Investigation of Analgesic, Anti-Inflammatory, Hypoglycaemic, Neuropharmacological and Cytotoxic Properties of Clerodendrum viscosum (Leaves)

Clerodendrum viscosum (CV) has been used traditionally to treat medical problems like asthma, ulcer, inflammation, pyrexia, diabetes, malaria, skin diseases, snakebite and tumor by folk practitioners. The present study evaluated the analgesic, antiinflammatory, neuropharmacological and cytotoxic properties C. viscosum (leaves) in rodents. Swiss albino mice of either sex weighing 25-30 gm and SD rats (150-180 mg) were divided into control (DW), standard (model specific) as well as test groups (n=6). Analgesic potential was evaluated using acetic acid-induced writhing and formalin induced pawlicking test. Anti-inflammatory properties were evaluated by xylene and croton oil induced ear edema test. Glucose tolerance was evaluated by OGTT in normal rats. Pentobarbital induced sleeping time test was applied to assess neuropharmacological activity. Also, Brine shrimp lethality bioassay method was employed for cytotoxicity evaluation. The alcoholic extracts showed significant antinociceptive activity in acetic acid test (p<0.01) and formalin test (p<0.05) at the dose of 1000mg/kg bw. The crude extract reduced inflammation significantly (p<0.01) in both xylene and croton oil induced ear edema test. At the dose of 1000mg/kg it increased glucose tolerance significantly (p<0.05) in normal rats. CV extract significantly (p<0.01) increased sleeping time indicating CNS depressant effect. The extract exhibited a potent cytotoxicity against brine shrimp (LC50=316.23μg/ml). C. viscosum leaves showed analgesic, antiinflammatory, hypoglycemic and CNS depressant effect against experimentally induced model mice. It also possessed cytotoxic properties and further studies are required to evaluate these effects and the potential of the plant.

microRNA Regulation of Nodule Zone-Specific Gene Expression in Soybean

Nitrogen is a paramount important essential element for all living organisms. It has been found to be a crucial structural component of proteins, nucleic acids, enzymes and other cellular constituents, which are inevitable for all forms of life. In the atmosphere, the percentage of nitrogen is very high (N2, 78%) compared to other inorganic gases. However, most organisms have practically no direct access to this nitrogen. While plants cannot directly uptake nitrogen from atmosphere, they are capable of assimilating other forms of nitrogen for example ammonium (NH4+) and nitrate (NO3-). For agricultural crop production, artificial fixation of nitrogen is heavily utilized and it is an expensive process that requires high temperatures (at least 400°C) and pressures (around 200atm). It has been conspicuously demonstrated that indiscriminate use of fertilizer hampers soil physical, chemical and micro biological properties and a potential risk to environment e.g. water quality. Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching. Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, only 1-2% of the nitrogen fixation in the world occurs through the natural process of lightening. Notably, microbial fixation is well characterized in diazotrophs for example: Rhizobia and Frankia, and blue-green algae. Against the backdrop, we are accentuated on an environmentally friendly and the most sustainable approach to increase productivity for legume and non-legume crops. Till today, the term Biological Nitrogen Fixation (BNF) has received much attention as a sustainable alternative; this process facilitates atmospheric nitrogen to convert into ammonia by rhizobia in specialized plan organs termed “root nodules”. This review article seeks to better understand plant mechanisms involved in the development of root nodules in soybean.

