Mature Sequence Research Articles

A short peptide exerts neuroprotective effects on cerebral ischemia–reperfusion injury by reducing inflammation via the miR-6328/IKKβ/NF-κB axis

BackgroundDespite considerable efforts, ischemic stroke (IS) remains a challenging clinical problem. Therefore, the discovery of effective therapeutic and targeted drugs based on the underlying molecular mechanism is crucial for effective IS treatment.MethodsA cDNA-encoding peptide was cloned from RNA extracted from Rana limnocharis skin, and the mature amino acid sequence was predicted and synthesized. Hemolysis and acute toxicity of the peptide were tested. Furthermore, its neuroprotective properties were evaluated using a middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats and an oxygen–glucose deprivation/reperfusion (OGD/R) model in neuron-like PC12 cells. The underlying molecular mechanisms were explored using microRNA (miRNA) sequencing, quantitative real-time polymerase chain reaction, dual-luciferase reporter gene assay, and western blotting.ResultsA new peptide (NP1) with an amino acid sequence of ‘FLPAAICLVIKTC’ was identified. NP1 showed no obvious toxicities in vivo and in vitro and was able to cross the blood–brain barrier. Intraperitoneal administration of NP1 (10 nmol/kg) effectively reduced the volume of cerebral infarction and relieved neurological dysfunction in MCAO/R model rats. Moreover, NP1 significantly alleviated the decrease in viability and increase in apoptosis of neuron-like PC12 cells induced by OGD/R. NP1 effectively suppressed inflammation by reducing interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels in vitro and in vivo. Furthermore, NP1 up-regulated the expression of miR-6328, which, in turn, down-regulated kappa B kinase β (IKKβ). IKKβ reduced the phosphorylation of nuclear factor-kappa B p65 (NF-κB p65) and inhibitor of NF-κB (I-κB), thereby inhibiting activation of the NF-κB pathway.ConclusionsThe newly discovered non-toxic peptide NP1 (‘FLPAAICLVIKTC’) exerted neuroprotective effects on cerebral ischemia–reperfusion injury by reducing inflammation via the miR-6328/IKKβ/NF-κB axis. Our findings not only provide an exogenous peptide drug candidate and endogenous small nucleic acid drug candidate but also a new drug target for the treatment of IS. This study highlights the importance of peptides in the development of new drugs, elucidation of pathological mechanisms, and discovery of new drug targets.

A novel approach to analyze the association characteristics between post-spliced introns and their corresponding mRNA.

Studies have shown that post-spliced introns promote cell survival when nutrients are scarce, and intron loss/gain can influence many stages of mRNA metabolism. However, few approaches are currently available to study the correlation between intron sequences and their corresponding mature mRNA sequences. Here, based on the results of the improved Smith-Waterman local alignment-based algorithm method (SW method) and binding free energy weighted local alignment algorithm method (BFE method), the optimal matched segments between introns and their corresponding mature mRNAs in Caenorhabditis elegans (C.elegans) and their relative matching frequency (RF) distributions were obtained. The results showed that although the distributions of relative matching frequencies on mRNAs obtained by the BFE method were similar to the SW method, the interaction intensity in 5'and 3'untranslated regions (UTRs) regions was weaker than the SW method. The RF distributions in the exon-exon junction regions were comparable, the effects of long and short introns on mRNA and on the five functional sites with BFE method were similar to the SW method. However, the interaction intensity in 5'and 3'UTR regions with BFE method was weaker than with SW method. Although the matching rate and length distribution shape of the optimal matched fragment were consistent with the SW method, an increase in length was observed. The matching rates and the length of the optimal matched fragments were mainly in the range of 60%-80% and 20-30bp, respectively. Although we found that there were still matching preferences in the 5'and 3'UTR regions of the mRNAs with BFE, the matching intensities were significantly lower than the matching intensities between introns and their corresponding mRNAs with SW method. Overall, our findings suggest that the interaction between introns and mRNAs results from synergism among different types of sequences during the evolutionary process.

