Natural and hybrid immunity after SARS-CoV-2 infection in children and adolescents.

PURPOSE: In contrast to adults, immune protection against SARS-CoV-2 in children and adolescents with natural or hybrid immunity is still poorly understood. The aim of this study was to analyze different immune compartments in different age groups and whether humoral immune reactions correlate with a cellular immune response. METHODS: 72 children and adolescents with a preceding SARS-CoV-2 infection were recruited. 37 were vaccinated with an RNA vaccine (BNT162b2). Humoral immunity was analyzed 3-26 months (median 10 months) after infection by measuring Spike protein (S), nucleocapsid (NCP), and neutralizing antibodies (nAB). Cellular immunity was analyzed using a SARS-CoV-2-specific interferon-γ release assay (IGRA). RESULTS: All children and adolescents had S antibodies; titers were higher in those with hybrid immunity (14,900 BAU/ml vs. 2118 BAU/ml). NCP antibodies were detectable in > 90%. Neutralizing antibodies (nAB) were more frequently detected (90%) with higher titers (1914 RLU) in adolescents with hybrid immunity than in children with natural immunity (62.5%, 476 RLU). Children with natural immunity were less likely to have reactive IGRAs (43.8%) than adolescents with hybrid immunity (85%). The amount of interferon-γ released by T cells was comparable in natural and hybrid immunity. CONCLUSION: Spike antibodies are the most reliable markers to monitor an immune reaction against SARS-CoV-2. High antibody titers of spike antibodies and nAB correlated with cellular immunity, a phenomenon found only in adolescents with hybrid immunity. Hybrid immunity is associated with markedly higher antibody titers and a higher probability of a cellular immune response than a natural immunity.

Characterization of a Unique Pair of Ferredoxin and Ferredoxin NADP+ Reductase Isoforms That Operates in Non-Photosynthetic Glandular Trichomes.

Our recent investigations indicated that isoforms of ferredoxin (Fd) and ferredoxin NADP+ reductase (FNR) play essential roles for the reductive steps of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of terpenoid biosynthesis in peppermint glandular trichomes (GTs). Based on an analysis of several transcriptome data sets, we demonstrated the presence of transcripts for a leaf-type FNR (L-FNR), a leaf-type Fd (Fd I), a root-type FNR (R-FNR), and two root-type Fds (Fd II and Fd III) in several members of the mint family (Lamiaceae). The present study reports on the biochemical characterization of all Fd and FNR isoforms of peppermint (Mentha × piperita L.). The redox potentials of Fd and FNR isoforms were determined using photoreduction methods. Based on a diaphorase assay, peppermint R-FNR had a substantially higher specificity constant (kcat/Km) for NADPH than L-FNR. Similar results were obtained with ferricyanide as an electron acceptor. When assayed for NADPH-cytochrome c reductase activity, the specificity constant with the Fd II and Fd III isoforms (when compared to Fd I) was slightly higher for L-FNR and substantially higher for R-FNR. Based on real-time quantitative PCR assays with samples representing various peppermint organs and cell types, the Fd II gene was expressed very highly in metabolically active GTs (but also present at lower levels in roots), whereas Fd III was expressed at low levels in both roots and GTs. Our data provide evidence that high transcript levels of Fd II, and not differences in the biochemical properties of the encoded enzyme when compared to those of Fd III, are likely to support the formation of copious amounts of monoterpene via the MEP pathway in peppermint GTs. This work has laid the foundation for follow-up studies to further investigate the roles of a unique R-FNR-Fd II pair in non-photosynthetic GTs of the Lamiaceae.

Association of known SARS-CoV-2 serostatus and adherence to personal protection measures and the impact of personal protective measures on seropositivity in a population-based cross-sectional study (MuSPAD) in Germany.