Recurrence Possibility of COVID-19 in India

Although nationwide lockdown was imposed in India amid COVID-19 outbreak since March 24, 2020, the COVID-19 infection is increasing day-by-day. India became world’s second most affected country. By 13th May, 2021 India recorded 23,703,665 cases with 258,351 deaths and 19,734,823 recovered cases. Here, we described the possibility of COVID-19 reactivation and disease recurrence through horizontal transmission in individuals after recovery from COVID-19 infection, in particularly in India. Additionally, the study will demonstrate how COVID-19 reactivation/reinfection could play a dominant role in disease burden. WHO (World Health Organization) recommended two times RT-PCR swabs test in discharged patients after clinical recovery from COVID-19 infection to confirm the disease recurrence globally. As per global data, the virus changing the molecular configuration and fluctuating time-by-time mainly because of viral load, occurrence of false-negative results at molecular test, inefficiency of sampling operator, even sampling procedure etc [1]. Recently, Ye et al. [2] mentioned that about 9% reactivation in COVID-19 patients occurred after discharge from the hospital. Further, they demonstrated host status, virologic features and steroid-induced immunosuppression as prime risk factor for the incidence of COVID-19 reactivation. Lombardi et al. [3] recommended domicile quarantine of 14 days after hospital discharge for safety purposes, but clear information about infectiousness time and virus shedding duration is still lacking. Rothe et al. [4] demonstrated that both pre-symptomatic and asymptomatic carriers might be responsible for COVID-19 reactivation, while Chen et al. [1] reported that convalescent might transmit the virus for further re-infection. Therefore, further investigations are needed to define appropriate quarantine period, to avoid transmission. Recently, COVID-19 reinfection among COVID-19 patients has dazed scientific community, but uncertainty exists whether this second wave is due to reinfection or new virus strain. Till now 64 cases of reinfection has been reported globally in individuals, recovered from COVID-19 with an estimate ranging from 7.35 to 21.4% [5]. Immunological analysis in this regard plays an important role to determine viral reinfection properly as traditional diagnostic methods like RT-qPCR, high through put sequencing, CT scan; blood sample analysis has some limitations. Additionally, different swab sample source, improper sampling, and variable sensitivity/specificity of nucleic acid tests can lead to false negative results implying disease persistence rather than recurrence. Generally, immunoglobulins alone are not enough for long-term immunity. Evidences showed that virus specific CD4+ T cells and CD8+ T cells plays a crucial role in long-term COVID-19 reactivation due to their persistency up to 6 years after SARS-CoV-1 virus infection [6]. Accordingly, Zhang et al. [7] observed lower concentrations of monocyte ACE2 (Angiotensin Converting Enzyme 2) in COVID-19 patients than healthy individuals, which necessitate further study to distinguish between reinfection and new infection. Further, the COVID-19 reactivation duration after first infection may vary between different virus clades of SARS-CoV-2 virus (e.g., A2a, B1), resulting distinct virulence as well. Accordingly, the nature of protective Neutralizing Antibodies (NAbs) varies for different strains and this indicated that NAbs of primary infection are not able to protect re-infection by other strains, resulting lower concentrations of NAbs during reinfection [6]. More recently, virus latency period is considered as potential factor to determine virus reactivation. Wang et al. [8] observed viral latency period 24 days for reinfection. In another study, Ye et al. [2] reported maximum latency period 17 days among 5 patients, but reactivation characteristics were not properly demarcated. They opined that virus is getting reactivated from a latent stage to a lytic stage with similar symptomatic manifestations. Additionally, SARSCoV- 2 virus can survive and replicate in neuronal cell lines [9], which indicated that there is possibility of reactivation through neuroinvasion of virus at later stage. In India, till now, three re-infection cases, one in Ahmedabad and two in Mumbai were brought under ICMR scanner, ICMR Director General Prof. Balram Bhargava said. Tillett et al. [10] demonstrated that individuals recovered from SARSCoV- 2 may not guarantee future immunity and second infection, if happens, was more severe with higher clinical symptoms than the first attack, the report says [10]. Whether the criteria to define a re-infection case is 90 days or 100 days, WHO is still not decided yet the cut-off point, says ICMR Director General Prof. Balram Bharagava. Further, Prof. Balram Bharagava demonstrated that India is considering cut-off about 100 day. But, till now ICMR did not revealed any data regarding those re-infected persons. Viral shedding is another potentially undetermined factor, which might cause reactivation or disease transmission from an apparently recovered individual or asymptomatic individual to healthy people [11]. Generally, viral shedding begins 2-3 days before symptoms appearance and it happens through non-respiratory or non-classical tract routes such as fecal-oral route, tears and conjunctival secretions etc. Virus remains unrecognized in all these non-respiratory or nonclassical routes during patient’s discharge, who are tested negative (nasopharyngeal RT-qPCR). But there is possibility of containing highly active viral titers in non-classical transmission sites of recovered patients, indicating that they not only reactivate themselves but also have capability to spread further infection. Accordingly, India should adopt more stringent public health emergency strategy along with exiting practices on urgent basis. Consequently, further research is also required simultaneously to fight the recurrence of COVID-19 symptoms in India. In particularly, infected persons should strictly be discharged after two consecutive RT-qPCR negative results of swab samples from various sources. Further, if possible, they should be monitored during post-discharge domiciliary quarantine period for 14 days followed by one additional RT-qPCR again. Finally, more precious policy should be adopted to stop the further spreading of COVID-19 infection in human population and need regularized monitoring of positive number of COVID-19 cases and death. In conclusion, considering the abovementioned facts, still there remains some unanswered questions: Are asymptomatic individuals more likely to spread the disease? Is proper treatment removes the virus completely from the system? Are the patients gained immunity for rest of their life? To address these facts, need immediate further study to prevent the COVID-19 infection vis-a-vis its reactivation.

First Report of Alternaria alternata Causing Leaf Spot on Beta vulgaris in North Dakota, USA

