Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant.

Fusarium wilt, caused by the fungus Fusarium buharicum, is an emerging disease of okra in Japan. The disease was first reported in Japan in 2015, causing significant damage to okra seedlings. Due to the potential threat in okra cultivation, the development of an accurate detection method for F. buharicum is needed for the surveillance and management of the disease. In this study, we designed a primer set and developed conventional and nested PCR assays for the specific detection of F. buharicum in infected okra plants and contaminated soil, respectively. We compared the diversity of the translation elongation factor 1 alpha (EF-1α) gene of F. buharicum with 103 other fungal species/isolates to design a species-specific primer. This primer pair successfully amplified approximately 400 bp of PCR product that was only detected in the F. buharicum isolate, not in the other fungal isolates. The developed nested PCR method was highly sensitive and could detect the fungus from a 0.01 fg DNA sample. The primer successfully detected the pathogen in artificially infected plants and soil by conventional and nested PCR, respectively. This is the first report of the development of the F. buharicum-specific primer set and detection assays, which can be used for the specific and sensitive detection of F. buharicum in field samples and for taking early control measures.

Zinc and Boron Soil Applications Affect Athelia rolfsii Stress Response in Sugar Beet (Beta vulgaris L.) Plants.

Generation of reactive oxygen species (ROS) constitutes an initial defense approach in plants during pathogen infection. Here, the effects of the two micronutrients, namely, zinc (Zn) and boron (B), on enzymatic and non-enzymatic antioxidant properties, as well as malondialdehyde (MDA) contents in leaves and roots challenged with Athelia rolfsii, which cause root rot disease, were investigated. The findings revealed that Zn and B application to the potting soil alleviated the adverse effect of A. rolfsii on sugar beet plants and increased the chlorophyll content in leaves. The increased enzymatic antioxidant activities such as catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), and non-enzymatic antioxidants such as ascorbic acid (AsA) were observed in Zn applied plants compared to both uninoculated and inoculated control plants. A significant rise in CAT activity was noted in both leaves (335.1%) and roots (264.82%) due to the Zn2B1.5 + Ar treatment, in comparison to the inoculated control plants. On the other hand, B did not enhance the activity of any one of them except AsA. Meanwhile, A. rolfsii infection led to the increased accumulation of MDA content both in the leaves and roots of sugar beet plants. Interestingly, reduced MDA content was recorded in leaves and roots treated with both Zn and B. The results of this study demonstrate that both Zn and B played a vital role in A. rofsii tolerance in sugar beet, while Zn enhances antioxidant enzyme activities, B appeared to have a less pronounced effect on modulating the antioxidant system to alleviate the adverse effect of A. rolfsii.

Morpho-molecular, cultural and pathological characterization of Athelia rolfsii causing southern blight disease on common bean.

Common bean (Phaseolus vulgaris), is a winter legume crop in Bangladesh and is considered an important vegetable with export potential. However, the production of common bean is severely affected by a newly reported soilborne fungal pathogen, Athelia rolfsii. This study aimed to characterize this new pathogen by morphological, molecular, cultural, and pathological analyses and determine the host range. The disease incidence in the affected field ranged between 6 and 13%. Initial disease symptoms were observed as brown sunken lesions at the point of infection and development of mycelia, followed by yellowing and quick wilting of the whole plant. A total of 10 fungal isolates were recovered from the infected plant samples, which were morphologically similar and produced white to brown mycelia and numerous brown sclerotia on the PDA medium. Two of them viz. BTCBSr3 and BTCBSr4 were used for the detailed study. Based on morphology and phylogenetic analyses of the sequenced data of internal transcribed spacer (ITS) and translation elongation factor 1 alpha (EF-1α), the pathogen was identified as A. rolfsii. Mycelial growth rate (3.6 cm/day) and fresh weight (107 mg) were higher in the PDA medium, whereas the number of sclerotia production (328/plate) was higher in OMA media. The isolates could grow in a wider range of incubation temperatures (15-35 °C) and media pH (3-9). In the cross-inoculation assay, both isolates were pathogenic on tomato, brinjal, and chickpea, but not on chili, soybean, and cowpea. This study has laid a foundation for further pathological research on the fungus in aid to develop an effective management practice against the pathogen.

Individualized Homeopathic Medicines in the Treatment of Tinea Corporis: Double-Blind, Randomized, Placebo-Controlled Trial.

