The giant diploid faba genome unlocks variation in a global protein crop.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.

Domestication has altered gene expression and secondary metabolites in pea seed coat.

The mature seed in legumes consists of an embryo and seed coat. In contrast to knowledge about the embryo, we know relatively little about the seed coat. We analyzed the gene expression during seed development using a panel of cultivated and wild pea genotypes. Gene co-expression analysis identified gene modules related to seed development, dormancy, and domestication. Oxidoreductase genes were found to be important components of developmental and domestication processes. Proteomic and metabolomic analysis revealed that domestication favored proteins involved in photosynthesis and protein metabolism at the expense of seed defense. Seed coats of wild peas were rich in cell wall-bound metabolites and the protective compounds predominated in their seed coats. Altogether, we have shown that domestication altered pea seed development and modified (mostly reduced) the transcripts along with the protein and metabolite composition of the seed coat, especially the content of the compounds involved in defense. We investigated dynamic profiles of selected identified phenolic and flavonoid metabolites across seed development. These compounds usually deteriorated the palatability and processing of the seeds. Our findings further provide resources to study secondary metabolism and strategies for improving the quality of legume seeds which comprise an important part of the human protein diet.

The loss of polyphenol oxidase function is associated with hilum pigmentation and has been selected during pea domestication.

Seed coats serve as protective tissue to the enclosed embryo. As well as mechanical there are also chemical defence functions. During domestication, the property of the seed coat was altered including the removal of the seed dormancy. We used a range of genetic, transcriptomic, proteomic and metabolomic approaches to determine the function of the pea seed polyphenol oxidase (PPO) gene. Sequencing analysis revealed one nucleotide insertion or deletion in the PPO gene, with the functional PPO allele found in all wild pea samples, while most cultivated peas have one of the three nonfunctional ppo alleles. PPO functionality cosegregates with hilum pigmentation. PPO gene and protein expression, as well as enzymatic activity, was downregulated in the seed coats of cultivated peas. The functionality of the PPO gene relates to the oxidation and polymerisation of gallocatechin in the seed coat. Additionally, imaging mass spectrometry supports the hypothesis that hilum pigmentation is conditioned by the presence of both phenolic precursors and sufficient PPO activity. Taken together these results indicate that the nonfunctional polyphenol oxidase gene has been selected during pea domestication, possibly due to better seed palatability or seed coat visual appearance.

Secret Recipe Revealed: Chemical Evaluation of Raw Colouring Mixtures from Early 19th Century Moravia.

An archaeological excavation in Prostejov (Czech Republic) revealed a workshop of a local potter with colourless, pink, and blue powders presumably used to produce faience/surface decoration. A comprehensive analytical study, which combined elemental and molecular analysis techniques, was performed to shed light on the chemical composition of these unique findings. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM EDX), inductively coupled-plasma mass spectrometry (ICP MS), flow injection analysis (FIA) with electrospray ionisation mass spectrometry (ESI MS), laser desorption ionisation mass spectrometry (LDI MS), and Raman spectroscopy were applied to reveal the elemental composition of the powders and identify the colouring agents in the pink and blue powders. The colouring agents in the pink powder were probably iron and the agent in the blue powder is Prussian blue. On top of that, it was also possible to determine the organic additives in these powders through pyrolysis gas chromatography with mass spectrometric detection (Py GC/MS), atmospheric solids analysis probe ion mobility mass spectrometry (ASAP IM MS), and LDI MS. The organic constituents were identified as plant resin, beeswax, and fats. These results point to the preparation of faience/pigment mixtures as oil paint.

A Rapid Nano-Liquid Chromatographic Method for the Analysis of Cannabinoids in Cannabis sativa L. Extracts.

Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H2O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization-mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia's protocol on medicinal plants, were carried out, and the results achieved were compared.

What about Dinner? Chemical and Microresidue Analysis Reveals the Function of Late Neolithic Ceramic Pans.

The Late Neolithic palafitte site, Ustie na Drim, in the northern part of Lake Ohrid (North Macedonia), excavated in 1962, offered ceramic fragments of large, flat, elongated pans. These artifacts could be dated by relative chronology to roughly around 5200-5000 BC. According to their shape and technological traits, the ceramic pans were probably used for baking. The attached materials on the surface of studied pan fragments were sampled for consequent chemical and microscopical analyses (i.e., analyses of starch, phytoliths, and microscopic animal remains). An immunological method revealed the presence of pork proteins in samples. The presence of organic residues of animal origin was, moreover, confirmed by the detection of cholesterol using gas chromatography coupled to mass spectrometry. Analysis of detected microscopic botanical objects revealed starch grains of several plants (i.e., oak, cattail, and grasses). An interesting find was the hair of a beetle larva, which could be interpreted contextually as the khapra beetle, a pest of grain and flour. Based on our data, we suppose that the ceramic pans from Ustie na Drim were used for the preparation of meals containing meat from common livestock in combination with cereals and wild plants.

Stereoisomer separation of flavanones and flavanone-7-O-glycosides by means of nanoliquid chromatography employing derivatized ß-cyclodextrins as mobile-phase additive.

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7-O-glycosides has been proposed employing a C18 capillary column and a chiral mobile-phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl-ß-cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether-ß-cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl-ß-cyclodextrin and 10 mM of sulfobutyl ether-ß-cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3-2.5 and 7.5-12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone-7-O-glycosides in Citrus juices after solid-phase extraction procedure.

Study of Tetrahydroxylated Anthraquinones-Potential Tool to Assess Degradation of Anthocyanins Rich Food.

Degradation of anthocyanins involves scission of the flavonoid skeleton yielding 2,4,6-trihydroxybenzaldehyde (phloroglucinaldehyde, PGA) and a phenolic acid. However, the process is not finished with the formation of PGA, as the consequent condensation of two PGA molecules providing colored hydroxylated anthraquinones was observed for the first time. This process was studied using a combination of preparative column chromatography, nuclear magnetic resonance, liquid chromatography/high resolution tandem mass spectrometry (LC/HRMS2), and quantum calculations using density functional theory. 1,3,5,7-tetrahydroxyanthraquinone (anthrachrysone) and its isomers were found to rise during heating (95 °C) in a buffered PGA model solution (phosphate buffer, pH 7). These compounds were detected in heated red wine after an increase of its pH value. The concentration of the identified anthrachrysone in the red wine reached 0.01 mg·L-1. Presence of those compounds could therefore indicate involvement of certain steps in the processing of plant materials rich in anthocyanins (e.g., utilization of a higher temperature and/or reduction of acidity) or long-term transformation of anthocyanins (potentially, for instance, in archaeological findings such as wine or fruit residues). Additionally, measurement of wine-soil suspensions proved an increase of their pH to the values suitable for anthocyanin cleavage (neutral to slightly alkaline; reached using soil from archaeologically well-known Bull Rock Cave). Although not found in artificially prepared samples (imitations) or authentic materials so far, according to our results the above mentioned conditions are suitable for the formation of tetrahydroxylated anthraquinone derivatives and their monitoring would be beneficial.

Correction to: Utilization of atmospheric solids analysis probe mass spectrometry for analysis of fatty acids on seed surface.

The authors would like to call the reader's attention to the following correction in the section "Semiquantitative analysis", page 1176, of the original publication.

Utilization of atmospheric solids analysis probe mass spectrometry for analysis of fatty acids on seed surface.

Atmospheric solids analysis probe mass spectrometry (ASAP-MS) was used for the first time for direct surface analysis of plant material. It can be readily used for surface analysis of whole and intact pea seeds and their seed coats, and for the study of the profile of fatty acids on the outer surface. Furthermore, ASAP-MS in combination with multivariate statistics allowed classification of pea genotypes with respect to physical dormancy and investigation of related biological markers. Hexacosanoic and octacosanoic acids were suggested to be important markers likely influencing water transport through the seed coat into the embryo (with the highest significance for dormant L100 genotype). ASAP-MS provided higher selectivity and better signal of fatty acids compared to (MA)LDI-MS (laser desorption ionization mass spectrometry either matrix free or matrix assisted) providing on the other hand spatial distribution information and results obtained by both methods are mutually supportive. The developed ASAP-MS method and obtained results can be widely utilized in biological, food, and agricultural research. Graphical abstract ᅟ.

Determination of Milk Products in Ceramic Vessels of Corded Ware Culture from a Late Eneolithic Burial.

In this study, a soil from two ceramic vessels belonging to Corded Ware culture, 2707⁻2571 B.C., found in a cremation grave discovered in Central Moravia, Czech Republic, was analyzed using matrix-assisted laser desorption/ionization⁻mass spectrometry (MALDI⁻MS) combined with advanced statistical treatment (principal component analysis, PCA, and orthogonal projection to latent structures discriminant analysis, OPLS-DA) and by enzyme-linked immunosorbent assay (ELISA). MALDI⁻MS revealed the presence of triacylglycerols in both vessels. This analytical technique was used for the analysis of the soil content from archaeological ceramic vessels for the first time. Targeted ELISA experiments consequently proved the presence of milk proteins in both ceramic vessels. These results represent the first direct evidence of the use of milk or dairy products in the Eneolithic period in Moravian Corded Ware Culture and help to better understand the diet habits and living conditions of Eneolithic populations in Central Europe.

Towards Better Understanding of Pea Seed Dormancy Using Laser Desorption/Ionization Mass Spectrometry.

Seed coats of six pea genotypes contrasting in dormancy were studied by laser desorption/ionization mass spectrometry (LDI-MS). Multivariate statistical analysis discriminated dormant and non-dormant seeds in mature dry state. Separation between dormant and non-dormant types was observed despite important markers of particular dormant genotypes differ from each other. Normalized signals of long-chain hydroxylated fatty acids (HLFA) in dormant JI64 genotype seed coats were significantly higher than in other genotypes. These compounds seem to be important markers likely influencing JI64 seed imbibition and germination. HLFA importance was supported by study of recombinant inbred lines (JI64xJI92) contrasting in dormancy but similar in other seed properties. Furthemore HLFA distribution in seed coat was studied by mass spectrometry imaging. HLFA contents in strophiole and hilum are significantly lower compared to other parts indicating their role in water uptake. Results from LDI-MS experiments are useful in understanding (physical) dormancy (first phases of germination) mechanism and properties related to food processing technologies (e.g., seed treatment by cooking).

Analytical and semipreparative chiral separation of cis-itraconazole on cellulose stationary phases by high-performance liquid chromatography.

cis-Itraconazole is a chiral antifungal drug administered as a racemate. The knowledge of properties of individual cis-itraconazole stereoisomers is vital information for medicine and biosciences as different stereoisomers of cis-itraconazole may possess different affinity to certain biological pathways in the human body. For this purpose, either chiral synthesis of enantiomers or chiral separation of racemate can be used. This paper presents a two-step high-performance liquid chromatography approach for the semipreparative isolation of four stereoisomers (two enantiomeric pairs) of itraconazole using polysaccharide stationary phases and volatile organic mobile phases without additives in isocratic mode. The approach used involves the separation of the racemate into three fractions (i.e. two pure stereoisomers and one mixed fraction containing the remaining two stereoisomers) in the first run and consequent separation of the collected mixed fraction in the second one. For this purpose, combination of cellulose tris-(4-methylbenzoate) and cellulose tris-(3,5-dimehylphenylcarbamate) columns with complementary selectivity for cis-itraconazole provided full separation of all four stereoisomers (with purity of each isomer > 97%). The stereoisomers were collected, their optical rotation determined and their identity confirmed based on the results of a previously published study. Pure separated stereoisomers are subjected to further biological studies.

Antiviral activity of porphyrins and porphyrin-like compounds against tick-borne encephalitis virus: Blockage of the viral entry/fusion machinery by photosensitization-mediated destruction of the viral envelope.

Tick-borne encephalitis virus (TBEV), the causative agent of tick-borne encephalitis (TBE), is a medically important flavivirus endemic to the European-Asian continent. Although more than 12,000 clinical cases are reported annually worldwide, there is no anti-TBEV therapy available to treat patients with TBE. Porphyrins are macrocyclic molecules consisting of a planar tetrapyrrolic ring that can coordinate a metal cation. In this study, we investigated the cytotoxicity and anti-TBEV activity of a large series of alkyl- or (het)aryl-substituted porphyrins, metalloporphyrins, and chlorins and characterized their molecular interactions with the viral envelope in detail. Our structure-activity relationship study showed that the tetrapyrrole ring is an essential structural element for anti-TBEV activity, but that the presence of different structurally distinct side chains with different lengths, charges, and rigidity or metal cation coordination can significantly alter the antiviral potency of porphyrin scaffolds. Porphyrins were demonstrated to interact with the TBEV lipid membrane and envelope protein E, disrupt the TBEV envelope and inhibit the TBEV entry/fusion machinery. The crucial mechanism of the anti-TBEV activity of porphyrins is based on photosensitization and the formation of highly reactive singlet oxygen. In addition to blocking viral entry and fusion, porphyrins were also observed to interact with RNA oligonucleotides derived from TBEV genomic RNA, indicating that these compounds could target multiple viral/cellular structures. Furthermore, immunization of mice with porphyrin-inactivated TBEV resulted in the formation of TBEV-neutralizing antibodies and protected the mice from TBEV infection. Porphyrins can thus be used to inactivate TBEV while retaining the immunogenic properties of the virus and could be useful for producing new inactivated TBEV vaccines.

Separation of Isomeric Tau Phosphopeptides from Alzheimer's Disease Brain by Cyclic Ion Mobility Mass Spectrometry.

Alzheimer's disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility-mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau-TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions (m/z 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at m/z 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.

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.

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.