The Growth Progression in Linear Body Measures among the Jeunkuruba, Kadukuruba and Yerava Tribal Boys of Karnataka

The present study has been carried out to analyze population-wise growth progression in linear body measures among the Jenukuruba, Kadukuruba, and Yerava (boys aged 10- 16 years) Scheduled tribes of Karnataka. A total of 475 subjects were measured cross-sectionally for 12 linear body measurements following the standard measurement techniques. Analysis results of the present study exhibit the prevalence of Cephalo-Caudal and Caudo-Cephalic sequence of maturation within the components of extremities among Jenukuruba and Kadukuruba boys, but in the case of Yerava boys shows only Cephalo-Caudal sequence of maturation throughout the age range studied. When taking account of within the subsegments of the upper extremities Jenukuruba boys at all ages show only CaudoCephalic sequence of maturation, where as Kadukuruba boys show Caudo-Cephalic as well as mixed gradients of maturational sequence, but in case of Yerava boys shows Cephalo-Caudal sequence besides mixed gradients of maturational sequence. And in the sub-segments of the lower extremities, Jenukuruba shows Caudo-Cephalic the sequence of growth, where as among Kadukuruba and Yerava exhibit Caudo-Cephalic as well as mixed gradients of maturational sequence throughout the age range studied.

The effects of short neuropeptide F on ovarian maturation and spawning in female giant freshwater prawn, Macrobrachium rosenbergii, and associated regulatory mechanisms

In the present study, we investigated the effects of two short neuropeptide F (Mro-sNPF) mature sequences, namely APALRLRFamide and DRTPALRLRFamide, on ovarian maturation and spawning in Macrobrachium rosenbergii females. At doses of 10−5 and 10−6 mol/prawn, both Mro-sNPF peptides significantly shortened the ovarian developmental period (P < 0.05), but enhanced the gonadosomatic index (GSI), oocyte diameter (OD), and cell proliferation, compared with control values (P < 0.05). However, at the lowest dose (10−7 mol/prawn), neither peptide significantly affected ovarian maturation, GSI, nor OD compared with the control values. Females treated with the two peptides at the dose of 10−5 or 10−6 mol/prawn attained mature oocyte stages faster than did control females. Treatment with both peptides significantly elevated hemolymph vitellogenin concentrations and upregulated MrEcR transcripts, compared with the control treatment (P < 0.05). Finally, both Mro-sNPFs induced spawning by shortening its duration by half (∼1.5 days) compared with the control duration (∼3.1 days) (P < 0.05). The number of eggs per spawn was approximately 15.4 × 104 eggs in the experimental group and in the control group (∼15.35 × 104 eggs), albeit without significance. Therefore, among the treatment groups, using APALRLRFamide at 10−5 mol/prawn showed promising effects on the induction of ovarian maturation and spawning. Taken together, Mro-sNPF peptides can promote ovarian development and spawning, indicating their potential application to stimulate gamete and larval production in this important crustacean aquaculture species.

In Silico Identification of Cassava Genome-Encoded MicroRNAs with Predicted Potential for Targeting the ICMV-Kerala Begomoviral Pathogen of Cassava.

