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Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human diseases. Graphical Abstract ᅟ.

BackgroundCohen Syndrome (COH1) is a rare autosomal recessive disorder, principally identified by ocular, neural and muscular deficits. We identified three large consanguineous Pakistani families with intellectual disability and in some cases with autistic traits.MethodsClinical assessments were performed in order to allow comparison of clinical features with other VPS13B mutations. Homozygosity mapping followed by whole exome sequencing and Sanger sequencing strategies were used to identify disease-related mutations.ResultsWe identified two novel homozygous deletion mutations in VPS13B, firstly a 1 bp deletion, NM_017890.4:c.6879delT; p.Phe2293Leufs*24, and secondly a deletion of exons 37-40, which co-segregate with affected status. In addition to COH1-related traits, autistic features were reported in a number of family members, contrasting with the "friendly" demeanour often associated with COH1. The c.6879delT mutation is present in two families from different regions of the country, but both from the Baloch sub-ethnic group, and with a shared haplotype, indicating a founder effect among the Baloch population.ConclusionWe suspect that the c.6879delT mutation may be a common cause of COH1 and similar phenotypes among the Baloch population. Additionally, most of the individuals with the c.6879delT mutation in these two families also present with autistic like traits, and suggests that this variant may lead to a distinct autistic-like COH1 subgroup.

BackgroundBreastfed human infants are predominantly colonized by bifidobacteria that thrive on human milk oligosaccharides (HMO). Two predominant species of bifidobacteria in infant feces are Bifidobacterium breve (B. breve) and Bifidobacterium longum subsp. infantis (B. infantis), both of which include avid HMO-consumer strains. Our laboratory has previously shown that B. infantis, when grown on HMO, increases adhesion to intestinal cells and increases the expression of the anti-inflammatory cytokine interleukin-10. The purpose of the current study was to investigate the effects of carbon source-glucose, lactose, or HMO-on the ability of B. breve and B. infantis to adhere to and affect the transcription of intestinal epithelial cells on a genome-wide basis.ResultsHMO-grown B. infantis had higher percent binding to Caco-2 cell monolayers compared to B. infantis grown on glucose or lactose. B. breve had low adhesive ability regardless of carbon source. Despite differential binding ability, both HMO-grown strains significantly differentially affected the Caco-2 transcriptome compared to their glucose or lactose grown controls. HMO-grown B. breve and B. infantis both downregulated genes in Caco-2 cells associated with chemokine activity.ConclusionThe choice of carbon source affects the interaction of bifidobacteria with intestinal epithelial cells. HMO-grown bifidobacteria reduce markers of inflammation, compared to glucose or lactose-grown bifidobacteria. In the future, the design of preventative or therapeutic probiotic supplements may need to include appropriately chosen prebiotics.

BackgroundIt is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5'-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.ResultsPhylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5'-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.ConclusionsLike the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5'-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

BackgroundBixin or annatto is a commercially important natural orange-red pigment derived from lycopene that is produced and stored in seeds of Bixa orellana L. An enzymatic pathway for bixin biosynthesis was inferred from homology of putative proteins encoded by differentially expressed seed cDNAs. Some activities were later validated in a heterologous system. Nevertheless, much of the pathway remains to be clarified. For example, it is essential to identify the methylerythritol phosphate (MEP) and carotenoid pathways genes.ResultsIn order to investigate the MEP, carotenoid, and bixin pathways genes, total RNA from young leaves and two different developmental stages of seeds from B. orellana were used for the construction of indexed mRNA libraries, sequenced on the Illumina HiSeq 2500 platform and assembled de novo using Velvet, CLC Genomics Workbench and CAP3 software. A total of 52,549 contigs were obtained with average length of 1,924 bp. Two phylogenetic analyses of inferred proteins, in one case encoded by thirteen general, single-copy cDNAs, in the other from carotenoid and MEP cDNAs, indicated that B. orellana is closely related to sister Malvales species cacao and cotton. Using homology, we identified 7 and 14 core gene products from the MEP and carotenoid pathways, respectively. Surprisingly, previously defined bixin pathway cDNAs were not present in our transcriptome. Here we propose a new set of gene products involved in bixin pathway.ConclusionThe identification and qRT-PCR quantification of cDNAs involved in annatto production suggest a hypothetical model for bixin biosynthesis that involve coordinated activation of some MEP, carotenoid and bixin pathway genes. These findings provide a better understanding of the mechanisms regulating these pathways and will facilitate the genetic improvement of B. orellana.

BackgroundThe role of vectors in the transmission of avian malaria parasites is currently understudied. Many studies that investigate parasite-vector relationships use limited trapping techniques and/or identify potential competent vectors in the field in such ways that cannot distinguish between an infected or infectious vector. Without the use of multiple trapping techniques that address the specific biology of diverse mosquito species, and without looking at the infection status of individual mosquitoes, it is not possible to make dependable conclusions on the role of mosquitoes in the transmission of avian malaria parasites.MethodsWe conducted two years of mosquito collections at a riparian preserve in California where a wide diversity of species were collected with multiple trap types. We hypothesized that competent mosquito species can influence the distribution and diversity of avian malaria parasites by acting as a compatibility filter for specific Plasmodium species. To determine the infection status of all individual mosquitoes for Plasmodium species/lineages, amplification within the cytochrome b gene was carried out on over 3000 individual mosquito thoraxes, and for those that tested positive we then repeated the same process for abdomens and salivary glands.ResultsOur data show heterogeneity in the transmissibility of Plasmodium among ornithophillic mosquito species. More specifically, Culex stigmatosoma appears to not be a vector of Plasmodium homopolare, a parasite that is prevalent in the avian population, but is a vector of multiple other Plasmodium species/lineages.ConclusionsOur results suggest that conclusions made on the role of vectors from studies that do not use different mosquito trapping methods should be re-evaluated with caution, as we documented the potential for trapping biases, which may cause studies to miss important roles of specific mosquito species in the transmission of avian malaria. Moreover, we document heterogeneity in the transmission of Plasmodium spp. by mosquitoes can influence Plasmodium diversity and prevalence in specific locations to Plasmodium-vector incompatibilities.

BackgroundLeucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive.ResultsThe LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants.ConclusionsLRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.

BackgroundN-terminal domains of BVU_4064 and BF1687 proteins from Bacteroides vulgatus and Bacteroides fragilis respectively are members of the Pfam family PF12985 (DUF3869). Proteins containing a domain from this family can be found in most Bacteroides species and, in large numbers, in all human gut microbiome samples. Both BVU_4064 and BF1687 proteins have a consensus lipobox motif implying they are anchored to the membrane, but their functions are otherwise unknown. The C-terminal half of BVU_4064 is assigned to protein family PF12986 (DUF3870); the equivalent part of BF1687 was unclassified.ResultsCrystal structures of both BVU_4064 and BF1687 proteins, solved at the JCSG center, show strikingly similar three-dimensional structures. The main difference between the two is that the two domains in the BVU_4064 protein are connected by a short linker, as opposed to a longer insertion made of 4 helices placed linearly along with a strand that is added to the C-terminal domain in the BF1687 protein. The N-terminal domain in both proteins, corresponding to the PF12985 (DUF3869) domain is a β-sandwich with pre-albumin-like fold, found in many proteins belonging to the Transthyretin clan of Pfam. The structures of C-terminal domains of both proteins, corresponding to the PF12986 (DUF3870) domain in BVU_4064 protein and an unclassified domain in the BF1687 protein, show significant structural similarity to bacterial pore-forming toxins. A helix in this domain is in an analogous position to a loop connecting the second and third strands in the toxin structures, where this loop is implicated to play a role in the toxin insertion into the host cell membrane. The same helix also points to the groove between the N- and C-terminal domains that are loosely held together by hydrophobic and hydrogen bond interactions. The presence of several conserved residues in this region together with these structural determinants could make it a functionally important region in these proteins.ConclusionsStructural analysis of BVU_4064 and BF1687 points to possible roles in mediating multiple interactions on the cell-surface/extracellular matrix. In particular the N-terminal domain could be involved in adhesive interactions, the C-terminal domain and the inter-domain groove in lipid or carbohydrate interactions.

Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼ 250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism's 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA(Leu), which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism's physiology and defines the essential gene set required for the growth of a photosynthetic organism.

BackgroundEukaryotic RNA polymerase II contains a C-terminal repeated domain (CTD) consisting of 52 consensus heptad repeats of Y1S2P3T4S5P6S7 that mediate interactions with many cellular proteins to regulate transcription elongation, RNA processing and chromatin structure. A number of CTD-binding proteins have been identified and the crystal structures of several protein-CTD complexes have demonstrated considerable conformational flexibility of the heptad repeats in those interactions. Furthermore, phosphorylation of the CTD at tyrosine, serine and threonine residues can regulate the CTD-protein interactions. Although the interactions of CTD with specific proteins have been elucidated at the atomic level, the capacity and specificity of the CTD-interactome in mammalian cells is not yet determined.ResultsA proteomic study was conducted to examine the mammalian CTD-interactome. We utilized six synthetic peptides each consisting of four consensus CTD-repeats with different combinations of serine and tyrosine phosphorylation as affinity-probes to pull-down nuclear proteins from HeLa cells. The pull-down fractions were then analyzed by MUDPIT mass spectrometry, which identified 100 proteins with the majority from the phospho-CTD pull-downs. Proteins pulled-down by serine-phosphorylated CTD-peptides included those containing the previously defined CTD-interacting domain (CID). Using SILAC mass spectrometry, we showed that the in vivo interaction of RNA polymerase II with the mammalian CID-containing RPRD1B is disrupted by CID mutation. We also showed that the CID from four mammalian proteins interacted with pS2-phosphorylated but not pY1pS2-doubly phosphorylated CTD-peptides. However, we also found proteins that were preferentially pulled-down by pY1pS2- or pY1pS5-doubly phosphorylated CTD-peptides. We prepared an antibody against tyrosine phosphorylated CTD and showed that ionizing radiation (IR) induced a transient increase in CTD tyrosine phosphorylation by immunoblotting. Combining SILAC and IMAC purification of phospho-peptides, we found that IR regulated the phosphorylation at four CTD tyrosine sites in different ways.ConclusionUpon phosphorylation, the 52 repeats of the CTD have the capacity to generate a large number of binding sites for cellular proteins. This study confirms previous findings that serine phosphorylation stimulates whereas tyrosine phosphorylation inhibits the protein-binding activity of the CTD. However, tyrosine phosphorylation of the CTD can also stimulate other CTD-protein interactions. The CTD-peptide affinity pull-down method described here can be adopted to survey the mammalian CTD-interactome in various cell types and under different biological conditions.

Scrub typhus is a potentially fatal rickettsiosis caused by Orientia species intracellular bacteria of the genus Orientia. Although considered to be restricted to the Asia Pacific region, scrub typhus has recently been discovered in southern Chile. We analyzed Orientia gene sequences of 16S rRNA (rrs) and 47-kDa (htrA) from 18 scrub typhus patients from Chile. Sequences were ≥99.7% identical among the samples for both amplified genes. Their diversity was 3.1%–3.5% for rrs and 11.2%–11.8% for htrA compared with O. tsusugamushi and 3.0% for rrs and 14.8% for htrA compared with Candidatus Orientia chuto. Phylogenetic analyses of both genes grouped the specimens from Chile in a different clade from other Orientia species. Our results indicate that Orientia isolates from Chile constitute a novel species, which, until they are cultivated and fully characterized, we propose to designate as Candidatus Orientia chiloensis, after the Chiloé Archipelago where the pathogen was identified.

Pigs are sometimes used in preclinical drug metabolism studies, with growing interest, and thus their drug-metabolizing enzymes, including the cytochromes P450 (P450 or CYP; EC 1.14.14.1), need to be examined. In the present study, novel CYP4A cDNAs were isolated and characterized, namely, pig CYP4A23 and CYP4A90; cat CYP4A37 and CYP4A106; and tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g. For comparison, the following known CYP4A cDNAs were also analyzed: pig CYP4A21 and dog CYP4A37, CYP4A38, and CYP4A39. These CYP4A cDNAs all contained open reading frames of 504-513 amino acids and had high amino acid sequence identity (74%-80%) with human CYP4As. Phylogenetic analysis of amino acid sequences revealed that these CYP4As were clustered in each species. All CYP4A genes contained 12 coding exons and formed a gene cluster in the corresponding genomic regions. A range of tissue types were analyzed, and these CYP4A mRNAs were preferentially expressed in liver and/or kidney, except for pig CYP4A90, which showed preferential expression in lung and duodenum. CYP4A enzymes, heterologously expressed in Escherichia coli , preferentially catalyzed lauric acid 12-hydroxylation and arachidonic acid 20-hydroxylation, just as human CYP4A11 does, with the same regioselectivity (i.e., at the ω -position of fatty acids). These results imply that dog, cat, pig, and tree shrew CYP4As have functional characteristics similar to those of human CYP4A11, with minor differences in lauric acid 12-hydroxylation. SIGNIFICANCE STATEMENT: Cytochrome P450 (P450, CYP) 4As are important P450s in human biological processes because of their fatty acid-metabolizing ability. Pig CYP4A21, CYP4A23, and CYP4A90; cat CYP4A37 and CYP4A106; tree shrew CYP4A11a, CYP4A11d, CYP4A11e, CYP4A11f, and CYP4A11g; and dog CYP4A37, CYP4A38, and CYP4A39 cDNAs were isolated and analyzed. These CYP4A cDNAs shared relatively high sequence identities with human CYP4A11 and CYP4A22. Pig, cat, tree shrew, and dog CYP4As in the liver and kidneys are likely to catalyze the ω -hydroxylation of fatty acids., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Hexosaminidases are key enzymes in glycoconjugate metabolism and occur in all kingdoms of life. Here, we have investigated the phylogeny of the GH20 glycosyl hydrolase family in nematodes and identified a β-hexosaminidase subclade present only in the Dorylaimia. We have expressed one of these, HEX-2 from Trichuris suis , a porcine parasite, and shown that it prefers an aryl β- N -acetylgalactosaminide in vitro . HEX-2 has an almost neutral pH optimum and is best inhibited by GalNAc-isofagomine. Toward N-glycan substrates, it displays a preference for the removal of GalNAc residues from LacdiNAc motifs as well as the GlcNAc attached to the α1,3-linked core mannose. Therefore, it has a broader specificity than insect fused lobe (FDL) hexosaminidases but one narrower than distant homologues from plants. Its X-ray crystal structure, the first of any subfamily 1 GH20 hexosaminidase to be determined, is closest to Streptococcus pneumoniae GH20C and the active site is predicted to be compatible with accommodating both GalNAc and GlcNAc. The new structure extends our knowledge about this large enzyme family, particularly as T. suis HEX-2 also possesses the key glutamate residue found in human hexosaminidases of either GH20 subfamily, including HEXD whose biological function remains elusive.

Antimicrobial peptides (AMPs) are compounds with a variety of bioactive properties. Especially promising are their antibacterial activities, often toward drug-resistant pathogens. Across different AMP sources, AMPs expressed within plants are relatively underexplored with a limited number of plant AMP families identified. Recently, we identified the novel AMPs CC-AMP1 and CC-AMP2 in ghost pepper plants ( Capsicum chinense x frutescens ), exerting promising antibacterial activity and not classifying into any known plant AMP family. Herein, AMPs related to CC-AMP1 and CC-AMP2 were identified within both Capsicum annuum and Capsicum baccatum . In silico predictions throughout plants were utilized to illustrate that CC-AMP1-like and CC-AMP2-like peptides belong to two broader AMP families, with three-dimensional structural predictions indicating that CC-AMP1-like peptides comprise a novel subfamily of α-hairpinins. The antibacterial activities of several closely related CC-AMP1-like peptides were compared with a truncated version of CC-AMP1 possessing significantly more activity than the full peptide. This truncated peptide was further characterized to possess broad-spectrum antibacterial activity against clinically relevant ESKAPE pathogens. These findings illustrate the value in continued study of plant AMPs toward characterization of novel AMP families, with CC-AMP1-like peptides possessing promising bioactivity.

The rubber tree (Hevea brasiliensis) is one of the major domesticated crops planted commercially for the production of natural rubber (NR) worldwide. In recent years, rubber trees in the Southern states of India and other rubber-producing countries have experienced a severe leaf spot disease, characterized by the appearance of several brown circular spots in the initial stage, which later spread all over the lamina of fully matured leaves, leading to yellowing and defoliation. The causal organism of this Circular Leaf Spot (CLS) disease has not been conclusively identified in any previous studies. In this study, we collected infected leaf samples from various locations in the South Indian states. We aimed to identify the actual fungal pathogen that causes the CLS disease on rubber trees. Based on the morphological and molecular analysis of the most frequently isolated fungi from infected leaf samples were identified as Colletotrichum siamense and Colletotrichum fructicola. Pathogenicity tests also confirmed the involvement of isolated Colletotrichum spp. in the development of CLS disease. These findings provide valuable insights into understanding the CLS disease and its impact on rubber cultivation. To our knowledge, it is the first report of C. siamense and C. fructicola associated with CLS disease of rubber trees in India., 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 © 2024. Published by Elsevier Ltd.)

Infection by the zoonotic fish-borne trematode, Opisthorchis viverrini, remains a crucial health issue in Thailand and neighboring countries. Recently, molecular analysis revealed two populations of putative O. viverrini: one found primarily in human hosts ("human-specific" population) and the other primarily in cats ("cat-specific" population). It is unclear how the infective stages (metacercariae) of these different populations circulate among definitive and reservoir hosts in nature. To gain an insight into this, mitochondrial cox1 and nad1 gene sequences of metacercariae from fish intermediate hosts were examined. None of 192 metacercariae from cyprinid fish in Lao PDR and Thailand had sequences typical of "cat-specific" O. viverrini, suggesting that cyprinid fish are not the main second intermediate hosts of this population. Interestingly, all 20 O. viverrini-like metacercariae from snakehead fish (Channa striata) shared 99.51-100% sequence identity with eggs from cats naturally infected in a previous study. Hence, we propose a modification of the known transmission dynamics of O. viverrini: consumption of metacercariae within snakehead fish provides another pathway for cats and (occasionally) humans to acquire infection. We also performed morphological comparisons of eggs, metacercariae, and adult flukes (raised in hamsters) of both Opisthorchis populations. The "cat-specific" population has eggs that are narrower and adults that are shorter and wider than in the human-specific population. The metacercaria of the "cat-specific" population is elliptical, while that of the "human-specific" population is oval, occasionally rounded. Our results confirmed that O. viverrini-like metacercariae from snakehead fish are the infective stages of the "cat-specific" fluke. This provides a new insight into the dissemination and transmission of each population in the second intermediate host. The identity of the cat-specific population is discussed., (Copyright © 2024 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Archaeorhizomyces is a diverse and ubiquitous genus of the subphylum Taphrinomycotina, which contains soil-inhabiting/root-associated fungi. Although ecological importance and root-associating lifestyles of Archaeorhizomyces can be postulated, morphological aspects of fungal body and root colonization are largely unknown due to the scarcity of cultures. We obtained three unidentified Archaeorhizomyces isolates from ericoid mycorrhizal (ErM) roots of Rhododendron scabrum and Rhododendron × obtusum collected in Japan. To advance our understanding of lifestyle of the genus, we investigated their general morphology, phylogeny, and in vitro root-colonizing ability in ericoid mycorrhizal hosts, Vaccinium virgatum and Rhododendron kaempferi. Some morphological characteristics, such as slow glowing white-to-creamy-colored colonies and formation of yeast-like or chlamydospore-like cells, were shared between our strains and two described species, Archaeorhizomycesfinlayi and Archaeorhizomyces borealis, but they were phylogenetically distant. Our strains were clearly distinguished as two undescribed species based on morphology and phylogenetic relationship. As seen in typical ErM fungi, both species frequently formed hyphal coils within vital rhizodermal cells of ErM plants in vitro. The morphology of hyphal coils was also different between species. Consequently, two novel species, Archaeorhizomyces notokirishimae sp. nov. and Archaeorhizomyces ryukyuensis sp. nov., were described., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2024 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Arceuthobium azoricum Wiens & Hawksw. is a rare Azorean endemic epiphytic hemiparasite (mistletoe), which typically parasitizes branches of the gymnosperm Juniperus brevifolia (Hochst. ex Seub.) Antoine (Cupressaceae), which is also an Azorean endemic. Here, we describe a population of A. azoricum , on Pico Island, parasitizing Erica azorica Hochst. ex Seub. (Ericaceae), which is also an Azorean endemic. Our molecular analysis (using nuclear ribosomal internal transcribed spacer) showed no differences between individuals parasitizing Erica and Juniperus species. Moreover, a preliminary analysis showed no differences in morphological characteristics between accessions sampled from different hosts. Given that this is the first bona fide record of Arceuthobium sp. parasitizing an angiosperm, this population represents a unique host-shifting event, and its conservation is important because it may allow new insights into host recognition mechanisms in mistletoes. Immediate attention should be given to characterizing this Pico Island population using appropriate molecular methods and additional morphological analyses., Competing Interests: The author(s) declare no conflict of interest.

In radiation-resistant bacteria belonging to the genus Deinococcus, transposition events of insertion sequences (IS elements) leading to phenotypic changes from a reddish color to white were detected following exposure to gamma irradiation and hydrogen peroxide treatment. This change resulted from the integration of IS elements into the phytoene desaturase gene, a key enzyme in the carotenoid biosynthesis pathway. To facilitate species identification and distinguish among Deinococcus strains, the gyrB gene encoding the B subunit of DNA gyrase was utilized. The s gnificance of the gyrB gene is well recognized not only in genome replication through the regulation of supercoiling but also in phylogenetic analysis providing support for 16S rRNA-based identification. Its mutation rate surpasses that of the 16S rRNA gene, offering greater resolution between closely related species, particularly those exhibiting >99% similarity. In this study, phylogenetic analysis was conducted comparing the 16S rRNA and gyrB gene sequences of Deinococcus species. Species-specific and genus-specific primers targeting Deinococcus species were designed and experimentally validated for selective amplification and rapid identification of the targeted species. This approach allows for the omission of 16S rRNA sequencing in the targeted Deinococcus species. Therefore, the gyrB gene is useful for identifying bacterial species and genus-level detection from individual microbes or microbial consortia using specialized primer sets for PCR amplification., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A female narrow-ridged finless porpoise ( Neophocaena asiaeorientalis ) stranded on a beach on Jeju Island showed epithelial proliferative skin lesions on its body. Two false killer whales ( Pseudorca crassidens ), caught using nets near Gangneung and Samcheok, respectively, had multiple plaques on their penile epidermis. Histological examination of the epidermis revealed that all the lesions had common features, including accentuated rete pegs, ballooning changes, and eosinophilic intranuclear inclusion (INI) bodies. Based on the histopathological results, herpesvirus infection was suspected, and thus further analysis was conducted using herpesvirus-specific primers. Based on nested polymerase chain reaction (PCR) tests using the herpesvirus-detectable primers, the PCR products demonstrated two fragments: a 222-base-pair (bp) sequence of the DNA polymerase gene, SNUABM_CeHV01, showing 96.4% identity with a bottlenose dolphin herpesvirus from the Jeju narrow-ridged finless porpoise; and a 222 bp sequence of the DNA polymerase gene, SNUABM_CeHV02, showing 95.95% identity with the same bottlenose dolphin herpesvirus from the Gangneung and Samcheok false killer whales. The significance of this study lies in its ability to demonstrate the existence of novel cetacean herpesviruses in South Korean seawater, representing an important step forward in studying potentially harmful pathogens that affect endangered whale and dolphin populations.

Ananteris is a scorpion genus that inhabits dry and seasonal areas of South and Central America. It is located in a distinctive morpho-group of Buthids, the ' Ananteris group', which also includes species distributed in the Old World. Because of the lack of information on venom composition, the study of Ananteris species could have biological and medical relevance. We conducted a venomics analysis of Ananteris platnicki , a tiny scorpion that inhabits Panama and Costa Rica, which shows the presence of putative toxins targeting ion channels, as well as proteins with similarity to hyaluronidases, proteinases, phospholipases A 2 , members of the CAP-domain family, and hemocyanins, among others. Venom proteolytic and hyaluronidase activities were corroborated. The determination of the primary sequences carried out by mass spectrometry evidences that several peptides are similar to the toxins present in venoms from Old World scorpion genera such as Mesobuthus , Lychas , and Isometrus , but others present in Tityus and Centruroides toxins. Even when this venom displays the characteristic protein families found in all Buthids, with a predominance of putative Na + -channel toxins and proteinases, some identified partial sequences are not common in venoms of the New World species, suggesting its differentiation into a distinctive group separated from other Buthids.

Bats are hosts for diverse Trypanosoma species, including trypanosomes of the Trypanosoma cruzi clade. This clade is believed to have originated in Africa and diversified in many lineages worldwide. In several geographical areas, including Cameroon, no data about trypanosomes of bats has been collected yet. In this study, we investigated the diversity and phylogenetic relationships of trypanosomes of different bat species in the central region of Cameroon. Trypanosome infections were detected in six bat species of four bat families, namely Hipposideridae, Pteropodidae, Rhinolophidae, and Vespertilionidae, with an overall prevalence of 29% and the highest infection rate in hipposiderid bat species. All trypanosomes were identified as belonging to the Trypanosoma livingstonei species group with one clade that might represent an additional subspecies of T. livingstonei. Understanding the prevalence, distribution, and host range of parasites of this group contributes to our overall knowledge of the diversity and host specificity of trypanosome species that phylogenetically group at the base of the T. cruzi clade., (© 2024. The Author(s).)

The North African hedgehog (Atelerix algirus) is an introduced species from Northwest Africa and is currently distributed in the Canary Islands. This species of hedgehog has been studied as a reservoir of enteropathogens, including Cryptosporidium spp. However, there are no data at species level. Therefore, the aim of the present study was to identify the Cryptosporidium species present in a population of hedgehogs (n = 36) in the Canary Islands. Molecular screening was performed using conventional polymerase chain reaction (PCR) targeting the small subunit ribosomal RNA (18S rRNA) gene of Cryptosporidium spp. Seven of the 36 fecal samples (19.45%) were positive and confirmed by nested PCR targeting the 18S rRNA gene and Sanger sequencing. Cryptosporidium parvum and Cryptosporidium muris were identified in 11.1% (4/36) and 5.6% (2/36) of the samples, respectively, while one sample could only be identified at the genus level. The zoonotic subtypes IIdA15G1 (n = 1), IIdA16G1b (n = 1), and IIdA22G1 (n = 1) of C. parvum were identified by nested PCR followed by analysis of the 60 kDa glycoprotein (gp60) gene sequence. This study is the first genetic characterization of Cryptosporidium spp. in A. algirus, identifying zoonotic species and subtypes of the parasite., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Perkinsus, a parasitic pathogen of marine bivalves, is widely distributed among various mollusks in numerous countries. However, the prevalence and diversity of Perkinsus species in the two economically important mussels, Mytilus coruscus and M. galloprovincialis, in China remain unknown. The presence of the Perkinsus species was identified in the two mussels sampled along the coast of the East China Sea and the Yellow Sea, using both the alternative Ray's fluid thioglycolate medium (ARFTM) and conventional polymerase chain reaction (PCR). The ARFTM test indicated the presence of Perkinsus-like hypnospores in the two mussels. The diameter of the hypnospores in M. coruscus was significantly smaller than that in M. galloprovincialis. The prevalence of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 37.5% and 0 to 25%, respectively. The mean intensity of Perkinsus in M. galloprovincialis and M. coruscus ranged from 0 to 5.14 and 0 to 4.92, respectively. The PCR assay showed that the prevalence of Perkinsus spp. in M. galloprovincialis and M. coruscus was 0 to 25.0% and 0 to 12.5%, respectively. The homology analysis of the newly obtained internal transcribed spacer (ITS) sequences of Perkinsus revealed the highest identity of 100% with P. beihaiensis. The phylogenetic analysis indicated that the Perkinsus isolates from the two mussels were clustered with P. beihaiensis. The results of the molecular biology indicated that only P. beihaiensis was detected in the two mussels. The highest prevalence of P. beihaiensis was observed in Liaoning province (Dalian, 20.83%), followed by Shandong province, Zhejiang province and Fujian province. Consequently, it is recommended that surveillance should be conducted in Dalian, where the prevalence and mean intensity of P. beihaiensis in M. galloprovincialis are the highest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The analysis of the structures of glycans present on glycoproteins is an essential component for determining glycoprotein function; however, detailed glycan structural assignment on glycopeptides from proteomics mass spectrometric data remains challenging. Glycoproteomic analysis by mass spectrometry currently can provide significant, yet incomplete, information about the glycans present, including the glycan monosaccharide composition and in some circumstances the site(s) of glycosylation. Advancements in mass spectrometric resolution, using high-mass accuracy instrumentation and tailored MS/MS fragmentation parameters, coupled with a dedicated definition of diagnostic fragmentation ions have enabled the determination of some glycan structural features, or glycotopes, expressed on glycopeptides. Here we present a collation of diagnostic glycan fragments produced by traditional positive-ion-mode reversed-phase LC-ESI MS/MS proteomic workflows and describe the specific fragmentation energy settings required to identify specific glycotopes presented on N- or O-linked glycopeptides in a typical proteomics MS/MS experiment.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

Over the past decades, proteomics has become increasingly important and a heavily discussed topic. The identification of intact proteins remains a major focus in this field. While most intact proteins are analyzed using high-resolution mass spectrometry, identifying them through low-resolution mass spectrometry continues to pose challenges. In our study, we investigated the capability of identifying various intact proteins using collision-induced dissociation (CID) and electron transfer without dissociation (ETnoD). Using myoglobin as our test protein, stable product ions were generated with CID, and the identities of the product ions were identified with ETnoD. ETnoD uses a short activation time (AcT, 5 ms) to create sequential charge-reduced precursor ion (CRI). The charges of the fragments and their sequences were determined with corresponding CRI. The product ions can be selected for subsequent CID (termed CID n ) combined with ETnoD for further sequence identification and validation. We refer to this method as CID n /ETnoD. The use of a multistage CID activation (CID n ) and ETnoD protocol has been applied to several intact proteins to obtain multiple sequence identifications.

An orange-pigmented, Gram-stain-negative, strictly aerobic, non-flagellated and rod-shaped bacterium, designated strain DF17 T , was isolated from coastal sediment collected from Jingzi Wharf, Weihai, PR China. The optimal growth conditions were determined to be at 30 °C, pH 7.5, and in 3 % (w/v) NaCl. According to phylogenetic analysis of the 16S rRNA gene sequence, strain DF17 T showed the highest sequence similarity of 96.9 % to Winogradskyella aquimaris KCTC 23502 T . The DNA G+C content was 35.8 mol%, and the major fatty acids were iso-C 15 : 1 G, iso-C 15 : 0 , and iso-C 17 : 0 3-OH. The major polar lipids were two aminoglycolipids, one phosphatidylethanolamine and four unidentified lipids. The predominant respiratory quinone was menaquinone-6 (MK-6). The average nucleotide identity, digital DNA-DNA hybridization, and amino acid identity values between strain DF17 T and other Winogradskyella species were below the species delineation thresholds of 69.35-72.95 %, 16.9-19.6 % and 71.25-78.93 %, respectively. On the basis of its phenotypic, genetic and physiological characteristics, strain DF17 T is suggested to represent a novel species of the genus Winogradskyella , for which the name Winogradskyella pelagia sp. nov. is proposed. The type strain is DF17 T (MCCC 1H00456 T =KCTC 82421 T ).

The planktonic dinoflagellate Prorocentrum compressum is widespread in warm and temperate seas. A strain identified as P. cf. compressum BEA 0681B isolated from the island of Gran Canaria, NE Atlantic Ocean, showed a divergence in rDNA/ITS phylogenies with respect to P. compressum. The Canarian strain was oval, with an average length-to-width ratio of 1.35, smooth thecal surface with less than 150 thecal pores, including oblique pores, sometimes with a bifurcated opening. In contrast, P. compressum was rounder, with a length-to-width ratio < 1.2, with reticulate-foveate ornamentation and 200-300 pores per valve. We propose Prorocentrum canariense sp. nov. These species clustered as the most early-branching lineage in the clade Prorocentrum sensu stricto. Although this clade mainly contains planktonic species, the closer relatives were the benthic species P. tsawwassenense and P. elegans. Interestingly, P. compressum and P. canariense sp. nov. are widely distributed in temperate and warm seas without an apparent morphological adaptation to planktonic life. The formation of two concentric hyaline mucilaginous walls could contribute to this success. We discuss the use of Prorocentrum bidens to solve the nomenclature issue of P. compressum that was described citing a diatom as basionym., (© 2024 International Society of Protistologists.)

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive genetic disorder caused by mutations in the DMD gene, which leads to a deficiency of the dystrophin protein. The main mutation types of this gene include exon deletions and duplications, point mutations, and insertions. These mutations disrupt the normal expression of dystrophin, ultimately leading to the disease. In this study, we reported a case of DMD caused by an insertion mutation in exon 59 (E59) of the DMD gene. The affected child exhibited significant abnormalities in related biochemical markers, early symptoms of DMD, and multiple gray hair. His mother and sister were carriers with slightly abnormal biochemical markers. The mother had mild clinical symptoms, while the sister had no clinical symptoms. Other family members were genetically and physically normal. Sequencing and sequence alignment revealed that the inserted fragment was an Alu element from the AluYa5 subfamily. This insertion produced two stop codons and a polyadenylate (polyA) tail. To understand the impact of this insertion on the DMD gene and its association with clinical symptoms, exonic splicing enhancer (ESE) prediction indicated that the insertion did not affect the splicing of E59. Therefore, we speculated that the insertion sequence would be present in the mRNA sequence of the DMD gene. The two stop codons and polyA tail likely terminate translation, preventing the production of functional dystrophin protein, which may be the mechanism leading to DMD. In addition to typical DMD symptoms, the child also exhibited premature graying of hair. This study reports, for the first time, a case of DMD caused by the insertion of an Alu element into the coding region of the DMD gene. This finding provides clues for studying gene mutations induced by Alu sequence insertion and expands the understanding of DMD gene mutations.

Agaricus is a genus with more than 500 species. Most of the new species reported since 2000 are tropical or subtropical. The study area, the Malakand region, located in the north of Pakistan, has a subtropical climate. In this study, nine species, including three new species, of Agaricus subgenus Pseudochitonia , are reported from this region. Description of the new species are based on morphological characteristics and phylogenetic analyses using three DNA regions: nuc ribosomal DNA internal transcribed spacers (ITS), fragments of the large subunit of nuc ribosomal DNA (28S), and the translation elongation factor 1-alpha gene ( TEF1 ). One new species, Agaricus lanosus , with wooly squamules on its cap, forms a lineage within Agaricus sect. Bivelares and cannot be classified with certainty in one of the two subsections ( Cupressorum and Hortenses ) of this section. Agaricus rhizoideus with rhizoid-like structure at the base of the stipe forms a basal clade in Agaricus sect. Hondenses . Specimens of the third new species, Agaricus malakandensis , form a species-level clade within Agaricus sect. Catenulati and exhibits the morphological characteristics of this section. Due to their similar ITS sequences, two previously unnamed specimens from Thailand ( A . sp. LD2012162 and CA799) are considered conspecific with A. malakandensis .

Mastophorus muris (Gmelin, 1790) is a globally distributed parasitic nematode of broad range mammals. The taxonomy within the genus Mastophorus and the cryptic diversity among the genus are controversial among taxonomists. This study provides a detailed morphological description of M. muris from Mus musculus combined with a molecular phylogenetic approach. Moreover, descriptions and molecular data of M. muris from non-Mus rodents and wildcats complement our findings and together provide new insights into their taxonomy. The analysis of M. muris was based on light microscopy and scanning electron microscopy. The morphological description focused on the dentition pattern of the two trilobed pseudolabia. Additionally, we described the position of the vulva, arrangement of caudal pairs of papillae, spicules and measured specimens from both sexes and the eggs. For the molecular phylogenetic approach, we amplified the small subunit ribosomal RNA gene and the internal transcribed spacer, and the cytochrome c oxidase subunit 1. Mastophorus morphotypes based on dentition patterns and phylogenetic clustering indicate a subdivision of the genus in agreement with their host. We recognize two groups without a change to formal taxonomy: One group including those specimens infecting Mus musculus, and the second group including organisms infecting non-Mus rodents. Our genetic and morphological data shed light into the cryptic diversity within the genus Mastopohorus. We identified two host-associated groups of M. muris. The described morphotypes and genotypes of M. muris allow a consistent distinction between host-associated parasites., (© 2024. The Author(s).)

A Gram-stain-negative, orange-yellow, rod-shaped bacterium, designated strain SCSIO 19198 T , was isolated from sediment of the Haima cold seep in the South China Sea, PR China. The strain was aerobic and non-motile. Growth of strain SCSIO 19198 T occurred at pH 7-9 (optimum, pH 7), 15-37 °C (optimum, 25-32 °C) and with 3-8 % (w/v) NaCl (optimum, 3-6 % NaCl). Phylogenetic analyses based on 16S rRNA sequences revealed that strain SCSIO 19198 T belonged to the genus Hwangdonia , having the highest similarity to Hwangdonia seohaensis HD-3 T (98.35 %), followed by Algibacter aquimarinus KYW589 T (95.17 %) and Gelatiniphilus marinus GYP-24 T (94.89 %). The DNA G+C content was 35.92 mol%. The average nucleotide identity value between the genome of strain SCSIO 19198 T and that of H. seohaensis HD-3 T was 88.49 %. The digital DNA-DNA hybridization value between strain SCSIO 19198 T and H. seohaensis HD-3 T was 36 %. The major fatty acids (>10 %) of strain SCSIO 19198 T were iso-C 15 : 0 , iso-C 15 : 1 G, summed feature 3 (C 16 : 1  ω 6 c /C 16 : 1  ω7 c ) and anteiso-C 15 : 0 . MK-6 was the only detected respiratory quinone. The polar lipids of strain SCSIO 19198 T included phosphatidylethanolamine, two aminolipids, glycolipid and two unidentified lipids. The phenotypic, phylogenetic, chemotaxonomic and genomic data clearly suggest that strain SCSIO 19198 T represents a novel species of the genus Hwangdonia , for which the name Hwangdonia lutea sp. nov. is proposed. The type strain is SCSIO 19198 T (=MCCC 1K08674 T =KCTC 102078 T ).

The hAT family transposable element, hopper, was originally discovered as a defective 3120-bp full-length element in a wild-type strain of the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), and subsequently a functional 3131-bp element, hopper Bdwe , was isolated from a white eye mutant strain. The latter study showed that closely related elements exist in melonfly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae), a closely related subgenus, suggesting that hopper could have a widespread presence in the Bactrocera genus. To further understand the distribution of hopper within and beyond the B. dorsalis species complex, primer pairs from hopper Bdwe and its adjacent genomic insertion site were used to survey the presence and relatedness of hopper in five species within the complex and four species beyond the complex. Based on sequence identity of a 1.94 kb internal nucleotide sequence, the closest relationships were with mutated elements from B. dorsalis s.s. and species synonymized with B. dorsalis including B. papayae, B. philippinensis and B. invadens, ranging in identity between 88.4% and 99.5%. Notably, Bactrocera carambolae (Drew & Hancock) (Diptera: Tephritidae), which is most closely related to B. dorsalis beyond the synonymized species, shared hopper identities of 97.3%-99.5%. Beyond the B. dorsalis complex, Z. cucurbitae, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) and Bactrocera zonata (Saunders) (Diptera: Tephritidae) shared identities of 83.1%-97.1%, while hopper was absent from the Bactrocera oleae (Gmelin) (Diptera: Tephritidae) strain tested. While the functional autonomous hopper Bdwe element was not detected in these species, another closely related hopper element isolated from a B. dorsalis genetic sexing strain has an uninterrupted transposase open reading frame. The discontinuous presence of hopper in the Bactrocera genus has implications for its use for genomic manipulation and understanding the phylogenetic relationship of these species., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Two Gram-stain-negative, rod-shaped, non-motile, strictly aerobic strains, forming yellow colonies and designated F6058 T and S2608 T , were isolated from marine sediment collected in Weihai, PR China. Both strains grow at 4-40 °C (optimum, 30-33 °C), pH 6.0-7.5 (optimum, pH 6.5) and in the presence of 0-7.0 % (w/v) NaCl. The optimum NaCl concentrations for strains F6058 T and S2608 T were 2.0 % and 2.5 %, respectively. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strains F6058 T and S2608 T share an evolutionary lineage with members of the genus Aequorivita . The isolates exhibited a 16S rRNA gene sequence similarity of 96.7 % to each other. Strains F6058 T exhibited the highest 16S rRNA gene sequence similarity to Aequorivita xiaoshiensis F64183 T (98.8 %), and S2608 T was most similar to Aequorivita capsosiphonis A71 T (96.9 %). Iso-C 15:0 , anteiso-C 15:0 and iso-C 17:0 3-OH were the major fatty acids of strains F6058 T and S2608 T . The sole respiratory quinone of both isolates was menaquinone 6 (MK-6). The polar lipid profiles of the isolates both consisted of phosphatidylethanolamine and phosphoglycolipids; however, strain F6058 T exhibited one glycolipid, one aminolipid and two unidentified polar lipids, and strain S2608 T also had two glycolipids and one unidentified polar lipid. The DNA G+C contents of strains F6058 T and S2608 T were 34.6 % and 37.7 mol%, respectively. Based on their phenotypic, chemotaxonomic and genomic characteristics, strains F6058 T and S2608 T were considered to represent novel species of the genus Aequorivita , for which the names Aequorivita sediminis sp. nov. and Aequorivita marina sp. nov. were proposed. The type strains are F6058 T (=KCTC 92653 T =MCCC 1H01358 T ) and S2608 T (KCTC 92652 T =MCCC 1H01361 T ).

Insects are the largest group of animals when it comes to the number and diversity of species. Yet, with the exception of Drosophila , no information is currently available on the primary structure of their sperm nuclear basic proteins (SNBPs). This paper represents the first attempt in this regard and provides information about six species of Neoptera: Poecillimon thessalicus, Graptosaltria nigrofuscata, Apis mellifera, Nasonia vitripennis, Parachauliodes continentalis , and Tribolium castaneum . The SNBPs of these species were characterized by acetic acid urea gel electrophoresis (AU-PAGE) and high-performance liquid chromatography fractionated. Protein sequencing was obtained using a combination of mass spectrometry sequencing, Edman N-terminal degradation sequencing and genome mining. While the SNBPs of several of these species exhibit a canonical arginine-rich protamine nature, a few of them exhibit a protamine-like composition. They appear to be the products of extensive cleavage processing from a precursor protein which are sometimes further processed by other post-translational modifications that are likely involved in the chromatin transitions observed during spermiogenesis in these organisms., Competing Interests: All authors have read and agreed to the published version of the manuscript and there is no conflict of interest, including specific financial interest and relationships and affiliations relevant to the subject of the manuscript.

Acanthamoeba castellanii (Douglas, 1930) Page, 1967 is the type species of a widespread genus of free-living amoebae, potentially pathogenic for humans and animals. The Neff strain is one of the most widely used in biological research, serving as a model for both A. castellanii and the whole genus in general. The Neff strain, isolated in California, closely resembles another strain found in France and originally described as a separate species, Acanthamoeba terricola Pussard, 1964, but both were successively synonymized with A. castellanii. Molecular sequence analysis has largely replaced morphological diagnosis for species identification in Acanthamoeba, and rDNA phylogenies show that the Neff strain forms a distinct lineage from that of the type strain of A. castellanii. In this study, we compared the type strain of A. terricola with the Neff strain and A. castellanii, and analysed the available molecular data including new sequences obtained from A. terricola. Here we provide molecular evidence to validate the species A. terricola. The Neff strain is therefore transferred to A. terricola and should no longer be considered as belonging to A. castellanii., 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 © 2024 Elsevier GmbH. All rights reserved.)

Prophages/phages are important components of the genome of ' Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas., Competing Interests: The author(s) declare no conflict of interest.

Sperm nuclear basic proteins (SNBPs) were isolated from extracted antheridia-rich male gametophytes raised from spores of the swordfern, Polystichum munitum . Electrophoretic (acetic acid-urea PAGE and SDS-PAGE) and chromatographic (rp-HPLC) characterization of the nuclear proteins exhibited the characteristics of the histone (H-type). In both types of gel electrophoresis, histones H1, H2A, and H2B showed an altered electrophoretic mobility corresponding to that which is routinely observed for the histones in other plants. Histones present during spermatogenesis of the fern P. munitum were compared with the few current SNBPs known to be present in higher and lower evolutionary plant clades. A transition from an early protamine (P-type) SNBPs in charophytes and bryophytes to the (H-type) SNBP observed here is reminiscent of similar reversions observed in the animal kingdom., Competing Interests: The authors have no conflicting interests or competing interests, financial or otherwise.

Actin filament networks assemble on cellular membranes in response to signals that locally activate neural Wiskott-Aldrich-syndrome protein (N-WASP) and the Arp2/3 complex. An inactive conformation of N-WASP is stabilized by intramolecular contacts between the GTPase binding domain (GBD) and the C helix of the verprolin-homology, connector-helix, acidic motif (VCA) segment. Multiple SH3 domain-containing adapter proteins can bind and possibly activate N-WASP, but it remains unclear how such binding events relieve autoinhibition to unmask the VCA segment and activate the Arp2/3 complex. Here, we have used purified components to reconstitute a signaling cascade driven by membrane-localized Src homology 3 (SH3) adapters and N-WASP, resulting in the assembly of dynamic actin networks. Among six SH3 adapters tested, Nck was the most potent activator of N-WASP-driven actin assembly. We identify within Nck a previously unrecognized activation motif in a linker between the first two SH3 domains. This linker sequence, reminiscent of bacterial virulence factors, directly engages the N-WASP GBD and competes with VCA binding. Our results suggest that animals, like pathogenic bacteria, have evolved peptide motifs that allosterically activate N-WASP, leading to localized actin nucleation on cellular membranes.

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase.

Retinal opsin photopigments initiate mammalian vision when stimulated by light. Most mammals possess a short wavelength-sensitive opsin 1 (SWS1) pigment that is primarily sensitive to either ultraviolet or violet light, leading to variation in colour perception across species. Despite knowledge of both ultraviolet- and violet-sensitive SWS1 classes in mammals for 25 years, the adaptive significance of this variation has not been subjected to hypothesis testing, resulting in minimal understanding of the basis for mammalian SWS1 spectral tuning evolution. Here, we gathered data on SWS1 for 403 mammal species, including novel SWS1 sequences for 97 species. Ancestral sequence reconstructions suggest that the most recent common ancestor of Theria possessed an ultraviolet SWS1 pigment, and that violet-sensitive pigments evolved at least 12 times in mammalian history. We also observed that ultraviolet pigments, previously considered to be a rarity, are common in mammals. We then used phylogenetic comparative methods to test the hypotheses that the evolution of violet-sensitive SWS1 is associated with increased light exposure, extended longevity and longer eye length. We discovered that diurnal mammals and species with longer eyes are more likely to have violet-sensitive pigments and less likely to possess UV-sensitive pigments. We hypothesize that (i) as mammals evolved larger body sizes, they evolved longer eyes, which limited transmittance of ultraviolet light to the retina due to an increase in Rayleigh scattering, and (ii) as mammals began to invade diurnal temporal niches, they evolved lenses with low UV transmittance to reduce chromatic aberration and/or photo-oxidative damage.

Symbioses include some of the clearest cases of coevolution, but their origin, loss or reassembly with different partners can rarely be inferred. Here we use ant/plant symbioses involving three plant clades to investigate the evolution of symbioses. We generated phylogenies for the big-eyed arboreal ants (Pseudomyrmecinae), including 72% of their 286 species, as well as for five of their plant host groups, in each case sampling more than 61% of the species. We show that the ant-housing Vachellia (Mimosoideae) clade and its ants co-diversified for the past 5 Ma, with some species additionally colonized by younger plant-nesting ant species, some parasitic. An apparent co-radiation of ants and Tachigali (Caesalpinioideae) was followed by waves of colonization by the same ant clade, and subsequent occupation by a younger ant group. Wide crown and stem age differences between the ant-housing genus Triplaris (Polygonaceae) and its obligate ant inhabitants, and stochastic trait mapping, indicate that its domatium evolved earlier than the ants now occupying it, suggesting previous symbioses that dissolved. Parasitic ant species evolved from generalists, not from mutualists, and are younger than the mutualistic systems they parasitize. Our study illuminates the macroevolutionary assembly of ant/plant symbioses, which has been highly dynamic, even in very specialized systems.

Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.

Neuronal excitability is strictly regulated by various mechanisms, including modulation of ion channel activity and trafficking. Stimulation of m1 muscarinic acetylcholine receptor (also known as CHRM1) increases neuronal excitability by suppressing the M-current generated by the Kv7/KCNQ channel family. We found that m1 muscarinic acetylcholine receptor stimulation also triggers surface transport of KCNQ subunits. This receptor-induced surface transport was observed with KCNQ2 as well as KCNQ3 homomeric channels, but not with Kv3.1 channels. Deletion analyses identified that a conserved domain in a proximal region of the N-terminal tail of KCNQ protein is crucial for this surface transport--the translocation domain. Proteins that bind to this domain were identified as α- and β-tubulin and collapsin response mediator protein 2 (CRMP-2; also known as DPYSL2). An inhibitor of casein kinase 2 (CK2) reduced tubulin binding to the translocation domain, whereas an inhibitor of glycogen synthase kinase 3 (GSK3) facilitated CRMP-2 binding to the translocation domain. Consistently, treatment with the GSK3 inhibitor enhanced receptor-induced KCNQ2 surface transport. M-current recordings from neurons showed that treatment with a GSK3 inhibitor shortened the duration of muscarinic suppression and led to over-recovery of the M-current. These results suggest that m1 muscarinic acetylcholine receptor stimulates surface transport of KCNQ channels through a CRMP-2-mediated pathway.

A peptide fragment of the human tau protein which stacks to form neat cross β-sheet fibrils, resembling that found in pathological aggregation, (273)GKVQIINKKLDL(284) (here "R2/WT"), was modified with a spin-label at the N-terminus. With the resulting peptide, R2/G273C-SL, we probed events at time scales spanning seconds to hours after aggregation is initiated using transmission electron microscopy (TEM), thioflavin T (THT) fluorescence, ion mobility mass spectrometry (IMMS), electron paramagnetic resonance (EPR), and Overhauser dynamic nuclear polarization (ODNP) to determine if deliberate changes to its conformational states and population in solution influence downstream propensity to form fibrillar aggregates. We find varying solution conditions by adding the osmolyte urea or TMAO, or simply using different buffers (acetate buffer, phosphate buffer, or water), produces significant differences in early monomer/dimer populations and conformations. Crucially, these characteristics of the peptide in solution state before aggregation is initiated dictate the fibril formation propensity after aggregation. We conclude the driving forces that accelerate aggregation, when heparin is added, do not override the subtle intra- or interprotein interactions induced by the initial solvent conditions. In other words, the balance of protein-protein vs protein-solvent interactions present in the initial solution conditions is a critical driving force for fibril formation.

While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27(-/-) cilia and revealed that β-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.

Observations from human microbiome studies are often conflicting or inconclusive. Many factors likely contribute to these issues including small cohort sizes, sample collection, and handling and processing differences. The field of microbiome research is moving from 16S rDNA gene sequencing to a more comprehensive genomic and functional representation through whole-genome sequencing (WGS) of complete communities. Here we performed quantitative and qualitative analyses comparing WGS metagenomic data from human stool specimens using the Illumina Nextera XT and Illumina TruSeq DNA PCR-free kits, and the KAPA Biosystems Hyper Prep PCR and PCR-free systems. Significant differences in taxonomy are observed among the four different next-generation sequencing library preparations using a DNA mock community and a cell control of known concentration. We also revealed biases in error profiles, duplication rates, and loss of reads representing organisms that have a high %G+C content that can significantly impact results. As with all methods, the use of benchmarking controls has revealed critical differences among methods that impact sequencing results and later would impact study interpretation. We recommend that the community adopt PCR-free-based approaches to reduce PCR bias that affects calculations of abundance and to improve assemblies for accurate taxonomic assignment. Furthermore, the inclusion of a known-input cell spike-in control provides accurate quantitation of organisms in clinical samples.