BACKGROUND: In 2020/2021 in Germany, several non-pharmacological interventions were introduced to lower the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated to what extent knowledge of prior infection with SARS-CoV-2 or vaccination status influenced the use of personal protection measures (PPM). Further, we were interested in the effect of compliance with PPM on SARS-CoV-2 serostatus. METHODS: Data was based on a sequential, multilocal seroprevalence study (MuSPAD), carried out in eight locations from July 2020 to August 2021. We estimated the association between a known SARS-CoV-2 serostatus (reported positive PCR test or vaccination) and self-reported PPM behavior (hand hygiene, physical distancing, wearing face mask), just as the association of PPM compliance with seropositivity against nucleocapsid (NC), receptor-binding domain (RBD), and spike protein (S) antigens. We identified relevant variables and deduced adjustment sets with directed acyclic graphs (DAG), and applied mixed logistic regression. RESULTS: Out of the 22,297 participants (median age: 54 years, 43% male), 781 were classified as SARS-CoV-2-infected and 3,877 had a vaccinated immune response. Vaccinated individuals were less likely to keep 1.5 m distance [OR = 0.74 (95% CI: 0.57-0.97)] and only partly physically distanced [OR = 0.71 (95% CI: 0.58-0.87)]. Participants with self-reported positive PCR test had a lower chance of adhering partly to physical distancing [OR = 0.70 (95% CI: 0.50-0.99)] in comparison to the reference group. Higher odds of additionally wearing a face mask was observed in vaccinated [OR = 1.28 (95% CI: 1.08-1.51)] even if it was not obligatory. Overall, among unvaccinated participants, we found little evidence of lower odds of seropositivity given mask wearing [OR: 0.91 (95% CI: 0.71-1.16)], physical distancing [OR: 0.84 (95% CI: 0.59-1.20)] and no evidence for completely adhering to hand cleaning [OR: 0.97 (95% CI: 0.29-3.22)]. CONCLUSIONS: A known confirmed prior infection and vaccination may have the potential to influence adherence to PPM.

Biochemical basis for the formation of organ-specific volatile blends in mint.

Above-ground material of members of the mint family is commercially distilled to extract essential oils, which are then formulated into a myriad of consumer products. Most of the research aimed at characterizing the processes involved in the formation of terpenoid oil constituents has focused on leaves. We now demonstrate, by investigating three mint species, peppermint (Mentha Ë£ piperita L.), spearmint (Mentha spicata L.) and horsemint (Mentha longifolia (L.) Huds.; accessions CMEN 585 and CMEN 584), that other organs - namely stems, rhizomes and roots - also emit volatiles and that the terpenoid volatile composition of these organs can vary substantially from that of leaves, supporting the notion that substantial, currently underappreciated, chemical diversity exists. Differences in volatile quantities released by plants whose roots had been dipped in a Verticillium dahliae-spore suspension (experimental) or dipped in water (controls) were evident: increases of some volatiles in the root headspace of mint species that are susceptible to Verticillium wilt disease (peppermint and M. longifolia CMEN 584) were detected, while the quantities of certain volatiles decreased in rhizomes of species that show resistance to the disease (spearmint and M. longifolia CMEN 585). To address the genetic and biochemical basis underlying chemical diversity, we took advantage of the newly sequenced M. longifolia CMEN 585 genome to identify candidate genes putatively coding for monoterpene synthases (MTSs), the enzymes that catalyze the first committed step in the biosynthesis of monoterpenoid volatiles. The functions of these genes were established by heterologous expression in Escherichia coli, purification of the corresponding recombinant proteins, and enzyme assays, thereby establishing the existence of MTSs with activities to convert a common substrate, geranyl diphosphate, to (+)-α-terpineol, 1,8-cineole, γ-terpinene, and (-)-bornyl diphosphate, but were not active with other potential substrates. In conjunction with previously described MTSs that catalyze the formation of (-)-ß-pinene and (-)-limonene, the product profiles of the MTSs identified here can explain the generation of all major monoterpene skeletons represented in the volatiles released by different mint organs.

Fluorescence-guided laser lithotripsy: Estimation of the potential effectiveness and safety increase based on first clinical data.

