Application of autologous platelet-rich plasma to graft donor sites to reduce pain and promote healing.

OBJECTIVE: Platelet-rich plasma (PRP) is widely used for wound healing in medical care because of the numerous growth factors it contains. Traditionally, donor sites are left to heal with a primary dressing so wounds are not left open. However, a delay in healing accompanied by pain at a donor site is often seen. This study primarily throws light on the use of autologous PRP over split-thickness skin graft (STSG) donor sites to promote healing and reduce pain. METHOD: The patients enrolled in this study in 2018-2019 were divided into two groups: the intervention group received autologous PRP applied topically at the donor site; in the control group, the wound was dressed traditionally. Pain scales were measured in the immediate postoperative period at six hours, 10 hours and 16 hours. The dressing was opened on the postoperative day 14 and observed for healing by an independent observer. RESULTS: A total of 100 patients were included in the study. Patients in the PRP group showed statistically significant faster healing at postoperative day 14 compared with the control group (p<0.05), who required dressings for 3-4 weeks postoperatively. Pain scale scores in the postoperative period were significantly less in the PRP group at six hours postoperatively compared with the control group (p<0.05). There was a reduced incidence of hypertrophic scar formation in the small number of patients in the PRP group who had developed hypertrophic scar previously. CONCLUSION: Application of PRP is a safe, cost-effective and easy method to achieve faster healing in graft donor site areas that are troublesome to both patients and doctors. It also reduces postoperative pain at donor sites. The authors recommend PRP is used more often in the management of donor sites for STSGs.

A multiplex PCR assay for rapid identification of major tospovirus vectors reported in India.

BACKGROUND: To date, four thrips vectors have been reported to transmit five different tospoviruses in India. Their identification at an early stage is crucial in formulating appropriate pest management strategies. Since morphometric key-based thrips identification based on the adult stage is time-consuming, there is a need to develop diagnostic tools which are rapid, accurate, and independent of developmental stages. Here, we report a multiplex PCR assay to identify four major thrips vectors viz. Thrips palmi, T. tabaci, Scirtothrips dorsalis, and Frankliniella schultzei present in India. RESULTS: Cytochrome oxidase subunit III and internal transcribed spacer region 2 were utilized to design species-specific primers. Of 38 pairs of primers tested, primer pairs AG35F-AG36R, AG47F-AG48R, AG87F-AG88R, and AG79F-AG80R amplified 568 bp, 713 bp, 388 bp, and 200 bp products from the DNA templates of T. palmi, S. dorsalis, T. tabaci, and F. schultzei, respectively at same PCR conditions. The specificity of the primer pairs was validated with a large number of known specimens and no cross-reactivity was observed with other thrips species. The multiplex PCR assay with a cocktail of all the four primer pairs detected four thrips vectors efficiently and could discriminate all of them concurrently in a single reaction. CONCLUSION: The multiplex PCR reported in this study could identify the major thrips vectors reported in India. The assay will be useful in ascertaining distribution profile of major thrips vectors, disease epidemiology, screening large samples, and quarantine.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

N-terminal in coat protein of Garlic virus X is indispensible for its serological detection.

Conserved coat protein region of plant viruses is often used as source of antigen for production of polyclonal antibodies for broad-based detection of closely related viruses. Antigenic region in coat protein is located either on N-terminal, and/or C-terminal or in the middle of coat protein. A study was undertaken to determine if antigenic region resides in N-terminal in Garlic virus X (GarV-X) of Allexivirus. In allexiviruses, N-terminal of coat protein region (1-57 amino acids) was highly variable. A complete coat protein of 27 kDa and a truncated protein without N-terminal (20 kDa) of GarV-X were expressed in pET expression vector and confirmed in western blotting using anti-His antisera. These expressed proteins were purified and used for antisera production. Specific and strong reaction was obtained for antisera generated against GarV-X full CP and GarV-X was detected in field-grown allium crops viz., onion, garlic, leek, and bunching onion and chives in ELISA. Antisera against GarV-X CPΔ1-61 (truncated CP) did not show reaction for GarV-X detection in immunoassay. Epitope mapping also indicated N-terminal as major antigenic determinant region with highest antigenic signal score. Our studies confirm that antigenic signals or epitopes reside in the N-terminal region of GarV-X which can be synthesized and used for production of monoclonal antibodies for specific detection purposes.

