Genetic diversity and molecular characterization of Cucumber mosaic cucumovirus (CMV) subgroup II infecting Spinach (Spinacia oleracea) and Pea (Pisum sativum) in Pothwar region of Pakistan

Cucumber mosaic virus (CMV) is a tremendous threat to vegetables across the globe, including in Pakistan. The present work was conducted to investigate the genetic variability of CMV isolates infecting pea and spinach vegetables in the Pothwar region of Pakistan. Serological-based surveys during 2016-2017 revealed 31.70% overall CMV disease incidence from pea and spinach crops. Triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) revealed that all the positive isolates belong to CMV subgroup II. Two selected cDNA from ELISA-positive samples representing each pea and spinach crops were PCR-amplified (ca.1100 bp) and sequenced corresponding to the CMV CP gene which shared 93.7% nucleotide identity with each other. Both the sequences of CMV pea (AAHAP) and spinach (AARS) isolates from Pakistan were submitted to GenBank as accession nos. MH119071 and MH119073, respectively. BLAST analysis revealed 93.4% sequence identity of AAHAP isolate with SpK (KC763473) from Iran while AARS isolate shared maximum identity (94.5%) with the strain 241 (AJ585519) from Australia and clustered with some reference isolates of CMV subgroup II from UK (Z12818) and USA (AF127976) in a Neighbour-joining phylogenetic reconstruction. A total of 59 polymorphic (segregating) sites (S) with nucleotide diversity (π) of 0.06218 was evident while no INDEL event was observed in Pakistani isolates. The evolutionary distance of Pakistani CMV isolates was recorded as 0.0657 with each other and 0.0574-0.2964 with other CMV isolates reported elsewhere in the world. A frequent gene flow (Fst = 0.30478 <0.33) was observed between Pakistani and earlier reported CMV isolates. In genetic differentiation analysis, the value of three permutation-based statistical tests viz; Z (84.3011), Snn (0.82456), and Ks* (4.04042) were non-significant. The statistical analysis revealed the values 2.02535, 0.01468, and 0.71862 of Tajima's D, Fu, & Li’s F* and D* respectively, demonstrating that the CMV population is under balancing selection.

Effects of Drilling Technology on Mini-Implant Primary Stability: A Comparison of the Mechanical Drilling and Femtosecond Laser Ablation

Objectives: Primary stability is a fundamental prerequisite in predicting the prognosis of a mini-implant (MI) as a skeletal anchorage. This study aims to evaluate the influence of implant site preparation technology on the primary stability of MI.Methods: A total of 108 bovine cortical bone samples were fabricated to three thicknesses (0.5, 1.0, and 1.5 mm). For each thickness group, the samples were divided into three subgroups: I (without site preparation), II (site preparation with a mechanical drill), and III (site preparation with femtosecond laser ablation). After MI insertion into these samples, the pull out strength of MI was measured by lateral pull out tests.Results: For the 0.5 mm thickness samples, the lateral pull-out strength was 9.9±2.7 N in subgroup I, 6.7±2.1 N in subgroup II, and 15.2±2.6 N in subgroup III. For the 1.0 mm thickness samples, the lateral pull-out strength was 39.3±2.5N in subgroup I, 38.2±2.7N in subgroup II, and 46.3±1.7 N in subgroup III. For the 1.5 mm thickness samples, the lateral pull-out strength was 73.9±4.8 N in subgroup I, 70.1±2.8 N in subgroup II, 75.0±2.2 N in subgroup III. No signs of carbonization or substantial cracking were visible in any of the bone samples.Conclusion: Site preparation with laser ablation significantly improved the lateral pull-out strength over mechanical preparation and control (no site preparation) in thinner cortical bone samples (1.0 and 0.5 mm). Such improvement in lateral pull-out strength decreases as the samples become thicker and diminishes in thick (1.5 mm) cortical bone samples.

