Tick-borne encephalitis virus modulates sphingolipid and phospholipid metabolism in infected human neuronal cells.

The life cycle of enveloped viruses is closely linked to host-cell lipids. However, changes in lipid metabolism during infections with the tick-borne encephalitis virus (TBEV) have not been described. TBEV is a medically important orthoflavivirus, which is endemic to many parts of Europe and Asia. In the present study, we performed targeted lipidomics with HPLC-MS/MS to evaluate changes in phospholipid and sphingolipid concentrations in TBEV-infected human neuronal SK-N-SH cells. TBEV infections significantly increased phosphatidylcholine, phosphatidylinositol, and phosphatidylserine levels within 48 h post-infection (hpi). Sphingolipids were slightly increased in dihydroceramides within 24 hpi. Later, at 48 hpi, the contents of sphinganine, dihydroceramides, ceramides, glucosylceramides, and ganglioside GD3 were elevated. On the other hand, sphingosine-1-phosphate content was slightly reduced in TBEV-infected cells. Changes in sphingolipid concentrations were accompanied by suppressed expression of a majority of the genes linked to sphingolipid and glycosphingolipid metabolism. Furthermore, we found that a pharmacological inhibitor of sphingolipid synthesis, fenretinide (4-HPR), inhibited TBEV infections in SK-N-SH cells. Taken together, our results suggested that both structural and signaling functions of lipids could be affected during TBEV infections. These changes might be connected to virus propagation and/or host-cell defense.

The Effect of Water Availability on the Carbon Content of Grain and Above- and Belowground Residues in Common and Einkorn Wheat.

The carbon (C) fixed by crops, which is exported with harvest and retained as postharvest residues in a field, is important for calculating the C balance. The aim of this study was to determine the effect of water availability on the C content in whole wheat plants. In a three-year field trial, the weights of grain, straw, chaff, stubble, and roots of two cultivars of winter wheat (Triticum aestivum L.) and one cultivar of einkorn wheat (Triticum monococcum L.) and their carbon contents were determined in water stress, irrigation, and rain-fed control treatments. The water availability, year, and cultivar had a significant influence on the C content in aboveground plant parts, but the effect of water on grain C was weak. The C content decreased with irrigation and increased with drought, but the differences were small (at most, 3.39% in chaff). On average, the C contents of grain, straw, chaff, and roots reached 45.0, 45.7, 42.6, and 34.9%, respectively. The amount of C exported with grain and left on the field in the form of postharvest residues depended on the weight of the total biomass and the ratio of grain to straw and residue. Whole plant C yield reached 8.99, 7.46, and 9.65 t ha-1 in rain-fed control, stressed, and irrigated treatments, respectively, and 8.91, 9.45, and 7.47 t ha-1 in Artix, Butterfly, and Rumona, respectively. Irrigation significantly increased the C content in grain and straw (but not in chaff, stubble, and roots) in comparison with water shortage conditions. On average, a grain yield of 1 t ha-1 corresponded to an average export of 0.447-0.454 t C ha-1 in the grain of all cultivars and inputs of 0.721, 0.832, and 2.207 t C ha-1 of residue to the soil in the form of straw and postharvest residue in the two cultivars of common wheat and one of einkorn. The results of the study provided reliable data for the calculation of the C balance of wheat under conditions of different water availability.

Efficacy and immunogenicity of a veterinary vaccine candidate against tick-borne encephalitis in dogs.

Tick-borne encephalitis (TBE) is a severe neuroinfection of humans. Dogs are also commonly infected with tick-borne encephalitis virus (TBEV). These infections are usually asymptomatic, but sometimes show clinical signs similar to those seen in humans and can be fatal. To date, there is no TBEV vaccine available for use in dogs. To address this need, a TBEV vaccine candidate for dogs based on inactivated whole virus antigen was developed. The safety, immunogenicity, and efficacy of the vaccine candidate were tested in mice as the preclinical model and in dogs as the target organism. The vaccine was well tolerated in both species and elicited the production of specific anti-TBEV antibodies with virus neutralising activity. Vaccination of mice provided complete protection against the development of fatal TBE. Immunisation of dogs prevented the development of viremia after challenge infection. Therefore, the developed vaccine candidate is promising to protect dogs from severe TBEV infections.

Membrane-Targeting Perylenylethynylphenols Inactivate Medically Important Coronaviruses via the Singlet Oxygen Photogeneration Mechanism.

