T is an aerobic, non-motile and non-spore-forming Gram-positive coccus isolated from the stools of a Burkinabe woman. In this report, we present its phenotypic description including MALDI-TOF mass spectrometry analysis and genome sequencing. Strain Marseille-Q0835T; 2.9768-Mb genome exhibited a 41.9 mol% G+C content and 2699 predicted genes. Considering phenotypic features and comparative genome studies, we propose the strain Marseille-Q0835T as the type strain of Enterococcus burkinafasonensis sp. nov., a new species within the family Enterococcaceae.Strain Marseille-Q0835 Enterococcus species purposely named Enterococcus burkinafasonensis.Culturomics strategy is a high-throughput culturing method . This stBiotyper 3.0 software  and analysed against the main spectra of the bacteria included in the database.In September 2018, a fresh stool sample was collected from an apparently healthy 28-year-old Burkinabe woman who was admitted for diagnosis check-up in the Regional Tuberculosis Control Centre, Bobo-Dioulasso, Burkina Faso. A stool sample was sent to the collaborative laboratory at IHU in Marseille, France for culturomics analysis, which isolated an unidentified bacterial strain from the stool. The study was validated by the Science and Health Research Ethics Committee of Bobo-Dioulasso, under number (N/Ref.002-2018-CEIRS). The bacterium here referred to as strain Marseille-Q0835 was isolated on Columbia sheep blood agar after a 24-hour incubation under aerobic atmosphere at 37°C and pH 7.5. Purified colonies could not be identified by MALDI-TOF MS. The screening was performed on a Microflex LT spectrometer , as previously described [CodonCode Aligner software (http://www.codoncode.com). BLASTn research was conducted using nucleotide databases for cross-species comparison (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). The search was limited to records that include sequences from type material, and exclude uncultured/environmental sample sequences. The result showed that strain Marseille-Q0835 exhibited a 97.80% sequence identity with Enterococcus gallinarum strain LMG 13129 (GenBank accession number NR_104559.2), the phylogenetically closest species with standing in nomenclature  and sequencing using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and ABI Prism 3130xl Genetic Analyzer capillary sequencer , as previously described . The 16Snclature . We consEnterococcus sp. Marseille- Q0835T showed negative catalase and oxidase activities. API 50CH and API ZYM tests  were performed at 37°C under aerobic conditions and the results are summarized in Enterococcus sp. nov. strain Marseille-Q0835T with other bacterial species  with the EZ1 DNA tissue kit and then sequenced on MiSeq technology  with the Nextera XT Paired end (Illumina), as previously described . The assVelvet , Spades  [Spades  and Soapp Denovo ) on trimmmomatic ) or raw  removed . The besEnterococcus sp. Marseille-Q0835T with closely related species was estimated using the OrthoANI software version 0.93.1 (https://www.ezbiocloud.net/tools/orthoani) [Enterococcus malodoratus strain DSM 20681 and Enterococcus gallinarum strain LMG 13129 to 91.81% for Enterococcus devriesei strain DSM 22802 and Enterococcus viikkiensis strain LMG 26075. When the isolate was compared with these closely related species, values ranged from 70.07% with E. gallinarum strain LMG 13129 to 73.41% with Enterococcus pseudoavium strain CBA7133. These values are lower than the 95% threshold used to discriminate bacterial species [.The degree of genomic similarity of rthoani) . Values  species .In silico DNA–DNA hybridization values obtained using the GGDC version 2.0 online tool (http://ggdc.dsmz.de/ggdc.php) are reported in T, these values ranged from 20% with E. devriesei strain DSM 22802 to 26.8% with Enterococcus faecium strain ISMMS VRE 1. Such values were lower than the 70% threshold recognized as delineating distinct species [. species ,17.TableT exhibited a 16S rRNA gene sequence divergence <98.65%, DNA–DNA hybridization values < 70% and an OrthoANI value < 95% with its phylogenetically closest species with standing in nomenclature, together with unique phenotypic features. We formally propose strain Marseille-Q0835T as the type strain of the new species named Enterococcus burkinafasonensis.Strain Marseille-Q0835Enterococcaceae within the phylum Firmicutes. The type strain Marseille-Q0835T (CSUR P0835) was isolated after a 24-hour incubation at 37°C and pH 7.5 in an anaerobic atmosphere of a fresh stool sample collected from a 28-year-old Burkinabe woman. Colonies were smooth, white with entire edges with an average diameter of 1 mm. Bacterial cells were Gram-positive, coccus-shaped, non-motile and non-spore-forming with negative catalase and oxidase activities . The bacterium belongs to the family ties see .Fig. 4HeT exhibits positive reaction for esterase lipase (C8), lipase (C14), naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase and N-acetyl-β-glucosaminidase, but negative reaction for alkaline phosphatase, esterase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase and α-fucosidase. Using an API 50CH strip, positive reactions were obtained for d-galactose, d-glucose, d-fructose, l-arabinose, d-ribose, d-mannose, l-rhammose, d-mannitol, N-acetylglucosamine, arbutin, esculin, salicin, d-cellobiose, d-maltose, d-trehalose and gentibiose.Using an APIZYM strip, strain Marseille-Q0835T genome is 2.9768 Mb long, with a G-C content of 41.9%.The strain Marseille-Q0835The 16S rRNA gene and genome sequences were deposited in GenBank under accession number LR742708 and NZ_CACSLH000000000, respectively.T has been deposited in the French culture collection centre, Collection de Souches de l’Unité des Rickettsies (CSUR), under the number Q0835.The strain Marseille-Q0835None to declare.10.13039/100007356IHU Méditerranée Infection  and by the French Government under the Investissements d'avenir (Investments for the Future) programme managed by the 10.13039/501100001665Agence Nationale de la Recherche , (reference: Méditerranée Infection 10-IAHU-03).This work was funded by the


E. coli. Terpene synthase activity was detected for 15 of these enzymes, and included mono-, sesqui- and diterpene synthase activities. A number of confirmed sesquiterpene synthases also exhibited promiscuous monoterpene synthase activity, suggesting that bacteria are potentially a richer source of monoterpene synthase activity then previously assumed. Several terpenoid products not previously detected in bacteria were identified, including aromandendrene, acora-3,7(14)-diene and longiborneol. Overall, we have identified promiscuous terpene synthases in bacteria and demonstrated that terpene synthases with substrate promiscuity are widely distributed in nature, forming a rich resource for engineering terpene biosynthetic pathways for biotechnology.Terpenes are the largest class of natural products with extensive structural diversity and are widely used as pharmaceuticals, herbicides, flavourings, fragrances, and biofuels. While they have mostly been isolated from plants and fungi, the availability and analysis of bacterial genome sequence data indicates that bacteria also possess many putative terpene synthase genes. In this study, we further explore this potential for terpene synthase activity in bacteria. Twenty two potential class I terpene synthase genes (TSs) were selected to represent the full sequence diversity of bacterial synthase candidates and recombinantly expressed in  The vast majority of terpenoids have been isolated from plants and fungi; however, bacteria are also known producers of volatile odoriferous metabolites. All terpenoids are synthesised from the universal C5 isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are joined by isoprenyl transferases to form isoprenyl diphosphate substrates of varying lengths, such as geranyl diphosphate , farnesyl diphosphate  and geranylgeranyl diphosphate . Terpene synthases (TSs) convert the linear isoprenyl diphosphate substrates into structurally diverse mono- (C10), sesqui- (C15) and diterpene (C20) scaffolds. Due to their structural diversity, terpenoids have a wide range of industrial applications as pharmaceuticals, flavourings, fragrances, antimicrobials, pesticides and alternative fuels . Al. AlSoranous work . When Scous work . These rubstrate .Streptomyces clavuligerus are well studied [S. clavuligerus accepts GPP and FPP [Kitasatospora setae with GPP yielded linalool as a major product and small amounts of several acyclic and cyclic monoterpenes [Until now, only two bacterial monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthases (bCinS) from  studied  and stru studied . Broad s studied , a bLinS and FPP , and incterpenes .CFM47198.1, ELQ82238.1, ACY98649.1, EXG82599.1, ABU58787.1, EJL71407.1, KFE96946.1 were tested for compound production with FPP and GPP but no terpenoids were detected. in vivo product formation, all identified TSs were cloned into the pBbB2a-GPPS  backbone plasmid and co-expressed with pMVA plasmid in E. coli [in vivo conditions ,4,11-germacratriene as seen in the in vitro assays 11-diene and (+)-γ-gurjunene when expressed in E. coli  from  E. coli . WhereasSM 43183  and EXG8SM 44712  producednditions .Streptomyces coelicolor A3(2) when expressed in E. coli produced various monoterpenes: β-myrcene, β-ocimene, linalool and geraniol  from E. coli with two mutations D2G, C155G (ispAM22), which can function as geranylgeranyl diphosphate synthase [in vivo. GGPP synthase activity using the variant prenyltransferase was confirmed by overexpression of spata-13, 17-diene synthase from Streptomyces xinghaiensis [ispAM22). This yielded the diterpene spata-13,17-diene, where the produced compound was extracted using a nonane layer  from Roseiflexus castenholzii DSM 13941  and Arctic Express (DE3) cells for recombinant protein expression.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableKnown TSs annotated in the tree in (DOCX)Click here for additional data file.S1 FigNeighbour-joining tree constructed from the amino acid sequences of characterized terpene synthases in this study (underlined) and in previous studies. Geosmin synthases (dark red), 2-methylisoborneol synthases (orange), monoterpene (light blue), diterpene (dark blue) and sesquiterpene synthases (purple) are shown and uncharacterized bacterial terpene synthases in this study (in red).