Cassava mosaic disease (CMD) is caused by several divergent species belonging to the genus Begomovirus (Geminiviridae) transmitted by the whitefly Bemisia tabaci cryptic species group. In India and other parts of Asia, the Indian cassava mosaic virus-Kerala (ICMV-Ker) is an emergent begomovirus of cassava causing damage that results in reduced yield loss and tuber quality. Double-stranded RNA-mediated interference (RNAi) is an evolutionary conserved mechanism in eukaryotes and highly effective, innate defense system to inhibit plant viral replication and/or translation. The objective of this study was to identify and characterize cassava genome-encoded microRNAs (mes-miRNA) that are predicted to target ICMV-Ker ssDNA-encoded mRNAs, based on four in silico algorithms: miRanda, RNA22, Tapirhybrid, and psRNA. The goal is to deploy the predicted miRNAs to trigger RNAi and develop cassava plants with resistance to ICMV-Ker. Experimentally validated mature cassava miRNA sequences (n = 175) were downloaded from the miRBase biological database and aligned with the ICMV-Ker genome. The miRNAs were evaluated for base-pairing with the cassava miRNA seed regions and to complementary binding sites within target viral mRNAs. Among the 175 locus-derived mes-miRNAs evaluated, one cassava miRNA homolog, mes-miR1446a, was identified to have a predicted miRNA target binding site, at position 2053 of the ICMV-Ker genome. To predict whether the cassava miRNA might bind predicted ICMV-Ker mRNA target(s) that could disrupt viral infection of cassava plants, a cassava locus-derived miRNA-mRNA regulatory network was constructed using Circos software. The in silico-predicted cassava locus-derived mes-miRNA-mRNA network corroborated interactions between cassava mature miRNAs and the ICMV-Ker genome that warrant in vivo analysis, which could lead to the development of ICMV-Ker resistant cassava plants.

A conserved SNP variation in the pre-miR396c flanking region in Oryza sativa indica landraces correlates with mature miRNA abundance

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the ‘A10234287911’ allele may promote better processing vis-à-vis the structure conferred by ‘G10234287911’ allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

Functional Role of Vitelline Glands and Mehlis Gland in the Process of Resistant Egg Shell Formation in Trematodes

The study of the functional morphology of the reproductive system of trematodes, as well as its individual parts, provides an opportunity to solve an important problem that plays a huge role in theoretical and practical terms as the directed regulation of the fecundity of endoparasites. The article is devoted to the study of the functional role of vitelline glands and Mehlis glands in the process of formation of resistant egg shells on the example of the Parastrigea robusta trematode. The functional mechanisms are revealed on the basis of histomorphological and ultrastructural studies, underlying the provision of trematode eggshell formation processes and the role of each of the mentioned departments. The sequence of appearance and maturation of shell material in the vitelline follicles during vitellogenesis has been determined. The role of the Mehlis gland is substantiated on the basis of electron microscopy methods and their functional morphological analysis of ultrastructural and micromorphological data. The obtained data reveals information on the structural and functional unity of the mechanisms underlying the formation of a resistant trematode eggshell, the role of vitelline glands, and Mehlis gland in providing a "large number of eggs". According to the results of histological and electron microscopic studies of vitelline glands and Mehlis gland of the Parastrigea robusta trematode, a general scheme of the formation of trematode eggshell was formed. As a result, the obtained knowledge on the peculiarities of the structure and maturation of the vitelline glands and Mehlis gland of Parastrigea robusta will significantly complement the knowledge on the structural features of the female reproductive system of trematodes and the fertility of endoparasites. The results of the work can be used for further research in the selection of anthelmintic agents that affect vitelline glands and Mehlis glands, which will lead to directed regulation of the fertility level of endoparasites.

Potential implications of the glycosylation patterns in collagen α1(I) and α2(I) chains for fibril assembly and growth

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

Identification of potential proteins translated from circular RNA splice variants

Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.

The Human Pre-miRNA Distance Distribution for Exploring Disease Association.

MicroRNAs (miRNAs), playing an important role in cell differentiation, development, gene regulation, and apoptosis, have attracted much attention in recent years. miRNAs were shown to be involved in the mechanisms of various diseases, and certainly, they can be employed as useful disease biomarkers. The phylogenetic tree analysis of miRNA biomarkers is a useful tool to investigate the association between various diseases as well as the association between viruses and disease. In addition to the phylogenetic tree analysis, a more advanced study is to use the miRNA distance distribution to evaluate the similarity of the miRNA biomarkers. The mature miRNA distance distribution based on mature miRNA sequences has been derived. The averages of the pairwise distances of miRNA biomarkers for several associated diseases were shown to be smaller than the overall mean of all miRNAs, which indicates the high similarity of miRNA biomarkers for associated diseases. In addition to the mature miRNA, the precursor miRNA (pre-miRNA) may be more useful to explore the similarity of miRNAs because the mature miRNA duplex is released from the pre-miRNA. Therefore, in this study, the distance distributions based on human pre-miRNA stem-loop sequences were derived. The 1917 human miRNA stem-loop sequences in the miRBase dataset were used to derive the pre-miRNA distance distribution, and this is the first study to provide the distance distribution based on the human pre-miRNAs. The similarity of miRNA biomarkers for several associated diseases or vaccines was examined using the derived distribution, and the results show that the similarity of pre-miRNA biomarkers may be a feasible way to help explore the disease association.