Biotransformation of 4-chloro-2-nitrophenol into 5-chloro-2-methylbenzoxazole by a marine Bacillus sp. strain MW-1.

Decolourization, detoxification and biotransformation of 4-chloro-2-nitrophenol (4C2NP) by Bacillus sp. strain MW-1 were studied. This strain decolorized 4C2NP only in the presence of an additional carbon source. On the basis of thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole were identified as metabolites. Resting cells depleted 4C2NP with stoichiometric formation of 5-chloro-2-methyl benzoxazole. This is the first report of the formation of 5-chloro-2-methylbenzoxazole from 4C2NP by any bacterial strain.

Chryseomicrobium imtechense gen. nov., sp. nov., a new member of the family Planococcaceae.

A Gram-stain-positive, rod-shaped, yellow, non-motile, non-spore-forming, strictly aerobic bacterial strain, designated MW 10(T), was isolated from seawater of the Bay of Bengal, India, and was subjected to a polyphasic taxonomic study. Analysis of the 16S rRNA gene sequence revealed that strain MW 10(T) showed highest similarity to the type strains of Psychrobacillus psychrodurans (96.15 %) and Psychrobacillus psychrotolerans (96.01 %) and showed less than 96 % similarity to members of the genera Paenisporosarcina, Planococcus, Sporosarcina and Planomicrobium. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain MW 10(T) formed a clade separate from members of closely related genera. The morphological, physiological and chemotaxonomic characteristics of strain MW 10(T) differed from those of members of closely related genera. The major fatty acid in strain MW 10(T) was iso-C(15 : 0) and the menaquinones were MK-7 (48.4 %), MK-8 (32.3 %), MK-7(H(2)) (13.7 %) and MK-6 (5.6 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown phospholipid, an unknown lipid and an unknown glycolipid. The cell-wall peptidoglycan type was l-Lys-d-Asp. The genomic DNA G+C content (53.4 mol%) of strain MW 10(T) was significantly different from those of members of closely related genera. On the basis of its morphological, physiological and chemotaxonomic characteristics as well as our phylogenetic analysis, we conclude that strain MW 10(T) is a member of a novel genus and species, for which the name Chryseomicrobium imtechense gen. nov., sp. nov., is proposed. The type strain of Chryseomicrobium imtechense is MW 10(T) ( = MTCC 10098(T)  = JCM 16573(T)).

Pathway for degradation of 2-chloro-4-nitrophenol in Arthrobacter sp. SJCon.

Degradation of 2-Chloro-4-nitrophenol (2C4NP) was studied by Arthrobacter sp. SJCon, isolated from the soil of a pesticide contaminated site. This strain utilized 2C4NP as sole source of carbon and energy and degraded 2C4NP with stoichiometric release of nitrite and chloride ions. A metabolite was detected during the study of 2C4NP degradation and identified as chlorohydroquinone (CHQ) by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Inhibition study using 2,2'-dipyridyl showed that CHQ is a terminal aromatic compound in degradation pathway of 2C4NP. CHQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain SJCon that suggested the cleavage of the CHQ to maleylacetate (MA). Our study clearly showed that Arthrobacter sp. SJCon degraded 2C4NP via formation of CHQ that further cleaved to MA by CHQ dioxygenase. This mechanism of degradation of 2C4NP differs from previously reported degradation pathways of 2C4NP.

Molecular and biological characterization of soybean yellow mottle mosaic virus severe strain infecting soybean in India.

The complete nucleotide sequence and genome organization of soybean yellow mottle mosaic virus severe strain causing bright yellow mosaic, mottling and puckering symptoms in soybean (Glycine max) from India was determined. The monopartite single stranded genomic RNA is 3974 nuclotides long and has the potential to encode six viral proteins viz., p25, p83, p8, p10, p39 and p25. The SYMMV-Sb isolate differed from mungbean strain with 69 nucleotides and nine aminoacids dispersed over the various ORFs. Comparative sequence analysis revealed that SYMMV-Sb shared 98% nt sequence identity at complete genome level and 96-100% at all ORFs level with SYMMV mungbean strain from India and 71-92% identity with SYMMV Korean soybean isolate, whereas it showed very low sequence identity with other tombusviridae members (2-53%). The phylogenetic analysis showed the clustering of SYMMV-Sb along with other members of genus Gammacarmovirus. The SYMMV-Sb isolate produced chlorotic blotches, mild and veinal mottling, necrosis and puckering symptoms in various leguminous host plants. The symptomatalogy of the soybean isolate was differed from mungbean strain as earlier induced severe symptoms on soybean and mild symptoms on mungbean. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02925-2.