(DOCX)Click here for additional data file.S2 Fig-1) on a HP5 column and its retention time at 7.5 minutes. B. Mass spectra of geosmin.GC-MS traces showing the separation of geosmin (0.1 mg mL(DOCX)Click here for additional data file.S3 FigGC-MS chromatogram of geosmin synthase with 75 μM of FPP. Peak 1: geosmin (rt: 7.5), peak 2: Germacrene D (rt: 7.86), peak 3: germacradienol (rt: 8.84). B. Mass spectra of compounds observed in the extracts.(DOCX)Click here for additional data file.S4 Fig-1) produced in this study on a HP5 column. The internal standard, sec-butyl benzene , has a retention time of 9.5 minutes. Peak 1: R- Limonene (rt: 9.41). B: Mass spectra of limonene.A. GC-MS traces showing the separation of limonene (0.1 mg mL(DOCX)Click here for additional data file.S5 FigA. GC chromatogram of 10 μg of limonene synthase (pJBEI-6410) crude extracts with 75 μM of GPP. Peak 1: limonene (rt: 9.41). B. Mass spectra of limonene.(DOCX)Click here for additional data file.S6 FigA. GC-MS traces showing the separation of standard nerolidol mix (0.1 mg mL-1) on a HP5 column. B. MS spectra of -cis and -trans Nerolidol. C. GC-MS chromatogram of trans-nerolidol produced by AHY47823 with FPP. D. Mass spectra for trans-nerolidol produced by AHY47823.(DOCX)Click here for additional data file.S7 Fig-1) on a CP-Chirasil-DEX-CB column. B. GC-MS chromatogram of linalool isomers produced by AHY47823 with FPP. Samples were analyzed by GC on an Agilent Technologies 7890A GC system equipped with an FID detector, a 7693 auto sampler, and a CP-Chirasil-DEX-CB column . For linalool, the program initiated at a temperature of 70°C which was then increased to 90°C at 8°C/min. This was followed by an increase in temperature to 150°C at a rate of 2°C/min and then to 190°C at 40°C/min (1 min hold). The FID detector was maintained at a temperature of 200°C with a flow of hydrogen at 30 mL/min.A. GC-MS traces showing the separation of standard linalool mix (0.1 mg mL(DOCX)Click here for additional data file.S8 Fig-1) on a CP-Chirasil-DEX-CB column. B. GC-MS chromatogram of linalool isomers produced by AHY45426 with FPP. Samples were analyzed by gas chromatography on an Agilent Technologies 7890A GC system equipped with an FID detector, a 7693 auto sampler, and a CP-Chirasil-DEX-CB column . For linalool, the program initiated at a temperature of 70°C which was then increased to 90°C at 8°C/min. This was followed by an increase in temperature to 150°C at a rate of 2°C/min and then to 190°C at 40°C/min (1 min hold). The FID detector was maintained at a temperature of 200°C with a flow of hydrogen at 30 mL/min.A. GC-MS traces showing the separation of standard linalool mix (0.1 mg mL(DOCX)Click here for additional data file.S9 FigTotal ion chromatograms of products obtained from an incubation RrNerS (left) and RrBerS (right) with FPP for 3 hours at variable temperatures. Samples were analyzed by GC-QToF on HP5 column.(DOCX)Click here for additional data file.S10 FigA. α–bergamotene produced by RrBerS, B. Aromandendrene produced by ScAroS, C and D. cadinene and longiborneol produced by BpLonS, E. germacrene D produced byKaGerS, F and G. copaene and acora-3,7(14)–diene produced byAbAcoS, and H and I. β-elemene and 1(10),4,7(11)-germacra-triene produced by MiGerS.The reference spectra from the NIST library shown in blue and the compound spectra shown in red.(DOCX)Click here for additional data file.S11 FigA. GC-MS traces showing the separation of monoterpenoids (0.1 mg mL-1) standards used in this study on a HP5 column. The internal standard used, sec-butyl benzene , has a retention time of 9 minutes. Peak 1: cis-ocimene (rt: 9.56), peak 2: trans-ocimene (rt: 9.72), 3: linalool (rt: 10.43). B. Comparison of obtained mass spectra with NIST Library spectra and standard as shown in A. of products yielded by CAN96536 with GPP in in vitro assay.(DOCX)Click here for additional data file.S12 FigA. BpTPS; B. ScTPS1; C. CvTPS; D. SsTPS; E. ScTPS2; F. SniTPS; G.SnaTPS. The peaks in the chromatogram do not correspond to any terpenoids.GC-MS chromatograms of extracts profile with FPP are shown for (DOCX)Click here for additional data file.S13 FigA. GC-MS chromatogram for geosmin synthase products, B. mass spectra for Germacradienol, C—G. Comparison of obtained mass spectra with NIST Library spectra of products yielded by GeoS.(DOCX)Click here for additional data file.S14 FigA–B. SrGuaS C. TcCubS D. CaCubS.(DOCX)Click here for additional data file.S15 FigA. spata-13,17-diene produced by overexpression of ispAM22 and WP_095757924 in E. coli. B. Mass spectra of produced spata-13,17-diene.(DOCX)Click here for additional data file.S16 Fig(DOCX)Click here for additional data file.S17 Fig(DOCX)Click here for additional data file.S18 Fig(DOCX)Click here for additional data file.


Vitamin D (VitD) deficiency during pregnancy has been associated with adverse neonatal outcomes and increased risk of late pregnancy complications. We planned to correlate serum VitD biomarkers; 25-hydroxyvitamin D (25-OH-VitD) and 1,25-dihydroxyvitamin D  levels; and their ratio with the frequency of feto-maternal pregnancy complications. A prospective cross-sectional case-control study was conducted at Aljouf Maternity and Children Hospital, Sakaka, Saudi Arabia, during the period of September 1, 2017 to September 30, 2019. 322 pregnant women were stratified into 2 groups: controls (110 cases) and complicated group (212 cases). The later comprised severe preeclamptic toxemia associated with intrauterine growth restriction (58 cases), gestational diabetes mellitus , abortion (26 cases), undisturbed ectopic pregnancy (16 cases), premature rupture of membranes , and, inevitable preterm labour (16 cases). After clinical assessment, peripheral blood samples were collected. Serum biomarkers were measured using specific immunoassays. The direct 1,25-diOH-VitD/25-OH-VitD ratio was calculated. Serum 25-OH-VitD indicated widely spreading VitD deficiency among participants with significantly higher levels in controls vs. GDM subgroup only. 1,25-diOH-VitD levels and the ratio were markedly reduced in the six complicated subgroups vs. controls, with non-significant differences amongst the complicated subgroups. ROC analysis showed very high sensitivity and specificity, to differentiate patients from controls, only for 1,25-diOH-VitD  followed by the ratio but not 25-OH-VitD. In conclusions, 25-OH-VitD did not show significant changes except for GDM. 1,25-diOH-VitD levels and the ratio showed strong associations with pregnancy complications. Serum 1,25-di-OH-VitD and its ratio to 25-OH-VitD are more reliable and physiologically relevant biomarkers for VitD status in pregnancy. Vitamin D (VitD) deficiency became a global epidemic, particularly for women of reproductive age. Nuclear receptor-mediated, the dihydoxy active form of VitD, 1,25-diOH-VitD, controls cellular proliferation, differentiation and apoptosis through targeting ≥1000 genes. Nevertheless, pathogenetic role of hypovitaminosis D in a number of comorbidities is still hotly debated Linked to hypovitaminosis D, adverse fetal outcomes included abortion, intrauterine growth restriction, fetal death and congenital malformations. Maternal adverse effects involved preeclampsia, gestational diabetes mellitus (GDM) and increased risk of preterm labor We planned to assess the relationship between hypovitaminosis D and feto-maternal pregnancy complications and morbidities, using the more patho-physiologically relevant biomarkers. We measured serum 25-OH-VitD, as the established VitD biomarker, and, 1,25-diOH-VitD as a functional VitD biomarker, and their ratio, and, correlated them with the existing pregnancy feto-maternal status.This prospective cross-sectional case control study was carried out at Aljouf Maternity and Children Hospital, Sakaka, Saudi Arabia, during the period between September 1, 2017 and September 30, 2019. It was approved by the local bioethical committees of Jouf University and Ministry of Health (#6-16-4/40). Written informed consents were obtained. The exclusion criteria were uncertain diagnosis, obesity, anemia, endocrinal disorders , immobilization for any reason, on anti-convulsion drug, acute infections, malabsorption syndromes, comorbid chronic medical disorder , multiple pregnancy, antepartum hemorrhage and superimposed preeclampsia.Among 450 pregnant women attending the hospital, 322 voluntary participants fulfilling the inclusion criteria were recruited. The 322 participants were classified into 2 groups; control group of 110 women who had normal feto-maternal pregnancy, and, complicated pregnancy group comprising 212 participants. The latter group was further subdivided into: severe preeclamptic toxemia (PET) associated with intrauterine growth restriction , GDM (82 cases), abortion (26 cases), undisturbed ectopic pregnancy (16 cases), premature rupture of membranes , and, inevitable preterm labor (16 cases).After history taking  and general examination , the head, face, neck, chest, heart, back, lower limbs and abdomen were systematically examined. The pregnancy was assessed ultra-sonographically for viability and fetal biometry to determine pregnancy duration and the expected fetal weight, condition of amniotic fluid and for diagnosing fetal anomalies, if present. Women with routine laboratory findings that contradict the inclusion criteria were excluded.oC. Specific quantitative ELISA immunoassay kits  were used to measure total 25-OH-VitD  and 1,25-DiOH-VitD . The direct 1,25-DiOH-VitD/25-OH-VitD ratio was calculated for each patient. Participants were stratified using the clinically relevant 25-OH-VitD cutoff levels of high/toxic as ≥50/>80 ng/mL, normal as ≥30-50 ng/mL, insufficient as ≥20 - <30 ng/mL, deficient as ≥10 - <20 ng/mL and severely deficient as <10 ng/mL Serum was recovered from peripheral blood samples by centrifugation and was aliquotted and stored till used at -80 Data was analyzed using SPSS . We expressed descriptive qualitative data as frequency and percentage, and, quantitative data as range and mean ± standard deviation (SD). Normal distribution was checked by Kolmogorov-Sminov test. One-way ANOVA with Tukey's post-test was employed for multiple comparisons. Correlation among variables was assessed by Spearman correlation coefficient test. Receiver Operating Characteristic (ROC) curve analysis was used to determine the area under curve (AUC) for 25-OH-VitD, 1,25-DiOH-VitD and their ratio to check their sensitivity and specificity to differentiate the cases from controls. P value of <0.05 at a confidence level of 95% was considered significant.Age was