Smilax auriculata: A new host for Orchid fleck dichorhavirusvirus identified in Florida, USA

In June 2020, Orchid fleck virus (OFV) was detected in a species of Liriope in Leon and Alachua County, Florida (Fife et al; 2021). In October of the same year, four adjacent dune/ear-leaf greenbrier vines, Smilax auriculata (Smilaceae: Liliales), showed yellowing and mottling symptoms (Figure 1). Infected and healthy S. auriculata leaves samples were collected in Alachua County by the Florida Department of Agriculture and Consumer Services, Gainesville, Florida. OFV primers successfully detected in four Smilax samples by conventional RT-PCR assay. Amplicon sequences (Acc. No. MZ645935 and MZ645938) shared 99% nucleotide identity with OFV infecting orchids (LC222629) and citrus (MK522804). The OFV subgroup I (OFV-Orc1) and subgroup II (OFV-Orc2) specific primers (Kondo et al 2017) were utilized to confirm the presence of OFV type strains infecting Smilax. Sanger sequencing of subgroup I specific amplicons (MZ645934) shared 99% nucleotide identity with OFV-Orc1 (LC222629) whereas subgroup II specific amplicon sequence (MZ645930) shared 98-99 % nucleotide identity with OFV-Orc2 (AB244417). Further confirmation was done by USDA-APHIS-PPQ-Plant Pathogen Confirmatory Diagnostics Laboratory utilizing optimized conventional RT-PCR protocols (Roy et al. 2020) and deep sequencing on a on a NextSeq550 Illumina platform. Assembled reads identified seven non-overlapping viral contigs. Five RNA1 and two RNA2 contigs covered more than 97% of the bipartite OFV genome with average coverage depth of 5297.61 and 5186.04, respectively. Contigs of RNA1 and RNA2 shared 98-99% nt identity to OFV-Orc2-RNA1 (AB244417) and OFV-Orc-RNA2 (AB244418 and LC222630). No other pathogen sequences were identified. This is the first time the genus Smilax has been identified as a natural host of OFV. Very recent findings of OFV-Orc in Florida in Liriope, Aspidistra, and Ophiopogon among the Asparagaceae family members (Fife et al; 2021) and now in the Smilacaceae suggest a broader host range of the virus than previously known; further research should be conducted to better characterize the potential risk of introduction into citrus in Florida.

Fecal Calprotectin as early predictor for screening and relapse in pediatric chronic diarrhea in India

: IBS and IBD are two abdomen ailments commonly seen in children. IBD is a life-long disorder that includes two major forms of chronic illness UC and CD, IBS may occur when the bowel is sensitive to specific foods or other triggers like stress. The aims of the study is to investigate clinical usefulness of FC as early predictor for screening and differentiating IBD and IBS and to monitor the treatment for relapse and remission in pediatric Indian population. This was hospital based observational cohort study, conducted over a period of twelve months from January 2019 to December 2019 Total 325 patients attending OPD were included in the study, in children between 1-18years in which FC was measured using commercially available CLIA kit. HB, ESR, Hscrp were also assessed, these patients were followed up. Patients were grouped asGroup I: IBS: 115(50:65M/F), Group II: 185 IBD, had Subgroup I:116 CD(48:68M/F);Group A: 62.7% presented with relapse Group B: 37.3% had disease in remission Subgroup II: 69 patients had UC(31:38 M/F); Group A: 60.9% had relapse and Group B: 39.1% in remission after follow up. Subgroup III: Others were 25(7.7%). In Study I: Levels of FC were significantly lower in patients with group I when compared to group II. In Subgroup I & Subgroup II Group A had higher FC levels when compared to group B. In Study II: A significant difference (P&#60;0.001) and lower values of the FC, ESR, Hs-crp, frequency of stools in IBS than in CD and UC patients were observed. In Study III: FC in IBD has positive correlation (p&#60;0.01), with the activity of the inflammatory disorder, HB, ESR, Hs-CRP and with increased frequency of diarrhea. In Study IV Fecal calprotectin value of ≥100 ug/g was diagnostic of IBD with sensitivity of 94.1%, specificity of 82.2%, PPV of 86.32%, NPV of 80.39%. The present study showed that the determination of FC assists to differentiate between IBD and IBS also useful in monitoring of remission and in early prediction of relapse in pediatric IBD.