Reducing split-thickness skin grafting donor site agony; faster healing and decreased pain-role of platelet-rich plasma.

INTRODUCTION: The split-thickness skin graft harvested donor site is associated with prolonged healing, discomfort, and pain. Platelet-rich plasma (PRP) contains platelet-derived growth factors and has been widely used in chronic wounds and skin graft donor sites. PRP application is known to accelerate wound epithelialization rates, and also reduce postoperative wound site pain. MATERIALS AND METHODS: We assessed 20 patients admitted to our hospital service who underwent split-thickness skin grafting (STSGs) with proximal half of the donor site treated with PRP. The dressing was conducted on postoperative day 7, 14, and 21. The donor site healing was assessed with serial photographs and donor site pain measured by numerical rating scale. RESULTS: Complete healing of wounds (epithelialization) was present in 12 (60%) patients dressed with PRP. Pain on opening dressing was an average of 3.5 in PRP dressed wounds and 6.35 in control wounds. Patients dressed without PRP, none of them had complete epithelialization. All patients had partial healing and were less than the donor site dressed with PRP. Based on these results, skin graft donor site with PRP showed accelerated healing and reduced pain and discomfort compared to control without PRP. CONCLUSION: PRP is a beneficial adjunct for reducing donor site pain and increased healing of donor site following STSG harvest.

Association of cucurbit aphid-borne yellows virus with cucumber plants in India.

Symptoms like bright yellowing, puckering of the leaf, vein banding, and vein thickening were observed on different cucurbit hosts at the experimental farm of Indian Agricultural Research Institute, New Delhi during Kharif 2019. Leaf-dip electron microscopy of the symptomatic leaves revealed the association of isometric virus particles measuring ~ 25 nm with bitter gourd and cucumber samples. The RT-PCR assay using polerovirus generic primers covering the partial RdRp, intergenic region, and partial CP region was resulted the amplicons of ~ 1.1 kb. Subsequent cloning, sequencing, and sequence analysis revealed the association of cucurbit aphid-borne yellows virus (CABYV) with bitter gourd (Momordica charantia) and cucumber (Cucumis sativus) plants. These results constitute the first report of CABYV infection on cucumber plants from India. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s13337-020-00645-4) contains supplementary material, which is available to authorized users.

Coverage of residual defects by using previous flaps: Flip flap puzzle flap.

INTRODUCTION: Terminal flap necrosis and flap retraction is a difficult problem faced by surgeons. These require some other flap for coverage of defect with limited local tissue availability. Flip flap puzzle flap is an option for small defect coverage using previous flap. METHODOLOGY: This flap is harvested from previous flap and raised at sub-dermal level. The flap is flipped 180° on itself to cover the defect. The residual area is finally covered with split skin graft. RESULTS: This is a prospective study conducted on 10 patients, with 9 flap survival and 1 flap failure. In 7 out of 10 cases partial graft failure was observed, but all the defects healed eventually. DISCUSSION AND CONCLUSION: This flap is a less morbid and rapid option for coverage of small sized defects. It has no donor site morbidity as flap is raised from the previously harvested flap.

Present Status and Future Management Strategies for Sugarcane Yellow Leaf Virus: A Major Constraint to the Global Sugarcane Production.

Sugarcane yellow leaf virus (SCYLV) is a distinct member of the Polerovirus genus of the Luteoviridae family. SCYLV is the major limitation to sugarcane production worldwide and presently occurring in most of the sugarcane growing countries. SCYLV having high genetic diversity within the species and presently ten genotypes are known to occur based on the complete genome sequence information. SCYLV is present in almost all the states of India where sugarcane is grown. Virion comprises of 180 coat protein units and are 24-29 nm in diameter. The genome of SCYLV is a monopartite and comprised of single-stranded (ss) positive-sense (+) linear RNA of about 6 kb in size. Virus genome consists of six open reading frames (ORFs) that are expressed by sub-genomic RNAs. The SCYLV is phloem-limited and transmitted by sugarcane aphid Melanaphis sacchari in a circulative and non-propagative manner. The other aphid species namely, Ceratovacuna lanigera, Rhopalosiphum rufiabdominalis, and R. maidis also been reported to transmit the virus. The virus is not transmitted mechanically, therefore, its transmission by M. sacchari has been studied in different countries. SCYLV has a limited natural host range and mainly infect sugarcane (Sachharum hybrid), grain sorghum (Sorghum bicolor), and Columbus grass (Sorghum almum). Recent insights in the protein-protein interactions of Polerovirus through protein interaction reporter (PIR) technology enable us to understand viral encoded proteins during virus replication, assembly, plant defence mechanism, short and long-distance travel of the virus. This review presents the recent understandings on virus biology, diagnosis, genetic diversity, virus-vector and host-virus interactions and conventional and next generation management approaches.

Expression, purification, crystallization and preliminary X-ray analysis of maleylacetate reductase from Burkholderia sp. strain SJ98.

Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 A. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 A resolution, respectively.

Development of Soybean Yellow Mottle Mosaic Virus-Based Expression Vector for Heterologous Protein Expression in French Bean.

Plant virus-based vectors provide attractive and valuable tools for rapid production of recombinant protein in large quantities as they produce systemic infections in differentiated plant tissues. In the present study, we engineered the Soybean yellow mottle mosaic virus (SYMMV) as a gene expression vector which is a promising candidate for systemic expression of foreign proteins in French bean plants. Full virus vector strategy was exploited for insertion of foreign gene by inserting MCS through PCR in the circular pJET-SYMMV clone. To examine the ability of the SYMMV vector system, GFP gene was cloned after the start codon of coat protein (CP) so that its expression was driven by the SYMMV-CP subgenomic promoter. When in vitro run off SYMMV-GFP transcript was mechanically inoculated to French bean leaves, good level of GFP expression was observed through confocal microscopy up to 40 dpi. Expression of heterologous protein was also confirmed through ISEM, DAC-ELISA and RT-PCR with specific primers at 20 dpi. The recombinant SYMMV construct was stable in in vitro runoff transcript inoculated plants but the inserted GFP was lost in progeny virion inoculated plants. The system developed here will be useful for further studies of SYMMV gene functions and exploitation of SYMMV as a gene expression vector.

De novo genome sequencing and secretome analysis of Tilletia indica inciting Karnal bunt of wheat provides pathogenesis-related genes.

Tilletia indica is an internationally quarantined fungal pathogen causing Karnal bunt of wheat. The present study carried out that the whole genome of T. indica was sequenced and identified transposable elements, pathogenicity-related genes using a comparative genomics approach. The T. indica genome assembly size of 33.7 MB was generated using Illumina and Pac Bio platforms with GC content of 55.0%. A total of 1737 scaffolds were obtained with N50 of 58,667 bp. The ab initio gene prediction was performed using Ustilago maydis as the reference species. A total number of 10,113 genes were predicted with an average gene size of 1945 bp out of which functionally annotated genes were 7262. A total number of 3216 protein-coding genes were assigned in different categories. Out of a total number of 1877 transposable elements, gypsy had the highest count (573). Total 5772 simple sequence repeats were identified in the genome assembly, and the most abundant simple sequence repeat type was trinucleotide having 42% of total SSRs. The comparative genome analysis suggested 3751 proteins of T. indica had orthologs in five fungi, whereas 126 proteins were unique to T. indica. Secretome analysis revealed the presence of 1014 secretory proteins and few carbohydrate-active enzymes in the genome. Some putative candidate pathogenicity-related genes were identified in the genome. The whole genome of T. indica will provide a window to understand the pathogenesis mechanism, fungal life cycle, survival of teliospores, and novel strategies for management of Karnal bunt disease of wheat.

First Draft Genome Sequence of a Pearl Millet Blast Pathogen, Magnaporthe grisea Strain PMg_Dl, Obtained Using PacBio Single-Molecule Real-Time and Illumina NextSeq 500 Sequencing.

