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Micropropagated Catharantus roseus plants infected with ' Candidatus Phytoplasma asteris' showed virescence symptoms, witches' broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.

Purpose: to assess the vasculature and thickness of the macula using OCT-A in patients who had experienced a previous episode of Leber hereditary optic neuropathy (LHON) or non-arteritic anterior ischemic optic neuropathy (NA-AION)., Methods: twelve eyes with chronic LHON and ten eyes with chronic NA-AION and eight NA-AION fellow eyes were examined using OCT-A. The vessel density was measured in the superficial and deep plexus of the retina. Moreover, the full and inner thicknesses of the retina were assessed., Results: There were significant differences in all sectors between the groups in regard to the superficial vessel density and the inner and full thicknesses of the retina. The nasal sector of the macular superficial vessel density was affected more in LHON than in NA-AION; the same with the temporal sector of the retinal thickness. There were no significant differences between the groups in the deep vessel plexus. There were no significant differences between the vasculature of the inferior and superior hemifields of the macula in all groups and no correlation with the visual function., Conclusions: The superficial perfusion and structure of the macula assessed with OCT-A are affected both in chronic LHON and NA-AION, but more in LHON eyes, especially in the nasal and temporal sectors., Competing Interests: The authors declare no conflict of interest.

This study investigated effects of dietary fumonisins (FBs) on gut and faecal microbiota of weaned pigs. In total, 18 7-week-old male pigs were fed either 0, 15 or 30 mg FBs (FB 1 + FB 2 + FB 3 )/kg diet for 21 days. The microbiota was analysed with amplicon sequencing of the 16S rRNA gene V3-V4 regions (Illumina MiSeq). Results showed no treatment effect ( p > 0.05) on growth performance, serum reduced glutathione, glutathione peroxidase and malondialdehyde. FBs increased serum aspartate transaminase, gamma glutamyl-transferase and alkaline phosphatase activities. A 30 mg/kg FBs treatment shifted microbial population in the duodenum and ileum to lower levels (compared to control ( p < 0.05)) of the families Campylobacteraceae and Clostridiaceae , respectively, as well as the genera Alloprevotella , Campylobacter and Lachnospiraceae Incertae Sedis (duodenum), Turicibacter (jejunum), and Clostridium sensu stricto 1 (ileum). Faecal microbiota had higher levels of the Erysipelotrichaceae and Ruminococcaceae families and Solobacterium , Faecalibacterium , Anaerofilum , Ruminococcus , Subdoligranulum , Pseudobutyrivibrio , Coprococcus and Roseburia genera in the 30 mg/kg FBs compared to control and/or to the 15 mg/kg FBs diets. Lactobacillus was more abundant in the duodenum compared to faeces in all treatment groups ( p < 0.01). Overall, the 30 mg/kg FBs diet altered the pig gut microbiota without suppressing animal growth performance.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Recently, in vitro gene preservation has gained ground thanks to its lower cost and higher stability compared to in vivo techniques. One of the methods that can preserve female-specific W chromosome-linked genes is primordial germ cell (PGC) freezing. PGCs can be isolated from Hamburger-Hamilton stage 14-16 embryos via blood sampling. In our experiment, we used two newly established Black Transylvanian naked neck chicken cell lines and four cell lines from our gene bank. We compared two different freezing media (FAM1 and FAM2) in this study. The cell number and viability of the PGCs were measured before freezing (BF) and after thawing on Day 0, Day 1, and Day 7 of cultivation. We analyzed the germ cell-specific chicken vasa homologue (CVH) expression profile in PGCs using RT-qPCR. We found that on Day 0, immediately after thawing, the cell number in cell lines frozen with the FAM2 medium was significantly higher than in the FAM1-treated ones. On Day 1 and Day 7, the cell number and viability were also higher in most cell lines frozen with FAM2, but the difference was insignificant. The freezing also affected the chicken vasa homologue gene expression in male lines treated with both freezing media.

Key Message: The bs5 resistance gene against bacterial spot was identified by map-based cloning. The recessive bs5 gene of pepper (Capsicum annuum L.) conditions a non-hypersensitive resistance trait, characterized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria infection. The isolation of the bs5 gene by map-based cloning revealed that the bs5 protein was shorter by 2 amino acids as compared to the wild type Bs5 protein. The natural 2 amino acid deletion occurred in the cysteine-rich transmembrane domain of the tail-anchored (TA) protein, Ca&#95;CYSTM1. The protein products of the wild type Bs5 and mutant bs5 genes were shown to be located in the cell membrane, indicating an unknown function in this membrane compartment. Successful infection of the Bs5 pepper lines was abolished by the 6 bp deletion in the TM encoding domain of the Ca&#95;CYSTM1 gene in bs5 homozygotes, suggesting, that the resulting resistance might be explained by the lack of entry of the Xanthomonas specific effector molecules into the plant cells., (© 2023. The Author(s).)

During muscle cell differentiation, the alternatively spliced, acidic β-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the β-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid β-domain variants, as well as by a disordered β-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the β-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that β-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process., (© 2023. The Author(s).)

In recent years, our understanding of the epigenetic mechanisms involved in tumor pathology has improved greatly. DNA and histone modifications, such as methylation, demethylation, acetylation, and deacetylation, can lead to the up-regulation of oncogenic genes, as well as the suppression of tumor suppressor genes. Gene expression can also be modified on a post-transcriptional level by microRNAs that contribute to carcinogenesis. The role of these modifications has been already described in many tumors, e.g., colorectal, breast, and prostate cancers. These mechanisms have also begun to be investigated in less common tumors, such as sarcomas. Chondrosarcoma (CS) is a rare type of tumor that belongs to sarcomas and is the second most common malignant bone tumor after osteosarcoma. Due to unknown pathogenesis and resistance to chemo- and radiotherapies of these tumors, there is a need to develop new potential therapies against CS. In this review, we summarize current knowledge on the influence of epigenetic alterations in the pathogenesis of CS by discussing potential candidates for future therapies. We also emphasize ongoing clinical trials that use drugs targeting epigenetic modifications in CS treatment.

The current report describes the chemical investigation and biological activity of extracts produced by three fungal strains Fusarium oxysporum, Penicillium simplicissimum, and Fusarium proliferatum isolated from the roots of Piper nigrum L. growing in Vietnam. These fungi were namely determined by morphological and DNA analyses. GC/MS identification revealed that the EtOAc extracts of these fungi were associated with the presence of saturated and unsaturated fatty acids. These EtOAc extracts showed cytotoxicity towards cancer cell lines HepG2, inhibited various microbacterial organisms, especially fungus Aspergillus niger and yeast Candida albicans (the MIC values of 50-100 μg/mL). In α-glucosidase inhibitory assay, they induced the IC 50 values of 1.00-2.53 μg/mL were better than positive control acarbose (169.80 μg/mL). The EtOAc extract of F. oxysporum also showed strong anti-inflammatory activity against NO production and PGE-2 level. Four major compounds linoleic acid (37.346 %), oleic acid (27.520 %), palmitic acid (25.547 %), and stearic acid (7.030 %) from the EtOAc extract of F. oxysporum were selective in molecular docking study, by which linoleic and oleic acids showed higher binding affinity towards α-glucosidase than palmitic and stearic acids. In subsequent docking assay with inducible nitric oxide synthase (iNOS), palmitic acid, oleic acid and linoleic acid could be moderate inhibitors., (© 2022 Wiley-VHCA AG, Zurich, Switzerland.)

Dermacentor reticulatus (Fabr., 1794) (Acari: Ixodidae) is parasite that spreads many diseases which are dangerous to humans and animals. Microelement lithium was found to have promising potential against the detrimental bee pest Varroa destructor. Furthermore, its effectiveness was confirmed against Dermanyssus gallinae, a major parasite of poultry, in vitro. In the present study, we investigated whether the efficacy of lithium chloride extends to other parasitic species, such as D. reticulatus . Our results revealed, for the first time, that the effectiveness of lithium chloride extends to D. reticulatus , confirmed to have 100% mortality at a relatively high minimum concentration of 1.38 M in vitro. The 24 h and 48 h median lethal concentration (LC 50 ) values proved to be 0.654 M and 0.481 M, respectively, for this species. Our pilot study may contribute to a better understanding of the properties of lithium ion. Furthermore, it may elicit further studies aiming to reveal whether the different environmental mineral conditions may influence the D. reticulatus population. Further studies might reveal whether lithium has any possible veterinary relevance.

Dravet syndrome (DRVT) is a rare form of neurodevelopmental disorder with a high risk of sudden unexpected death in epilepsy (SUDEP), caused mainly (>80% cases) by mutations in the SCN1A gene, coding the Nav1.1 protein (alfa-subunit of voltage-sensitive sodium channel). Mutations in SCN1A are linked to heterogenous epileptic phenotypes of various types, severity, and patient prognosis. Here we generated iPSC lines from fibroblasts obtained from three individuals affected with DRVT carrying distinct mutations in the SCN1A gene (nonsense mutation p.Ser1516*, missense mutation p.Arg1596His, and splicing mutation c.2589+2dupT). The iPSC lines, generated with the non-integrative approach, retained the distinct SCN1A gene mutation of the donor fibroblasts and were characterized by confirming the expression of the pluripotency markers, the three-germ layer differentiation potential, the absence of exogenous vector expression, and a normal karyotype. The generated iPSC lines were used to establish ventral forebrain organoids, the most affected type of neurons in the pathology of DRVT. The DRVT organoid model will provide an additional resource for deciphering the pathology behind Nav1.1 haploinsufficiency and drug screening to remediate the functional deficits associated with the disease.

Ralstonia solanacearum ( Rs ), the causal agent of bacterial wilt disease in an unusually wide range of host plants, including potato ( Solanum tuberosum ), is one of the most destructive phytopathogens that seriously reduces crop yields worldwide. Identification of defence mechanisms underlying bacterial wilt resistance is a prerequisite for biotechnological approaches to resistance breeding. Resistance to Rs has been reported only in a few potato landraces and cultivars. Our in vitro inoculation bioassays confirmed that the cultivars 'Calalo Gaspar' (CG) and 'Cruza 148' (CR) are resistant to Rs infection. Comparative transcriptome analyses of CG and CR roots, as well as of the roots of an Rs -susceptible cultivar, 'Désirée' (DES), were carried out two days after Rs infection, in parallel with their respective noninfected controls. In CR and DES, the upregulation of chitin interactions and cell wall-related genes was detected. The phenylpropanoid biosynthesis and glutathione metabolism pathways were induced only in CR, as confirmed by high levels of lignification over the whole stele in CR roots six days after Rs infection. At the same time, Rs infection greatly increased the concentrations of chlorogenic acid and quercetin derivatives in CG roots as it was detected using ultra-performance liquid chromatography - tandem mass spectrometry. Characteristic increases in the expression of MAP kinase signalling pathway genes and in the concentrations of jasmonic, salicylic, abscisic and indoleacetic acid were measured in DES roots. These results indicate different Rs defence mechanisms in the two resistant potato cultivars and a different response to Rs infection in the susceptible cultivar., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jose, Éva, Bozsó, Hamow, Fekete, Fábián, Bánfalvi and Sági.)

Recent discoveries of various noncanonical RNA caps, such as dinucleoside polyphosphates (Np n N), coenzyme A (CoA), and nicotinamide adenine dinucleotide (NAD) in all domains of life have led to a revision of views on RNA cap function and metabolism. Enzymes from the NudiX family capable of hydrolyzing a polyphosphate backbone attached to a nucleoside are the strongest candidates for degradation of noncanonically capped RNA. The model plant organism Arabidopsis thaliana encodes as many as 28 NudiX enzymes. For most of them, only in vitro substrates in the form of small molecules are known. In our study, we focused on four A. thaliana NudiX enzymes (AtNUDT6, AtNUDT7, AtNUDT19 and AtNUDT27), and we studied whether these enzymes can cleave RNA capped with Np n Ns (Ap 2-5 A, Gp 3-4 G, Ap 3-5 G, m 7 Gp 3 G, m 7 Gp 3 A), CoA, ADP-ribose, or NAD(H). While AtNUDT19 preferred NADH-RNA over other types of capped RNA, AtNUDT6 and AtNUDT7 preferentially cleaved Ap 4 A-RNA. The most powerful decapping enzyme was AtNUDT27, which cleaved almost all types of capped RNA at a tenfold lower concentration than the other enzymes. We also compared cleavage efficiency of each enzyme on free small molecules with RNA capped with corresponding molecules. We found that AtNUDT6 prefers free Ap 4 A, while AtNUDT7 preferentially cleaved Ap 4 A-RNA. These findings show that NudiX enzymes may act as RNA-decapping enzymes in A. thaliana and that other noncanonical RNA caps such as Ap 4 A and NADH should be searched for in plant RNA., Competing Interests: Authors declare no conflicts of interests., (This journal is © The Royal Society of Chemistry.)

Studies conducted on large populations show a lack of connection between vaccination and serious neurological symptoms. However, there are isolated cases that indicate such a relationship. These reports on adverse effects following immunization (AEFI) reduce social confidence in vaccination; however, their background may be rare genetic defects. The aim of the presented study was to examine if neurological AEFI in children may be associated with variants in genes related to neurodevelopment. To identify such possible associations, a descriptive study of the Polish case series was conducted. We performed next-generation sequencing in patients who, up to 4 weeks of injection of any vaccine, manifested neurological AEFI. We included 23 previously normally developing children with first seizures that occurred after vaccination. We identified pathogenic/likely pathogenic variants in genes engaged in neurodevelopment in nine patients and variants of uncertain significance in another nine patients. The mutated genes belonged to the group of genes related to epilepsy syndromes/epileptic encephalopathy. We showed that AEFI might have a genetic background. We hypothesized that in some AEFI patients, the vaccine might only trigger neurological symptoms that would have been manifested anyway as a result of a pathogenic variant in a gene engaged in neurodevelopment.

The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a posttranscriptional process that occurs in the nucleus by canonical poly(A) polymerases (PAPs). In some specific instances, the poly(A) tail can also be extended in the cytoplasm by noncanonical poly(A) polymerases (ncPAPs). This epitranscriptomic regulation of mRNA recently became one of the most interesting aspects in the field. Advances in RNA sequencing technologies and software development have allowed the precise measurement of poly(A) tails, identification of new ncPAPs, expansion of the function of known enzymes, discovery and a better understanding of the physiological role of tail heterogeneity, and recognition of a correlation between tail length and RNA translatability. Here, we summarize the development of polyadenylation research methods, including classic low-throughput approaches, Illumina-based genome-wide analysis, and advanced state-of-art techniques that utilize long-read third-generation sequencing with Pacific Biosciences and Oxford Nanopore Technologies platforms. A boost in technical opportunities over recent decades has allowed a better understanding of the regulation of gene expression at the mRNA level. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico., (© 2022 The Authors. WIREs RNA published by Wiley Periodicals LLC.)

In life-science research isogenic B-lymphoblastoid cell lines (LCLs) are widely known and preferred for their genetic stability - they are often used for studying mutations for example, where genetic stability is crucial. We have shown previously that phenotypic variability can be observed in isogenic B-lymphoblastoid cell lines. Isogenic LCLs present well-defined phenotypic differences on various levels, for example on the gene expression level or the chromatin level. Based on our investigations, the phenotypic variability of the isogenic LCLs is accompanied by certain genetic variation too. We have developed a compendium of LCL datasets that present the phenotypic and genetic variability of five isogenic LCLs from a multiomic perspective. In this paper, we present additional datasets generated with Next Generation Sequencing techniques to provide genomic and transcriptomic profiles (WGS, RNA-seq, single cell RNA-seq), protein-DNA interactions (ChIP-seq), together with mass spectrometry and flow cytometry datasets to monitor the changes in the proteome. We are sharing these datasets with the scientific community according to the FAIR principles for further investigations., (© 2022. The Author(s).)

Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Volatile organic compounds (VOCs), a bouquet of chemical compounds released by all life forms, play essential roles in trophic interactions. VOCs can facilitate a large number of interactions with different organisms belowground. VOCs-regulated plant-plant or plant-insect interaction both below and aboveground has been reported extensively. Nevertheless, there is little information about the role of VOCs derived from soilborne pathogenic fungi and beneficial fungi, particularly mycorrhizae, in influencing plant performance. In this review, we show how plant VOCs regulate plant-soilborne pathogenic fungi and beneficial fungi (mycorrhizae) interactions. How fungal VOCs mediate plant-soilborne pathogenic and beneficial fungi interactions are presented and the most common methods to collect and analyze belowground volatiles are evaluated. Furthermore, we suggest a promising method for future research on belowground VOCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Duc, Vo, van Doan, Hamow, Le and Posta.)

Photodynamic therapy (PDT) is a low-invasive treatment method that can be used to treat VIN patients. A photosensitizer (PS) applied to a patient is activated with use of the appropriate wavelength of light, which in an oxygen environment leads to the formation of a reactive oxygen species (ROS) that destroys the tumor. However, cells can protect themselves against these cytotoxic products by increasing their antioxidant mechanisms and repair capacity. Changes in the cytoskeleton may also influence resistance to PDT. Our results revealed that PDT-resistant cells changed the amount of ROS. Cells resistant to PDT A-431 exhibited a decreased ROS level and showed higher viability after oxidizing agent treatment. Resistant Cal-39 cells exhibited a decreased O 2 level but increased other ROS. This provides protection from PDT but not from other oxidizing agents. Moreover, PDT leads to alterations in the cytoskeleton that may result in an epithelial-mesenchymal transition (EMT) or increased adhesion. Both EMT and cell adhesion may activate signaling pathways involved in survival. This means that resistance to PDT in vulvar cancer may be at least in part a result of changes in ROS level and alterations in the cytoskeleton.- level but increased other ROS. This provides protection from PDT but not from other oxidizing agents. Moreover, PDT leads to alterations in the cytoskeleton that may result in an epithelial-mesenchymal transition (EMT) or increased adhesion. Both EMT and cell adhesion may activate signaling pathways involved in survival. This means that resistance to PDT in vulvar cancer may be at least in part a result of changes in ROS level and alterations in the cytoskeleton.

The aim of this study was to determine the breed boundary of the Hungarian Short-haired Vizsla (HSV) dog breed. Seventy registered purebred HSV dogs were genotyped on approximately 145,000 SNPs. Principal Component Analysis (PCA) and Admixture analysis certified that they belong to the same population. The outer point of the breed demarcation was a single Hungarian Wire-haired Vizsla (HWV) individual, which was the closest animal genetically to the HSV population in the PCA analysis. Three programs were used for the breed assignment calculations, including the widely used GeneClass2.0 software and two additional approaches developed here: the 'PCA-distance' and 'IBS-central' methods. Both new methods calculate a single number that represents how closely a dog fits into the actual reference population. The former approach calculates this number based on the PCA distances from the median of HSV animals. The latter calculates it from identity by state (IBS) data, measuring the distance from a central animal that is the best representative of the breed. Having no mixed-breed dogs with known HSV genome proportion, admixture animals were simulated by using data of HSV and HWV individuals to calibrate the inclusion/exclusion probabilities for the assignment. The numbers generated from these relatively simple calculations can be used by breeders and clubs to keep their populations under genetic supervision.

This work aimed to study the plant conditioning effect and mode of action of a plant-based biostimulant used in organic farming. This new generation plant biostimulant, named ELICE16INDURES ® , is rich in plant bio-active ingredients containing eleven supercritical botanical extracts encapsulated in nano-scale liposomes. The dose-response (10 to 240 g ha -1 ) relationship was tested in a field population of autumn barley ( Hordeum vulgare) test crop, and underlying molecular mechanisms were studied. Applying nanotechnology, cell-identical nanoparticles may help the better uptake and delivery of active ingredients increasing resilience, vitality, and crop yield. The amount of harvested crops showed a significant increase of 27.5% and 39.9% interconnected to higher normalized difference vegetation index (NDVI) of 20% and 25% after the treatment of low and high dosages (20 and 240 g ha -1 ), respectively. Illumina NextSeq 550 sequencing, gene expression profiling, and KEGG-pathway analysis of outstanding dosages indicated the upregulation of pathogenesis-related (PR) and other genes-associated with induced resistance-which showed dose dependency as well.

The poultry red mite ( Dermanyssus gallinae ) is the main pest of poultry, causing severe problems by being a vector of several animal and human pathogens. The number of miticides is few, and their efficacy in practice implies problems of residues and resistance; therefore, the demand for a new and safe agent is constant. The present publication investigated the effectiveness of lithium chloride under in vitro conditions on poultry red mites. This chemical currently appears to be one of the most promising alternatives to study amongst potential applicants to treat varroosis, a fatal disease of honey bees. In Experiment I, the previously used experimental doses (5.52 M, 2.76 M, 1.38 M) on Varroa mites confirmed their in vitro activity on the poultry red mite. Three event times (uncontrolled movement, immobilisation and death) were recorded to base the response to treatment for each concentration. In Experiment II, the LD 50 value was calculated, i.e., the value at which 50% of the mites were killed by the treatment. This Experiment showed that the LD50 of lithium chloride = 0.265 M in the poultry red mite. It is to note that the study remained restricted to in vitro confirmation of lithium chloride's effectiveness on the parasite. Thus, further extensive studies are needed to decide whether it has any relevance in practice against D. gallinae , and also to assess potential residue problems that could affect poultry products.

Duchenne muscular dystrophy (DMD) is the most common inherited muscle dystrophy. Patients are characterized by muscle weakness, gross motor delay, and elevated serum creatinine kinase (CK) levels. The disease is caused by mutations in the DMD gene located on the X chromosome. Due to the X-linked recessive inheritance pattern, DMD most commonly affects males, who are generally diagnosed between the age of 3-5 years. Here we present an ultra-rare manifestation of DMD in a female patient. Cytogenetic examination showed that she has a t(X;10)(p21.1;p12.1) translocation, which turned out to affect the DMD gene with one of the breakpoints located in exon 54 (detected by genome sequencing). The X-inactivation test revealed skewed X-inactivation (ratio 99:1). Muscle histology and dystrophin immunohistochemistry showed severe dystrophic changes and highly reduced dystrophin expression, respectively. These results, in accordance with the clinical picture and a highly elevated serum CK, led to the diagnosis of DMD. In conclusion, although in very rare cases, DMD can manifest in female patients as well. In this case, a balanced X-autosome reciprocal translocation disrupts the DMD gene and skewed X-inactivation leads to the manifestation of the DMD phenotype.

The aim of this study was metagenomics analyses of acquired antibiotic-resistance genes (ARGs) in the intestinal microbiome of two important food-animal species in Hungary from a One Health perspective. Intestinal content samples were collected from 12 domestic pigs ( Sus scrofa ) and from a common carp ( Cyprinus carpio ). Shotgun metagenomic sequencing of DNA purified from the intestinal samples was performed on the Illumina platform. The ResFinder database was applied for detecting acquired ARGs in the assembled metagenomic contigs. Altogether, 59 acquired ARG types were identified, 51 genes from domestic pig and 12 genes from the carp intestinal microbiome. ARG types belonged to the antibiotic classes aminoglycosides (27.1%), tetracyclines (25.4%), β-lactams (16.9%), and others. Of the identified ARGs, tet (E), a bla OXA - 48-like β-lactamase gene, as well as cphA4 , ampS , aadA2 , qnrS2 , and sul1 , were identified only in carp but not in swine samples. Several of the detected acquired ARGs have not yet been described from food animals in Hungary. The tet (Q), tet (W), tet (O), and mef (A) genes detected in the intestinal microbiome of domestic pigs had also been identified from free-living wild boars in Hungary, suggesting a possible relationship between the occurrence of acquired ARGs in domestic and wild animal populations.

A digestibility trial was conducted with African catfish hybrid juveniles in order to determine the apparent digestibility coefficients (ADCs) of different nutrients. The experimental diets contained defatted black soldier fly (BSL), yellow mealworm (MW), or fully fat blue bottle fly (BBF) meals, in a 70 : 30 ratio between the control diet and the tested insect meals. The indirect method for the digestibility study was performed using 0.1% yttrium oxide as an inert marker. Fish juveniles of 217.4 ± 9.5 g initial weight were distributed in 1 m 3 tanks (75 fish/tank) of a recirculating aquaculture system (RAS), in triplicates, and fed until satiation for 18 days. The average final weight of the fish was 346 ± 35.8 g. The ADCs of the dry matter, protein, lipid, chitin, ash, phosphorus, amino acids, fatty acids, and gross energy for the test ingredients and diets were calculated. A six-month storage test was carried out to evaluate the shelf life of the experimental diets, while the peroxidation and microbiological status of the diets were also assessed. The ADC values of the test diets differed significantly ( p < 0.001) compared to those of the control for most of the nutrients. Altogether, the BSL diet was significantly more digestible for protein, fat, ash, and phosphorus than the control diet but less digestible for essential amino acids. Significant differences were found between the ADCs of the different insect meals evaluated ( p < 0.001) for practically all nutritional fractions analyzed. The African catfish hybrids were able to digest BSL and BBF more efficiently than MW, and the calculated ADC values agreed with those of other fish species. The lower ADCs of the tested MW meal correlated ( p < 0.05) with the markedly higher acid detergent fiber (ADF) levels present in the MW meal and MW diet. Microbiological evaluation of the feeds revealed that mesophilic aerobic bacteria in the BSL feed were 2-3 orders of magnitude more abundant than those in the other diets and their numbers significantly increased during storage. Overall, BSL and BBF proved to be potential feed ingredients for African catfish juveniles and the shelf life of the produced diets with 30% inclusion of insect meal retained the required quality during a six-month period of storage., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 Zsuzsanna J. Sándor et al.)

Arabidopsis thaliana is one of the most studied model organisms of plant biology with hundreds of geographical variants called ecotypes. One might expect that this enormous genetic variety could result in differential response to pathogens. Indeed, we observed previously that the Bur ecotype develops much more severe symptoms (upward curling leaves and wavy leaf margins) upon infection with two positive-strand RNA viruses of different families (turnip vein-clearing virus, TVCV, and turnip mosaic virus, TuMV). To find the genes potentially responsible for the ecotype-specific response, we performed a differential expression analysis of the mRNA and sRNA pools of TVCV and TuMV-infected Bur and Col plants along with the corresponding mock controls. We focused on the genes and sRNAs that showed an induced or reduced expression selectively in the Bur virus samples in both virus series. We found that the two ecotypes respond to the viral infection differently, yet both viruses selectively block the production of the TAS3-derived small RNA specimen called tasiARF only in the virus-infected Bur plants. The tasiARF normally forms a gradient through the adaxial and abaxial parts of the leaf (being more abundant in the adaxial part) and post-transcriptionally regulates ARF4, a major leaf polarity determinant in plants. The lack of tasiARF-mediated silencing could lead to an ectopically expressed ARF4 in the adaxial part of the leaf where the misregulation of auxin-dependent signaling would result in an irregular growth of the leaf blade manifesting as upward curling leaf and wavy leaf margin. QTL mapping using Recombinant Inbred Lines (RILs) suggests that the observed symptoms are the result of a multigenic interaction that allows the symptoms to develop only in the Bur ecotype. The particular nature of genetic differences leading to the ecotype-specific symptoms remains obscure and needs further study., Competing Interests: The authors have declared that no competing interests exist.

Mutations in the SCN1A gene can cause a variety of phenotypes, ranging from mild forms, such as febrile seizures and generalized epilepsy with febrile seizures plus, to severe, such as Dravet and non-Dravet developmental epileptic encephalopathies. Until now, more than two thousand pathogenic variants of the SCN1A gene have been identified and different pathogenic mechanisms (loss vs. gain of function) described, but the precise molecular mechanisms responsible for the deficits exhibited by patients are not fully elucidated. Additionally, the phenotypic variability proves the involvement of other genetic factors in its final expression. This is the reason why animal models and cell line models used to explore the molecular pathology of SCN1A -related disorders are only of limited use. The results of studies based on such models cannot be directly translated to affected individuals because they do not address each patient's unique genetic background. The generation of functional neurons and glia for patient-derived iPSCs, together with the generation of isogenic controls using CRISPR/Cas technology, and finally, the 3D brain organoid models, seem to be a good way to solve this problem. Here, we review SCN1A -related encephalopathies, as well as the stem cell models used to explore their molecular basis.

Kae1 is a subunit of the highly evolutionarily conserved KEOPS/EKC complex, which is involved in universal (t6A 37 ) tRNA modification. Several reports have discussed the participation of this complex in transcription regulation in yeast and human cells, including our previous observations of KaeA, an Aspergillus nidulans homologue of Kae1p. The aim of this project was to confirm the role of KaeA in transcription, employing high-throughput transcriptomic (RNA-Seq and ChIP-Seq) and proteomic (LC-MS) analysis. We confirmed that KaeA is a subunit of the KEOPS complex in A. nidulans. An analysis of kaeA19 and kaeA25 mutants showed that, although the (t6A 37 ) tRNA modification is unaffected in both mutants, they reveal significantly altered transcriptomes compared to the wild type. The finding that KaeA is localized in chromatin and identifying its protein partners allows us to postulate an additional nuclear function for the protein. Our data shed light on the universal bi-functional role of this factor and proves that the activity of this protein is not limited to tRNA modification in cytoplasm, but also affects the transcriptional activity of a number of nuclear genes. Data are available via the NCBI's GEO database under identifiers GSE206830 (RNA-Seq) and GSE206874 (ChIP-Seq), and via ProteomeXchange with identifier PXD034554 (proteomic).

HAX1 is a human protein with no known homologues or structural domains. Mutations in the HAX1 gene cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported the RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here, we report the transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in translation, ribosome biogenesis, and rRNA processing. Using CRISPR knockouts, we find that HAX1 RNA targets partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. The functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

Mycotoxins are bioaccumulative contaminants impacting animals and humans. The simultaneous detection of frequent active exposures and accumulated mycotoxin level (s) in exposed organisms would be the most ideal to enable appropriate actions. However, few methods are available for the purpose, and there is a demand for dedicated, sensitive, reliable, and practical assays. To demonstrate the issue, mice were exposed to a relevant agent Ochratoxin A (OTA), and accumulated OTA was measured by fine-tuned commercial assays. Quantitative high-performance liquid chromatography with fluorescence detection, enzyme-linked immunosorbent assay, and flow cytometry assays have been developed/modified using reagents available as commercial products when appropriate. Assays were performed on excised samples, and results were compared. Accumulated OTA could be detected and quantified; positive correlations (between applied doses of exposure and accumulated OTA levels and the results from assays) were found. Dedicated assays could be developed, which provided comparable results. The presence and accumulation of OTA following even a short exposure could be quantitatively detected. The assays performed similarly, but HPLC had the greatest sensitivity. Blood contained higher levels of OTA than liver and kidney. We demonstrate that specific but flexible and practical assays should be used for specific/local purposes, to measure the exposure itself and accumulation in blood or organs.

In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between 'self' and 'non-self' in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2'-O-methylation of the second transcribed nucleotide within the mRNA 5' cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2'-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as 'self' and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Numtogenesis is observable in the mammalian genomes resulting in the integration of mitochondrial segments into the nuclear genomes (numts). To identify numts in rabbit, we aligned mitochondrial and nuclear genomes. Alignment significance threshold was calculated and individual characteristics of numts were analysed. We found 153 numts in the nuclear genome. The GC content of numts were significantly lower than the GC content of their genomic flanking regions or the genome itself. The frequency of three mammalian-wide interspersed repeats were increased in the proximity of numts. The decreased GC content around numts strengthen the theory which supposes a link between DNA structural instability and numt integration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Extreme temperatures during heat waves can induce mass-mortality events, but can also exert sublethal negative effects by compromising life-history traits and derailing sexual development. Ectothermic animals may, however, also benefit from increased temperatures via enhanced physiological performance and the suppression of cold-adapted pathogens. Therefore, it is crucial to address how the intensity and timing of naturally occurring or human-induced heat waves affect life-history traits and sexual development in amphibians, to predict future effects of climate change and to minimize risks arising from the application of elevated temperature in disease mitigation. We raised agile frog (Rana dalmatina) and common toad (Bufo bufo) tadpoles at 19 °C and exposed them to a simulated heat wave of 28 or 30 °C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 °C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 °C decreased body mass at metamorphosis. Furthermore, exposure to 30 °C during any period and to 28 °C late in development caused female-to-male sex reversal, skewing sex ratios strongly towards males. In common toads, high temperature only slightly decreased survival and did not influence phenotypic sex ratio, while it reduced metamorph mass and length of larval development. Juvenile body mass measured 2 months after metamorphosis was not adversely affected by temperature treatments in either species. Our results indicate that heat waves may have devastating effects on amphibian populations, and the severity of these negative consequences, and sensitivity can vary greatly between species and with the timing and intensity of heat. Finally, thermal treatments against cold-adapted pathogens have to be executed with caution, taking into account the thermo-sensitivity of the species and the life stage of animals to be treated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The removal of RNA primers is essential for mitochondrial DNA (mtDNA) replication. Several nucleases have been implicated in RNA primer removal in human mitochondria, however, no conclusive mechanism has been elucidated. Here, we reconstituted minimal in vitro system capable of processing RNA primers into ligatable DNA ends. We show that human 5'-3' exonuclease, EXOG, plays a fundamental role in removal of the RNA primer. EXOG cleaves short and long RNA-containing flaps but also in cooperation with RNase H1, processes non-flap RNA-containing intermediates. Our data indicate that the enzymatic activity of both enzymes is necessary to process non-flap RNA-containing intermediates and that regardless of the pathway, EXOG-mediated RNA cleavage is necessary prior to ligation by DNA Ligase III. We also show that upregulation of EXOG levels in mitochondria increases ligation efficiency of RNA-containing substrates and discover physical interactions, both in vitro and in cellulo, between RNase H1 and EXOG, Pol γA, Pol γB and Lig III but not FEN1, which we demonstrate to be absent from mitochondria of human lung epithelial cells. Together, using human mtDNA replication enzymes, we reconstitute for the first time RNA primer removal reaction and propose a novel model for RNA primer processing in human mitochondria., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Plants, like animals, are constantly exposed to abiotic and biotic stresses, which often inhibit plant growth and development, and cause tissue damage, disease, and even plant death. Efficient and timely response to stress requires appropriate co- and posttranscriptional reprogramming of gene expression. Alternative pre-mRNA splicing provides an important layer of this regulation by controlling the level of factors involved in stress response and generating additional protein isoforms with specific features. Recent high-throughput studies have revealed that several defence genes undergo alternative splicing that is often affected by pathogen infection. Despite extensive work, the exact mechanisms underlying these relationships are still unclear, but the contribution of alternative protein isoforms to the defence response and the role of regulatory factors, including components of the splicing machinery, have been established. Modulation of gene expression in response to stress includes alternative splicing, chromatin remodelling, histone modifications, and nucleosome occupancy. How these processes affect plant immunity is mostly unknown, but these facets open new regulatory possibilities. Here we provide an overview of the current state of knowledge and recent findings regarding the growing importance of alternative splicing in plant response to biotic stress., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Background: PLA2G6-Associated Neurodegeneration, PLAN, is subdivided into: Infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, and adult-onset dystonia parkinsonism [1]. It is elicited by a biallelic pathogenic variant in phospholipase A2 group VI (PLA2G6) gene. In this study we describe new cases and provide a comprehensive review of previously published cases., Methods: Eleven patients, from four different institutions and four different countries. All underwent a comprehensive chart review., Results: Ages at onset ranged from 1 to 36 years, with a median of 16 and a mean of 16.18 ± 11.91 years. Phenotypic characteristics were heterogenous and resembled that of patients with infantile neuroaxonal dystrophy (n = 2), atypical neuroaxonal dystrophy (n = 1), adult-onset dystonia parkinsonism (n = 1), complex hereditary spastic paraparesis (n = 3), and early onset Parkinson's disease (n = 2). Parental genetic studies were performed for all patients and confirmed with sanger sequencing in five. Visual evoked potential illustrated optic atrophy in P4. Mineralization was evident in brain magnetic resonance imaging of P1, P2, P4, P5, P7, and P11. Single photon emission computed tomography was conducted for three patients, revealed decreased perfusion in the occipital lobes for P10. DaTscan was performed for P11 and showed decreased uptake in the deep gray matter, bilateral caudate nuclei, and bilateral putamen. Positive response to Apomorphine was noted for P10 and to Baclofen in P2, and P3., Conclusions: PLAN encompasses a wide clinical spectrum. Age and symptom at onset are crucial when classifying patients. Reporting new variants is critical to draw more attention to this condition and identify biomarkers to arrive at potential therapeutics., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The E. coli element IS30 has adopted the copy-out-paste-in transposition mechanism that is prevalent in a number of IS-families. As an initial step, IS30 forms free circular transposition intermediates like IS minicircles or tandem IS-dimers by joining the inverted repeats of a single element or two, sometimes distantly positioned IS copies, respectively. Then, the active IR-IR junction of these intermediates reacts with the target DNA, which generates insertions, deletions, inversions or cointegrates. The element shows dual target specificity as it can insert into hot spot sequences or next to its inverted repeats. In this study the pathways of rearrangements of transposition-derived cointegrate-like structures were examined. The results showed that the probability of further rearrangements in these structures depends on whether the IS elements are flanked by hot spot sequences or take part in an IR-IR junction. The variability of the deriving products increases with the number of simultaneously available IRs and IR-IR joints in the cointegrates or the chromosome. Under certain conditions, the parental structures whose transposition formed the cointegrates are restored and persist among the rearranged products. Based on these findings, a novel dynamic model has been proposed for IS30, which possibly fits to other elements that have adopted the same transposition mechanism. The model integrates the known transposition pathways and the downstream rearrangements occurring after the formation of different cointegrate-like structures into a complex network. Important feature of this network is the presence of "feedback loops" and reversible transposition rearrangements that can explain how IS30 generates variability and preserves the original genetic constitution in the bacterial population, which contributes to the adaptability and evolution of host bacteria., Competing Interests: The authors have declared that no competing interests exist.

Varroosis is one of the most dangerous threats to the bee industry but means of its treatment are still unsatisfactory. Lithium-based anti-Varroa treatments may provide an alternative, as this trace element can be a natural component of honey and is well tolerated by adult bees. However, it can be toxic to larvae and its use in beekeeping practice is not yet well understood. The present study aimed to investigate the efficacy of relevant application methods of acaricides used in beekeeping practice in brood-free conditions for lithium. Vaporisation proved to be an inefficient method of lithium treatment and killed only 9.9 ± 3.3% (mean ± SD) of mites in the hive. Lithium-impregnated paper strips showed moderate efficiency by killing 55.1 ± 26.2% of mites. The most effective way of applying lithium was the trickling method; different trickling treatments decreased the abundance of mites on average by 65 to 99.7%, depending on the applied dosage and the number of treatments. Repeated trickling treatments were more effective than single treatments, and they generally provided >90% efficiency. Experiments also proved that adding sugar to the trickling solution does not influence treatment efficiency. Thus, it is suggested that repeated and sugar-free trickling treatments with moderate lithium dosage could be the most rational methodology. Since lithium is not yet legalised in beekeeping practice, comprehensive studies are also needed to uncover the amount of lithium residue in bee products, depending on the treatment parameters.

Kisspeptin neurons in the mediobasal hypothalamus (MBH) are critical targets of ovarian estrogen feedback regulating mammalian fertility. To reveal molecular mechanisms underlying this signaling, we thoroughly characterized the estrogen-regulated transcriptome of kisspeptin cells from ovariectomized transgenic mice substituted with 17β-estradiol or vehicle. MBH kisspeptin neurons were harvested using laser-capture microdissection, pooled, and subjected to RNA sequencing. Estrogen treatment significantly ( p.adj . < 0.05) up-regulated 1,190 and down-regulated 1,139 transcripts, including transcription factors, neuropeptides, ribosomal and mitochondrial proteins, ion channels, transporters, receptors, and regulatory RNAs. Reduced expression of the excitatory serotonin receptor-4 transcript ( Htr4 ) diminished kisspeptin neuron responsiveness to serotonergic stimulation. Many estrogen-regulated transcripts have been implicated in puberty/fertility disorders. Patients ( n = 337) with congenital hypogonadotropic hypogonadism (CHH) showed enrichment of rare variants in putative CHH-candidate genes (e.g., LRP1B , CACNA1G , FNDC3A ). Comprehensive characterization of the estrogen-dependent kisspeptin neuron transcriptome sheds light on the molecular mechanisms of ovary-brain communication and informs genetic research on human fertility disorders.

Chytridiomycosis, an emerging infectious disease caused by Batrachochytrium dendrobatidis (Bd), poses a serious threat to amphibians. The thermal optimum of Bd is lower than that of most amphibians, providing an opportunity to cure infected individuals with elevated temperature. However, this approach presupposes detailed knowledge about the thermal tolerance of the fungus. To determine the temperature that may effectively reduce infection burdens in vivo, detailed in vitro studies are needed to characterize thermal tolerance of the fungus without complexities introduced by the species-specific characteristics of hosts' immune systems. The aim of our study was to evaluate the thermal tolerance of a hypervirulent isolate of Bd, considering the limits of its thermal tolerance and its capacity to rebound following heat treatment. We incubated Bd cell cultures at five different temperatures (21, 25.5, 27, 29, or 30.5 C) for one of six exposure durations (3, 4, 5, 6, 7, or 8 days) and subsequently counted the number of zoospores to assess the temperature dependence of Bd growth. We observed intensive Bd growth at 21 C. At 25.5 C, the number of zoospores also increased over time, but the curve plateaued at about half of the maximum values observed in the lower temperature treatment. At temperatures of 27 C and above, the fungus showed no measurable growth. However, when we moved the cultures back to 21 C after the elevated temperature treatments, we observed recovery of Bd growth in all cultures previously treated at 27 C. At 29 C, a treatment duration of 8 days was necessary to prevent recovery of Bd growth, and at 30.5 C a treatment duration of 5 days was needed to achieve the same result, revealing that these moderately elevated temperatures applied for only a few days have merely a fungistatic rather than a fungicidal effect under in vitro conditions.

A new tobamovirus named tomato brown rugose fruit virus (ToBRFV) overcomes the effect of the Tm-1, Tm-2, and Tm-2 2 resistance genes introgressed from wild Solanum species into cultivated tomato (Solanum lycopersicum). Here, we report the isolation and molecular characterization of a spontaneous mutant of ToBRFV that breaks resistance in an unknown genetic background, demonstrated recently in Solanum habrochaites and Solanum peruvianum. The wild isolate ToBRFV-Tom2-Jo and the mutant ToBRFV-Tom2M-Jo were fully sequenced and compared to each other and to other ToBRFV sequences available in the NCBI GenBank database. Sequence analysis revealed five nucleotide substitutions in the ToBRFV-Tom2M-Jo genome compared to ToBRFV-Tom2-Jo. Two substitutions were located in the movement protein (MP) gene and resulted in amino acid changes in the 30-kDa MP (Phe22 → Asn and Tyr82 → Lys). These substitutions were not present in any of the previously described ToBRFV isolates. No amino acid changes were found in the 126-kDa and 183-kDa replicase proteins or the 17.5-kDa coat protein. Our data strongly suggest that breaking the newly discovered resistance in wild tomatoes is associated with one or two mutations on the MP gene of ToBRFV., (© 2022. The Author(s).)

Parkinson's disease (PD) is generally considered a sporadic disorder, but a strong genetic background is often found. The aim of this study was to identify the underlying genetic cause of PD in two affected siblings and to subsequently assess the role of mutations in Cathepsin B (CTSB) in susceptibility to PD. A typical PD family was identified and whole-exome sequencing was performed in two affected siblings. Variants of interest were validated using Sanger sequencing. CTSB p.Gly284Val was genotyped in 2077 PD patients and 615 unrelated healthy controls from the Czech Republic, Ireland, Poland, Ukraine, and the USA. The gene burden analysis was conducted for the CTSB gene in an additional 769 PD probands from Mayo Clinic Florida familial PD cohort. CTSB expression and activity in patient-derived fibroblasts and controls were evaluated by qRT-PCR, western blot, immunocytochemistry, and enzymatic assay. The CTSB p.Gly284Val candidate variant was only identified in affected family members. Functional analysis of CTSB patient-derived fibroblasts under basal conditions did not reveal overt changes in endogenous expression, subcellular localization, or enzymatic activity in the heterozygous carrier of the CTSB variant. The identification of the CTSB p.Gly284Val may support the hypothesis that the CTSB locus harbors variants with differing penetrance that can determine the disease risk.