Perylenylethynyl derivatives have been recognized as broad-spectrum antivirals that target the lipid envelope of enveloped viruses. In this study, we present novel perylenylethynylphenols that exhibit nanomolar or submicromolar antiviral activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and feline infectious peritonitis virus (FIPV) in vitro. Perylenylethynylphenols incorporate into viral and cellular membranes and block the entry of the virus into the host cell. Furthermore, these compounds demonstrate an ability to generate singlet oxygen when exposed to visible light. The rate of singlet oxygen production is positively correlated with antiviral activity, confirming that the inhibition of fusion is primarily due to singlet-oxygen-induced damage to the viral envelope. The unique combination of a shape that affords affinity to the lipid bilayer and the capacity to generate singlet oxygen makes perylenylethynylphenols highly effective scaffolds against enveloped viruses. The anticoronaviral activity of perylenylethynylphenols is strictly light-dependent and disappears in the absence of daylight (under red light). Moreover, these compounds exhibit negligible cytotoxicity, highlighting their significant potential for further exploration of the precise antiviral mechanism and the broader scope and limitations of this compound class.

A combination of two resistance mechanisms is critical for tick-borne encephalitis virus escape from a broadly neutralizing human antibody.

Tick-borne encephalitis virus (TBEV) is a flavivirus that causes human neuroinfections and represents a growing health problem. The human monoclonal antibody T025 targets envelope protein domain III (EDIII) of TBEV and related tick-borne flaviviruses, potently neutralizing TBEV in vitro and in preclinical models, representing a promising candidate for clinical development. We demonstrate that TBEV escape in the presence of T025 or T028 (another EDIII-targeting human monoclonal antibody) results in virus variants of reduced pathogenicity, characterized by distinct sets of amino acid changes in EDII and EDIII that are jointly needed to confer resistance. EDIII substitution K311N impairs formation of a salt bridge critical for T025-epitope interaction. EDII substitution E230K is not on the T025 epitope but likely induces quaternary rearrangements of the virus surface because of repulsion of positively charged residues on the adjacent EDI. A combination of T025 and T028 prevents virus escape and improves neutralization.

Exploring Wild Hordeum spontaneum and Hordeum marinum Accessions as Genetic Resources for Fungal Resistance.

Crop Wild Relatives (CWRs), as potential sources of new genetic variants, are being extensively studied to identify genotypes that will be able to confer resistance to biotic stresses. In this study, a collection of barley wild relatives was assessed in the field, and their phenotypic variability was evaluated using a Barley Description List, reflecting the identified ecosites. Overall, the CWRs showed significant field resistance to various fungal diseases. To further investigate their resistance, greenhouse tests were performed, revealing that several CWRs exhibited resistance against Fusarium culmorum, Pyrenophora teres, and Puccinia hordei G.H. Otth. Additionally, to characterize the genetic diversity within the collection, DNA polymorphisms at 21 loci were examined. We successfully employed barley-specific SSR markers, confirming their suitability for identifying H. spontaneum and even H. marinum, i.e., perennial species. The SSR markers efficiently clustered the investigated collection according to species and ecotypes, similarly to the phenotypic assessment. Moreover, SSR markers associated with disease resistance revealed different alleles in comparison to those found in resistant barley cultivars. Overall, our findings highlight that this evaluated collection of CWRs represents a valuable reservoir of genetic variability and resistance genes that can be effectively utilized in breeding programs.

Origin and genetic variability of populations of the invasive plant Rumex alpinus L. in the Giant (Krkonose) Mountains.

Monk's rhubarb, Rumex alpinus L. (R. alpinus), is a perennial plant native to the mountains of Central and Southern Europe. Currently, the distribution of R. alpinus has been partly affected by its utilization as a vegetable and a medicinal herb. In the mountains of the Czech Republic, it is considered an invasive plant, probably introduced into the Krkonose Mountains by colonists from the Alps. This study's main aim was to verify whether R. alpinus was introduced into the Krkonose Mountains by alpine colonists or whether it was anthropogenically introduced from the Carpathians. Furthermore, the genetic structure of native and introduced populations of R. alpinus was determined. For the evaluation of genetic structure, 417 samples of R. alpinus were collected from the Alps, Carpathians, Balkan, Pyrenees, and Czech Mountains. In total, 12 simple sequence repeat (SSR) markers were applied. The results of AMOVA showed a high 60% variation within populations, 27% variation among groups, and 13% among the population within groups. The overall unbiased gene diversity was high (^h = 0.55). The higher level of genetic differentiation among populations (FST = 0.35; p < .01) indicated restricted gene flow between populations. Compared to native populations, limited genetic variability was observed in the nonnative populations. It was concluded that local adaptation, low gene exchange, and genetic drift affected the genetic diversity of nonnative R. alpinus. The results support a genetic link between Alpine and Czech genotypes of R. alpinus, while the Carpathians genotypes corresponded to the Balkan genotype.