In clinically approved laser lithotripsy systems, there is no automatic monitoring of fiber position to date. We investigated whether detecting stone autofluorescence, excited by a green aiming beam, is possible via the fiber during fragmentation by continuously recording the fluorescence signal in 12 ureterosopic lithotripsy procedures. We estimated which threshold the fluorescence signal's amplitude exceeds before laser pulses with visible stone removal by retrospective inspection of the endoscope's video data. For all procedures, blocking the laser when the fluorescence amplitude is below a threshold corresponding to the signal's baseline plus its range (maximum-minimum value) would have been appropriate to suppress ineffective pulses-the energy input could have been reduced by a mean of 14% (1%-29%) without changing the operation time. Ablation of the PTFE coating of the guidewire could have been prevented three times and cutting of a wire of the retrieval basket two times.

Controlled environments for cannabis cultivation to support "omics" research studies and production.

The cannabis (Cannabis sativa L.) genome is highly heterozygous and, to retain genetic identity, clonal propagation of cultivars is very common. Establishing controlled environments, often involving multiple locations throughout a single grow, is critical for reliably generating materials to be used in research and production. In this article, we break down different periods of the grow cycle, such as cloning, hardening (optional), vegetative growth, flowering growth, and harvest, into individual steps. We are including images and videos for an in-depth coverage of methodological details. We are providing a list of equipment, supplies, reagents, and other resources to help with planning a grow experiment. Finally, we are discussing considerations for different aspects of controlled environments, including lighting, fertilizer regimes, and integrated pest management. With this article, it is our goal to empower researchers to reliably generate disease-free cannabis material suitable for genetic and biochemical studies that require full control of environmental factors.

Standard operating procedures for the comprehensive and reliable analysis of cannabis terpenes.

Terpenes are the primary determinants of cannabis flower aroma, and ongoing research tests their potential for impacting the overall experience. Frustratingly, despite the importance of terpenes in cannabis physiology and commercial uses, literature reports vary widely regarding the major constituents of volatile blends and the concentrations of individual terpenes. In this article, we provide detailed descriptions of complementary approaches that will allow researchers to determine the identity and quantity of cannabis terpenes unequivocally and reliably. These standard operating procedures will guide decisions about which method to employ to address specific analytical goals. We are including two application examples to illustrate the utility of different approaches for tackling the analysis of terpenes in cannabis flower samples.

The different forms of the left brachiocephalic vein visualised during cardiovascular implantable electronic device implantation procedures.

BACKGROUND: Among the factors ensuring successful completion of such minimally invasive procedures as cardiac implantable electronic device (CIED) implantation and central venous catheter (CVC) placement are the morphometry and topography of the vessels used for cardiac lead or catheter advancement. Venous access through the left clavipectoral triangle makes use of the left brachiocephalic vein (BCV). The purpose of this study was to present the radiology images of various individual forms of this vessel observed during CIED implantation procedures. MATERIALS AND METHODS: Our analysis included 100 venography recordings illustrating the left BCV, obtained during de novo CIED implantation procedures. We assessed the mediastinal course of the left BCV, with its natural angles, including angle α (in the middle section of the vessel) and the two angles created by the left BCV and the left subclavian vein (angle ß) and the left BCV and the superior vena cava (angle γ). RESULTS: The mean values of angle α tended to be higher (approximately 141°) than those of the two remaining angles (γ and ß), which were comparable at 123° and 127°, respectively. An increase in mean angle α values were accompanied by increased mean angle γ and ß values (p = 0.05), with only 5% of ß and γ angles, in total, having values close to those of a right angle (90 ± 10º). CONCLUSIONS: Individual variability of left BCV topography and morphology comes from developmental formation of the physiological angle in the middle section of this mediastinal vessel's course. The presence of near-right angles along the course of left BCV may potentially result in injuries to the vessel during intravascular procedures.

Improving the chemical and sensory characteristics of red and white wines with pectinase-producing non-Saccharomyces yeasts.