nonsignificantly different among all groups except for preeclamptics vs. each of controls (p = 0.011) and PROMs (p = 0.014), and, PROMs vs. GDM (p = 0.035). BMI showed nonsignificant difference among the investigated groups except comparing GDM vs. each of controls (p = 0.002), abortion (p = 0.02), ectopic pregnancy (p <0.001) and PROMs (p <0.001); and preeclamptics vs. each of ectopic pregnancy (p <0.028) and PROM (p = 0.045). Gravidity showed nonsignificant difference among the investigated groups except comparing GDM vs. each of controls (p <0.001), preeclamptics (p <0.001), abortion (p <0.036), ectopic pregnancy (p = 0.002) and PROMs (p = 0.002). Parity showed nonsignificant difference among the investigated groups except comparing GDM vs. each of preeclamptics (p <0.001), abortion (p = 0.035), ectopic pregnancy (p = 0.002) and PROMs (p <0.035). Duration of pregnancy showed very strong significant difference among the investigated groups with a few non-significant exceptions; it was significantly different comparing controls vs. all complication subgroups (p <0.01 - 0.001) except GDM, comparing preeclamptics vs. all other subgroups (p <0.05 - <0.001) except preterm labour, comparing GDM vs. the others (p <0.001), comparing abortion vs. the others (p <0.001) except ectopic pregnancy, and, comparing ectopic pregnancy vs. the others (p <0.001) except PROMs.Serum 25-OH-VitD levels showed nonsignificant difference among the investigated groups except comparing GDM vs. each of controls (p <0.001) and ectopic pregnancy (p = 0.01). Serum 1,25-diOH-VitD levels showed nonsignificant difference among the investigated groups of patients. However, controls had markedly higher levels vs. each of the six complicated groups (p <0.001). Similarly, the direct 1,25-diOH-VitD/25-OH-VitD ratio showed very marked significant reduction comparing controls vs. each of the six patients' groups (p <0.001), albeit, with nonsignificant difference among the complicated groups.Using serum 25-OH-VitD as the biomarker for VitD status, 43.636% of controls were deficient (≥10 ng/mL), 38.181% were insufficient (≥20 ng/mL), and, equal proportions (9.09%) were either normal (≥30 ng/mL) or having toxic levels (>80 ng/mL). 82.759% of preeclamptic cases were insufficient, 13.793% were deficient and 3.448% had normal levels. 82.927% of GDM cases were deficient, 12.195% were insufficient and 4.878 had normal levels. 84.615% of abortion cases deficient, and equal proportions (7.692%) had either insufficient or toxic levels. 50.0% of ectopic pregnancy cases were deficient, 37.5% were insufficient and the rest (12.5%/2) had toxic levels. 71.429%/ of PROMs cases were deficient and 28.571% were insufficient. 75% of preterm labor cases were insufficient and 25.0% were deficient. Fortunately, severe deficiency (<10 ng/mL) was not observed in our cases. Therefore, VitD insufficiency/deficiency is widely spreading among the study participants . Controls presented a better picture only considering insufficiency/deficiency proportion, and, % of normal/super-normal levels.ROC curve analysis showed that 1,25-DiOH-VitD level is the most sensitive and most specific biomarker for differentiating between cases and controls; with an AUC of 0.965 . It is followed by 1,25-DiOH-VitD25-OH-VitD ratio; with AUC of 0.843 . 25-OH-VitD levels fell near the diagonal line with no effect, i.e., showing least sensitivity and least specificity  , parity (0.468/<0.001) and BMI (0.837/<0.001), and negative relationship with the ratio (-0.155/<0.05). Gravidity showed positive relationship vs. parity (0.958/<0.001) and BMI (0.480/<0.001). Parity correlated positively with BMI (0.448/<0.001). BMI correlated negatively vs. the ratio (-0.184/= 0.027). Pregnancy duration correlated negatively with 1,25-DiOH-VitD (-0.241/<0.006) and ratio (-0.192/= 0.022). 25-OH-VitD correlated positively vs. 1,25-DiOH-VitD (0.157/<0.05) and negatively vs. the ratio (-0.647/<0.001). 1,25-DiOH-VitD correlated positively with the ratio (0.562/<0.001).For preeclamptic cases, age had positive correlation vs. gravidity (0.682/<0.001), parity (0.608/<0.001) and BMI (0.690/<0.001), and, negative correlation vs. 1,25-DiOH-VitD (-0.267/= 0.021). Gravidity related had positively with parity (0.962/<0.001) and BMI (0.497/<0.001), but, negatively vs. 1,25-DiOG-VitD (-0.282/= 0.016). Parity correlated positively vs. BMI (0.424/<0.001), and, negatively with 1,25-DiOH-VitD (-0.218/<0.05). BMI correlated positively vs. duration of pregnancy (0.333/= 0.005) and negatively vs. 1,25-DiOH-VitD (-0.275/= 0.017). 25-OH-VitD correlated negatively with the ratio (-0.490/<0.001). 1,25-DiOH-VitD correlated positively vs. the ratio (0.853/<0.001).For GDM cases, age correlated positively vs. each of gravidity (0.680/<0.001), parity (0.732/<0.001), BMI (0.657/<0.001) and 25-OH-VitD (0.239/= 0.015), but, negatively vs. each of 1,25-DiOH-VitD (-0.179/<0.05) and the ratio (-0.320/<0.002). Gravidity correlated positively with parity (0.865/<0.001), BMI (0.671/<0.001) and 25-OH-VitD (0.216/<0.026), and, negatively vs. duration of pregnancy (-0.294/<0.004) and the ratio (-0.183/<0.05). Parity correlated positively vs. BMI (0.725/<0.001) and 25-OH-VitD (0.187/0.047), and, negatively vs. duration of pregnancy (-0.179/<0.05). BMI correlated positively vs. 25-OH-VitD (0.412/<0.001) and negatively vs. the ratio (-0.225/<0.021). 25-OH-VitD correlated negatively with the ratio (-0.757/<0.001). 1,25-DiOH-VitD had positive correlation vs. the ratio (0.592/<0.001).For abortion cases, age correlated positively with gravidity (0.406/<0.02), parity (0.535/= 0.0024), BMI (0.757/<0.001), and pregnancy duration (0.337/= 0.046), and, negatively vs. 1,25-DiOH-VitD (-0.669/<0.001) and the ratio (-0.380/<0.028). Gravidity correlated positively with parity (0.815/<0.001) and duration of pregnancy (0.565/= 0.0013), and, negatively vs. each of 1,25-DiOH-VitD (-0.333/= 0.048) and the ratio (-0.381/ = 0.027). Parity related positively with duration of pregnancy (0.358/= 0.036). BMI associated negatively with 1,25-DiOH-VitD (-0.385/= 0.026). Pregnancy duration correlated negatively vs. 1.25-DiOH-VitD (-0.371/<0.031) and the ratio (-0.490/<0.006). 25-OH-VitD correlated negatively vs. the ratio (-0.773/<0.001). 1,25-DiOH-VitD correlated positively vs. the ratio (0.665/<0.001).For the ectopic pregnancy cases, age correlated positively vs. gravidity (0.884/<0.001), parity (0.927/<0.001), and BMI (0.922/<0.001), but, negatively vs. 1,25-DiOH-VitD (-0.639/<0.005) and the ratio (-0.527/<0.02). Gravidity correlated positively vs. parity (0.906/<0.001) and BMI (0.854/<0.001), and, negatively vs. 1,25-DiOH-VitD (-0.565/<0.013). Parity correlated positively with BMI (0.933/<0.001), and, negatively vs. 1,25-DiOH-VitD (-0.624/<0.006) and the ratio (-0.437/<0.05). BMI correlated positively vs. duration of pregnancy (0.433/<0.05), and, negatively vs. 1,25-DiOH-VitD (-0.443/= 0.044). Pregnancy duration related negatively vs. 1,25-DiOH-VitD (-0.504/<0.027). 25-OH-VitD correlated negatively vs. the ratio (-0.690/= 0.002). 1,25-DiOH-VitD correlated positively with the ratio (0.647/= 0.004).Among PROMs cases, age correlated positively vs. gravidity (0.578/<0.018), parity (0.578/<0.018), BMI (0.727/= 0.002), 1,25-DiOH-VitD (0.500/<0.038) and the ratio (0.474/<0.05). Gravidity correlated positively vs. parity (1.0/<0.001), BMI (0.517/= 0.03) and duration of pregnancy (0.459/<0.05), but, negatively vs. 25-OH-VitD (-0.543/<0.026). Parity associated positively vs. BMI (0.517/= 0.03) and pregnancy duration (0.467/<0.05), but, negatively vs. 25-OH-VitD (-0.543/<0.026). BMI positively correlated with 1,25-DiOH-VitD (0.800/<0.001) and the ratio (0.655/= 0.007). 25-OH-VitD correlated positively with 1,25-DiOH-VitD (0.468/<0.048). 1,25-DiOH-VitD correlated positively vs. the ratio (0.786/<0.001).Within the preterm labor cases, age correlated positively vs. gravidity (0.898/<0.001), parity (0.908/<0.001), BMI (0.952/<0.001), and the ratio (0.452/<0.042), but, negatively vs. 25-OH-VitD (-0.439/<0.05). Gravidity correlated positively vs. parity (0.933/<0.001), BMI (0.802/<0.001) and the ratio (0.503/= 0.025). Parity correlated positively vs. BMI (0.835/<0.001) and the ratio (0.454/= 0.04), but, negatively with 25-OH-VitD (-0.454/= 0.04). BMI associated negatively with 25-OH-VitD (-0.500/<0.027), but, positively vs. the ratio (0.548/= 0.016). 1,25-DiOH-VitD correlated positively vs. the ratio (0.786/<0.001).st trimester of pregnancy, PROMs occurs after 37th weeks and before onset of labor and GDM is common at beginning of the last trimester), it showed significant differences comparing all groups Age was insignificantly different among our complicated groups. It had significant difference only when controls are compared to preeclamptics. Preeclampsia usually attacks extremely aged mothers; mainly primigravida. Gravidity showed a significant difference between the controls and GDM groups. Repeated pregnancy is a definite risk factor for GDM development. BMI was significantly different only comparing GDM and controls. GDM usually associates with higher BMI. As the gestational age is unique for the occurring complication  the traditionally low VitD foods, 2) a scalding local summer sun and ice-cold winter that give very narrow room for sun exposure, 3) low supplement intake, and, 4) absence of a national mandatory VitD food fortification program.The difficulties faced us carrying out this study included: 1) Absence of a reasonable number of other pregnancy complications such as congenital anomalies, stillbirth, isolated IUGR, 2) we could not correlate findings with comorbid rate of infection and inflammation, length of hospital stays, and long-term outcomes, & 3) Longitudinal analysis was not accessible.VitD deficiency/insufficiency is widely spreading in the studied population of pregnant women. Levels of 25-OH-VitD were not different among controls and complicated pregnancies except for a significantly lower level in GDM. 25-OH-VitD levels did not correlate with most of the characteristics of participants and pregnancy complications. 1,25-diOH-VitD and its ratio to 25-OH-VitD revealed very highly significant reduction in complicated pregnancy and were highly sensitive and specific in differentiating controls from complicated pregnancies. They also correlate with most of the characteristics of the participating women. 1,25-diOH-VitD and its ratio to 25-OH-VitD are potentially more functionally relevant biomarkers for VitD status particularly during pregnancy.