INTRODUCTION: Tinea corporis (TC; ringworm or dermatophytosis) is a superficial skin infection caused by Microsporum, Epidermophyton and Trichophyton genera of dermatophytes. We compared the effects of individualized homeopathic medicines (IHMs) in fifty-millesimal (LM) potencies against placebo in TC. METHODS: A double-blind, randomized, placebo-controlled, two parallel arms trial was conducted on 62 individuals suffering from TC at the National Institute of Homoeopathy, India. Participants were randomized in a 1:1 ratio to receive either IHMs in LM potencies or identical-looking placebos for a period of 3 months. The primary outcome measure was the number of participants showing complete disappearance of skin lesions after 3 months. Secondary outcomes were a numeric rating scale (NRS) measuring intensity of itching and the Skindex-29 questionnaire (overall, and three sub-scales - degree of symptoms, psychological functioning, emotional status). All were assessed at baseline and every month, up to 3 months. The intention-to-treat sample was analyzed to detect inter-group differences using two-way repeated measures analysis of variance after adjusting for baseline differences. RESULTS: The primary outcome revealed no improvement in either of the groups (χ 2 = 0.012, p = 0.999). Inter-group differences in some of the secondary outcomes favored IHMs against placebo - itching NRS (mean group difference after 3 months: -0.7 (95% confidence interval [CI], -1.1 to -0.4; p = 0.001); Skindex-29 overall (mean group difference after 3 months: 3.2 [95% CI, -0.6 to 7.0; p = 0.009]); Skindex-29 degree of symptoms (mean group difference after 3 months: 0.9 [95% CI, -0.2 to 1.9; p = 0.007]); and Skindex-29 psychological functioning (mean group difference after 3 months: 1.7 [95% CI, 0-3.4; p = 0.002]). CONCLUSION: Results were negative on the primary outcome; however, secondary outcomes included some statistically significant results favoring IHMs against placebo after 3 months. TRIAL REGISTRATION: CTRI/2019/11/021999; UTN: U1111-1242-0070.

Individualized Homeopathic Medicines in Treatment of Vitiligo: Double-Blind, Randomized, Placebo-Controlled Pilot Trial.

Objective: The feasibility of a definitive trial was tested to evaluate individualized homeopathic medicines (IHMs) for the treatment of vitiligo. Design: This was a double-blind randomized (1:1) placebo-controlled pilot trial conducted at the National Institute Homeopathy, India. Sixty patients with vitiligo were included in the study. Interventions: IHMs and identical-looking placebos at 50-millesimal (LM) potencies. Outcome measures: Feasibility issues and scores from the Vitiligo Area Scoring Index (VASI), Vitiligo-specific Quality-of-life instrument (VitiQoL), and Dermatology Life Quality Index (DLQI) were measured at baseline and after 3 and 6 months. Results: The recruitment and retention rates were satisfactory. Mean reductions in the outcome measures were higher in the IHM group than placebo. Conclusions: Definitive efficacy trials are warranted. Clinical Trials Registry-India: CTRI/2018/10/016160; secondary identifier UTN: U1111-1221-7704.

Safe drinking water and toilet facility in public places in India: What we need to do!

There is abundant literature on domestic safe water supply and hygienic sanitation. Yet, there is a paucity of research studies on adequate supply of safe drinking water and toilet facilities in places of population movement and congregation in India. This study stresses on the importance of availability of safe water and sanitation facilities in all the places of human congregation and movement in India. 49 research studies were identified from 169 potentially relevant publications. Studies were selected: first, all protocols of water and toilet facilities among published literature were meticulously searched. Second, information sources on sanitation facilities in public life, viz., railways, roadways, waterways, market places and shopping complexes, schools, and other higher educational institutions, fairs and festivals, entertainment establishments, healthcare facilities, were explored from publications of various resources of different levels. Third, published reports from apex bodies of national and international importance like Indian Council of Medical Research, World Health Organization (WHO), UNICEF, USAID, etc. were given due weightage. Of late, the concept of cleanliness and making communities free from open defecation are in limelight as the Government of India has taken up Swachh Bharat Mission (SBM); even then, contextual gaps remain. This is the cause for concern as both safe water supply and basic sanitation are directly linked to health promotion of the community. Further, this research group have noted that due importance has not been integrated in the planning of SBM regarding availability of safe water and scientific sanitation facilities in all the places of human movement and congregation. Hence, there is more need for creating awareness among general population as well as stakeholders regarding this aspect of cleanliness. It is noted that despite considerable improvement in safe water and sanitation facilities in domestic life, there is considerable population left, who are still lacking access to these facilities in public space.

Oil induces chlorophyll deficient propagules in mangroves.

In Australia, some trees of the mangrove, Avicennia marina, growing in a chronic oil polluted site, produce chlorophyll deficient (albino) propagules. We tested the hypothesis that albinism was due to an oil-induced mutant allele that controls photosynthesis. We determined whether there are genetic differences between normal and chlorophyll deficient propagules. Four gene regions (nuclear 18S-26S cistron; chloroplast - trnH-psbA, rsp16 and matK) were sequenced and analysed for normal and albino propagules. Mutations occurred in both nuclear (ITS) and coding chloroplast (matK) genes of albino propagules. There were 10 mutational differences between normal and albino propagules in the matK samples. Analysis of molecular variation (AMOVA) of the matK dataset indicated highly significant genetic differentiation between normal and albino propagules. Our study suggests for the first time that PAHs from a chronic oil polluted site resulted in mutations in both nuclear and chloroplast genes, resulting in the production of albino propagules.

