy resistance through transferring drug efflux pumps and posing multiple drug resistances. Such EVs can also help as biomarkers for different cancer types and stages, which ultimately help with cancer diagnosis at early stages. In this review, we will shed light on EVs' role in performing normal functions of the body and their position in different hallmarks of cancer, in altering the genetics of a normal cell in a tumor microenvironment, and their role in therapy resistance, as well as the importance of EVs as diagnostic tools.

Antioxidant activity of Anethum graveolens L. essential oil constituents and their chemical analogues.

In the present study, Dill (Anethum graveolens) seed essential oil, its nonpolar and polar fractions, compounds isolated and derivatized were evaluated for their antioxidant potential using different in vitro assays. The major compounds carvone, limonene, and camphor were isolated from dill seed essential oil using column chromatography and characterized using spectroscopic techniques. Among all the tested components for antioxidant activity, carveol and perillyl alcohol were most effective (IC50 values < 0.16 mg/ml), whereas camphor was least effective (IC50 values > 10 mg/ml). All the tested compounds exhibited lower antioxidant potential than the standard. PRACTICAL APPLICATIONS: Oxidation of food products was delayed by compounds known as antioxidants. The use of synthetic antioxidant is restricted because of carcinogenicity in human servings and plant-based natural antioxidant are preferred due to safety and less toxicity. The aim of this in vitro study was to assess the antioxidant activity of the different constituents of dill seed essential oil. The present study revealed that carvone and its derivatives are potent scavengers of free radicals which might be due to the presence of unsaturated hydroxyl group. Thus, natural antioxidants are the important source of alternative medicines and natural therapy in the pharmaceutical industry.

Microfluidic-Enabled Print-to-Screen Platform for High-Throughput Screening of Combinatorial Chemotherapy.

Since the 1960s, combination chemotherapy has been widely utilized as a standard method to treat cancer. However, because of the potentially enormous number of drug candidates and combinations, conventional identification methods of the effective drug combinations are usually associated with significantly high operational costs, low throughput screening, laborious and time-consuming procedures, and ethical concerns. In this paper, we present a low-cost, high-efficiency microfluidic print-to-screen (P2S) platform, which integrates combinatorial screening with biomolecular printing for high-throughput screening of anticancer drug combinations. This P2S platform provides several distinct advantages and features, including automatic combinatorial printing, high-throughput parallel drug screening, modular disposable cartridge, and biocompatibility, which can potentially speed up the entire discovery cycle of potent drug combinations. Microfluidic impact printing utilizing plug-and-play microfluidic cartridges is experimentally characterized with controllable droplet volume and accurate positioning. Furthermore, the combinatorial print-to-screen assay is demonstrated in a proof-of-concept biological experiment which can identify the positive hits among the entire drug combination library in a parallel and rapid manner. Overall, this microfluidic print-to-screen platform offers a simple, low-cost, high-efficiency solution for high-throughput large-scale combinatorial screening and can be applicable for various emerging applications in drug cocktail discovery.

Dissipation kinetics of trifloxystrobin and tebuconazole on wheat leaves and their harvest time residues in wheat grains and soil.

Following single application of Nativo 75 WG (trifloxystrobin 25% + tebuconazole 50%) on wheat crop @ 300 and 600 g ha(-1), resulting in active application of trifloxystrobin @ 75.0 and 150.0 g a.i. ha(-1) and tebuconazole @ 150 and 300 g a.i. ha(-1), the average initial deposits of trifloxystrobin on wheat leaves were 5.54 and 8.30 mg kg(-1), and that of tebuconazole were 14.66 and 27.94 mg kg(-1), respectively. Half-life values for trifloxystrobin were observed to be 2.80 and 2.51 days whereas those for tebuconazole were found to be 2.46 and 1.85 days at recommended and double the recommended dosages, respectively. The residues of trifloxystrobin and tebuconazole dissipated to the extent of >89% at both the dosages after 7 days. Wheat grain samples at harvest having pre harvest interval of 140 days did not show the presence of trifloxystrobin and tebuconazole at their determination limit of 0.01 mg kg(-1).

Persistence of fipronil and its risk assessment on cabbage, Brassica oleracea var. capitata L.

Persistence of fipronil in cabbage was studied following three applications of Jump 80 WG at 75 and 150 g a.i. ha(-1) at 7 day interval. The average initial deposits of total fipronil (fipronil and its metabolites) were 1.226 and 2.704 mg kg(-1) on the heads following 3rd application of fipronil at single and double the dosages, respectively. Desulfinyl was found to be the main metabolite followed by sulfone and sulfide. Metabolite amide was not detected in cabbage samples. Half-life periods for fipronil were found to be 3.43 and 3.21 day at single and double the application rates, respectively. Risk assessment of fipronil to the consumers was calculated on the basis of per capita 80 g consumption of cabbage and comparing it to its ADI for an adult of 55 kg which was found to be less than its ADI on 10th day at both the dosages.