Noroviruses  are a common cause of acute gastroenteritis in all age groups. These small non-envelope viruses with a single-stranded (+)RNA genome are characterized by high genetic variability. Continuous changes in the genetic diversity of co-circulating noroviruses and the emergence of new recombinant variants are observed worldwide. Recently, new recombinant noroviruses with a novel GII.P16 polymerase associated with different capsid proteins VP1 were reported. As a part of the surveillance study of sporadic cases of acute gastroenteritis in Novosibirsk, a total of 46 clinical samples from children with diarrhea were screened in 2016. Norovirus was detected in six samples from hospitalized children by RT-PCR. The identified noroviruses were classified as recombinant variants GII.P21/GII.3, GII. Pe/GII.4_Sydney_2012, and GII.P16/GII.4_Sydney_2012 by sequencing of the ORF1/ORF2 junction. In Novosibirsk, the first appearance of the new recombinant genotypeGII.P16/ GII.4_Sydney_2012 was recorded in spring 2016. Before this study, only four complete genome sequences of the Russian GII.P16/GII.3 norovirus strains were available in the GenBank database. In this work, the complete genome sequence of the Russian strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 (GenBank KY210980) was determined. A comparison of the nucleotide and the deduced amino acid sequences showed a high homology of the Russian strain with GII.P16/GII.4_Sydney_2012 strains from other parts of the world. A comparative analysis showed that several unique substitutions occurred in the GII.P16 polymerase, N-terminal p48 protein, and minor capsid protein VP2 genes, while no unique changes in the capsid VP1 gene were observed. A functional significance of these changes suggests that a wide distribution of the strains with the novel GII.P16 polymerase may be associated both with several amino acid substitutions in the polymerase active center and with the insertion of glutamic acid or glycine in an N-terminal p48 protein that blocks the secretory immunity of intestinal epithelial cells. Further monitoring of genotypes will allow determining the distribution of norovirus recombinants with the polymerase GII.P16 in Russia. Noroviruses  areconsidered to be one of the common causes of outbreaks andsporadic cases of acute gastroenteritis (AGE) in humans ofall ages . Norovirus infection can causesevere outcomes of the disease in very young and elderlyindividuals, as well as chronic diarrhea, lasting from severalmonths to several years, in immunocompromised and cancerpatients, and humans after organ transplantation . Dueto the low infectious dose  and highresistance in the environment, noroviruses are rapidly transmittedperson-to-person, by food and water . A meta-analysis of epidemiological datafrom many countries showed that the incidence of norovirusinfection among patients with AGE regardless of their age was17–20 % in 2008–2014 . The prevalenceof asymptomatic norovirus infection is estimated from 4 to18 % in different regions .The polyadenylated single-stranded (+)RNA genome ofnorovirus (~7.5 kb) contains three overlapping open readingframes (ORF1–ORF3) . ORF1 encodes alarge polyprotein that is post-translationally cleaved by viralprotease into six nonstructural proteins, including RNAdependentRNA polymerase (RdRp); ORF2 and ORF3 encodemajor (VP1) and minor (VP2) capsid proteins, respectively.Two mechanisms of norovirus genetic variability have beenidentified: point mutations and recombination . Due to recombination events occurring in the norovirusgenome near the overlapping region of the 3ʹ-end of ORF1(RdRp) and 5ʹ-end of ORF2 (VP1), a dual nomenclature ofnoroviruses defining the RdRp/VP1 genotypes was recentlydeveloped .https://www.rivm.nl/mpf/typingtool/norovirus/). The average duration ofgenotype-specific immunity after norovirus infection can befrom 4 to 8 years , however, due to theexistence of a wide range of genetic variants, subsequent norovirusinfection with other antigenic variants or “immunotypes”can occur in a shorter time .Noroviruses exhibit significant genetic and antigenic diversity.Based on the VP1 amino acid sequence, noroviruses arecurrently classified into at least seven genogroups (GI–GVII),which are further sub-divided into more than forty genotypes. It has been established that GI, GIIand GIV noroviruses can cause disease in humans . In the most common genogroup GII,at least 31 RdRp genotypes and 23 VP1 genotypes are distinguished,and their combination is designated as GII.Px/GII.x. In 2012, a newrecombinant GII.4 norovirus classified as GII.Pe/GII.4_Sydney_2012 appeared and later became the dominant strainworldwide . However, changes in themolecular epidemiology of norovirus have been observed inrecent years. In the winter season 2014/2015, a new GII.P17/GII.17 strain, which was first registered in China, quicklyreplaced the GII.Pe/GII.4_Sydney_2012 variant and initiallyspread to Asia, and later, to other regions . Recently, the prevalence of new recombinant norovirusstrains with the GII.P16 polymerase associated with multipleVP1 genotypes, including GII.4_Sydney_2012, has beenreported in different regions .In Novosibirsk, long-term monitoring of the genetic diversityof enteric viruses showed that noroviruses GII.P4/GII.4were a common cause of sporadic AGE cases in 2003–2012,while noroviruses with the GII.P16 polymerase were rarelydetected . In the spring of2016, we recorded the emergence of a new recombinant variantGII.P16/GII.4_Sydney_2012 in Novosibirsk. Before thisstudy, only four complete genome sequences of recombinantGII.P16/GII.3 noroviruses from Russia were available in theGenBank database . Theaim of this study was complete genome sequencing of the new Russian GII.P16/GII.4_Sydney_2012 strain and comparativeanalysis with similar strains from other regions and with Russian2005–2012 strains in which the GII.P16 polymerase wasin association with various other VP1 genotypes.Origin of virus strains. As a part of the surveillance studygenetic diversity of enteric viruses, clinical samples werecollected from children with diarrhea who were hospitalizedat Children’s City Clinical Hospital No. 3 and were onoutpatient treatment in 2016. Written informed consent wasobtained from each parent/guardian of the child to participatein the study, in compliance with voluntariness in accordancewith the Federal Law “On the Principles of the Protection ofCitizens’ Health in the Russian Federation”. Detection anddifferentiation of viral RNA were performed by RT-PCR usinga verified laboratory primer panel, as previously described.Sequencing. The detected noroviruses were characterizedby sequencing of the genome region (~1400 nt), includingthe ORF1/ORF2 junction (20 nt). The nucleotide sequenceswere determined by the Sanger method using the BigDye™Terminator v.3.1 Cycle Sequencing Kit and 3500 GeneticAnalyzer . The completegenome sequencing of the strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 was performed by the “primerwalking”method using a panel of newly designed primers.The obtained data were analyzed by FinchTV . Partial fragments were assembled into a full-length genomesequence using SeqMan from the Lasergene EvolutionSuite software package .Norovirus genotype was determined using the Norovirus TypingTool v. 2.0  .Phylogenetic analysis. Reference norovirus sequenceswere obtained by search on BLAST 2.9.0+ (https://www.ncbi.nlm.nih.gov/). ClustalW alignment and phylogenetic analysisof nucleotide sequences were performed using MEGA 7(https://www.megasoftware.net/). Phylogenetic trees wereconstructed using the Neighbor-Joining method with theKimura 2-parameter model. The analysis was performed with abootstrap of 1000 replicas; only values >80 % were indicated.The identity of the nucleotide and amino acid sequences was calculated using BioEdit v7.2.6 software (http://www.mbio.ncsu.edu/bioedit/bioedit.html). The variability of the deducedamino acid sequences of the polyprotein comprising GII.P16RdRp, as well as the capsid proteins VP1 and VP2 of variantGII.4_Sydney_2012 was determined using the Sequence DataExplorer from MEGA 7.The nucleotide sequences obtained in this study were annotatedand deposited in the GenBank database with accessnumbers KY210919, KY210976–KY210980, KY210983,MG892912 и MG892914.https://www.ncbi.nlm.nih.gov/) and RIVM (https://www.rivm.nl/mpf/typingtool/norovirus/) showed that the identified noroviruses belongedto three recombinant variants (Table 1).A total of 46 fecal samples from children aged 1 monthto 8 years were tested by RT-PCR. Enteric viruses weredetected in 15 (32.6 %) samples. Norovirus infection wasidentified in six hospitalized children aged 1 to 9 months.Analysis of nucleotide sequences (~1400 nt), including theORF1/ORF2 junction, by BLAST (The nucleotide sequences of the norovirus strains that weremost homologous to the sequences obtained in this studywere determined by search on BLAST. Isolate NS16-C32related to GII.P21/GII.3 genotype had high homology withstrains circulating in Novosibirsk (97.6–98.9 %) in 2010–2012 and in Europe (96.5–97.2 %) in2014–2016 . Two isolates NS16-C12and NS16-C13 genotyped as GII.Pe/GII.4_Sydney_2012had a high similarity (99.3–99.5 %) with GII.Pe/GII.4 strains(2014–2016) from Southeast Asia and Great Britain  and 96.9–97.1 % identity with strains that previouslycirculated in Novosibirsk .Nucleotide sequences of three remaining isolates NS16-C36,NS16-C37, and NS16-C38 related to the new genotype GII.P16/GII.4_Sydney_2012 had 100 % identity to each otherand 96.2 % homology with isolates NS16-C12 and NS16-C13. Genetic similarity of isolates NS16-C36, NS16-C37 andNS16-C38 with strains GII.P16/GII.4_Sydney_2012 fromother regions was 97.4–98.9 %.Based on complete norovirus sequences available in theGenBank database, several sets of original primers were designedfor complete sequencing of ORF1, RdRp, ORF2, and ORF3 of different genotypes. For two isolates, NS16-C13and NS16-C32, nucleotide sequences (~4300 nt), includingRdRp, ORF2 and ORF3, were identified and depositedin the GenBank database as strains Hu/GII.Pe-GII.4/RUS/Novosibirsk/NS16-C13/2016 (GenBank KY210977) andHu/GII.P21-GII.3/RUS/Novosibirsk/NS16-C23/2016 (Gen-Bank KY210919).For isolate NS16-C38, complete genome sequence(7560 nt) including the 3ʹ-untranslated region (47 nt) wasdetermined and deposited in the GenBank database as thestrain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016(GenBank KY210980). Analysis of the deduced amino acidsequences showed that ORF1 (5100 nt) encoded a polyproteinof 1700 amino acid (aa) residues of length; ORF2 (1623 nt) andORF3 (807 nt) encoded the capsid proteins VP1 (541 aa) andVP2 (269 aa), respectively. The complete nucleotide sequenceof the Russian strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 had 98–99 % homology with recombinantGII.P16/GII.4_Sydney_2012 strains that appeared in theUSA and the UK in the winter season 2015/2016. Since thestrain studied was recombinant, a