Antiviral activity of singlet oxygen-photogenerating perylene compounds against SARS-CoV-2: Interaction with the viral envelope and photodynamic virion inactivation.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted great interest in novel broad-spectrum antivirals, including perylene-related compounds. In the present study, we performed a structure-activity relationship analysis of a series of perylene derivatives, which comprised a large planar perylene residue, and structurally divergent polar groups connected to the perylene core by a rigid ethynyl or thiophene linker. Most of the tested compounds did not exhibit significant cytotoxicity towards multiple cell types susceptible to SARS-CoV-2 infection, and did not change the expressions of cellular stress-related genes under normal light conditions. These compounds showed nanomolar or sub-micromolar dose-dependent anti-SARS-CoV-2 activity, and also suppressed the in vitro replication of feline coronavirus (FCoV), also termed feline infectious peritonitis virus (FIPV). Perylene compounds exhibited high affinity for liposomal and cellular membranes, and efficiently intercalated into the envelopes of SARS-CoV-2 virions, thereby blocking the viral-cell fusion machinery. Furthermore, the studied compounds were demonstrated to be potent photosensitizers, generating reactive oxygen species (ROS), and their anti-SARS-CoV-2 activities were considerably enhanced after irradiation with blue light. Our results indicated that photosensitization is the major mechanism underlying the anti-SARS-CoV-2 activity of perylene derivatives, with these compounds completely losing their antiviral potency under red light. Overall, perylene-based compounds are broad-spectrum antivirals against multiple enveloped viruses, with antiviral action based on light-induced photochemical damage (ROS-mediated, likely singlet oxygen-mediated), causing impairment of viral membrane rheology.

Nutritional Evaluation of Quinoa Genetic Resources Growing in the Climatic Conditions of Central Europe.

Quinoa displays huge genetic variability and adaptability to distinct climatic conditions. Quinoa seeds are a good source of nutrients; however, the overall nutritional composition and nutrient content is influenced by numerous factors. This study focused on the nutritional and morphologic evaluation of various quinoa genotypes grown in the Czech Republic. Significant differences between years were observed for morphological traits (plant height, inflorescence length, weight of thousand seeds). The weather conditions in the year 2018 were favorable for all the morphological traits. The protein content of quinoa accessions ranged between 13.44 and 20.01% and it was positively correlated to mauritianin. Total phenolic content varied greatly from year to year, while the antioxidant activity remained relatively stable. The most abundant phenolic compounds were the flavonoids miquelianin, rutin, and isoquercetin. Isoquercetin, quercetin, and N-feruoloyl octopamine showed the highest stability under variable weather conditions in the analyzed years. A total of six compounds were detected and quantified in quinoa for the first time. Most varieties performed well under Central European conditions and can be considered a good source of nutrients and bioactive compounds. These data can be used as a source of information for plant breeders aiming to improve the quality traits of quinoa.

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Functional characterization and immunogenicity of a novel vaccine candidate against tick-borne encephalitis virus based on Leishmania-derived virus-like particles.