Completely self-contained cell culture system: from storage to use.

In vitro cell culture systems provide researchers the appropriate tools for effectively studying cell growth and differentiation, understanding cellular response to specific environmental stimuli, and elucidating the function of heterologous biological molecules produced from expression systems. All in vitro cell culture systems require a specific culture media formulated to the nutritional and metabolic requirements of the particular cell type to be cultured. However, the complexity of these systems varies depending on the model organism origin of the cells being cultured (e.g., bacteria, plants, yeast or animal). Unlike bacteria and yeast, mammalian cell cultures require sophisticated auxiliary technologies (e.g., controlled gas mixtures and pressure flow systems, specialized facilities and equipment) and careful handling by trained personnel. These complex requirements pose a limitation to transferring cells to and from remote field locations for investigations. Furthermore, this limitation is a technical hurdle in the development of technologies involving use of live cells (e.g., cytosensors). We identified a novel and unrealized feature in the conventional cell culture system that may be exploited to adapt simple existing technologies to form a portable apparatus for storing and growing cells. The approach we describe is a completely self-contained cell culture system that not only will bring down the cost of culturing cells but also will expand cell culture applications in medicine, research, environmental health, and safety.

Multiplex-PCR assay for the deletions causing hereditary persistence of fetal hemoglobin.

INTRODUCTION: Hereditary persistence of fetal hemoglobin (HPFH) is a benign condition caused by the failure of normal switching from the fetal to the adult beta-globin gene, resulting in continuous production of fetal hemoglobin beyond the perinatal period. To date, eight deletions of variable size and position have been reported for HPFH. Southern hybridization and PCR are the most common methods used to detect each deletion. AIM: Our aim was to develop a multiplex-PCR assay to detect these deletions in a single tube in order to facilitate rapid and accurate molecular diagnosis. METHODS AND RESULTS: This report is the first application of multiplex-gap-PCR to detect all HPFH deletions simultaneously to expedite diagnosis. The deletion breakpoints were precisely identified for each deletion and primers were designed in the unique regions across the breakpoints of HPFH-1 (Black), HPFH-2 (Ghanaian), HPFH-3 (Asian Indian), HPFH-4 (Italian), HPFH-5 (Italian), HPFH-6 (Vietnamese), HPFH-7 (Kenyan), and SEA-HPFH (Southeast Asian). As many as 16 primers were used in a single amplification reaction by adjusting the relative primer concentrations. The multiplex-PCR approach was standardized on known positive control samples. We identified unique deletion-specific products for each deletion. The results were confirmed by sequence analysis. CONCLUSIONS: We conclude that our multiplex-gap PCR strategy provides the most rapid and accurate diagnosis for the deletions in the beta-globin gene cluster causing HPFH.

Molecular genetic confirmatory testing from newborn screening samples for the common African-American, Asian Indian, Southeast Asian, and Chinese beta-thalassemia mutations.

beta-Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with beta-thalassemia. Our purpose was to develop single-tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for beta-thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic-specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for beta-thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African-American descent. Specimens from African-American neonates were tested for two mutations (-88 and -29); Asian Indians for five mutations (IVSI-1, IVSI-5, codons (Cd) 41/42, Cd 8/9, and 619-bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, -28, IVSII-654, Cd 71/72, IVSI-5, and IVSI-1). We identified each of these beta-thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with beta-thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic-specific beta-thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation.

DNA diagnosis confirms hemoglobin deletion in newborn screen follow-up.

Molecular genetic confirmatory testing with polymerase chain reaction amplification is integral to neonatal hemoglobinopathy screening programs. In this study, we demonstrate applicability of polymerase chain reaction-based testing for the common deletions in blacks responsible for hereditary persistence of fetal hemoglobin. This approach will provide rapid diagnostic clarification in newborn screening follow-up.

Rapid confirmation of Southeast Asian and Filipino alpha-thalassemia genotypes from newborn screening specimens.

Alpha(+)-thalassemia (-alpha) is prevalent throughout tropical and subtropical regions of world, whereas alpha(0)-thalassemia (--) occurs at higher frequency in Southeast Asia. Homozygosity for the alpha(0)-thalassemia deletion (--/--) is a serious health problem in Southeast Asia and the Philippines and is responsible for the majority of hydrops fetalis in this region. Asian immigration in United States has impacted the demography of genetic disease in U.S. As Asians comprise of 10% of all California births and represent a population at highest risk for alpha(0)-thalassemia, we developed a rapid and efficient PCR approach to detect the common alpha(0)-thalassemia deletions in Southeast Asians and Filipinos using newborn screening specimen.