comparative analysis wasperformed separately for each ORF.Comparative analysis of the ORF1Phylogenetic analysis of complete ORF1 nucleotide sequenceswith GII.P16 RdRp available in GenBank showed that theanalyzed strains were divided into three clusters ; separate clades (supported on >85 %) within them wereformed by strains with the same VP1 genotype .Cluster III contained contemporary recombinant strains withGII.P16 RdRp associated with VP1 of four genotypes, GII.1,GII.2, GII.3, and GII.4_Sydney_2012. The ORF1 homology ofthe Russian strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 with other strains of cluster III was 97.9–99 %.Comparative analysis showed that the complete ORF1sequences of the GII.P16 RdRp strains varied from 5094 nt(reference strain AY772730_Hu/GII.P16-GII.16/DEU/Neustrelitz/2000) to 5100 nt (cluster III). When aligned, theGAA insert in the region encoding the N-terminal protein p48was found in recombinant GII.P16/GII.17 strains (cluster I)and in all strains from cluster II. Additional insertion of GAAor GGA was detected in contemporary recombinant strainsfrom cluster III in the same region of the p48 gene. For ORF1,1317 (25.8 %) variable sites were determined, of which906 (17.7 %) were informative, i. e. were in two or more strains.For polyprotein, 182 (10.7 %) variable sites were found,104 (6.1 %) of which were informative (Table 2). Comparativeanalysis showed that 14 variable sites were unique to thenovel GII.P16 RdRp lineage (cluster III), and the substitutionsin seven positions 52, 53, 644, 845, 853, 1546, and 1549resulted in a change in amino acid chemistry. An assessmentof the functional significance of the changes revealed thatthree non-synonymous substitutions  and77E/G insert were found in p48, which plays a role in virusentry through the host cell membrane . Four of the five non-synonymous substitutions  within GII.P16 RdRp occurredin the active center (see Table 2), and this may have affectedthe norovirus transmission.Comparative analysis of the ORF2In addition to the sequences determined in this study, phylogeneticanalysis of ORF2 included strains of different capsidgenotypes, which were identified in combination with theGII. P16 RdRp. On phylogenetic tree of the partial ORF2sequences,the analyzed strains formed separate clusters inaccordance with the capsid genotype and were further subdividedinto separate clades depending on the RdRp genotype.The GII.4 nucleotide sequences divided into two majorclades. The most polymorphic clade includes strains withthe GII.P4 RdRp, which previously circulated in Novosibirskand belonged to six GII.4 epidemic variants: Farmington_Hills_2002, Hunter_2004, Yerseke_2006a, Den_Haag_2006b,Apeldoorn_2007 and New-Orleans_2009 . The second clade was formed by GII.4_Sydney_2012 strains, which were further divided into two separateclusters, depending on the RdRp genotype – GII.Pe or GII. P16(supported on 99–100 %).The ORF2 similarity of the Russian strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 to the GII.P16/GII.4_Sydney_2012 strains from other regions was 98.8–99 %.Comparison of complete ORF2 sequences of the GII. P16/GII.4_Sydney_2012 strains with the GII.Pe/GII.4_Sydney_2012 and GII.P4/GII.4_New_Orleans_2009 strains revealed236 (14.5 %) variable sites, of which 132 (8.1 %) were informative.In deduced amino acid sequences of VP1, eightvariable sites were unique to the GII.4_Sydney_2012 variant, which distinguishedthem from the GII.4_New_Orleans_2009 variant,and only two of these changes (at 368 and 373) were locatedin the hypervariable epitope A (Table 3). Notably, only onevariable site at position 540 was unique to the new lineage ofGII.P16/GII.4_Sydney_2012, however, it was not located inantigenic regions of the major capsid protein VP1.Comparative analysis of the ORF3Phylogenetic analysis of complete ORF3 sequences ofstrains with the GII.P16 RdRp showed that the analyzedsequences were divided into separate clusters depending onthe VP1 genotype . Within each VP1 genotype, strainswith different RdRp genotypes were grouped into separateclades. Comparative analysis of complete ORF3 sequencesof GII.P16/GII.4_Sydney_2012 strains with those of GII.Pe/GII.4_Sydney_2012 and GII.P4/GII.4_New_Orleans_2009strains revealed 109 (13.5 %) variable sites, and 55 (6.8 %)of them were informative.In the deduced amino acid sequences of the minor capsidprotein VP2 of GII.4_Sydney_2012 strains, eight uniquevariable sites at positions 81, 108, 148, 149, 158, 164, 205,and 241 were identified (Table 4), which differed them fromGII.4_New_Orleans_2009 strains. In addition, two other variablesites (at 155 and 157) were unique to the new lineage ofGII.P16/GII.4_Sydney_2012.This work is a part of long-term monitoring of the geneticdiversity of noroviruses associated with sporadic AGE cases inNovosibirsk, Russia. In March 2016, a new variant of GII. P16/GII.4_Sydney_2012 norovirus was first isolated in feces fromhospitalized children. In samples from hospitalized adults,this variant was first identified in autumn 2016 . GenBank searchshowed that in the European part of Russia, similar GII.4_Sydney_2012 noroviruses (GenBank MK033810–MK033811)were found in samples of children from Nizhny Novgorod alsoat the end of 2016, unfortunately, the RdRp genotype was notdetermined for those isolates.Until recently, noroviruses with the GII.P16 RdRp wereconsidered uncommon, although local outbreaks associatedwith GII.P16/GII.2 (2009/2010 and 2012/2014) were reportedin Japan , and thosecaused by GII.P16/GII.13 (2009/2010) in Nepal . During the 10-year (2003–2012) monitoring ofnorovirus genotypes in Novosibirsk, Russia , GII.P16 RdRp was identified in five samples:GII.P16/GII.16 , GII.P16/GII.3  and GII.P16/GII.5(GenBank HM596590). Until 2016 in the Russian Federation,except Novosibirsk, GII.P16/GII.3 noroviruses were rarelydetected in Omsk  andSmolensk (GenBank KF895841), and GII.P16/GII.16 in Moscowand St. Petersburg . InNovosibirsk, recombinant noroviruses with the novel GII.P16RdRp, which differed from RdRp of variant 2010–2012 andwas in combination with multiple capsid genotypes , were often found in samplesfrom adult AGE patients since 2016 (data not published).Our results confirmed the hypothesis of the spread of newlyemerged recombinant norovirus strains with the novel GII. P16RdRp in different regions of the world .Before this study, only four complete genome sequencesof recombinant GII.P16/GII.3 norovirus strains from Russianwere available in the GenBank database . In this work, complete genome sequenceof the Russian strain Hu/GII.P16-GII.4/RUS/Novosibirsk/NS16-C38/2016 related to the newly emerged recombinantgenotype GII.P16/GII.4_Sydney_2012 was determined. Thecomparative analysis showed that unique changes occurred inthe amino acid sequences of two non-structural proteins – theN-terminal protein p48 and GII.P16 RdRp, as well as in the minor capsid protein VP2; at the same time, no significantchanges were detected in the major capsid protein VP1 ofGII.4_Sydney_2012.RNA-dependent RNA polymerase plays a crucial role inthe replication of the norovirus genome. Recent studies haveshown that the RdRp coding region is changing quickly;however, variable mutation rates were observed in differentRdRp genotypes . Our findings are consistentwith the hypothesis of Ruiz et al. (2017) that unusualworldwide distribution of the novel GII.P16 lineage is mainlydue to changes in the polymerase active center, which couldincrease the norovirus transmission. However, we assumethat changes in the N-terminal protein p48 have also playedsome role in the wide distribution of these new recombinantstrains. It was previously shown that p48 can bind the hostrestriction factors in an infected cell, allowing norovirus toavoid the host immune response, and its coding region hasa higher evolution rate than the complete norovirus genome. In addition, it is known that p48 is ableto block the local secretory immunity of intestinal epithelialcells, induce the disintegration of the Golgi apparatus anddisrupt the intracellular traffic of proteins . We assume that the insertion ofglutamic acid residue into the region, which already containsfour consecutive glutamic acid residues, increases the negativecharge at the N-terminus of p48, and this may affect boththe norovirus entry into the intestinal epithelial cells and theGolgi disintegration rate.The minor capsid protein VP2, playing an important role invirus replication  and viral particlestability , is also involved in modulation ofthe host immune response . The mutationrate of VP2 is higher than that for the major capsid protein VP1. Identified amino acid substitutionscould affect the ability of VP2 to suppress the presentationof antigens on cell membranes and the induction of humanprotective immunity.As the result of long-term monitoring of noroviruses RdRp/VP1 genotypes, the emergence of the novel GII.P16/GII.4_Sydney_2012 recombinant was recorded in Russia. Theanalysis showed that the distribution of the newly emergedrecombinant GII.P16/GII.4_Sydney_2012 is not associatedwith changes in the antigenic profile of the major capsid proteinVP1, which usually led to the emergence of new epidemicGII.4 variants. In GII.P16/GII.4_Sydney_2012 strains, a certainrole was probably played by changes in the minor proteinVP2 that might affect the antigenic composition of the viralparticle and help to avoid the cellular immune response. 