The phosphodiesterase DibA interacts with the c-di-GMP receptor LapD and specifically regulates biofilm in Pseudomonas putida.

The ubiquitous bacterial second messenger c-di-GMP is synthesized by diguanylate cyclase and degraded by c-di-GMP-specific phosphodiesterase. The genome of Pseudomonas putida contains dozens of genes encoding diguanylate cyclase/phosphodiesterase, but the phenotypical-genotypical correlation and functional mechanism of these genes are largely unknown. Herein, we characterize the function and mechanism of a P. putida phosphodiesterase named DibA. DibA consists of a PAS domain, a GGDEF domain, and an EAL domain. The EAL domain is active and confers DibA phosphodiesterase activity. The GGDEF domain is inactive, but it promotes the phosphodiesterase activity of the EAL domain via binding GTP. Regarding phenotypic regulation, DibA modulates the cell surface adhesin LapA level in a c-di-GMP receptor LapD-dependent manner, thereby inhibiting biofilm formation. Moreover, DibA interacts and colocalizes with LapD in the cell membrane, and the interaction between DibA and LapD promotes the PDE activity of DibA. Besides, except for interacting with DibA and LapD itself, LapD is found to interact with 11 different potential diguanylate cyclases/phosphodiesterases in P. putida, including the conserved phosphodiesterase BifA. Overall, our findings demonstrate the functional mechanism by which DibA regulates biofilm formation and expand the understanding of the LapD-mediated c-di-GMP signaling network in P. putida.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Effect of diisobutyl adipate on the expression of biomarker genes that respond to endocrine disruption and on gonadal sexual differentiation in Japanese medaka (Oryzias latipes).

Phthalate and non-phthalate plasticizers are used in polymer materials, such as plastic and rubber. It has recently been found that diisobutyl adipate (DIBA), which is considered an environmentally safe non-phthalate plasticizer, potentially acts as a thyroid disruptor in fish. Here, we investigated the sexual hormone effects of DIBA based on the expression levels of genes that respond to endocrine disruption and sexual hormone activity in the livers and gonads, and on gonadal sexual differentiation in Japanese medaka. Compared with the control group, the mRNA expression of chgH, vtg1, vtg2, and esr1 was significantly suppressed in the livers of DIBA exposed XX individuals. Furthermore, the mRNA expression of gsdf was significantly upregulated and downregulated in the gonads of XX and XY individuals, respectively. The mRNA expressions of esr1 and esr2b were significantly suppressed by DIBA exposure in the gonads of both XX and XY individuals. These observations suggest that DIBA has potential androgenic activity in Japanese medaka. However, normal testes and ovaries were observed in respective XY and XX medaka after DIBA exposure; therefore, these results suggest that DIBA may have weak androgenic activity.

Prevalence and Associated Factors of Human Immune Deficiency Virus and Tuberculosis Co-Infection in Patients Attending Kolla Diba Health Center, Dembia District, Northwest Ethiopia.

BACKGROUND: TB-HIV co-infection is the most common problem of African countries, especially, Sub-Saharan countries including Ethiopia. So this study aimed to assess TB-HIV co-infection with its associated factors in patients with Tuberculosis in Northwest Ethiopia. Although the prevalence of TB-HIV was low, the need for strengthening the health extension program especially in urban dwellers also needed to include TB-HIV testing. OBJECTIVE: This study aimed to assess TB-HIV co-infection with its associated factors in patients with Tuberculosis in Northwest Ethiopia. METHODOLOGY: Institutional based cross-sectional study has been done and a total of 638 subjects participated in the study. The data of the study subjects were collected from the tuberculosis logbook using two trained data collectors who were work in the TB DOTS program and by using a well-prepared checklist and SPSS was used for analyzing data. RESULTS: 9.7% (62/638) of TB patients were found to be co-infected with HIV. Among these 32 (11.4%) were females and 30 (8.4%) were males. More infected individuals were found in urban residents 44 (20%) than rural residents and age groups 30-40 years 31 (22.5%) are more infected than the other age group. TBforms, age, and residence were associated with HIV/TB co-infection significantly. CONCLUSIONS AND RECOMMENDATIONS: Although the prevalence of TB-HIV was low, the need for strengthening the health extension program especially in urban dwellers is needed to include TB-HIV testing. Further surveys involving HIV infected TB patients to strengthen and scale-up for TB and HIV is needed.

Prevalence and Associated Factors of Pterygium Among Adults Living in Kolla Diba Town, Northwest Ethiopia.