Tick-borne encephalitis virus (TBEV) is a major cause of neurological infections in many regions of central, eastern and northern Europe and northern Asia. In approximately 15% of cases, TBEV infections lead to the development of severe encephalitis or meningitis. The main route of TBEV transmission is tick bites; however, ingestion of dairy products from infected animals (goats, cattle and sheep) is also a frequent cause of the disease. Therefore, vaccination of livestock in virus endemic regions could also contribute to the decrease in TBEV infection among humans. Although few vaccines against TBEV based on inactivated viruses are available for humans, due to high costs, vaccination is not mandatory in most of the affected countries. Moreover, there is still no vaccine for veterinary use. Here, we present a characterization and immunogenicity study of a new potential TBEV vaccine based on virus-like particles (VLPs) produced in Leishmania tarentolae cells. VLPs, which mimic native viral particles but do not contain genetic material, show good immunogenic potential. For the first time, we showed that the protozoan L. tarentolae expression system can be successfully used for the production of TBEV virus-like particles with highly efficient production. We confirmed that TBEV recombinant structural proteins (prM/M and E) from VLPs are highly recognized by neutralizing antibodies in in vitro analyses. Therefore, VLPs in combination with AddaVax adjuvant were used in immunization studies in a mouse model. VLPs proved to be highly immunogenic and induced the production of high levels of neutralizing antibodies. In a challenge experiment, immunization with VLPs provided full protection from lethal TBE in mice. Thus, we suggest that Leishmania-derived VLPs may be a good candidate for a safe alternative human vaccine with high efficiency of production. Moreover, this potential vaccine candidate may constitute a low-cost candidate for veterinary use.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Safety of Ustekinumab and Vedolizumab During Pregnancy-Pregnancy, Neonatal, and Infant Outcome: A Prospective Multicentre Study.

BACKGROUND AND AIMS: Evidence on the safety of newer biologics during pregnancy is limited. We aimed to assess the safety of ustekinumab and vedolizumab treatment during gestation on pregnancy and infant outcome. Furthermore, we evaluated the placental transfer of these agents. METHODS: We performed a prospective, multicentre, observational study in consecutive women with inflammatory bowel disease exposed to ustekinumab or vedolizumab 2 months prior to conception or during pregnancy. Pregnancy, neonatal, and infant outcomes were evaluated and compared with the anti-tumour necrosis factor [TNF]-exposed control group. Drug levels were assessed in maternal and cord blood at delivery. RESULTS: We included 54 and 39 pregnancies exposed to ustekinumab and vedolizumab, respectively. In the ustekinumab group, 43 [79.9%] resulted in live births, and 11 [20.4%] led to spontaneous abortion. Thirty-five [89.7%] pregnancies on vedolizumab ended in a live birth, two [5.1%] in spontaneous, and two [5.1%] in therapeutic abortion. No significant difference in pregnancy outcome between either the vedolizumab or the ustekinumab group and controls was observed [p >0.05]. Similarly, there was no negative safety signal in the postnatal outcome of exposed children regarding growth, psychomotor development, and risk of allergy/atopy or infectious complications. The median infant-to-maternal ratio of ustekinumab levels was 1.67 and it was 0.59 in vedolizumab. CONCLUSIONS: Use of ustekinumab and vedolizumab in pregnancy seems to be safe, with favuorable pregnancy and postnatal infant outcomes. Placental transfer differed between these two drugs, with ustekinumab having similar and vedolizumab having inverse infant-to-maternal ratio of drug levels compared with anti-TNF preparations.

Experimental and Natural Infections of Tick-Borne Encephalitis Virus in Dogs.

Dogs are frequently infected with the tick-borne encephalitis virus (TBEV). However, to date, only a few clinically manifest cases of tick-borne encephalitis (TBE) have been reported in dogs. In this study, three-month-old beagle dogs were infected with TBEV through a subcutaneous injection. Body temperature, clinical signs, blood haematology, blood biochemistry, and immune responses were monitored for up to 28 days postinfection (p.i.). No changes in body temperature or clinical signs were observed in the infected dogs. Most haematology and blood biochemistry parameters were unchanged after the infection, except for a slight reduction in blood lymphocyte counts, but they were within the physiological range. Low-titre viraemia was detected in 2/4 infected dogs between days 1 and 3 p.i. All infected dogs developed a robust immune response, in terms of neutralising antibodies. Thus, TBEV infections lead to effective seroconversion in dogs. Next, to assess TBEV exposure in dogs in the TBEV-endemic region of the Czech Republic, we conducted a serosurvey. Virus neutralisation tests revealed TBEV-specific antibodies in 17 of 130 (13.07%) healthy dogs, which confirmed a high, but clinically inappreciable TBEV exposure rate in the endemic area. The seropositivity rate was similar (12.7%; 41 positives out of 323) in a subgroup of dogs with various clinical disorders, and it was 13.4% (23 out of 171) in a subgroup of dogs with signs of acute neurological disease. Two dogs with fatal acute meningoencephalitis showed positive results for TBEV-specific IgM and IgG antibodies. These data extended our understanding of the clinical presentation of TBEV infections.

Non-Nucleotide RNA-Dependent RNA Polymerase Inhibitor That Blocks SARS-CoV-2 Replication.

SARS-CoV-2 has caused an extensive pandemic of COVID-19 all around the world. Key viral enzymes are suitable molecular targets for the development of new antivirals against SARS-CoV-2 which could represent potential treatments of the corresponding disease. With respect to its essential role in the replication of viral RNA, RNA-dependent RNA polymerase (RdRp) is one of the prime targets. HeE1-2Tyr and related derivatives were originally discovered as inhibitors of the RdRp of flaviviruses. Here, we present that these pyridobenzothiazole derivatives also significantly inhibit SARS-CoV-2 RdRp, as demonstrated using both polymerase- and cell-based antiviral assays.

Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum.

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Differences in the placental pharmacokinetics of vedolizumab and ustekinumab during pregnancy in women with inflammatory bowel disease: a prospective multicentre study.

BACKGROUND: Vedolizumab demonstrated different placental pharmacokinetics than other immunoglobulin G1 antibodies, leading to lower drug levels in cord blood in contrast to maternal blood at the time of delivery. The placental transfer of ustekinumab seems to have a pattern similar to anti-tumour necrosis factor agents. Current evidence on the placental pharmacokinetics of vedolizumab and ustekinumab is limited. We aimed to assess the placental transfer of ustekinumab and vedolizumab in pregnant patients with inflammatory bowel disease. METHODS: Consecutive women from a prospective observational study who were exposed to ustekinumab or vedolizumab within 2 months prior to conception or during pregnancy were included. Ustekinumab and vedolizumab levels were measured in maternal and cord blood at the time of delivery. RESULTS: Drug levels were available in 31 infant-mother pairs (15 exposed to ustekinumab and 16 to vedolizumab). The median maternal and newborn ustekinumab levels were 5.3 mg/l and 10.3 mg/l, respectively (the median infant-to-maternal ratio was 1.7), while the median maternal and cord vedolizumab levels were 7.3 mg/l and 4.5 mg/l (the median infant-to-maternal ratio was 0.66). The ustekinumab levels in cord blood positively correlated with the maternal levels at delivery (ρ = 0.751, p = 0.001). However, no correlation with the timing of the last drug administration was found. In contrast, the vedolizumab levels in cord blood demonstrated significant positive correlation with the maternal levels (ρ = 0.831, p < 0.001) along with the gestational week of the last infusion (ρ = 0.736, p = 0.001). CONCLUSION: Vedolizumab demonstrated different placental pharmacokinetics, leading to lower drug levels in cord blood compared to maternal blood at delivery; in contrast, the placental transfer of ustekinumab seems to have a pattern similar to anti-tumour necrosis factor (TNF) agents.

Breeding Buckwheat for Nutritional Quality in the Czech Republic.

Buckwheat is a nutritionally valuable crop, an alternative to common cereals also usable in gluten-free diets. The selection of buckwheat genotypes suitable for further breeding requires the characterization and evaluation of genetic resources. The main objective of this work was to evaluate selected phenotypic and morphological traits using international buckwheat descriptors, including total phenolic content and antioxidant activity, on a unique set of 136 common buckwheat accessions grown in 2019-2020 under Czech Republic conditions. In addition, UHPLC-ESI- MS/MS was used to analyze a wide spectrum of 20 phenolic compounds in buckwheat seeds, including four flavanols, three phenolic acids, seven flavonols, four flavones, and two flavanones. Significant differences among years and genotypes were observed for morphological traits (plant height and 1000-seed weight) and antioxidant activity, as well as levels of observed chemical compounds. Antioxidant activity, crude protein content, plant height and rutin content were characterized by higher mean values in 2020 than in 2019 and vice versa for total polyphenol content and 1000-seed weight. Crude protein content was the most stable across years, while total polyphenol content and rutin content varied greatly from year to year. The most abundant phenolic compounds were rutin, hyperoside, epicatechin, catechin, vitexin, isovitexin, orientin and isoorientin. Protein content was negatively correlated with plant height, catechin and epicatechin content. On the other hand, AA and TPC were positively correlated with rutin, hyperoside and chlorogenic acid. Five accessions showed high stability of the evaluated traits under changing conditions within both years of observation. These materials can be used in breeding programmes aimed at improving buckwheat genotypes with emphasis on quality traits.

OpiumPlex is a novel microsatellite system for profiling opium poppy (Papaver somniferum L.).

Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625-1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28-15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3-14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).

Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease.

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.