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Cnidium monnieri (L.) Cuss (C. monnieri) is one of the most widely used traditional herbal medicines, exhibiting a wide range of pharmacological functions for treating asynodia, trichomonas vaginitis, and osphyalgia. Its important medicinal value comes from its abundance of coumarins. To identify genes involved in coumarin biosynthesis and accumulation, we analyzed transcriptome data from flower, leaf, root and stem tissues of C. monnieri. A total of 173,938 unigenes with a mean length of 1,272 bp, GC content of 38.79%, and N50 length of 2,121 bp were assembled using the Trinity program. Of these, 119,177 unigenes were annotated in public databases. We identified differentially expressed genes (DEGs) based on expression profile analysis. These DEGs exhibited higher expression levels in flower tissue than in leaf, stem or root tissues. We identified and analyzed numerous genes encoding enzymes involved in coumarin biosynthesis, and verified genes encoding key enzymes using quantitative real-time PCR. Our transcriptome data will make great contributions to research on C. monnieri and provide clues for identifying candidate genes involved in coumarin metabolic pathways. Cnidium monnieri (L.) Cuss, an annual plant of the Umbelliferae family native to China and Vietnam, exhibits a diverse set of pharmacological activities including anti-osteoporotic  has been widely applied as a powerful method for discovering and predicting functional genes, and revealing gene expression patterns, especially in non-model species for which reference genome sequences are insufficient . To dateus albus , Artemisia argyi , Arisaemophyllum , Clinopochinense  and Polyyrtonema . HoweverC. monnieri using BGI-500 sequencing technology. Numerous potential genes and differentially expressed genes (DEGs) involved in coumarin biosynthesis were screened using de novo transcriptome sequencing. Our findings will increase our understanding of the molecular mechanisms of coumarin biosynthesis in C. monnieri and provide clues for exploring functional genes in other plants having close relationships with C. monnieri.The purpose of this study was to uncover functional genes associated with or playing regulatory roles in coumarin biosynthesis. We conducted transcriptome sequencing and gene expression profiling in C. monnieri plants were collected from Tongcheng city in Anhui Province, China in May 2018, with verbal permission of the Manager Zhongquan Fang , and authenticated by Professor Dequn Wang (Anhui University of Chinese Medicine). Prior to the experiment, the plants were grown at a temperature of 22−26 °C/14−18 °C (day/night) and relative humidity of 65–80%. All samples were rinsed in ultrapure water, and leaves, roots, flowers and stems, which were harvested from three individual C. monnieri plants, were placed in centrifuge tubes, frozen in liquid nitrogen immediately and stored at −80 °C.Whole Total RNA was extracted from each tissue using RNA plant Plus Reagent  according to the manufacturer’s instructions. The extracted RNA was checked using a NanoDrop 2000 , and the concentration of the isolated RNA, 28S/18S and integrity (RIN) were estimated using an RNA Nano 6000 Assay Kit and the Agilent Bioanalyzer 2100 system  .C. monnieri samples from flowers, roots, leaves and stems were used for isolation of osthole as previously reported  to remove DNA residues and then mixed with magnetic beads containing oligo (dT) to purify mRNA. After purification, the mRNA was fragmented into small pieces under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Second-strand cDNA was synthesized using DNA Polymerase I and RNase H. These cDNA fragments had the addition of a single adenine at the 3′ end for subsequent ligation of adapters. The products were then purified and enriched using PCR amplification. The quality of each sample library was evaluated using an Agilent 2100 Bioanalyzer system . Each cDNA library was sequenced using the BGISEQ-500 system at Beijing Genomics Institute (BGI)  with paired-end (PE) sequencing length of 100 bp.i − A 0.25). In the absence of a reference genome, clean reads were subjected to transcriptome assembly using Trinity software (version 2.5.1) applying the following parameters: –min_contig_length 150 –CPU 8 –min_kmer_cov 3 –min_glue 3 –bfly_opts ’-V 5 –edge-thr =0.1 –stderr’  , NCBI non-redundant protein sequences (NR) (update date: 23 June 2019) , clusters of euKaryotic Orthologous Groups (KOG) (http://www.ncbi.nlm.nih.gov/KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) (update date: 1 July 2018) (http://www.genome.jp/kegg/) (http://www.uniprot.org/). Assembled unigenes were annotated against NR database using the software BLAST . And the results of NR annotation were used for Gene Ontology (GO) (update date: 1 January 2019) annotation using Blast2GO (version v2.5.0) program (Assembled unigenes were annotated against the NCBI nucleotide sequences (NT) (p/kegg/) , and a m program . Pfam  using Power SYBR® Green PCR Master Mix . The actin gene (CL3748.Contig7) of C. monnieri was used as a reference. Primers for all selected genes were designed using Premier 6.0 and are listed in ® Green Master Mix. Reactions were conducted under the following conditions: denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 63 °C for 25 s. Each qRT-PCR was performed using three biological replicates. The 2Ct−ΔΔ method was used to calculate the relative expression level of genes . qRT-PCR analysis of each gene was performed on a QuantStudioof genes .After assembly, clean reads were mapped to unigenes using Bowtie2 (version 2.2.5) , and theThe open reading frames (ORFs) of unigenes were initially detected using getorf (version EMBOSS:6.5.7.0)  and furtP value <0.05 was considered to be statistically significant.The contents of osthole and all data from qRT-PCR were presented as mean ± standard deviation. Statistics were done by GraphPad Prism 6.0. Heatmap and boxplot were performed using R software (version 3.4.4). The one-way analysis of variance (ANOVA) was used to compare the data between groups with Duncan t test. C. monnieri. Osthole content was highest in flowers (1.372%) and lowest in stems (0.283%)  . OstholeC. monnieri using BGISEQ-500 sequencing technology. After eliminating low-quality reads, we obtained a total of 10.6 Gb, 10.47 Gb, 10.81 Gb and 10.38 Gb of clean reads from 83.42 Mb, 81.65 Mb, 84.27 Mb and 82.14 Mb sets of raw data from leaf, stem, flower and root tissue, respectively, with all Q30 values greater than 85%  against seven public databases , and a total of 110,992 (63.81%), 95,603 (54.96%), 80,216 (46.12%), 86,568 (49.77%), 87,523 (50.32%), 79,335 (45.61%) and 56,429 (32.44%) unigenes were aligned, respectively (Daucus carota subsp. sativus (82.2%) and others (17.8%)  (e <10ectively . We annoectively . We focuectively . Additio (17.8%) .We determined expression values of transcripts with fragments per kilobase of transcript per million mapped reads (FPKM) >1 for each tissue and found 55,421, 52,787, 48,287 and 44,717 expressed unigenes in flower, leaf, stem and root tissues, respectively . The oveC. monnieri, 86,568 (49.77%) unigenes were annotated and assigned to 135 KEGG pathways (19 subcategories) , cinnamate 4-hydroxylase , 4-coumarate–CoA ligase , shikimate hydroxycinnamoyl transferase , 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase , caffeoyl-CoA O-methyltransferase  and feruloyl-CoA ortho-hydroxylase   . Fourteeegories) . Phenylpnigenes) . These dnigenes) .C. monnieri (C4H), CL16061.Contig1 (4CL) and CL8379.Contig6 (F6H1) genes was highest in root tissue, whereas relative expression of the CL11351.Contig3 (PAL) and CL2358.Contig9 (BGA) genes was highest in flower tissue. Unigene 20867 (BGA) had a high expression level in leaf tissue. The expression levels determined by qRT-PCR were consistent with the FPKM values.We selected six unigenes encoding five key enzymes and used quantitative real-time PCR (qRT-PCR) to determine their relative expression levels in the leaf, stem, flower and root tissues of monnieri . RelativWe identified 12,635 unigenes uniquely expressed in flowers and 77,762 shared unigenes exhibiting expression in all four tissues . Total DAll DEGs were annotated in the KEGG database to further describe and evaluate their biological functions. This analysis revealed 32,060 DEGs in flower versus leaf associated with 137 pathways; these DEGs were mainly enriched in the “plant hormone signal transduction,” “plant-pathogen interaction,” “circadian rhythm - plant” and “photosynthesis - antenna proteins” pathways . In flowA total of 18,881 up-regulated DEGs showed flower-specific expression with |log2 (fold changes) | >2. We inferred the nature of these DEGs via GOSlim functional analysis. Sequence homology revealed that each of the 18,881 flower-specific DEGs mapped to at least one ontology term, including 2,814 for biological processes, 3,232 for cellular components and 4,508 for molecular function; many genes were enriched in “cellular process” and “catalytic activity” subcategories within the biological processes and molecular function categories, respectively .To evaluate the biological functions of these DEGs, the 18,881 flower-specific DEGs were annotated in the KEGG database. KEGG enrichment analysis showed that these DEGs were significantly enriched in the “ribosome,” “starch and sucrose metabolism,” “pentose and glucuronate interconversions” and “phenylpropanoid biosynthesis” categories . AdditioC. monnieri transcriptome database, including 1,169, 1,032 and 920 unigenes up-regulated in flowers compared with leaves, stems and roots, respectively (Transcription factors (TFs) play an important role in regulating secondary metabolites by modulating the expression of genes related to biosynthetic pathways. We identified 3,799 unigenes encoding putative TFs in the ectively . The majectively . Of thesC. monnieri. Their structures and physicochemical properties have been well studied  genes were mapped in at least one public database, while 54,761 (31.48%) genes remained unannotated because of the lack of available genomic information.Coumarins are the most important bioactive components of  studied . HoweverPAL), CL2358.Contig9 (BGA), Unigene 20867 (BGA), CL3766.Contig1 (C4H), CL16061.Contig1 (4CL) and CL8379.Contig6 (F6H1) examined using qRT-PCR were consistent with the FPKM values determined by RNA-Seq. Coumarins are a major group of natural plant products derived from the phenylpropanoid pathway  in Angelica gigas , 4CL (2 DEGs), HCT (13 DEGs), COMT (7 DEGs) and F6′H1 (8 DEGs) were related to coumarin biosynthesis . These kArabidopsis results in increased biosynthesis and accumulation of scopoletin, which belongs to the coumarins  Standard curve of osthole at 322 nm. (B) Osthole content from flowers, leaves, roots and stems of Click here for additional data file.10.7717/peerj.10157/supp-2Figure S2Click here for additional data file.10.7717/peerj.10157/supp-3Figure S3Click here for additional data file.10.7717/peerj.10157/supp-4Figure S4Click here for additional data file.10.7717/peerj.10157/supp-5Figure S5Click here for additional data file.10.7717/peerj.10157/supp-6Figure S6Click here for additional data file.10.7717/peerj.10157/supp-7Figure S7Red boxes represent enzymes annotated in the KEGG database. Photo credit: Kanehisa Laboratories.Click here for additional data file.10.7717/peerj.10157/supp-8Figure S8actin gene (CL3748.Contig7) as a reference gene for normalization. FPKM values of these genes are shown as red lines, qRT-PCR results of these genes are shown as blue bars.Relative expression of (A) CL11351.Contig3 (PAL), (B) CL2358.Contig9 (BGA), (C) Unigene 20867 (BGA), (D) CL3766.Contig1 (C4H), (E) CL16061.Contig1 (4CL) and (F) CL8379.Contig6 (F6H1) was analyzed by qRT-PCR using the Click here for additional data file.10.7717/peerj.10157/supp-9Figure S9Click here for additional data file.10.7717/peerj.10157/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.10157/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.10157/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.10157/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.10157/supp-14Supplemental Information 14Click here for additional data file.10.7717/peerj.10157/supp-15Supplemental Information 15Click here for additional data file.10.7717/peerj.10157/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.10157/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.10157/supp-18Supplemental Information S1Click here for additional data file.


This study examined the diagnostic accuracy of 18F-DCFPyL (PSMA) PET/CT for lymph-node staging in primary PCa.The detection of lymph-node metastases (N1) with conventional imaging such as magnetic resonance imaging (MRI) and computed tomography (CT) is inadequate for primarily diagnosed prostate cancer (PCa). Prostate-specific membrane antigen (PSMA) PET/CT is successfully introduced for the staging of  recurrent PCa. Besides the frequently used 18F-DCFPyL PET/CT prior to robot-assisted radical prostatectomy (RARP) with extended pelvic lymph-node dissection (ePLND). Patients were included between October 2017 and January 2020. A Memorial Sloan Kettering Cancer Centre (MSKCC) nomogram risk probability of ≥ 8% of lymph-node metastases was set to perform ePLND. All images were reviewed by two experienced nuclear physicians, and were compared with post-operative histopathologic results.This was a prospective, multicentre cohort study. Patients with primary PCa underwent 18F-DCFPyL PET/CT detection of pelvic lymph-node metastases on a patient level were 41.2% : 19.4–66.5%), 94.0% (CI 86.9–97.5%), 53.8% (CI 26.1–79.6%) and 90.4% (CI 82.6–95.0%), respectively.A total of 117 patients was analysed. Lymph-node metastases (N1) were histologically diagnosed in 17/117 patients (14.5%). The sensitivity, specificity, positive predictive value and negative predictive value for the 18F-DCFPyL PET/CT showed a high specificity (94.4%), yet a limited sensitivity (41.2%) for the detection of pelvic lymph-node metastases in primary PCa. This implies that current PSMA PET/CT imaging cannot replace diagnostic ePLND. Further research is necessary to define the exact place of PSMA PET/CT imaging in the primary staging of PCa. Prostate cancer (PCa) is the most frequently diagnosed cancer in men in the Western world , 2. Init68gallium-labelled PSMA tracers. High detection rates for metastases were demonstrated even at low prostate-specific antigen (PSA) values (i.e. 45% for PSA < 0.5 ng/mL and over 95% for PSA ≥ 2.0 ng/mL) fluoro-pyridine-3-carbonyl)-amino]-pentyl)-ureido)-pentanedioic acid)  112–123 min) of a median dose of 311 MBq 18F-DCFPyL (IQR 297–324 MBq) within a median of 4.1 weeks (IQR 2.1–6.6) after prostate biopsy and within a median of 5.9 weeks (IQR 3.6–12.0) prior to surgery. Image acquisitions were performed using a Philips Ingenuity TF  and a Siemens Biograph-16 TruePoint  PET/CT system. The scan trajectory included mid-thigh to skull base, with 4 min (Philips) and 5 min (Siemens) per bed position. All PET scans were combined with a low-dose CT (33/117 patients) or contrast-enhanced CT scan (84/117 patients) . Images were corrected for decay, scatter, random coincidences and photon attenuation.Patients were staged with Images were reconstructed with a BLOB-based Ordered-Subsets Expectations Maximization algorithm   and the All scans were clinically and prospectively interpreted in the participating centres by one of two nuclear medicine physicians  with ample experience in PSMA PET interpretation (> 300 scans). Upon completion of the study, all scans were reviewed by a second independent reader who was blinded to initial scan interpretation, surgery and histopathology results . A joint re-evaluation was performed in case of incongruent scan interpretation (consensus read), and used for final analysis. Lymph-node metastases were defined as increased PSMA expression, higher than the background, incompatible with physiological uptake, and in a typical site of PCa. A significant CT substrate was not an absolute prerequisite. The following parameters were recorded: detection of the primary tumour, tumour stage and presence of pelvic lymph-node metastases (N1). Pelvic lymph-node metastases were further classified in accordance with the four sections of the ePLND .To assess inter-observer variability, the proportional agreement was calculated, based on the two individual scan interpretations . Proportpeak), with correction for background uptake [peak is defined as the highest local intensity of uptake with a 6-mm-radius sphere [peak of the prostate tumour for patients with and without lymph-node metastases, the Mann-Whitney U test was used (significance set at p < 0.05). To compare the median SUVpeak of the prostate tumour with the corresponding Gleason score of the lesion, the Kruskall-Wallis test was used (significance set at p < 0.05). A linear regression was run to predict PSA from SUVpeak of the prostate index lesion (significance set at p < 0.05).PET/CT scans with PSMA-avid lesions in the prostate were delineated according to the reports of the nuclear medicine physicians. Semi-automatic delineation on the PET scans from both participating centres was performed with the in-house developed ACCURATE tool© . The ACCd uptake . SUVpeaks sphere . To compThe ePLND surgical template includes removal of fatty lymphoid tissue overlying the common and external iliac vessels and within the obturator fossa . The medProstate specimens and resected lymph-node templates were fixated in formaldehyde (10%) directly after surgery and processed according to routine clinical standards . Individ18F-DCFPyL PET/CT to detect pelvic lymph-node metastases. The diagnostic accuracy of 18F-DCFPyL PET/CT was calculated with histopathologic evaluation of ePLND as a reference. The sensitivity, specificity and positive and negative predictive values  of 18F-DCFPyL PET/CT for the detection of pelvic lymph-node metastases (pN1) were calculated both on a patient level and on a surgical template level. The surgical template analysis was based on the abovementioned 4 surgical templates of the ePLND, and was applied to approximate lesion based-detection using 18F-DCFPyL PET/CT. To compare the median diameter of PET/CT-detected lymph-node metastases vs. PET/CT-undetected lymph-node metastases, the Mann-Whitney U test was used (significance set at p < 0.05).The primary outcome of this study was the patient-based sensitivity of 18F-DCFPyL PET/CT to differentiate local advancement  from prostate-confined disease (T2). This study did not investigate the exact location of local PCa advancement, only the presence of extracapsular or seminal vesicle invasion was noted. Local advancement was defined as PSMA expression outside the borders of the prostate gland, not suspect for overprojection or bladder/urethral physiological activity. Numerical variables were summarized with median values and interquartile ranges (IQR), categorical variables with proportions (%). Statistical analysis was done with IBM® SPSS® Statistics for Windows®, version 26.For the assessment for the local tumour stage (pT), we measured the accuracy of 18F-DCFPyL PET/CT, as presented in Fig. A total of 120 patients were included in this study, and scheduled for ePLND with RALP after 18F-DCFPyL PET/CT suspicious for lymph-node metastases. Hence, the patient-based sensitivity to detect lymph-node metastases using 18F-DCFPyL PET/CT was 41.2%  19.4–66.5), with a specificity of 94.0% (95%CI 86.9–97.5), a PPV of 53.8% (95%CI 26.1–79.6) and a NPV of 90.4% (95%CI 82.6–95.0), as shown in Table Pathological features after RARP and ePLND are listed in Table 18F-DCFPyL PET/CT preoperatively identified 38 PSMA-avid regions suspect for lymph-node metastases in 18 surgical ePLND templates. Therefore, the template-based sensitivity for the detection of lymph-node metastases using 18F-DCFPyL PET/CT was 34.7% (95%CI 17.1–57.1), with a specificity of 97.7% (95%CI 95.7–98.9), a PPV of 44.4 (95%CI 22.4–68.6) and a NPV of 96.6% (95%CI 94.4–98.0) as seen in Table 18F-DCFPyL PET/CT, please see Fig. In the 17 patients with lymph-node metastases, 31 lymph-node metastases were histologically identified in 23 surgical ePLND templates. n = 12) had a median tumour size of 5.5 mm (IQR 2.4–6.6), whereas the PET/CT-undetected lymph-node metastases (n = 19) had a significantly smaller median tumour size of 1.5 mm (IQR 1.0–4.5) (p = 0.03). A clinical example of a patient with both missed and detected lymph-node metastases is shown in Fig. The median diameter of resected lymph-node metastases was 2.5 mm (IQR 1.0–6.0). The PET/CT-detected lymph-node metastases  p = 0.0. A clinimm IQR 2.–6.6, whe18F-DCFPyL PET/CT to detect locally advanced tumour growth (pT3-4) were 45.2% (95%CI 32.7–58.2%), 94.4% (95%CI 83.7–98.6%), 90.3% (95%CI 73.1–97.5%) and 60.0% (95%CI 48.8–70.7%), respectively, as seen in Table 18F-DCFPyL PET/CT were 18.1% (95%CI 8.7–33.2), 97.2% (95%CI 89.4–99.5), 80.0% (95%CI 44.2–96.5) and 66.0% (95%CI 56.1–74.8), respectively. For the detection of pT3b sub-stage, the sensitivity, specificity, PPV and NPV of 18F-DCFPyL PET/CT were 52.9% (95%CI 28.5–76.1), 89.9% (95%CI 81.8–94.8), 47.4% (95%CI 25.2–70.5) and 91.8% (95%CI 83.9–96.1), respectively. A clinical example of seminal vesicle (pT3b) PCa involvement on PET/CT is presented in Fig. A total of 116/117 patients (99.1%) showed PSMA expression in the prostate at PET/CT. The sensitivity, specificity, PPV and NPV of owth pT3- were 45.peak of the PSMA-avid prostate lesions was found between patients with and without lymph-node metastases  (p = 0.46). A Gleason score of 4 + 5 = 9 or higher was associated with a higher median SUVpeak when compared with the lower Gleason scores . A Gleason score of 4 + 4 = 9 or higher was not associated with a higher median SUVpeak when compared with the lower scores . No correlation was found between SUVpeak of the PSMA-avid prostate lesions and PSA (R2 = 0.12).No significant difference in the median SUVProportional agreement for the detection of lymph-node metastases using PET/CT was present in 94.7% (95%CI 89.8–97.7) overall, with a positive agreement of 78.6% (95%CI 53.4–93.9) and a negative agreement of 97.0% (95%CI 92.4–99.2). Proportional agreement for locally advanced tumours was observed in 79.1% (95%CI 71.7–85.3), with a positive agreement of 55.6% (95%CI 38.2–72.0), and a negative agreement of 86.2% (95%CI 77.9–91.5).18F-DCFPyL PET/CT for primary staging of PCa, assessing the diagnostic accuracy for the detection of pelvic lymph-node metastases. The results of a total of 117 patients with intermediate and high-risk PCa that underwent 18F-DCFPyL PET/CT and ePLND were analysed.PSMA PET/CT imaging is currently the imaging technique of choice for patients with biochemically recurrent disease after initial curative local treatment (EAU guidelines) . Its val18F-DCFPyL PET/CT imaging demonstrated a limited sensitivity for pelvic lymph-node metastases of 41.2%, at 94.0% specificity. The limited sensitivity indicates that 18F-DCFPyL PET/CT does not detect all lymph-node metastases. Therefore, albeit invasive, ePLND remains the gold standard for nodal staging. The 90% NPV might suggest that a negative test is reliable in the majority of times, though we should mind the low prevalence of lymph-node metastases in this cohort (14.5%). This prevalence was congruent with the median MSKCC risk for lymph-node metastases of 14.4% (IQR 10.1–30.2). Although specificity for lymph-node metastases was favourable in this study, the PPV was moderate at 53.8% . In this clinical setting, not all positive 18F-DFCPyL PET/CT results for pelvic lymph-node metastases represent actual metastatic disease. Good intra-observer agreement for the detection of pelvic lymph-node metastases using 18F-DFCPyL PET/CT was observed (95%).In this study, 68Ga-PSMA study from van Kalmthout et al. [n = 103 patients), applying the same standardized ePLND techniques, histopathology analyses and PET positivity criteria. It revealed a patient-based sensitivity for lymph-node metastases of 41.5% (95%CI 26.7–57.8) and a specificity of 90.9% (95%CI 79.3–96.6) [18F-DCFPyL PET/CT is at least comparable with 68Ga-PSMA imaging.Our results appear to be in line with a recent prospective t et al. . This st.3–96.6) . Althoug68Ga PSMA ligands, which reported a specificity of 90% and higher [18F-DCFPyL PET/CT as an imaging tool for initial staging of PCa [The high specificity presented in the current study confirms results from previous retrospective studies with d higher , 29, 30.g of PCa . In 25 pg of PCa . PotentiU test: p = 0.03). This may explain the imperfect imaging sensitivity reported in this study. The 5-mm spatial resolution offered by PET is still an improvement compared with the detection limits of CT and MRI (i.e. > 10 mm [68Ga-PSMA PET/CT vs. MRI (65% vs. 41%) [PET/CT-detected lymph-node metastases were larger than lymph-node metastases that were not detected by PET/CT . This d > 10 mm . This st18F-DCFPyL PET/CT should still be followed by curative treatment in the form of a RALP with an ePLND. However, the threshold to perform ePLND with certain amounts of detected number of lymph-node metastases remains unclear.The therapeutic consequence of PSMA-detected pelvic lymph-node metastases remains a matter of debate. Previous research showed that patients with lymph-node metastases detected intra-operatively (with frozen sections) or preoperatively (with CT) still benefit from radical prostatectomy and complete lymph-node dissection –34. This18F-DCFPyL PET/CT, yet the sensitivity was limited at 45.2% (95%CI 32.7–58.2). Moreover, the promising specificity of the detection of pT3a-b using 18F-DCFPyL PET/CT of 94.4% (95%CI 83.7–98.6) is in line with previous reports on 68Ga-PSMA (specificity > 90% for T3b) [18F-DCFPyL PET/CT is comparable with that of mpMRI, for which a meta-analysis revealed a specificity of 88% (95%CI 85–97%) compared with our 94.4% (95%CI 83.7–98.6) [A total of 116/117 patients (99.1%) showed PSMA expression in the prostate at PET/CT. A promising PPV for the detection of pT3a-b of 90.3% (95%CI 73.1–97.5) was observed using for T3b) , 35, 36..7–98.6) . The sen.7–98.6) . Altoget18F-DCFPyL PET/CT for detecting distant metastases. Only patients undergoing RARP and ePLND were considered for analysis, which naturally excludes patients with distant metastases in which radical surgery is forgone. As such, our results should be interpreted as the accuracy of 18F-DCFPyL PET/CT for N-staging in patients (expected to be) free from distant metastases . Therefore, we decided to focus our study on determining the accuracy of 18F-DCFPyL PET/CT for N-staging, as hereto a solid reference standard is available (ePLND). Determining the accuracy of M-staging is certainly of interest, yet any such analysis is limited to providing a PPV, as the true prevalence of distant metastases cannot be known.Our study has inherent limitations. Firstly, this study did not assess the accuracy of 18F-DCFPyL PET images were obtained, again detecting positive lymph nodes in the surgical template. Metastasis-directed radiotherapy to these lesions was followed by a PSA-response. The ePLND may possibly have missed these lesions initially that were rightfully detected on the first PET/CT scan.Since the PET/CT resolution is confined at 5 mm, limited diagnostic accuracy for micro metastases is to be expected. Secondly, this study might not have been adequately powered, since the expected prevalence was higher than the actual prevalence (30% vs. 14.5%). Moreover, the sensitivity used for the power analysis was higher than actually realized (90% vs. 41.2%) due to the high expectations for the sensitivity of PSMA PET/CT. Lastly, we should consider that the golden standard (ePLND) is not always flawless: in two patients with PET-positive lymph nodes, the ePLND was reported to be technically challenging. Histopathological analysis did not reveal any lymph-node metastases, yet these patients soon developed a biochemical recurrence. Repeated 18F-DCFPyL PET/CT scan could be used to withhold PCa patients from an ePLND. Future studies are therefore needed to assess whether the diagnostic accuracy of 18F-DCFPyL PET/CT, its high specificity in particular, could assist in proper treatment planning of patients with intermediate and high-risk stages of disease.The follow-up data of this cohort is necessary to investigate whether a specific risk profile in combination with a negative 18F-DCFPyL PET/CT imaging for the detection of lymph-node metastatic disease in men with intermediate and high-risk prostate cancer, undergoing radical surgery. We found a limited sensitivity of 41.2% (95%CI 19.4–66.5) at excellent specificity (94.0%). Based on current results, 18F-DCFPyL PET/CT imaging should not replace ePLND.In this prospective cohort study, we evaluated the accuracy of


Ulmus parvifolia cvs. Allee and Drake in Lake County, Florida. Nematode species were identified using both molecular analysis and morphology of perineal patterns. Meloidogyne enterolobii and M. javanica were identified from U. parvifolia cv. Allee. Meloidogyne arenaria and M. javanica were identified from U. parvifolia cv. Drake. This is a first report of these nematode species infecting Chinese Elm in Florida.Samples of galled roots, resembling those induced by root-knot nematodes, and rhizosphere soil were collected from potted plants of  Ulmus parvifolia Jacq.: Ulmaceae: Rosales) are valued for their tough lumber, resistance to some elm pests and pathogens  were collected directly from the rhizosphere of U. parvifolia. These samples were designated with internal FDACS-DPI sample identifiers N20-110, N20-113 (both from U. parvifolia cv. Allee), and N20-115 (from U. parvifolia cv. Drake). Round galls, resembling those commonly induced by M. enterolobii, were observed on secondary and tertiary roots of U. parvifolia cv. Allee in one of the samples  and used for qPCR, conventional PCR, and sequencing. The cytochrome c oxidase subunit I (COI) and intergenic spacer 2 (IGS2) qPCR assays were repeated with DNA from J2 extracted directly from the roots of each cultivar. Standard PCRs targeted NADH-ubiquinone oxidoreductase chain 5 (NADH5) and cytochrome c oxidase subunit II (COXII) using the primers NAD5F2/NADH5R1 and COX2F/COX2R, respectively, and thermocycle conditions described by R assays , isozymeR assays . DNA wasMeloidogyne isolates with both NADH5 and COXII sequences were further analyzed. The alignments of NADH5 (448 bp) and COXII (323 bp) were trimmed until data were 100% complete for each terminal taxon. Alignments were then concatenated  and analyzed simultaneously. K2P . Newly generated sequences  were aligned in MEGA7  using thsly. K2P  distancesly. K2P .Meloidogyne spp. found infecting U. parvifolia is provided (n = 6) and were positively identified as M. enterolobii from both qPCR assays. All other samples had undetermined Ct values for the qPCR assays. COXII sequences from N20-110 samples were 100% BLASTn matches to previously published M. enterolobii data. With the concatenated matrix, isolates N20-115-1A, N20-115-6A, N20-115-10A, N20-115-16B, N20-113-1B, N20-113-14B, N20-113-18B were 100% matches to M. javanica  Chitwood, 1949  Chitwood, 1949 (M. sp. n. 1 (n = 26 for each sample) and morphology of perineal patterns were consistent with those reported for M. enterolobii  isolated singly from U. parvifolia cv. Allee (N20-110) and M. javanica  also found singly on this same cultivar (N20-113), and both M. arenaria  and M. javanica  identified as mixed species infecting U. parvifolia cv. Drake (N20-115). To our knowledge this is the first report of Ulmus parvifolia as a host of M. arenaria, M. enterolobii, and M. javanica in Florida.A summary of identified provided . Samples


Escherichia coli (STEC) O26:H11 strain, MBT-5 , and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1  and 59 , respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.The complete genome sequence of a Shiga toxin-producing  Escherichia coli (STEC) O26:H11 strain, MBT-5 , and two draft genome sequences of Listeria monocytogenes strains MBT-6 and MBT-7 belonging to the virulent sequence types 1  and 59 , respectively, were determined. The strains were isolated in 2015 from ready-to-eat mixed greens in Germany.The complete genome sequence of a Shiga toxin-producing  Escherichia coli (STEC) serotype O26 can lead to life-threatening infections and is the second most common serotype (after O157) found in clinical and food samples in Europe  for E. coli. The AX Bacteria+ kit is suitable for getting high-molecular-weight DNA. DNA quantification and paired-end  and long-read  sequencing were performed as previously described (https://github.com/s-andrews/FastQC). Hybrid assembly of short and long sequence reads of STEC strain MBT-5, obtained from Illumina and MinION sequencing, respectively, was performed with Unicycler v0.4.8 (L. monocytogenes strains MBT-6 and MBT-7 was done with SPAdes v3.10.0 (Cultures were grown overnight at 37°C in brain heart infusion (BHI) broth, and genomic DNA was extracted using the ZR fungal/bacterial DNA miniprep kit  for escribed . For sho v3.10.0 .dnaA as the origin with Geneious v9.2. Plasmids were not rotated. Strain MBT-5 was identified as serotype O26:H11, sequence type 21 (ST21), and adhesin fimH type 440 using SerotypeFinder v2.0, MLST v2.0, and FimTyper v1.0, respectively (–ehxA (enterohemolysin), espA (extracellular serine protease), toxB (toxin B), and katP , while all other virulence genes were located on the chromosome.The hybrid assembly of STEC strain MBT-5 generated a single circular chromosome of 5,705,580 bp, with 6 circular plasmids . The circular chromosome of MBT-5 was rotated to ectively –12. Viructively – 13). In. IndnaA L. monocytogenes strains MBT-6 and MBT-7 generated 21 and 24 contigs, respectively. No plasmid sequences could be determined. Genome characteristics are shown in L. monocytogenes strains showed genetic characteristics similar to those of previously reported clinical, food, and outbreak-associated populations   , indicatPRJNA643697. The sequences of STEC strain MBT-5 and L. monocytogenes strains MBT-6 and MBT-7 were deposited at DDBJ/ENA/GenBank under the accession numbers CP058682 (chromosome), CP058683 (plasmid 5.1), CP058684 (plasmid 5.2), CP058685 (plasmid 5.3), CP058686 (plasmid 5.4), CP058687 (plasmid 5.5), CP058688 (plasmid 5.6), JACBGM000000000, and JACBGL000000000. The raw sequencing data were also deposited at the SRA with the accession numbers shown in The genome sequences have been deposited in GenBank under the BioProject number


Correction to: Pilot and Feasibility Studies 6, 69 (2020)https://doi.org/10.1186/s40814-020-00613-1Following publication of the original article , the autStudies into TFT for non-refugee traumatized populations show higher effects (d = 1.08—1.40) [7], than for refugee populations (g = .25—1.01) [8].Which will be replaced by:There is evidence that non-refugee traumatized populations benefit more from TFT than traumatized groups with a refugee background .This also involves changes in reference [8].Epidemiology and psychiatric sciences, 28(4), 376–388.doi.org/10.1017/S2045796019000027”.“Turrini, G., Purgato, M., Acarturk, C., Anttila, M., Au, T., Ballette, F., ... & Hall, J. (2019). Efficacy and acceptability of psychosocial interventions in asylum seekers and refugees: systematic review and meta-analysis. should replace:“Lambert JE, Alhassoon OM. Trauma-focused therapy for refugees: Meta-analytic findings. Journal of counseling psychology. 2015;62(1):28. DOI: 10.1037/cou0000048”.The publishers apologise for this error.