Improved Coverage of the N-Terminome by Combining ChaFRADIC with Alternative Proteases.

Many proteolytic cleavage events cannot be covered with conventional trypsin-based N-terminomics workflows. These typically involve the derivatization of protein N-termini and Lys residues as an initial step, such that trypsin will cleave C-terminal of arginine but not lysine residues (ArgC-like cleavage). From 20,422 reviewed human protein sequences in Uniprot, 3597 have known N-terminal signal peptides. An in silico ArgC-like digestion of the corresponding 3597 mature protein sequences reveals that-even for these well-known and well-studied proteolytic events-trypsin-based N-terminomics workflows may miss up to 50% of signaling cleavage events as the corresponding neo-N-terminal peptides will have an unfavorable length of <7 (875 peptides) or >30 (911 peptides) amino acids. In this chapter, we provide a protocol that can be applied to all kinds of samples to improve access to this "inaccessible" N-terminome, by making use of the alternative, broad-specificity protease subtilisin for fast and reproducible digestion of proteins.

Cone snail species off the Brazilian coast and their venoms: a review and update.

The genus Conus includes over 900 species of marine invertebrates known as cone snails, whose venoms are among the most powerful described so far. This potency is mainly due to the concerted action of hundreds of small bioactive peptides named conopeptides, which target different ion channels and membrane receptors and thus interfere with crucial physiological processes. By swiftly harpooning and injecting their prey and predators with such deadly cocktails, the slow-moving cone snails guarantee their survival in the harsh, competitive marine environment. Each cone snail species produces a unique venom, as the mature sequences of conopeptides from the venoms of different species share very little identity. This biochemical diversity, added to the numerous species and conopeptides contained in their venoms, results in an immense biotechnological and therapeutic potential, still largely unexplored. That is especially true regarding the bioprospection of the venoms of cone snail species found off the Brazilian coast - a region widely known for its biodiversity. Of the 31 species described in this region so far, only four - Conus cancellatus, Conus regius, Conus villepinii, and Conus ermineus - have had their venoms partially characterized, and, although many bioactive molecules have been identified, only a few have been actually isolated and studied. In addition to providing an overview on all the cone snail species found off the Brazilian coast to date, this review compiles the information on the structural and pharmacological features of conopeptides and other molecules identified in the venoms of the four aforementioned species, paving the way for future studies.

Comprehensive, integrative genomic analysis of microRNA expression profiles in different tissues of two wheat cultivars with different traits.

Wheat is one of the most important food sources on Earth. MicroRNAs (miRNA) play important roles in wheat productivity. To identify wheat miRNAs, we constructed and sequenced sRNA libraries from leaves and roots of two wheat cultivars (RAC875 and Kukri) with many different traits. Given that available miRNA wheat complement in the plant-specific database PmiREN ( https://pmiren.com ) does not include root tissues and root-associated miRNAs might thus be missing, we performed first the prediction of novel miRNAs using the sRNAbench tool. We found a total of 150 putatively novel miRNA genes with expression of both arms from 289 unique mature sequences and nearly 30% of all miRNA reads in roots corresponded to novel miRNAs. In contrast, this figure in leaves dropped to under 3%, confirming the undersampling of roots in the complement of known miRNAs. By using 120 publicly available wheat datasets, 598 Zea mays small RNA libraries, 64 plant species genomes, wheat degradome library, and functional enrichment analysis, a subset of novel miRNAs were confirmed as bona-fide miRNAs. Of the total 605 miRNAs identified in this study inclusive of 316 known miRNAs, 528 miRNAs were shared by both cultivars, 429 miRNAs were shared by both root tissues and 329 miRNAs were shared by both leaf tissues. In addition, 32 miRNAs were specific to Kukri while 45 miRNAs were specific to RAC875. These miRNAs had diverse functions, such as regulation of gene transcription, protein translation, energy metabolism, and cell cycle progression. Our data provide a genome-wide miRNA expression profile in these two wheat cultivars and help functional studies of wheat genomics.