The first draft genome sequence of the pearl millet blast pathogen Magnaporthe grisea PMg_Dl from India is presented. The genome information of M. grisea will be useful to understand the Magnaporthe speciation, genetic diversity, environmental adaptation, and pathogenic and host range determinants.

Field-usable lateral flow immunoassay for the rapid detection of a macluravirus, large cardamom chirke virus.

A simple and rapid lateral flow immunoassay (LFIA) was developed by utilizing gold nanoparticles conjugated to a polyclonal antibody against coat protein of large cardamom chirke virus (LCCV). The LFIA based on the principle of sandwich immunoassay detected LCCV within ∼10 min and the result could be evaluated visually. The colloidal gold (CG) was made using 1% gold chloride solution. The LCCV IgG (1 µg/µl) and Mouse IgG (0.5 µg/µl) were conjugated with CG individually and coated onto a conjugate pad at 1:1 ratio. A sample extraction procedure was optimized in order to get adequate clear leaf sap of large cardamom leaf within few minutes. The sensitivity limit of the detection was 1:40 dilution of LCCV infected leaf sap. The diagnostic performance of LFIA was compared with ELISA using field samples. The LFIA was free from false positive as no visible test line was developed with healthy and potyviruses such as papaya ringspot virus and potato virus Y. The diagnostic specificity and sensitivity of LFIA was 100% and 90%, respectively. The Cohen's kappa coefficient (0.701) suggested a very good agreement between the ELISA and LFIA. Receiver operating characteristic analysis indicated that LFIA was a robust method as the area under the curve (0.950) is significantly (P <0.0001) broader.

A Constellation of Rare Findings in a Case of Goldenhar Syndrome.

An 18-month-old child presented with right macrostomia, bilateral preauricular skin tags, bilateral CTEV, squint in bilateral eyes, thoracic vertebral anomalies, right sided aortic arch, and associated left pulmonary agenesis. The patient did not have any associated respiratory symptoms. Ipsilateral pulmonary agenesis is considered as a rare association with Goldenhar syndrome and a case of contralateral pulmonary aplasia has been described as an even rarer association.

Genome characterization, infectivity assays of in vitro and in vivo infectious transcripts of soybean yellow mottle mosaic virus from India reveals a novel short mild genotype.

Nucleotide sequence of a distinct soybean yellow mottle mosaic virusisolate from Vignaradiata (mungbean isolate, SYMMV-Mb) from India was determined and compared with othermembers of the family Tombusviridae. The complete monopartite single-stranded RNA genome of SYMMV-Mb consisted of 3974nt with six putative open reading frames and includes 5' and 3' untranslated regions of 35 and 254nt, respectively. SYMMV-Mb genome shared 75% nt sequence identity at complete genome level and 67-92% identity at all ORFs level with SYMMV Korean and USA isolates (soybean isolates) followed by CPMoV, whereas it shared very low identity with other tombusviridae members (5-41%). A full-length infectious cDNA clone of the SYMMV-Mb placed under the control of the T7 RNA polymerase and the CaMV35S promoters was generated and French bean plants on mechanical inoculation with in vitro RNA transcripts, p35SSYMMV-O4 plasmid and agroinoculation with p35SSYMMV-O4 showed symptoms typical of SYMMV-Mb infection. The infection was confirmed by DAC-ELISA, ISEM, RT-PCR and mechanical transmission to new plant species. Further testing of different plant species with agroinoculation of p35SSYMMV-O4 showed delay in symptoms but indistinguishable from mechanical sap inoculation and the infection was confirmed by DAC-ELISA, RT-PCR and mechanical transmission to new plants. The system developed here will be useful for further studies on pathogenecity, viral gene functions, plant-virus-vector interactions of SYMMV-Mb and to utilize it as a gene expression and silencing vector.

Genome Sequence of a Unique Magnaporthe oryzae RMg-Dl Isolate from India That Causes Blast Disease in Diverse Cereal Crops, Obtained Using PacBio Single-Molecule and Illumina HiSeq2500 Sequencing.

The whole-genome assembly of a unique rice isolate from India, Magnaporthe oryzae RMg-Dl that causes blast disease in diverse cereal crops is presented. Analysis of the 34.82 Mb genome sequence will aid in better understanding the genetic determinants of host range, host jump, survival, pathogenicity, and virulence factors of M. oryzae.