This study examined the influence of pectinase-producing non-Saccharomyces yeasts on the chemical and sensory attributes of red and white wines with added pectin. Merlot and Chardonnay wines were produced with or without a mixture of pectinase-producing non-Saccharomyces yeasts (Cryptococcus adeliensis, Issatchenkia orientalis, and Pichia kluyveri) added to the must prior to alcoholic fermentation conducted by a commercial strain of Saccharomyces cerevisiae. To ensure sufficient substrate was present, varying concentrations of apple pectin (up to 1.25 g/L for red wines and 1.00 g/L for white wine) were added at the start of fermentation. After bottling, trained panelists (n = 10) analyzed these wines for aroma, flavor, taste, and mouthfeel attributes. For both wines, significant interactions were noted between the presence of non-Saccharomyces yeasts and pectin addition which affected pH, titratable acidity, and concentrations of D-galacturonic acid. While no significant sensory differences were observed among the red wines, limited changes were noted for white wines. However, a strong positive correlation was found between the D-galacturonic acid and buttery aroma for Chardonnay and with flavor for Merlot. Increasing D-galacturonic acid concentrations, through utilization of non-Saccharomyces yeasts, may improve the wine quality as a buttery aroma is often associated with high-quality Chardonnay. For both red and white wines, the utilization of these particular non-Saccharomyces yeasts significantly influenced chemical properties but yielded minor sensory changes without any faults. PRACTICAL APPLICATION: With the recent trend to reduce alcohol content in commercial wines, the interest in non-Saccharomyces yeasts has grown. This study showed that the addition of non-Saccharomyces yeasts, perhaps due to their pectinase activity, influenced the chemical characteristics of red and white wines with limited sensory differences, making these yeasts a useful tool for winemakers to modify wine properties.

The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

Chromosome-level genome assembly of Mentha longifolia L. reveals gene organization underlying disease resistance and essential oil traits.

Mentha longifolia (L.) Huds., a wild, diploid mint species, has been developed as a model for mint genetic and genomic research to aid breeding efforts that target Verticillium wilt disease resistance and essential oil monoterpene composition. Here, we present a near-complete, chromosome-scale mint genome assembly for M. longifolia USDA accession CMEN 585. This new assembly is an update of a previously published genome draft, with dramatic improvements. A total of 42,107 protein-coding genes were annotated and placed on 12 chromosomal scaffolds. One hundred fifty-three genes contained conserved sequence domains consistent with nucleotide binding site-leucine-rich-repeat plant disease resistance genes. Homologs of genes implicated in Verticillium wilt resistance in other plant species were also identified. Multiple paralogs of genes putatively involved in p-menthane monoterpenoid biosynthesis were identified and several cases of gene clustering documented. Heterologous expression of candidate genes, purification of recombinant target proteins, and subsequent enzyme assays allowed us to identify the genes underlying the pathway that leads to the most abundant monoterpenoid volatiles. The bioinformatic and functional analyses presented here are laying the groundwork for using marker-assisted selection in improving disease resistance and essential oil traits in mints.

Selectivity of enzymes involved in the formation of opposite enantiomeric series of p-menthane monoterpenoids in peppermint and Japanese catnip.

Peppermint (Mentha x piperita L.) and Japanese catnip (Schizonepeta tenuifolia (Benth.) Briq.) accumulate p-menthane monoterpenoids with identical functionalization patterns but opposite stereochemistry. In the present study, we investigate the enantioselectivity of multiple enzymes involved in monoterpenoid biosynthesis in these species. Based on kinetic assays, mint limonene synthase, limonene 3-hydroxylase, isopiperitenol dehydrogenase, isopiperitenone reductase, and menthone reductase exhibited significant enantioselectivity toward intermediates of the pathway that proceeds through (-)-4S-limonene. Limonene synthase, isopiperitenol dehydrogenase and isopiperitenone reductase of Japanese catnip preferred intermediates of the pathway that involves (+)-4R-limonene, whereas limonene 3-hydroxylase was not enantioselective, and the activities of pulegone reductase and menthone reductase were too low to acquire meaningful kinetic data. Molecular modeling studies with docked ligands generally supported the experimental data obtained with peppermint enzymes, indicating that the preferred enantiomer was aligned well with the requisite cofactor and amino acid residues implicated in catalysis. A striking example for enantioselectivity was peppermint (-)-menthone reductase, which binds (-)-menthone with exquisite affinity but was predicted to bind (+)-menthone in a non-productive orientation that positions its carbonyl functional group at considerable distance to the NADPH cofactor. The work presented here lays the groundwork for structure-function studies aimed at unraveling how enantioselectivity evolved in closely related species of the Lamiaceae and beyond.

Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis.

Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq., Nepeta tenuifolia Briq., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (-)-pulegone reductase from Nepeta tenuifolia (NtPR), which can catalyze (-)-pulegone to (+)-menthone and (-)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from M. piperita (MpPR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of MpPR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.

Taxanes and taxoids of the genus Taxus - A comprehensive inventory of chemical diversity.

The pseudoalkaloid diterpene Taxol® (paclitaxel) emerged as a best-selling anti-cancer drug in the mid-1990s. The compound attracted considerable interest because of its unique mechanism to stabilize microtubules, thus reducing dynamicity and ultimately promoting mitotic arrest. Taxol was originally isolated from members of the genus Taxus. Over the last 50 years, close to 600 metabolites with taxane scaffolds were isolated from various Taxus species and their structures reported. The present review article provides an overview of the known chemical diversity of taxanes, with an emphasis on the functionalization of diterpene scaffolds. The implications of the occurrence of chemically diverse taxane metabolites for unraveling Taxol biosynthesis and enabling pathway engineering are discussed as well.

Corrigendum: Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis.

[This corrects the article DOI: 10.3389/fpls.2021.780970.].

Crop Wild Relatives as Germplasm Resource for Cultivar Improvement in Mint (Mentha L.).

Mentha is a strongly scented herb of the Lamiaceae (formerly Labiatae) and includes about 30 species and hybrid species that are distributed or introduced throughout the globe. These fragrant plants have been selected throughout millennia for use by humans as herbs, spices, and pharmaceutical needs. The distilling of essential oils from mint began in Japan and England but has become a significant industrial product for the US, China, India, and other countries. The US Department of Agriculture (USDA), Agricultural Research Service, National Clonal Germplasm Repository (NCGR) maintains a mint genebank in Corvallis, Oregon. This facility preserves and distributes about 450 clones representing 34 taxa, hybrid species, advanced breeder selections, and F1 hybrids. Mint crop wild relatives are included in this unique resource. The majority of mint accessions and hybrids in this collection were initially donated in the 1970s by the A.M. Todd Company, located in Kalamazoo, Michigan. Other representatives of diverse mint taxa and crop wild relatives have since been obtained from collaborators in Australia, New Zealand, Europe, and Vietnam. These mints have been evaluated for cytology, oil components, verticillium wilt resistance, and key morphological characters. Pressed voucher specimens have been prepared for morphological identity verification. An initial set of microsatellite markers has been developed to determine clonal identity and assess genetic diversity. Plant breeders at private and public institutions are using molecular analysis to determine identity and diversity of the USDA mint collection. Evaluation and characterization includes essential oil content, disease resistance, male sterility, and other traits for potential breeding use. These accessions can be a source for parental genes for enhancement efforts to produce hybrids, or for breeding new cultivars for agricultural production. Propagules of Mentha are available for distribution to international researchers as stem cuttings, rhizome cuttings, or seed, which can be requested through the GRIN-Global database of the US National Plant Germplasm System, subject to international treaty and quarantine regulations.

[A boy with pain in the popliteal fossa]. / Een jongen met pijn in de knieholte.

A 12-year-old boy was referred by the general practitioner with a 3-week history of pain in the popliteal fossa. There was no sign of trauma or infection, physical examination was normal, and his CRP level was mildly elevated. X-ray and MRI revealed a Brodie's abscess, which was treated surgically and with antibiotics and he made a good recovery.