Sugar beet is an economically important crop which is contributing 55% of the total sugar in the USA. In June 2018, irregular dark brown somewhat circular spots were observed on sugar beet leaves in Hickson, North Dakota. The symptoms covered approximately 5% on the lower leaves. Symptomatic leaf tissue were excised from the junction of diseased and healthy tissue. Small pieces (5 mm²) were surface sterilized with 10% sodium hypochlorite for 1 min, rinsed thrice with sterile distilled water, air dried and transferred to Potato Dextrose Agar (PDA), and incubated at 24°C with a 12-h photoperiod for 5 days. Dark-green velvety colony appeared in two weeks. Three isolates were developed by the single spore isolation technique. Conidia were club-shaped, two to four transverse septa, and pale brown, without any beak, often in chains (4 to 8 conidia) and or solitary. The dimension of conidia varied from 25-40x7-14 μm [1,2]. Based on the morphological characters, the fungus was tentatively identified as Alternaria species. Genomic Deoxyribonucleic Acids (gDNAs) were extracted from the culture generated from a single spore using Qiagen kit. ITS4/ITS5 were used to amplify the fragments of the Internal Transcribed Spacer (ITS) region. The amplified PCR products were cleaned and sent for Sanger sequencing by GenScript (GenScript, Piscataway, NJ). The sequences from GenScript were congruence to the reference sequence ID MT126620.1. The entire sequences were deposited at NCBI (GenBank accession nos. MK441717). Koch postulates were followed by spraying conidia suspension (5×105 conidia/ml) to 8-week age of 20 plants of and kept in humidity chamber at 28-30 °C, 80-85 % RH. Mock-inoculated seeds were also sown as a control. Three weeks of post inoculation, the similar irregular dark brown symptoms observed in twelve plants. No symptoms were found in the mock. The experiment was conducted twice. The fungus was reisolated from the diseased leaf tissue, as described above. Macroscopic and microscopic analysis indicated the similar dark-green colony and morphology, respectively. Molecular detection performed using the same ITS primers and sent for Sanger sequencing by GenScript, this study further confirmed that the isolate was similar to A. alternata [3]. Another close species of Alternaria was recently reported in sugar beet to cause leaf spot in Minnesota [4,5]. To our best knowledge, this is the first report of A. alternata causing leaf spot on sugar beet in North Dakota.

First Report of Globisporangium ultimum Causing Constriction on Hypocotyl of Beta vulgaris in North Dakota, USA

Sugar beet (Beta vulgaris) sowing to post-harvest is continuously encountered with wide range of soil-borne pathogens including Rhizoctonia, Fusarium, Aphanomyces, Rhizopus, Pythium, Talaromyces, Clonostachys, and Geotrichum. These evident to cause substantial qualitative and quantitative losses. In May/2019, dark brown to black irregular water-soaked lesions were observed near the soil-line (constriction of hypocotyl) on sugar beet seedlings in Prosper (46.9630° N, 97.0198° W), North Dakota (Figure 1). Approximately 2-5% of disease incidence was recorded. Small pieces (10 mm²) of symptomatic root tissues were cut from the margins between infected and healthy tissue, and disinfected with 10% sodium hypochlorite for 1 min, rinsed three times with autoclaved water, and then placed on Corn Meal Agar (CMA) amended with pimaricin-vancomycin- Pentachloronitrobenzene (PCNB) [1] for incubation at 20°C in the dark for 5 days. Subsequently, single hyphal tips were transferred to Water Ager (WA) (Figure 2). In total, 8 isolates were developed from diseased beets. Isolates were developed light fungal biomass on WA in 14 days. Microscopically, hyphal swelling and globular or lemon shaped sporangia were present (Figure 3). Based on macroscopic and microscopic characteristics, the fungus was speculated to be Oomycetes genus [2]. Determining the species, genomic DNAs of 8-isolates were used for Polymerase Chain Reaction (PCR) using ITS- 6 and ITS-7 primer. PCR amplicons were cleaned with E.Z.N.A ®Cycle Pure Kit, OMEGA, and 8 samples were sent for Sanger sequencing to GenScript (Piscataway, NJ) [3]. The sequences were identical, and standard nucleotide blast search showed 99% sequence identity to Globisporangium ultimum (Genbank accessions: MK794771.1). The nucleotide sequence was deposited in Genbank (MN086365.1). Phyto-pathogenicity testing was performed with 2-week old sugar beet susceptible cultivar which was inoculated with a 2-week old WA culture (5 mm² mycelial cut). This plug/mycelial cut was placed at a 1/2-inch depth close to seedlings in each plastic pot (27 x 13 x 13 cm, T.O. Plastics, Inc.; Clearwater, MN, USA) which was filled with vermiculite and perlite mixer (PRO-MIX FLX) amended with osmocote (N-P-K:15-9-12) fertilizer (Scotts Company; Marysville, OH). The pots were arranged in completely randomized design. The study was done twice with four replicates and 10 plants per replicate. One week of post-inoculation, 40% of the inoculated plants developed similar damping-off symptoms as discussed above (Figure 4), whereas mock-inoculated plants were healthy. Pathogen re-isolated from the infected hypocotyl tissue and was further confirmed morphologically and molecularly, fulfilled Koch’s postulates. Globisporangium species complex were previously reported to cause damping-off on aleppo pine (Pinus halepensis Mill) in Australia, Africa, Mediterranean, rootcrown rot of pepper (Capsicum annuum) in Turkey and alfalfa [4- 7]. To our best knowledge, this is the first report of Globisporangium ultimum causing constriction on hypocotyl of sugar beet seedlings in North Dakota, USA.