An open-label randomized pragmatic non-inferiority pilot trial to compare the effectiveness of Dysentery compound with individualized homeopathic medicines in irritable bowel syndrome.

Background Irritable Bowel Syndrome (IBS) is a prevalent gastro-intestinal disorder characterized by recurrent abdominal pain, bloating, altered bowel function and myriad of gastro-intestinal symptoms. Dysentery compound (DC), a 'bowel nosode', is one of the homeopathic medicines to treat IBS, but remained under-researched. We hypothesized that DC would be non-inferior to individualized homeopathy (IH) in treatment of IBS. Method An open, randomized (1:1), parallel arms, pragmatic, non-inferiority, pilot trial was conducted to compare the effectiveness of DC with IH medicines in 60 IBS patients. IBS Quality of Life (IBS-QOL) questionnaire was used as the outcome measure; assessed at baseline and after 3 months. Comparative analysis was carried out on the primary outcome to detect non-inferiority by one-tailed t test at alpha=5% with a prefixed margin (Δ) of 1.0 based on assumption. Results Six subjects dropped out. Groups were comparable at baseline (all p>0.01). Though intra-group changes were higher favoring IH over DC, group differences were statistically non-significant (all p>0.01). Non-inferiority was not demonstrated by DC against IH over 3 months (mean difference= -3.3, SE=5.2, lower 95% confidence limit -11.9, t= -0.453, p=0.674). No adverse events were reported from either group. Conclusion Non-inferiority of DC against IH in treatment of IBS was not demonstrated though it appeared as safe; still, being a pilot trial, no definite conclusion could be drawn. Further exploration of both efficacy and effectiveness of either of the therapies is necessary by adequately powered trials and independent replications. Trial registration: CTRI/2017/05/008480; UTN: U1111-1196-1004.

Public health crisis of road traffic accidents in India: Risk factor assessment and recommendations on prevention on the behalf of the Academy of Family Physicians of India.

Roads are considered a sign of development bringing colossal benefits to community as socioeconomic and logistic facilitator. Yet, growth of road network has brought road crashes leading to civic pain from premature deaths of productive age group. In 2017, 16 citizens were killed and 53 injured every hour on Indian roads as per officially reported data, while a fair number go unreported. This is unacceptably high when compared with international standards. Risk correlates of road traffic injuries (RTIs) need to be redefined so as to form a continuum with other confounding factors that impact to take lives on road. Risk factors impacting RTIs vary from human components to the roles and responsibilities of healthcare stakeholders. We should have made roads safer for all citizens because a large percentage of population - children, pedestrians, cyclists, motorcyclists, and the elderly - are most vulnerable. A taskforce was set up by the Academy of Family Physicians of India to scientifically analyze the literature available to assess risks and put forward appropriate recommendations.

Acute coronary syndrome-related mortality audit in a teaching hospital at Port Blair, India.

BACKGROUND: India has a growing trend of acute myocardial infarction (AMI) due to shifting lifestyle. OBJECTIVE: To study the profile of patients died due to AMI and to find its risk correlates. METHODS: A study was conducted on consecutive AMI cases admitted in the teaching hospital at Port Blair from April 2011 to March 2016. During inpatients management, outcomes were followed up from admission till discharge or expiry. RESULTS: Of the total 491 cases, majority (75.99%) had ST-elevated myocardial infarction (STEMI); mean age of 73 deaths was 58.01 ± 13.60, mortality probability among females was less; in the age group 41-50 years the case fatality rate was the lowest (7.58%). Mean age of survival was 56.75 ± 10.47; great majorities were males across all age groups in cases and deaths; highest number of cases were in the age group 51-60 (34.21%); reportedly 83.10% had some physical activities; 6.52% were vegetarian; 34.22% were smokers; 10.39% had family history of AMI, majority (59.06%) were from white collar profession (teacher, clerical, etc.); 52.95% were diabetics; and 47.45% were hypertensives. Lifestyle-related risk factors, physical activity, and vegetarian diet were not protective; family history and addiction to smoking were significantly associated with AMI deaths. Thrombolytic intervention helped the survival of 73.68%, and the odds ratio of survival showed benefit. CONCLUSIONS: Acute STEMI had male and middle-age predominance with a common risk factor of family history, smoking, diabetes, and hypertension.