Indicators of vitamin D in case of Coronavirus infection COVID-19 in children

Purpose — to determine and compare vitamin D levels in children diagnosed with COVID-19-associated pneumonia and healthy children. Materials and methods. The study involved 90 children. The main group consisted of 75 inpatient children with a diagnosis of COVID-19-associated pneumonia (positive PCR — polymerase chain reaction with reverse transcription). Patients in the main group were divided into 2 subgroups: subgroup I consisted of 49 patients with a pneumonia of moderate course, and subgroup II — 26 patients with a pneumonia of severe course. The control group consisted of 15 practically healthy children. The scheme of examination of sick children included anamnestic data, the use of clinical, instrumental and laboratory, immunological examination methods. The concentration of vitamin D in the blood serum of sick and healthy children was determined by the method of enzyme3linked immunosorbent assay (ELISA). The indicators were measured on a Stat Fax 4700 device using a Pishqaman reactive kit (made in Germany). Results. The concentration of vitamin D in blood serum was 1.8 times lower in subgroup I and 2.0 times in subgroup II, compared with the control group. The average values of vitamin D in blood serum compared with the control group were <1.1 times lower in the age group <1 year, 1.1 times lower in the age group 1–3 years, and 2.3 times lower in the age group >3 years. Conclusions. Based on our research, it can be concluded that there is a relationship between vitamin D deficiency and COVID-19. Thus, the average level of vitamin D was lower in children with COVID-19-associated pneumonia, and varied depending on the severity of the disease (more pronounced changes were observed in severe pneumonia). There are also differences in vitamin D levels in the age groups of children with COVID-19-associated pneumonia: in children from age groups under 1 year and 1–3 years old the decrease in serum vitamin D level is less pronounced than in the age group over 3 years old. The main mechanisms of vitamin D involvement in the immune modulation process in COVID-19 require further study. The research was carried out in accordance with the principles of the Helsinki declaration. The study protocol was approved by the Local ethics committee of the participating institution. The informed consent of the patient was obtained for conducting the studies. No conflict of interest was declared by the authors. Key words: children, COVID-19, vitamin D.

The Weak Small RNA-Binding Activity of the 2b Proteins of Subgroup II Cucumber Mosaic Virus Strains Is Insufficient for RNA Silencing Suppression

The 2b proteins encoded by cucumber mosaic virus (CMV) subgroup I strains suppress RNA silencing primarily by competitively binding small RNAs (sRNAs) in the host cell cytoplasm. Interestingly, 2b proteins encoded by CMV subgroup II strains accumulate predominantly in nuclei. Here we determined that whereas the 2b protein (Fny2b) of subgroup IA strain Fny-CMV is highly effective in suppressing both sense RNA-induced and inverted repeat-induced posttranscriptional gene silencing, the 2b protein (LS2b) of the subgroup II strain LS-CMV was not as effective. Reducing nuclear accumulation of LS2b by mutating a residue in its nuclear localization sequence had no effect on RNA silencing suppressor activity, while attenuated viral symptoms. Electrophoretic mobility shift assays showed that the sRNA binding of LS2b was weaker and more selective than that of Fny2b. The domain determining the differential sRNA-binding ability was delimited to the putative helix α1 region. Moreover, LS2b mutants that completely lost suppressor activity still retained their weak sRNA-binding ability, suggesting that sRNA binding is not sufficient for LS2b to suppress RNA silencing. Considering the subgroup I strain-encoded 2b proteins that require sRNA-binding ability for the suppression of RNA silencing, we suggest that in addition to binding sRNA, the 2b proteins of subgroup II CMV strains would require extra biological activities to achieve RNA silencing inhibition.

Chemical Variability and In Vitro Anti-Inflammatory Activity of Leaf Essential Oil from Ivorian Isolona dewevrei (De Wild. & T. Durand) Engl. & Diels

The chemical variability and the in vitro anti-inflammatory activity of the leaf essential oil from Ivorian Isolona dewevrei were investigated for the first time. Forty-seven oil samples were analyzed using a combination of CC, GC(RI), GC-MS and 13C-NMR, thus leading to the identification of 113 constituents (90.8–98.9%). As the main components varied drastically from sample to sample, the 47 oil compositions were submitted to hierarchical cluster and principal components analyses. Three distinct groups, each divided into two subgroups, were evidenced. Subgroup I−A was dominated by (Z)-β-ocimene, β-eudesmol, germacrene D and (E)-β-ocimene, while (10βH)-1β,8β-oxido-cadina-4-ene, santalenone, trans-α-bergamotene and trans-β-bergamotene were the main compounds of Subgroup I−B. The prevalent constituents of Subgroup II−A were germacrene B, (E)-β-caryophyllene, (5αH,10βMe)-6,12-oxido-elema-1,3,6,11(12)-tetraene and γ-elemene. Subgroup II−B displayed germacrene B, germacrene D and (Z)-β-ocimene as the majority compounds. Germacrene D was the most abundant constituent of Group III, followed in Subgroup III−A by (E)-β-caryophyllene, (10βH)-1β,8β-oxido-cadina-4-ene, germacrene D-8-one, and then in Subgroup III−B by (Z)-β-ocimene and (E)-β-ocimene. The observed qualitative and quantitative chemical variability was probably due to combined factors, mostly phenology and season, then harvest site to a lesser extent. The lipoxygenase inhibition by a leaf oil sample was also evaluated. The oil IC50 (0.020 ± 0.005 mg/mL) was slightly higher than the non-competitive lipoxygenase inhibitor NDGA IC50 (0.013 ± 0.003 mg/mL), suggesting a significant in vitro anti-inflammatory potential.

Identification of the Molecular Subgroups in Idiopathic Pulmonary Fibrosis by Gene Expression Profiles

Background. Idiopathic Pulmonary Fibrosis (IPF) is one of the most common idiopathic interstitial pneumonia, which can occur all over the world. The median survival time of patients is about 3-5 years, and the mortality is relatively high. Objective. To reveal the potential molecular characteristics of IPF and deepen the understanding of the molecular mechanism of IPF. In order to provide some guidance for the clinical treatment, new drug development, and prognosis judgment of IPF. Although the preliminary conclusion of this study has certain guiding significance for the treatment of IPF and so on, it needs more accurate analytical approaches and large sample clinical trials to verify. Methods. 220 patients with IPF were divided into different subgroups according to the gene expression profiles, which were obtained from the Gene Expression Omnibus (GEO) database. In addition, these subgroups present different expression forms and clinical features. Therefore, weighted gene coexpression analysis (WGCNA) was used to seek the differences between subtypes. And six subgroup-specific WGCNA modules were identified. Results. Combined with the characteristics of WGCNA and KEGG enrichment modules, the autophagic pathway was only upregulated in subgroup I and enriched significantly. The differentiation pathways of Th1 and Th2 cells were only upregulated and enriched in subgroup II. At the same time, combined with clinical information, IPF patients in subgroup II were older and more serious, which may be closely related to the differentiation of Th1 and Th2 cells. In contrast, the neuroactive ligand-receptor interaction pathway and Ca+ signaling pathway were significantly upregulated and enriched in subgroup III. Although there was no significant difference in prognosis between subgroup I and subgroup III, their intrinsic biological characteristics were very different. These results suggest that the subtypes may represent risk factors of age and intrinsic biological characteristics and may also partly reflect the severity of the disease. Conclusion. In conclusion, current studies have improved our understanding of IPF-related molecular mechanisms. At the same time, because the results show that patients from different subgroups may have their own unique gene expression patterns, it reminds us that patients in each subgroup should receive more personalized treatment.

Genomic diversity, life strategies and ecology of marine HTVC010P-type pelagiphages

SAR11 bacteria dominate ocean surface bacterioplankton communities, and play an important role in marine carbon and nutrient cycling. The biology and ecology of SAR11 are impacted by SAR11 phages (pelagiphages) that are highly diverse and abundant in the ocean. Among the currently known pelagiphages, HTVC010P represents an extremely abundant but under-studied phage group in the ocean. In this study, we have isolated seven new HTVC010P-type pelagiphages, and recovered 77 nearly full-length HTVC010P-type metagenomic viral genomes from marine metagenomes. Comparative genomic and phylogenomic analyses showed that HTVC010P-type pelagiphages display genome synteny and can be clustered into two major subgroups, with subgroup I consisting of strictly lytic phages and subgroup II mostly consisting of phages with potential lysogenic life cycles. All but one member of the subgroup II contain an integrase gene. Site-specific integration of subgroup II HTVC010P-type pelagiphage was either verified experimentally or identified by in silico genomic sequence analyses, which revealed that various SAR11 tRNA genes can serve as the integration sites of HTVC010P-type pelagiphages. Moreover, HTVC010P-type pelagiphage integration was confirmed by the detection of several Global Ocean Survey (GOS) fragments that contain hybrid phage–host integration sites. Metagenomic recruitment analysis revealed that these HTVC010P-type phages were globally distributed and most lytic subgroup I members exhibited higher relative abundance. Altogether, this study significantly expands our knowledge about the genetic diversity, life strategies and ecology of HTVC010P-type pelagiphages.