BACKGROUND: Pterygium is a disfiguring disease that can potentially lead to blindness. It is more common in warm, windy and dry climates of tropical and sub-tropical regions of Africa. Globally, the prevalence ranging from 0.07% to 53%. Studies conducted on the prevalence of pterygium in developing countries were limited with a wider discrepancy between them. In this study, we aimed to assess the prevalence of pterygium and its associated factors among adults in Kolla Diba town. METHODS: A community-based cross-sectional study was done in Kolla Diba town from May 30-June 16, 2019. A systematic random sampling technique was used to select 627 study participants. The basic ophthalmic examination was performed using portable slit lamp, 3x magnifying loop with torch light and a pretested structured questionnaire was completed. The data entered into EPI INFO version 7 and analyzed using SPSS version 20. Descriptive statistics and binary logistic regression analysis were employed. P-values of <0.05 were considered statistically significant. RESULTS: A total of 605 study participants were involved with a response rate of 96.5%. Among them, 317 (52.4%) participants were males. The mean age of the respondents was 38.18 ± 15.56 with a range of (18-95) in years. The overall prevalence of pterygium was 112 (18.5% (95% CI (15.6-21.7)). Being widowed (AOR = 7.32 (95% CI: 2.88, 18.57)), outdoor occupation (AOR = 2.50 (95% CI: 1.46, 4.29)), sun exposure (AOR = 2.38 (95% CI: 1.28, 4.43)), wind exposure (AOR = 1.97 (95% CI: 1.04, 3.72)), alcohol drinking (AOR = 2.26 (95% CI: 1.48, 4.63)), and severe blepharitis (AOR = 2.45 (95% CI: 1.48, 4.05)) had statistically significant positive association with pterygium. CONCLUSION: The prevalence of pterygium was relatively higher. Being widowed, outdoor occupation, sun exposure, wind exposure, alcohol drinking, and severe blepharitis were significantly associated with the development of pterygium.

Development of a gold-nano particle based novel dot immunobinding assay for rapid and sensitive detection of Banana bunchy top virus.

An improved gold nanoparticle based Dot immunobinding assay (DIBA) was developed for the detection of Banana bunchy top virus (BBTV), that is more efficient, sensitive, rapid and simpler than conventional DIBA and ELISA. Instead of enzyme conjugates, gold nanoparticles were used as reporters owing to their unique optical properties. Antibody was raised against expressed recombinant coat protein of BBTV. The gold nanoparticles were conjugated to primary / detection antibody raised following immunization with recombinant coat protein, making it highly specific for the virus. Gold nanoparticle conjugated primary antibody (GCPab) based DIBA developed in this study has a detection efficiency comparable to ELISA. The results of using this assay format for detection of BBTV in banana plants from four geographical regions of India are also presented in this report. The test could detect the virus at sap dilution up-to 10-2. Using this improved DIBA, any lab with basic amenities can perform indexing on large numbers of samples.

Utilization of insecticide treated bed net and associated factors among households of Kola Diba town, North Gondar, Amhara region, Ethiopia.

OBJECTIVE: The primary aim of this study was to assess the utilization of an insecticide-treated bed net on the preceding night and associated factors among households of Kola Diba town in 2017. RESULTS: Of the 260 households, 239 (91.9%) (95% CI = 88.5-95%) utilized insecticide-treated bed net on the night preceding the interview. 242 (93.1%) households didn't treat the bed net whereas 18 (6.9%) treat the bed net by chemical regularly. Household structure and knowledge about malaria transmission were found to be associated with insecticide-treated bed net utilization.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

First report of lily mottle virus on lily (Lilium sp.) in southern India.

Lily samples collected from commercial cut flower growing field at Nilgiris, Tamil Nadu, India were serologically tested through direct antigen coating-ELISA and dot immuno binding assay for the infection of potyvirus through potyvirus group specific polyclonal antibody. The RT-PCR using potyvirus universal degenerate primers (PNIbF1/PCPR1) showed the presence of a potyvirus and the sequencing results showed the association of lily mottle virus (LMoV). Further LMoV was confirmed by RT-PCR amplification using newly designed primer pair covering entire coat protein gene and sequence results showed 100% nucleotide identity with the LMoV reported from South Korean. To our knowledge, this is the first confirmed evidence for the occurrence of LMoV on lily in southern India.

Rice Black-streaked Dwarf Virus Preparation and Infection on Rice.

Rice black-streaked dwarf virus (RBSDV), a member of genus Fijivirus in the family Reoviridae, infects rice, maize, barley and wheat, and can seriously affect crop yields. RBSDV is transmitted by the small brown planthopper (Laodelphax striatellus, SBPH) in a persistent manner. RBSDV has 10 linear dsRNA genomic segments, making it difficult to construct infectious clones for functional studies in plants. Here we describe a method for inoculating and maintaining RBSDV on rice in a greenhouse for use in laboratory research. The protocol uses SBPHs mass reared in the laboratory. We also describe in detail the propagation of a healthy planthopper population, the preparation of plant material, RBSDV inoculation and the evaluation of the rice after inoculation.

Highly efficient immunodiagnosis of Large cardamom chirke virus using the polyclonal antiserum against Escherichia coli expressed recombinant coat protein.

Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renaturation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom.