Uncharacterized Protein Research Articles

Genetic characterization of tetracycline-resistant Staphylococcus aureus with reduced vancomycin susceptibility using whole-genome sequencing.

This study aimed to analyze the genetic characteristics of Staphylococcus aureus with reduced vancomycin susceptibility (RVS-SA). Whole-genome sequencing was performed on 27 RVS-SA clinical isolates, and comparative genomic analysis was performed using S. aureus reference strains. Pan-genome orthologous groups (POGs) were identified that were present in RVS-SA but absent in the reference strains, but further analysis showed that the presence of these POGs was influenced by tetracycline resistance rather than vancomycin resistance. Therefore, we restricted our analysis to tetracycline-resistant (tetR) RVS-SA and tetR vancomycin-susceptible S. aureus (VSSA). Phylogenomic analysis showed them to be closely related, and further analysis revealed the presence of an uncharacterized protein SAB0394 and the absence of lytA in tetR RVS-SA, which are involved in cell wall thickening. In summary, using whole-genome sequencing we identified gain or loss of genes in tetR RVS-SA strains. These findings provide insights into the investigation of mechanisms associated with reduced vancomycin susceptibility and have the potential to contribute to the development of molecular biomarkers for the rapid and efficient detection of RVS-SA.

Functional Annotation of Uncharacterised Proteins Whose Expression Patterns Affect the Lifespan under Metformin Treatment in Fission Yeast

Functional Annotation of Uncharacterised Proteins Whose Expression Patterns Affect the Lifespan under Metformin Treatment in Fission Yeast

A bacterial sensor taxonomy across earth ecosystems for machine learning applications.

Microbial communities have evolved to colonize all ecosystems of the planet, from the deep sea to the human gut. Microbes survive by sensing, responding, and adapting to immediate environmental cues. This process is driven by signal transduction proteins such as histidine kinases, which use their sensing domains to bind or otherwise detect environmental cues and "transduce" signals to adjust internal processes. We hypothesized that an ecosystem's unique stimuli leave a sensor "fingerprint," able to identify and shed insight on ecosystem conditions. To test this, we collected 20,712 publicly available metagenomes from Host-associated, Environmental, and Engineered ecosystems across the globe. We extracted and clustered the collection's nearly 18M unique sensory domains into 113,712 similar groupings with MMseqs2. We built gradient-boosted decision tree machine learning models and found we could classify the ecosystem type (accuracy: 87%) and predict the levels of different physical parameters (R2 score: 83%) using the sensor cluster abundance as features. Feature importance enables identification of the most predictive sensors to differentiate between ecosystems which can lead to mechanistic interpretations if the sensor domains are well annotated. To demonstrate this, a machine learning model was trained to predict patient's disease state and used to identify domains related to oxygen sensing present in a healthy gut but missing in patients with abnormal conditions. Moreover, since 98.7% of identified sensor domains are uncharacterized, importance ranking can be used to prioritize sensors to determine what ecosystem function they may be sensing. Furthermore, these new predictive sensors can function as targets for novel sensor engineering with applications in biotechnology, ecosystem maintenance, and medicine.IMPORTANCEMicrobes infect, colonize, and proliferate due to their ability to sense and respond quickly to their surroundings. In this research, we extract the sensory proteins from a diverse range of environmental, engineered, and host-associated metagenomes. We trained machine learning classifiers using sensors as features such that it is possible to predict the ecosystem for a metagenome from its sensor profile. We use the optimized model's feature importance to identify the most impactful and predictive sensors in different environments. We next use the sensor profile from human gut metagenomes to classify their disease states and explore which sensors can explain differences between diseases. The sensors most predictive of environmental labels here, most of which correspond to uncharacterized proteins, are a useful starting point for the discovery of important environment signals and the development of possible diagnostic interventions.

Venomics of Leiurus abdullahbayrami, the most lethal scorpion in the Levant region of the Middle East

The scorpion Leiurus abdullahbayrami has been associated with severe/lethal envenomings throughout the Levant region of the Middle East, encompassing Turkey, Syria, and Lebanon, and only scarce information is available on its venom composition, activity, and antigenicity. We report on the composition of L. abdullahbayrami venom collected from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality, through LD50 determination in mice (intraperitoneal), was also assessed (0.75 (0.16–1.09) mg/kg), confirming L.abdullahbayrami venom vertebrate toxicity. Fifty-four peaks were detected using RP-HPLC, half of which eluted in the gradient region between 20 and 40% acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 24.4, 43.1, and 48.9 kDa. Venom mass fingerprint by MALDI-TOF detected 21 components within the 1000–12,000 m/z range. Whole venom ‘shotgun’ bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 113 different components belonging to 15 venom families and uncharacterized proteins, with ion channel-active components (K+ channel toxins (28); Na+ channel toxins (42); Cl− channel toxins (4); Ca+2 toxins (2)) being predominant. A single match for a L. adbullahbayrami NaTx was found in the UniProt database with other congeneric species, toxin h3.1 from Leiurus hebraeus, suggesting this might be an indication of venom divergence within Leiurus, eventhough this warrants further investigation involving venom proteomics and transcriptomics of relevant species. Considering such potential interspecific venom variation, future work should address whether preparation of a specific anti-L. abdullahbayrami antivenom is justified.

The Golgi-localized transporter OsPML4 contributes to manganese homeostasis in rice

Manganese (Mn), an indispensable plant micronutrient, functions as a vital enzyme co-factor in numerous biochemical reactions. In rice, the Golgi-localized PHOTOSYNTHESIS-AFFECTED MUTANT 71-LIKE 3 (OsPML3), a member of the UNCHARACTERIZED PROTEIN FAMILY (UPF0016), plays a pivotal role in Mn homeostasis, particularly in rapidly developing tissues. This study focused on the functional characterization of another UPF0016 family member in rice, OsPML4, to elucidate its involvement in Mn homeostasis. OsPML4 had a 73% sequence identity with OsPML3 and exhibited expression in both shoots and roots, albeit at a lower transcriptional level than OsPML3. Furthermore, subcellular localization studies confirmed that OsPML4 localizes in the Golgi apparatus. Notably, heterologous expression of OsPML4 restored growth in the Mn uptake-deficient yeast strain Δsmf1 under Mn-limited conditions. Under Mn-deficient conditions, OsPML4 knockout exacerbated the decline in shoot dry weight and intensified necrosis in young leaves of OsPML3 knockout lines, which displayed stunted growth. The Mn concentration in OsPML3PML4 double knockout lines was lower than in wild-type (WT) and OsPML3 knockout lines. At the reproductive phase, OsPML3PML4 double knockout lines exhibited reduced fertility and grain yield compared to WT and OsPML3 knockout lines. Notably, reductions were observed in the deposition of cell wall polysaccharides and the content of Lea (Lewis A structure)-containing N-glycans in the young leaves of OsPML3PML4 double knockout lines, surpassing the reductions in WT and OsPML3 knockout lines. These findings underscore the significance of OsPML4 in Mn homeostasis in the Golgi apparatus, where it co-functions with OsPML3 to regulate cell wall polysaccharide deposition and late-stage Golgi N-glycosylation.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Abstract C058: Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men

Abstract The incidence of penile squamous cell carcinoma (PSCC) in Puerto Rico is nearly three times higher when compared with other racial and ethnic groups in the United States. Mortality rate for PSCC is also significantly higher among Puerto Rican men when compared to the US population. Human papillomavirus (HPV) is a common virus, and its infection may increase the risk of developing PeCa. Despite the high incidence, the molecular mechanism of PSCC remains poorly understood. Our laboratory found that the prevalence of HPV in PSCC in Puerto Rican men is 49%. Due to the high rates of HPV infection and increased incidence of penile cancer in Puerto Rican men, there is a need to understand the pathogenesis of penile cancer. DNA Methylation is an epigenetic alteration that can be found in cancer cells, which can lead to changes in gene expression and cellular function. This study aims to analyze the DNA methylation patterns in HPV-positive and HPV-negative PSCC tissues from Puerto Rican men. DNA was extracted from twenty-four fresh PSCC tissue samples from Puerto Rican patients. HPV status and genotyping were determined using the DNA ELISA kit HPV SPF10 protocol. DNA was amplified and hybridized using PCR. We perform a DNA methylation assay using the Infinium Methylation EPIC v2.0 BeadChip Kit to identify and compare average DNA methylation levels (%) in the 9 HPV-positive and 15 HPV-negative penile cancer samples. Our results demonstrated 43 loci significantly different between HPV-positive and HPV-negative samples, with an adjusted p-value &amp;lt;0.1 (Benjamini-Hochberg method). Most of the hypermethylated loci were present in HPV-negative tumor samples. Additionally, we identified four methylated-differentially expressed genes: ANXA4, CACNA1C-AS1, UBXN2A, and DUOX1, which are found as uncharacterized proteins (open reading frames, ORFs) of the human genome with adjusted p-value &amp;lt;0.05. Studies have revealed that specific DNA methylation patterns could be linked with cancer development and progression. Identification of differentially methylated regions in HPV positive (HPV+) PSCC compared with HPV negative (HPV-) cases will provide much needed data to aid studies on early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Identification of differentially expressed genes and pathways in HPV positive (HPV+) PeCa compared with HPV negative (HPV-) cases will provide much needed data to aid studies on prevention, early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Citation Format: Yarelis M. Roque Reyes, María Sánchez-Vázquez, Josué Perez, Luis R. Llanos, Ingrid Montes, Jasmine Figueroa-Díaz, Carlos Rivera-Lopez, María Marcos, Antonio Puras, Magaly Martínez-Ferrer. Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C058.

Identification and preliminary characterization of conserved uncharacterized proteins from Chlamydomonas reinhardtii, Arabidopsis thaliana, and Setaria viridis.

The rapid accumulation of sequenced plant genomes in the past decade has outpaced the still difficult problem of genome-wide protein-coding gene annotation. A substantial fraction of protein-coding genes in all plant genomes are poorly annotated or unannotated and remain functionally uncharacterized. We identified unannotated proteins in three model organisms representing distinct branches of the green lineage (Viridiplantae): Arabidopsis thaliana (eudicot), Setaria viridis (monocot), and Chlamydomonas reinhardtii (Chlorophyte alga). Using similarity searching, we identified a subset of unannotated proteins that were conserved between these species and defined them as Deep Green proteins. Bioinformatic, genomic, and structural predictions were performed to begin classifying Deep Green genes and proteins. Compared to whole proteomes for each species, the Deep Green set was enriched for proteins with predicted chloroplast targeting signals predictive of photosynthetic or plastid functions, a result that was consistent with enrichment for daylight phase diurnal expression patterning. Structural predictions using AlphaFold and comparisons to known structures showed that a significant proportion of Deep Green proteins may possess novel folds. Though only available for three organisms, the Deep Green genes and proteins provide a starting resource of high-value targets for further investigation of potentially new protein structures and functions conserved across the green lineage.

The protein interactome of the citrus Huanglongbing pathogen Candidatus Liberibacter asiaticus

The bacterium Candidatus Liberibacter asiaticus (CLas) causes citrus Huanglongbing disease. Our understanding of the pathogenicity and biology of this microorganism remains limited because CLas has not yet been cultivated in artificial media. Its genome is relatively small and encodes approximately 1136 proteins, of which 415 have unknown functions. Here, we use a high-throughput yeast-two-hybrid (Y2H) screen to identify interactions between CLas proteins, thus providing insights into their potential functions. We identify 4245 interactions between 542 proteins, after screening 916 bait and 936 prey proteins. The false positive rate of the Y2H assay is estimated to be 2.9%. Pull-down assays for nine protein-protein interactions (PPIs) likely involved in flagellar function support the robustness of the Y2H results. The average number of PPIs per node in the CLas interactome is 15.6, which is higher than the numbers previously reported for interactomes of free-living bacteria, suggesting that CLas genome reduction has been accompanied by increased protein multi-functionality. We propose potential functions for 171 uncharacterized proteins, based on the PPI results, guilt-by-association analyses, and comparison with data from other bacterial species. We identify 40 hub-node proteins, including quinone oxidoreductase and LysR, which are known to protect other bacteria against oxidative stress and might be important for CLas survival in the phloem. We expect our PPI database to facilitate research on CLas biology and pathogenicity mechanisms.

The social and structural architecture of the yeast protein interactome

Cellular functions are mediated by protein–protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification coupled to mass spectrometry is an ideal tool for such mapping, but it has been difficult to identify low copy number complexes, membrane complexes and complexes that are disrupted by protein tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive high-throughput method using highly reproducible affinity enrichment coupled to mass spectrometry combined with a quantitative two-dimensional analysis strategy to comprehensively map the interactome of Saccharomyces cerevisiae. Thousand-fold reduced volumes in 96-well format enabled replicate analysis of the endogenous GFP-tagged library covering the entire expressed yeast proteome1. The 4,159 pull-downs generated a highly structured network of 3,927 proteins connected by 31,004 interactions, doubling the number of proteins and tripling the number of reliable interactions compared with existing interactome maps2. This includes very-low-abundance epigenetic complexes, organellar membrane complexes and non-taggable complexes inferred by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 16 interactors. Similar to social networks between humans, the average shortest distance between proteins is 4.2 interactions. AlphaFold-Multimer provided novel insights into the functional roles of previously uncharacterized proteins in complexes. Our web portal (www.yeast-interactome.org) enables extensive exploration of the interactome dataset.

Transcriptome-Based Comparative Expression Profiling of Sweet Potato during a Compatible Response with Root-Knot Nematode Meloidogyne incognita Infection.

M. incognita, a root-knot nematode (RKN), infects the roots of several important food crops, including sweet potato (Ipomoea batatas Lam.), and severely reduces yields. However, the molecular mechanisms underlying infection remain unclear. Previously, we investigated differential responses to RKN invasion in susceptible and resistant sweet potato cultivars through RNA-seq-based transcriptome analysis. In this study, gene expression similarities and differences were examined in RKN-susceptible sweet potato cultivars during the compatible response to RKN infection. Three susceptible cultivars investigated in previous research were used: Dahomi (DHM), Shinhwangmi (SHM), and Yulmi (YM). Of the three cultivars, YM had the highest number of genes with altered expression in response to infection. YM was also the cultivar with the highest susceptibility to RKN. Comparisons among cultivars identified genes that were regulated in more than one cultivar upon infection. Pairwise comparisons revealed that YM and DHM shared the most regulated genes, whereas YM and SHM shared the lowest number of regulated genes. Five genes were up-regulated, and two were down-regulated, in all three cultivars. Among these, four genes were highly up-regulated in all cultivars: germin-like protein, anthranilate synthase α subunit, isocitrate lyase, and uncharacterized protein. Genes were also identified that were uniquely regulated in each cultivar in response to infection, suggesting that susceptible cultivars respond to infection through shared and cultivar-specific pathways. Our findings expand the understanding of the compatible response to RKN invasion in sweet potato roots and provide useful information for further research on RKN defense mechanisms.

Membrane-specific and calcium-dependent binding of the Arabidopsis C2 domain protein CaLB revealed by ATR-FTIR spectroscopy

C2 domain-containing proteins bind to cellular membranes and mediate diverse cellular processes. Although many of these membrane-interacting proteins have been identified, the molecular mechanisms of protein-membrane interactions and conformational dynamics are often poorly understood and remain to be investigated with appropriate methods. Here, we used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and biomimetic membrane systems to analyse CalB, a yet uncharacterized Arabidopsis C2 domain protein. We studied membrane binding, lipid specificity and calcium dependency with solid-supported lipid membranes (SSLB) and small unilamellar lipid vesicles (SUVs). Membranes were composed of pure POPC lipids or of POPC/PI(3)P lipid mixtures. A significantly increased protein binding affinity was observed with membranes containing 1% PI(3)P indicating the high binding specificity of CaLB for PI(3)P. Furthermore, membrane binding occurs in a calcium-dependent manner with a higher calcium concentration increasing the binding of CaLB to the POPC/PI(3)P membrane. Secondary structure analysis of IR-spectra reveals that only minor conformational changes take place upon binding with a slight increase in the helical and disordered regions of CaLB.

Abstract 18695: C5ORF51/RIMOC1 - A Novel Regulator of Cardiogenesis

Introduction: Adult human cardiomyocytes (CMs) have poor capacity for proliferation and functional recovery after heart attack, thus leading to the high morbidity and mortality of cardiovascular diseases. Thus, there is great need to understand the underlying mechanisms of adult CM turnover and maturation. While screening for modulators of TEAD1, the downstream transcriptional effector of Hippo pathway, my lab identified a novel factor—C5ORF51/RIMOC1—as a potential cardiomyogenic modulator. C5ORF51 (hereinafter referred to as C5x) is a previously uncharacterized protein of nearly unknown function, which appears to play critical roles in the regulation of CM homeostasis and heart size. Hypothesis: C5x is a novel cardiomyogenic regulator which determines CM turnover, maturation, and function. Methods and Results: (1) Ex vivo C5x deletion in mouse neonatal CMs leads to significantly increased percentages of cells in S-G2-M phases, suggesting that C5x mediates CM cell cycle arrest in a cell-autonomous manner; (2) In vivo CM-specific C5x-knockout mouse model shows significantly enhanced CM proliferation in adult mice as well as increased CM endowment and mononuclear diploid populations, indicating that C5x has an essential role in cell replication and division in vivo; (3) With age, C5x-CM-knockout mice develop systolic dysfunction, massive cardiomegaly, late-onset dilated cardiomyopathy, and premature mortality, demonstrating a critical role of C5x in maintaining normal cardiac function; (4) RNA sequencing analysis further reveals that C5x loss-of-function activates fetal gene expression while its gain-of-function suppresses fetal gene expression, suggesting C5x as a gatekeeper of the fetal gene program. Conclusions: C5x is required for CM cell cycle arrest, and its loss results in fetal gene activation leading to a primitive cell state and immature CM expansion.

PSXI-11 Microbiome Taxa Involved in Carbohydrate Breakdown and Synthesis of Short Chain Fatty Acids in the Piglet Gut

Abstract The gut microbiome plays a critical role in swine health and growth, and diet is a key factor which shapes microbiome communities. In particular, the fermentation of indigestible carbohydrates by microbes has important consequences for microbiome composition and host health. Pigs do not encode all the enzymes needed to breakdown dietary polysaccharides, but bacteria contain numerous carbohydrate-active enzymes (CAZys) which contribute to this task. In addition, microbial fermentation of carbohydrates produces many bioactive metabolites such as short chain fatty acids (SCFAs). The relationships between microbes, dietary components, and the pig are complex, but a thorough understanding of these interactions would promote the design of interventions which target the microbiome and improve swine growth, especially during the critical weaning transition. At weaning, the microbiome undergoes rapid compositional changes as the diet shifts from milk to plant based. Here, we use quantitative (TMT)-based liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantitate piglet and microbial proteins in the gut during the weaning transition, with a focus on those involved in carbohydrate breakdown and nutrient synthesis. We analyzed 6 fecal samples from healthy, nursery piglets at weaning [day 21 postnatal (PN)] and 6 samples taken two weeks later, after acclimation to nursery diet (day 49 PN). In total, we detected 14,319 bacterial and fungal protein groups and 1,835 pig protein groups, and abundances of roughly one-half of the protein groups changed significantly over time (P = 0.05, fold change &amp;gt; 1.5). As expected, from weaning to day 49 PN, expression of glycoside hydrolases (GHs) which degrade milk oligosaccharides decreased, while the abundance of most enzymes which degrade plant carbohydrates such as xylan, cellulose, and starch, increased. Some plant-degrading enzymes were most abundant during weaning, which may reflect the tendency for piglets to steal solid feed from their mothers during nursing. Subdoligranulum was one of the top producers of multiple GHs capable of degrading milk or plant carbohydrates, while other genera specialized in the production of enzymes which targeted either milk oligosaccharides (Blautia, Lachnoclostridium Clostridium, Fournierella, Faecalibacterium, Eisenbergiella) or plant carbohydrates (Prevotella, Selemonas, Anaerovibrio). Aspergillus produced an uncharacterized protein predicted to degrade plant xyloglucan, highlighting the contributions of fungi in swine digestion. Host glycan-degrading enzymes decreased over time. Last, we detected acetate and butyrate production enzymes including acetate kinase (26 genera) and phosphate acetyltransferase (9 genera), and butyrate kinase and phosphate butyryltransferase from Subdoligranulum. Together, these data identify specific enzymes and the microbes that produce them during the transition from a milk to plant-based diet. This information provides a more robust understanding of digestion in the weanling pig and will ultimately provide a basis for effective design of probiotics and other therapeutics which target the gut microbiome to enhance swine growth and productivity.

In silico and experimental analysis of the repeated domains in BvaP, a protein important for GBS vaginal colonization.

Streptococcus agalactiae (group B strep, GBS) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Previously, we highlighted the importance of a formerly uncharacterized protein, BvaP, in GBS vaginal colonization. BvaP is highly conserved across GBS and is made up of repeated domains, with a variable number of repeats between strains. Here, we evaluate the prevalence of BvaP repeated domains and their relevance in phenotypes previously associated with vaginal colonization. Using in silico analysis, we found that the number of repeats in the BvaP protein does not generally appear to be associated with serotype, isolation site, or host. Using BvaP truncations in GBS strain A909, we determined that a smaller number of repeats was correlated with decreased bacterial chain length, but adherence to vaginal epithelial cells was complemented using BvaP containing one, two, three, or five repeats. Future research will be geared toward understanding the host immune response to BvaP in vivo and whether vaginal carriage or host response is dependent on the BvaP repeated domains.

Genome-wide association study of feed conversion ratio in turbot (Scophthalmus maximus) based on genome resequencing

The feed conversion rate (FCR) is a crucial economic trait in aquaculture fish, as feed costs account for the vast majority of aquaculture costs. Given the importance of this trait, there have little efforts to unravel the genomic architecture of FCR and identify key genes or genomic regions useful as selection makers. This situation may stem from the inherent swimming behavior of fish, which presents challenges in obtaining individual FCRs. In this work, we developed a specialized small cage culture system. Through this system, the Feed Conversion Ratio (FCR) of 300 individuals was successfully determined, marking the first successful determination of individual FCRs for turbot. The results revealed a range of FCR values from 35.3% to 192.6%, with an average of 126.5% ± 23.5%. On this basis, a genome-wide association study (GWAS) was conducted to identify single nucleotide polymorphisms (SNP) associated with FCR. A total of 2613,115 SNPs on 22 chromosomes were selected for GWAS analysis by Efficient Mixed-Model Association eXpedited (EMMAX) software. A linear mixed model was employed to test the association between each SNP and FCR, considering population structure and genetic relatedness among individuals. Two significant SNPs on Chr6 were identified to be correlated with FCR. According to their localization in genome, four candidate genes were identified on Chr6, including uncharacterized protein KIAA1755, Kinesin Family Member 21B (KIF21B), Discs large homolog-associated protein 4B (DLGAP4B), and Copine 5B (CPNE5B), all of which are closely located in the genome and exhibit significant functional similarities. Our results will facilitate the implementation of marker-assisted selection in breeding programs and serve as a foundation for future research in this area.

African swine fever virus B175L inhibits the type I interferon pathway by targeting STING and 2'3'-cGAMP.

African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.

Genome-wide identification and characterisation of Aquaporins in Rosa chinensis

ABSTRACT Aquaporins (AQPs) are small integral membrane proteins that facilitate the efficient transport of small solutes like water, metalloids, urea, and CO2 across the membrane. Plant aquaporins are known to be involved in vital physiological processes like seed germination, stomatal movement, cell elongation, and reproductive growth. In the present study, 36 AQPs were identified through genome-wide computational analysis performed in the rose genome. Subsequent phylogenetic analysis classified the AQPs into five subfamilies, including 10 Plasma membrane intrinsic proteins (PIPs), 8 Tonoplast intrinsic proteins (TIPs), 9 Nodulin intrinsic proteins (NIPs), 3 Small intrinsic proteins (SIPs), and 6 uncharacterised intrinsic proteins (XIPs). Based on the prediction of silicon solute specificity, one of the NIPs is predicted to be a silicon transporter. Structural analysis revealed the characteristic six trans-membrane domains and two-half alpha helix harbouring two conserved NPA motifs in the majority of the predicted AQPs. The protein tertiary structures predicted with homology-based modelling were used to study the pore lining remains and to predict the solute transport activity of the AQPs. Expression profiling of the rose AQPs showed tissue-specific expression of many members. The data provided here will help in understanding the AQP-based solute transport system in roses and other related species.

A conserved complex of microneme proteins mediates rhoptry discharge in Toxoplasma.

Apicomplexan parasites discharge specialized organelles called rhoptries upon host cell contact to mediate invasion. The events that drive rhoptry discharge are poorly understood, yet essential to sustain the apicomplexan parasitic life cycle. Rhoptry discharge appears to depend on proteins secreted from another set of organelles called micronemes, which vary in function from allowing host cell binding to facilitation of gliding motility. Here we examine the function of the microneme protein CLAMP, which we previously found to be necessary for Toxoplasma gondii host cell invasion, and demonstrate its essential role in rhoptry discharge. CLAMP forms a distinct complex with two other microneme proteins, the invasion-associated SPATR, and a previously uncharacterized protein we name CLAMP-linked invasion protein (CLIP). CLAMP deficiency does not impact parasite adhesion or microneme protein secretion; however, knockdown of any member of the CLAMP complex affects rhoptry discharge. Phylogenetic analysis suggests orthologs of the essential complex components, CLAMP and CLIP, are ubiquitous across apicomplexans. SPATR appears to act as an accessory factor in Toxoplasma, but despite incomplete conservation is also essential for invasion during Plasmodium falciparum blood stages. Together, our results reveal a new protein complex that mediates rhoptry discharge following host-cell contact.

Antiviral type III CRISPR signalling via conjugation of ATP and SAM

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine–aspartate (HD) nuclease domain3–5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6–9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an ‘off switch’ analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.