Multistep regulation of protein kinase A in its localization, phosphorylation and binding with a regulatory subunit in fission yeast.

The cAMP-PKA is the major glucose-sensing pathway that controls sexual differentiation in Schizosaccharomyces pombe. Sequencing from the pka1 locus of recessive sam mutants, in which cells are highly inclined to sexual differentiation, led to the identification of mutations in the pka1 locus in sam5 (pka1-G441E) and sam7 (pka1-G441R). Rst2 and Ste11 proteins were induced and localized to the nucleus of sam5 and sam7 mutants even under rich glucose conditions, indicating that the function of Pka1 was completely abolished by mutations. Pka1-G441E and Pka1-G441R mutant proteins reside in the cytoplasm, even under glucose-rich conditions, while wild-type Pka1 resides in the nucleus, indicating that the functionality of Pka1 is important for its nuclear localization. This is supported by the observation that the Pka1-T356A mutant, which partially lacks Pka1 function, was localized to both the cytoplasm and the nucleus, but an active phosphomimetic Pka1-T356D mutant prtotein was localized to the nucleus under glucose-rich conditions. In addition to the basal phosphorylation of Pka1 at T356, hyperphosphorylation of Pka1 was observed under glucose-starved conditions, and such hyperphosphorylation was not observed in pka1-G441E, pka1-G441R, pka1-T356A or pka1-T356D mutants. As these mutant proteins failed to interact with a regulatory subunit Cgs1, hyperphosphorylation of Pka1 mutant proteins was considered to be dependent on Cgs1 interaction. Consistent with a role for Cgs1 in Pka1 phosphorylation, we detected the formation of a Cgs1-Pka1 complex prior to Pka1 hyperphosphorylation. Together, these results indicate that nuclear localization of Pka1 depends on its activity and hyperphosphorylation of Pka1 depends on Cgs1 interaction.

Complex formation, phosphorylation, and localization of protein kinase A of Schizosaccharomyces pombe upon glucose starvation.

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.

A large complex mediated by Moc1, Moc2 and Cpc2 regulates sexual differentiation in fission yeast.

Sexual differentiation in Schizosaccharomyces pombe is triggered by nutrient starvation and is downregulated by cAMP. Screening programs have identified the moc1/sds23, moc2/ded1, moc3 and moc4/zfs1 genes as inducers of sexual differentiation, even in the presence of elevated levels of cAMP. To investigate possible interactions among Moc1, Moc2, Moc3 and Moc4 proteins, we first screened for individual Moc-interacting proteins using the yeast two-hybrid system and verified the interactions with other Moc proteins. Using this screening process, Cpc2 and Rpl32-2 were highlighted as factors involved in interactions with multiple Moc proteins. Cpc2 interacted with Moc1, Moc2 and Moc3, whereas the ribosomal protein Rpl32-2 interacted with all Moc proteins in the two-hybrid system. Physical interactions of Cpc2 with Moc1, Moc2 and Rpl32-2, and of Rpl32-2 with Moc2 were confirmed by coimmunoprecipitation. In addition, using Blue Native/PAGE, we revealed that each Moc protein exists as a large complex. Overexpression of Moc1, Moc2, Moc3, Moc4 and Rpl32-2 resulted in the efficient induction of a key transcription factor Ste11, suggesting that all proteins tested are positive regulators of Ste11. Considering that Moc2/Ded1 is a general translation factor and that Cpc2 associates with many ribosomal proteins, including Rpl32-2, it is possible that a large Moc-mediated complex, detected in this study, may act as a translational regulator involved in the control of sexual differentiation in S. pombe through the induction of Ste11.

Glutamyl tRNA synthetases and glutamic acid induce sexual differentiation of Schizosaccharomyces pombe.

The moc3 gene was screened out as an inducer of sexual differentiation in fission yeast Schizosaccharomyces pombe. We isolated a novel gene, named ers2, encoding mitochondrial glutamyl tRNA synthetase (mGluRS) as a Moc3 interacting element by the yeast two-hybrid system. Cytoplasmic glutamyl tRNA synthetase (cGluRS) also interacted with Moc3 in a yeast two-hybrid system. Disruption of ers1 (cGluRS) and of ers2 (mGluRS) indicated that these genes are both essential for the cell growth of S. pombe. We found that ers2 severely affected cell growth and decreased viability, but induced sexual differentiation of S. pombe when it was over-expressed. Over-expression of ers1 also stimulated sexual differentiation in S. pombe. These observations led us to test the effects of various amino acids on sexual differentiation. We found that glutamic acid, as well as other specific amino acids, such as tryptophan, methionine, and threonine, efficiently induced sexual differentiation in S. pombe. Our findings suggest a new regulatory mechanism where GluRSs and glutamic acid are involved in sexual differentiation in S. pombe.