Immunometabolic involvement of hepcidin genes in iron homeostasis, storage, and regulation in gilthead seabream (Sparus aurata)

Iron is essential for all living things, especially marine organisms, due to its low availability in the marine environment. Iron regulation is key in all vertebrates and is controlled by hepcidin–ferroportin. To improve the knowledge of iron homeostasis in fish, an iron overload was induced in gilthead seabream (Sparus aurata), which was chosen as a study species because of its high interest in Mediterranean aquaculture. The amount of iron in different tissues was measured to determine its biodistribution and/or bioaccumulation. Since the liver is directly involved in iron metabolism, the morphological changes induced in this organ as a consequence of the iron increase were studied. The bactericidal activity of fish skin mucus was also determined, observing that it decreased in fish with high iron levels compared to control fish. In addition, to better understand iron regulation, the gene expression of hepcidin, ferroportin, transferrin, and ferritin was evaluated in the head kidney (the main hematopoietic organ in this species) and in the liver. Special interest was taken in the study of the multiple copies of the hamp2 gene present in the gilthead seabream genome. Bioinformatic analysis of the protein sequences derived from these hepcidin genes allowed us to determine the presence of one type I hepcidin and 12 type II hepcidins, all of them with antimicrobial potential. This number of mature hepcidin sequences found in gilthead seabream is the highest within Eupercaria described to date. All the results obtained indicate that the modulation of iron in seabream seems to be much more complicated than in other vertebrates, probably due to the possible involvement of the different hepcidins as mediators between iron metabolism and host immune response.

Differential expression of selected microRNAs in cryopreserved sperm of Bos taurus sires with diverse fertility status

Our study aimed to evaluate the expression of four selected microRNAs (miRNA) in commercially produced cryopreserved semen of bulls with high vs. low fertility. The annual fertility index (FI) of 151 sires with ≥300 first services was recorded between November 2018 and November 2019. Sires lying at the extremes of the FI distribution (outside the range of mean FI±SD) were classified as low- (LF; n=13 bulls) or high-fertile (HF; n=7 bulls), with a non-return rate of 61.4±6.9% and 72.6±1.5%, respectively. For each bull, total RNA was isolated using a modified TRIzolTM-based protocol from two cryopreserved semen batches produced with 1-mo interval (40 sperm samples totally). Following preparation of first-strand cDNA, relative quantification of the mature sequences of hsa-miR-30d-5p, hsa-miR-92a-3p, hsa-miR-132-3p and hsa-miR-204-5p was carried out by real-time PCR. The four miRNAs were selected based on our previous studies showing their constant detection in sperm of sires with diverse age, breed and fertility. The quantification cycles (Cq) of each miRNA was normalized using hsa-miR-30d-5p and hsa-miR-132-3p combined as reference genes. The relative expression of the four miRNAs was quantified using the ΔCq and 2- ΔΔCq methods (Livak and Schmittgen, Methods, 25:402-8, 2001). In HF samples, the fluorescent signal of miR-92a, miR-204, miR-132 and miR-30d was detected after Cq 22.34±1.10, 26.19±5.58, 26.69±2.20 and 28.05±3.32, respectively; the Cq values recorded in the LF group were 22.72±1.30, 23.23±1.92, 26.97±2.50 and 27.43±2.81, respectively. Expression of miR-204 was approximately 18-fold lower in the HF than in the LF group; this difference was significant only for miR-204 when applying a mixed-effects linear model with fertility group as fixed effect and the semen batch nested within sire as random effect (b=18.36±6.48, adjusted P=0.014). The expression of miR-92a, miR-132 and miR-30d did not differ between the two groups (adjusted P>0.05 in all cases). Concluding, our study showed the differential expression of the mature sequence of hsa-miR-204, a miRNA involved in chromatin condensation during spermiogenesis, in cryopreserved sperm of sires with diverse fertility status.

Venomics survey of six myrmicine ants provides insights into the molecular and structural diversity of their peptide toxins

Among ants, Myrmicinae represents the most speciose subfamily. The venom composition previously described for these social insects is extremely variable, with alkaloids predominant in some genera while, conversely, proteomics studies have revealed that some myrmicine ant venoms are peptide-rich. Using integrated transcriptomic and proteomic approaches, we characterized the venom peptidomes of six ants belonging to the different tribes of Myrmicinae. We identified a total of 79 myrmicitoxins precursors which can be classified into 38 peptide families according to their mature sequences. Myrmicine ant venom peptidomes showed heterogeneous compositions, with linear and disulfide-bonded monomers as well as dimeric toxins. Several peptide families were exclusive to a single venom whereas some were retrieved in multiple species. A hierarchical clustering analysis of precursor signal sequences led us to divide the myrmicitoxins precursors into eight families, including some that have already been described in other aculeate hymenoptera such as secapin-like peptides and voltage-gated sodium channel (NaV) toxins. Evolutionary and structural analyses of two representatives of these families highlighted variation and conserved patterns that might be crucial to explain myrmicine venom peptide functional adaptations to biological targets.

KRGG1 function in RNA editing in Trypanosoma brucei.

Mitochondrial gene expression in trypanosomes requires numerous multiprotein complexes that are unique to kinetoplastids. Among these, the most well characterized are RNA editing catalytic complexes (RECCs) that catalyze the guide RNA (gRNA)-specified insertion and deletion of uridines during mitochondrial mRNA maturation. This post-transcriptional resequencing of mitochondrial mRNAs can be extensive, involving dozens of different gRNAs and hundreds of editing sites with most of the mature mRNA sequences resulting from the editing process. Proper coordination of the editing with the cognate gRNAs is attributed to RNA editing substrate-binding complexes (RESCs), which are also required for RNA editing. Although the precise mechanism of RESC function is less well understood, their affinity for binding both editing substrates and products suggests that these complexes may provide a scaffold for RECC catalytic processing. KRGG1 has been shown to bind RNAs, and although affinity purification co-isolates RESC complexes, its role in RNA editing remains uncertain. We show here that KRGG1 is essential in BF parasites and required for normal editing. KRGG1 repression results in reduced amounts of edited A6 mRNA and increased amounts of edited ND8 mRNA. Sequence and structure analysis of KRGG1 identified a region of homology with RESC6, and both proteins have predicted tandem helical repeats that resemble ARM/HEAT motifs. The ARM/HEAT-like region is critical for function as exclusive expression of mutated KRGG1 results in growth inhibition and disruption of KRGG1 association with RESCs. These results indicate that KRGG1 is critical for RNA editing and its specific function is associated with RESC activity.

High conopeptide diversity in Conus striatus: Revealed by integration of two transcriptome sequencing platforms

Marine cone snail venoms represent a vast library of bioactive peptides with proven potential as research tools, drug leads, and therapeutics. In this study, a transcriptome library of four different organs, namely radular sheath, venom duct, venom gland, and salivary gland, from piscivorous Conus striatus was constructed and sequenced using both Illumina next-generation sequencing (NGS) and PacBio third-generation sequencing (TGS) technologies. A total of 428 conotoxin precursor peptides were retrieved from these transcriptome data, of which 413 conotoxin sequences assigned to 13 gene superfamilies, and 15 conotoxin sequences were classified as unassigned families. It is worth noting that there were significant differences in the diversity of conotoxins identified from the NGS and TGS data: 82 conotoxins were identified from the NGS datasets while 366 conotoxins from the TGS datasets. Interestingly, we found point mutations in the signal peptide sequences of some conotoxins with the same mature sequence. Therefore, TGS broke the traditional view of the conservation of conotoxin signal peptides and the variability of mature peptides obtained by NGS technology. These results shed light on the integrated NGS and TGS technologies to mine diverse conotoxins in Conus species, which will greatly contribute to the discovery of novel conotoxins and the development of new marine drugs.

Moving the needle: Employing deep reinforcement learning to push the boundaries of coarse-grained vaccine models.

Highly mutable infectious disease pathogens (hm-IDPs) such as HIV and influenza evolve faster than the human immune system can contain them, allowing them to circumvent traditional vaccination approaches and causing over one million deaths annually. Agent-based models can be used to simulate the complex interactions that occur between immune cells and hm-IDP-like proteins (antigens) during affinity maturation-the process by which antibodies evolve. Compared to existing experimental approaches, agent-based models offer a safe, low-cost, and rapid route to study the immune response to vaccines spanning a wide range of design variables. However, the highly stochastic nature of affinity maturation and vast sequence space of hm-IDPs render brute force searches intractable for exploring all pertinent vaccine design variables and the subset of immunization protocols encompassed therein. To address this challenge, we employed deep reinforcement learning to drive a recently developed agent-based model of affinity maturation to focus sampling on immunization protocols with greater potential to improve the chosen metrics of protection, namely the broadly neutralizing antibody (bnAb) titers or fraction of bnAbs produced. Using this approach, we were able to coarse-grain a wide range of vaccine design variables and explore the relevant design space. Our work offers new testable insights into how vaccines should be formulated to maximize protective immune responses to hm-IDPs and how they can be minimally tailored to account for major sources of heterogeneity in human immune responses and various socioeconomic factors. Our results indicate that the first 3 to 5 immunizations, depending on the metric of protection, should be specially tailored to achieve a robust protective immune response, but that beyond this point further immunizations require only subtle changes in formulation to sustain a durable bnAb response.

Generation of myostatin gene-edited blotched snakehead (Channa maculata) using CRISPR/Cas9 system

Blotched snakehead (Channa maculata) is one of the most important freshwater fish in China. Genome editing is of great potential in generating new snakehead strains with valuable economic traits without integration of exogenous genes. Myostatin (Mstn) inhibits the development and growth of skeletal muscle in vertebrates, including teleosts. Mstn mutations induced by genome editing can stimulate muscle growth and development, therefore Mstn is an appropriate candidate to breed the fast-growing snakehead strain. For this study, one orthologous Mstn gene was characterized in C. maculata, designated as CmMstn, which possessed the relatively conserved gene and protein structure. CmMstn had the highest expression level and sexual dimorphism in muscles. Two CRISPR/Cas9 target sites were designed to generate knockout mutants within the mature mRNA sequence of CmMstn located in Exon I and Exon II. 1 nL mixture solution of 200 pg gRNA and 300 pg Cas9 protein was directly microinjected into each snakehead embryonic yolk at 1⁓4 cell stage. Mstn was efficiently edited by CRISPR/Cas9 system in shakehead, and all designed gRNAs effectively worked at their target sites with high mutagenesis efficiencies, from 40.0% to 53.3%. The majority of mutations were frame-shift and resulted in premature termination of transcription and disruption of the molecular function of CmMstn protein. Furthermore, we obtained four founders (F0) of snakehead with the mutated Mstn gene in their primordial germ cells, and nineteen F1 offspring of the F0 mutants mated with wild-type individuals were displayed to carry Mstn null alleles. It is the first application of genome editing technology in blotched snakehead, and CRISPR/Cas9 system successfully edit the primordial germ cells. Our findings will accelerate our understanding of the role of Mstn gene in snakehead growth, and speed up the cultivation of the fast-growing snakehead strain.