The parasite Trypanosoma brucei causes African sleeping sickness that is fatal to patients if untreated. Parasite differentiation from a replicative slender form into a quiescent stumpy form promotes host survival and parasite transmission. Long noncoding RNAs (lncRNAs) are known to regulate cell differentiation in other eukaryotes. To determine whether lncRNAs are also involved in parasite differentiation, we used RNA sequencing to survey the T. brucei genome, identifying 1428 previously uncharacterized lncRNA genes. We find that grumpy lncRNA is a key regulator that promotes parasite differentiation into the quiescent stumpy form. This function is promoted by a small nucleolar RNA encoded within the grumpy lncRNA. snoGRUMPY binds to messenger RNAs of at least two stumpy regulatory genes, promoting their expression. grumpy overexpression reduces parasitemia in infected mice. Our analyses suggest that T. brucei lncRNAs modulate parasite-host interactions and provide a mechanism by which grumpy regulates cell differentiation in trypanosomes.

Effective utilization of genetic diversity in wild relatives to improve wheat requires recombination between wheat and alien chromosomes. However, this is suppressed by the Pairing homoeologous gene, Ph1 , on the long arm of wheat chromosome 5B. A deletion mutant of the Ph1 locus ( ph1b ) has been used widely to induce homoeologous recombination in wheat × alien hybrids. However, the original ph1b mutation, developed in Chinese Spring (CS) background has poor agronomic performance. Hence, alien introgression lines are first backcrossed with adapted wheat genotypes and after this step, alien chromosome segments are introduced into breeding lines. In this work, the ph1b mutation was transferred from two CS ph1b mutants into winter wheat line Mv9kr1. Homozygous genotypes Mv9kr1 ph1b / ph1b exhibited improved plant and spike morphology compared to Chinese Spring. Flow cytometric chromosome analysis confirmed reduced DNA content of the mutant 5B chromosome in both wheat genotype relative to the wild type chromosome. The ph1b mutation in the Mv9kr1 genotype allowed wheat-alien chromosome pairing in meiosis of Mv9kr1 ph1b &#95;K ×  Aegilops biuncialis F 1 hybrids, predominantly with the M b -genome chromosomes of Aegilops relative to those of the U b genome. High frequency of wheat- Aegilops chromosome interactions resulted in rearranged chromosomes identified in the new Mv9kr1 ph1b  ×  Ae. Biuncialis amphiploids, making these lines valuable sources for alien introgressions. The new Mv9kr1 ph1b mutant genotype is a unique resource to support alien introgression breeding of hexaploid wheat., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Türkösi, Ivanizs, Farkas, Gaál, Kruppa, Kovács, Szakács, Szőke-Pázsi, Said, Cápal, Griffiths, Doležel and Molnár.)

Sequence-based predictors of the residue-level protein function and structure cover a broad spectrum of characteristics including intrinsic disorder, secondary structure, solvent accessibility and binding to nucleic acids. They were catalogued and evaluated in numerous surveys and assessments. However, methods focusing on a given characteristic are studied separately from predictors of other characteristics, while they are typically used on the same proteins. We fill this void by studying complementarity of a representative collection of methods that target different predictions using a large, taxonomically consistent, and low similarity dataset of human proteins. First, we bridge the gap between the communities that develop structure-trained vs. disorder-trained predictors of binding residues. Motivated by a recent study of the protein-binding residue predictions, we empirically find that combining the structure-trained and disorder-trained predictors of the DNA-binding and RNA-binding residues leads to substantial improvements in predictive quality. Second, we investigate whether diverse predictors generate results that accurately reproduce relations between secondary structure, solvent accessibility, interaction sites, and intrinsic disorder that are present in the experimental data. Our empirical analysis concludes that predictions accurately reflect all combinations of these relations. Altogether, this study provides unique insights that support combining results produced by diverse residue-level predictors of protein function and structure., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Pet127 is a mitochondrial protein found in multiple eukaryotic lineages, but absent from several taxa, including plants and animals. Distant homology suggests that it belongs to the divergent PD-(D/E)XK superfamily which includes various nucleases and related proteins. Earlier yeast genetics experiments suggest that it plays a nonessential role in RNA degradation and 5' end processing. Our phylogenetic analysis suggests that it is a primordial eukaryotic invention that was retained in diverse groups, and independently lost several times in the evolution of other organisms. We demonstrate for the first time that the fungal Pet127 protein in vitro is a processive 5'-to-3' exoribonuclease capable of digesting various substrates in a sequence nonspecific manner. Mutations in conserved residues essential in the PD-(D/E)XK superfamily active site abolish the activity of Pet127. Deletion of the PET127 gene in the pathogenic yeast Candida albicans results in a moderate increase in the steady-state levels of several transcripts and in accumulation of unspliced precursors and intronic sequences of three introns. Mutations in the active site residues result in a phenotype identical to that of the deletant, confirming that the exoribonuclease activity is related to the physiological role of the Pet127 protein. Pet127 activity is, however, not essential for maintaining the mitochondrial respiratory activity in C. albicans ., (© 2022 Łabędzka-Dmoch et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Mitochondria play a pivotal role in numerous cellular processes. One of them is regulation of the innate immune pathway. In this instance, mitochondria function in two different aspects of regulatory mechanisms. First, mitochondria are part of the antiviral signaling cascade that is triggered in the cytoplasm and transmitted to effector proteins through mitochondria-localized proteins. Second, mitochondria can become an endogenous source of innate immune stimuli. Under some pathophysiological conditions, mitochondria release to the cytoplasm immunogenic factors, such as mitochondrial nucleic acids. Here, we focus on immunogenic mitochondrial double-stranded RNA (mt-dsRNA) and its origin and metabolism. We discuss factors that are responsible for regulating mt-dsRNA and its escape from mitochondria, emphasizing the contribution of polynucleotide phosphorylase (PNPase, PNPT1). Finally, we review current knowledge of the role of PNPase in human health and disease. This article is categorized under: RNA in Disease and Development > RNA in Disease., (© 2021 Wiley Periodicals LLC.)

Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections.