Mutation profiling through whole genome sequencing of electron beam-induced black gram (Vigna mungo L. Hepper) mutant.

PURPOSE: Black gram (Vigna mungo [L.] Hepper) is an important annual legume with great economic, nutritional and ecological significance. Novel variations through induced mutagenesis can accelerate narrow genetic base-impeded black gram improvement. This is a first study on characterization of genome-wide mutation spectrum induced by electron beam (EB). MATERIALS AND METHODS: Black gram genotype 'Pant U-31' was irradiated with 400 Gy EB generated in a 10 MeV LINAC. A stable mutant PM-32 (M6) was re-sequenced by combining Illumina (BIOO Scientific, Inc., Austin, TX) and Nanopore Technologies (Oxford, UK). Variants were predicted in reference to the available whole genome scaffold level draft assembly of parent 'Pant U-31'. RESULTS: Genome analysis predicted a total of 76,893 genes of which 58,517 were annotated. The identified variants totaling 728,161, largely comprised (91.56%) of single base substitutions (SBSs) with a transition (Ti) to transversion (Tv) ratio of 1.95. Of the indels constituting 8.44% of total induced variants, insertions accounted for 4.29%, with preponderance of multiple bases (53.63%) and 2-5 bp insertions as the major class (33.71%). Multiple-base deletions (2-5 bases) formed the bulk (31.14%) of the total deletions. The genic variants (2438) with estimated high and moderate effects were located within 1271 predicted genes. A higher number of mutations were observed on chromosomes Vm1 (588) and Vm3 (428) with the highest frequency on chromosome Vm3 (every 0.07 Mb). CONCLUSIONS: Our study reiterated the mutagenic utility of EB for inducing SBSs and small indels genome-wide. The knowledge gained from SNP-level profiling of EB-induced mutations can expedite comparative mutation breeding studies in legumes.

The roles of Growth Hormone dependent JAK-STAT5 and Lyn kinase signalling in determining lifespan and cancer incidence.

In rodents, loss of GH or its receptor is associated with extended lifespan. We aimed to determine the signalling process resulting in this longevity using GH receptor (GHR) mutant mice with key signalling pathways deleted and correlate this with cancer incidence and expression of genes associated with longevity. GHR uses both canonical JAK2-STAT signalling as well as signalling via LYN-ERK1/2 pathway. We utilised C57BL/6 mice with loss of key receptor tyrosines and truncation resulting in (1) loss of most STAT5 response to GH or (2) total inability to generate STAT5 to GH or (3) loss of Box1 to prevent activation of JAK2 but not LYN kinase or (4) total knockout of the receptor. For each mutant we analysed lifespan, histopathology to determine likely cause of death, and hepatic gene and protein expression. The extended lifespan is evident in the Box1 mutant males (retains Lyn activation) have median lifespan of 1016 days compared to 890 days for the Ghr-/- males. In the females, GhrBox1-/- mice have a median lifespan of 970 days compared to 911 days for the knockout females. Sexually dimorphic GHR-STAT5 is repressive for longevity, since its removal results in a median lifespan of 1003 days in females compared to 734 days for wild type females. Numerous transcripts related to insulin sensitivity, oxidative stress response and mitochondrial function are regulated by GHR-STAT5, however LYN responsive genes involve DNA repair, cell cycle control, and anti-inflammatory response. There appears to be a yin-yang relationship between JAK2 and LYN that determines lifespan.

Elucidating the downstream pathways triggered by H2S signaling in Arabidopsis thaliana under drought stress via transcriptome analysis.

Hydrogen sulfide (H2S) is a crucial signaling molecule in plants. Recent studies have shown that H2S plays an equally important role as nitric oxide (NO) and hydrogen peroxide (H2O2) in plant signaling. Previous studies have demonstrated the involvement of H2S in regulating drought and other stressful environmental conditions, but the exact downstream molecular mechanisms activated by the H2S signaling molecule remain unclear. In this study, we conducted a comprehensive genome-wide transcriptomic analysis of both wild type (WT) and double mutant (lcd/des1). Arabidopsis thaliana plants were exposed to 40% polyethylene glycol (PEG) to induce drought stress and 20 µM sodium hydrosulfide (NaHS). The resulting transcriptome data were analyzed for differentially significant genes and their statistical enrichments in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The results indicated significant upregulation of genes related to photosynthesis, carbon fixation, plant secondary metabolite biosynthesis, inositol and phosphatidylinositol signaling pathways, and stress-responsive pathways in mutant plants under drought stress. Mutant plants with impaired H2S signaling mechanisms displayed greater susceptibility to drought stress compared to wild-type plants. In summary, all findings highlight the pivotal role of H2S signaling in stimulating other drought-responsive signaling pathways.

The dimer of human SVCT1 is key for transport function.

Humans and other primates lack the ability to synthesize the essential nutrient, Vitamin C, which is derived exclusively from the diet. Crucial for effective vitamin C uptake are the Na+ dependent Vitamin C transporters, SVCT1 and SVCT2, members of the nucleobase ascorbate transporter (NAT) family. SVCT1 and 2 actively transport the reduced form of Vitamin C, ascorbic acid, into key tissues. The recent structure of the mouse SVCT1 revealed the molecular basis of substrate binding and that, like the other structurally characterised members of the NAT family, it exists as a closely associated dimer. SVCT1 is likely to function via the elevator mechanism with the core domain of each protomer able to bind substrate and move through the membrane carrying the substrate across the membrane. Here we explored the function of a range of variants of the human SVCT1, revealing a range of residues involved in substrate selection and binding, and confirming the importance of the C-terminus in membrane localisation. Furthermore, using a dominant negative mutant we show that the dimer is essential for transport function, as previously seen in the fungal homologue, UapA. In addition, we show that a localisation deficient C-terminal truncation of SVCT1 blocks correct localisation of co-expressed, associated wildtype SVCT1. These results clearly show the importance of the dimer in both correct SVCT1 trafficking and transport activity.

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research.

This report describes the development and characterization of a comprehensive collection of CHO cell glycosylation mutants with significant potential for advancing glycobiology and biotechnology. EPO-Fc and trastuzumab, two model molecules, were produced using these mutants to assess the effects of mutated glycogenes, and LC-MS/MS analysis was employed to quantitatively analyse their N-glycans. EPO-Fc exhibited exclusively homogeneous Man9 glycans only when nearly all α-mannosidases in the genome were inactivated, except lysosomal MAN2B1. Some mutants lacking GnT-I activity produce mostly Man5 N-glycans, while their O-glycan and glycolipid profiles can differ due to other mutations in the cell. GnT-II deficiency prevents GnT-V from adding GlcNAc to the core N-glycan, resulting in branches attaching solely to the α1,3-linked mannose, leaving the α1,6-linked mannose free. The mutant-produced antibody's single-branched glycan contains more sialic acid than the dual-branched glycans produced in CHO-K1 cells. Trastuzumab produced in these mutants provided insights into how Fc N-glycans impact the antibody's interaction with FcγR1 and FcγR2a, FcγR3a, and their influence on antibody-dependent cellular cytotoxicity (ADCC). In the study of Fc glycans in Fc-FcγR1 and FcγR2a interactions, we observed a consistent glycan-related impact on binding to both receptors, indicating a common interaction mechanism between Fc glycans and both FcγRI and FcγRIIa. CHO mutants produced trimeric gp120 demonstrated distinct reactivity with multiple broadly neutralizing anti-HIV antibodies, confirming the involvement of gp120 glycans in interactions with specific broadly neutralizing antibodies. Finally, one of the mutants produced human ß-glucocerebrosidase with uniform Man5 N-glycans, showcasing its potential for glycoengineered production and enhancement in therapeutic efficacy.

Structural investigation, computational analysis, and theoretical cryoprotectant approach of antifreeze protein type IV mutants.

Antifreeze proteins (AFPs) have unique features to sustain life in sub-zero environments due to ice recrystallization inhibition (IRI) and thermal hysteresis (TH). AFPs are in demand as agents in cryopreservation, but some antifreeze proteins have low levels of activity. This research aims to improve the cryopreservation activity of an AFPIV. In this in silico study, the helical peptide afp1m from an Antarctic yeast AFP was modeled into a sculpin AFPIV, to replace each of its four α-helices in turn, using various computational tools. Additionally, a new linker between the first two helices of AFPIV was designed, based on a flounder AFPI, to boost the ice interaction activity of the mutants. Bioinformatics tools such as ExPASy Prot-Param, Pep-Wheel, SOPMA, GOR IV, Swiss-Model, Phyre2, MODFOLD, MolPropity, and ProQ were used to validate and analyze the structural and functional properties of the model proteins. Furthermore, to evaluate the AFP/ice interaction, molecular dynamics (MD) simulations were executed for 20, 100, and 500 ns at various temperatures using GROMACS software. The primary, secondary, and 3D modeling analysis showed the best model for a redesigned antifreeze protein (AFP1mb, with afp1m in place of the fourth AFPIV helix) with a QMEAN (Swiss-Model) Z score value of 0.36, a confidence of 99.5%, a coverage score of 22%, and a p value of 0.01. The results of the MD simulations illustrated that AFP1mb had more rigidity and better ice interactions as a potential cryoprotectant than the other models; it also displayed enhanced activity in limiting ice growth at different temperatures.

Microbiota responses to mutations affecting NO homeostasis in Arabidopsis thaliana.

Interactions between plants and microorganisms are pivotal for plant growth and productivity. Several plant molecular mechanisms that shape these microbial communities have been identified. However, the importance of nitric oxide (NO) produced by plants for the associated microbiota remains elusive. Using Arabidopsis thaliana isogenic mutants overproducing NO (nox1, NO overexpression) or down-producing NO (i.e. nia1nia2 impaired in the expression of both nitrate reductases NR1/NIA1 and NR2/NIA2; the 35s::GSNOR1 line overexpressing nitrosoglutathione reductase (GSNOR) and 35s::AHB1 line overexpressing haemoglobin 1 (AHB1)), we investigated how altered NO homeostasis affects microbial communities in the rhizosphere and in the roots, soil microbial activity and soil metabolites. We show that the rhizosphere microbiome was affected by the mutant genotypes, with the nox1 and nia1nia2 mutants causing opposite shifts in bacterial and fungal communities compared with the wild-type (WT) Col-0 in the rhizosphere and roots, respectively. These mutants also exhibited distinctive soil metabolite profiles than those from the other genotypes while soil microbial activity did not differ between the mutants and the WT Col-0. Our findings support our hypothesis that changes in NO production by plants can influence the plant microbiome composition with differential effects between fungal and bacterial communities.

Screening and Validation of Leaf Width-Related Genes in Inbred Maize Lines.

Leaf width is a key determinant of planting density and photosynthetic efficiency. In an effort to determine which genes regulate maize plant leaf width, we performed a genome-wide association study (GWAS) of 1.49 × 106 single nucleotide polymorphisms (SNPs) in 80 sequenced backbone inbred maize lines in Jilin Province, China, based upon phenotypic leaf width data from two years. In total, 14 SNPs were identified as being significantly related to leaf width (p < 0.000001), with these SNPs being located on chromosomes 1, 2, 3, 5, 6, 7, 8, and 9. A total of five candidate genes were identified within a mean linkage disequilibrium (LD) distance of 9.7 kb, with a significant SNP being identified within the Zm00001d044327 candidate gene. RNA was then isolated from 12 different inbred maize lines from this GWAS study cohort and was used to conduct qPCR analyses which revealed significant differences in Zm00001d044327 expression among strains exhibiting significant differences in leaf width. Based on an assessment of EMS mutant lines harboring a conserved amino acid stop mutation and two non-synonymous mutations in Zm00001d044327 that exhibited a narrow leaf width, these data suggested that Zm00001d044327 is a key regulator of maize leaf width.

Photonanozyme-Kras-ribosome combination treatment of non-small cell lung cancer after COVID-19.

With the outbreak of the coronavirus disease 2019 (COVID-19), reductions in T-cell function and exhaustion have been observed in patients post-infection of COVID-19. T cells are key mediators of anti-infection and antitumor, and their exhaustion increases the risk of compromised immune function and elevated susceptibility to cancer. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer with high incidence and mortality. Although the survival rate after standard treatment such as surgical treatment and chemotherapy has improved, the therapeutic effect is still limited due to drug resistance, side effects, and recurrence. Recent advances in molecular biology and immunology enable the development of highly targeted therapy and immunotherapy for cancer, which has driven cancer therapies into individualized treatments and gradually entered clinicians' views for treating NSCLC. Currently, with the development of photosensitizer materials, phototherapy has been gradually applied to the treatment of NSCLC. This review provides an overview of recent advancements and limitations in different treatment strategies for NSCLC under the background of COVID-19. We discuss the latest advances in phototherapy as a promising treatment method for NSCLC. After critically examining the successes, challenges, and prospects associated with these treatment modalities, their profound prospects were portrayed.

Two new mouse alleles of Ocm and Slc26a5.

The genes Ocm (encoding oncomodulin) and Slc26a5 (encoding prestin) are expressed strongly in outer hair cells and both are involved in deafness in mice. However, it is not clear if they influence the expression of each other. In this study, we characterise the auditory phenotype resulting from two new mouse alleles, Ocmtm1e and Slc26a5tm1Cre. Each mutation leads to absence of detectable mRNA transcribed from the mutant allele, but there was no evidence that oncomodulin regulates expression of prestin or vice versa. The two mutants show distinctive patterns of auditory dysfunction. Ocmtm1e homozygotes have normal auditory brainstem response thresholds at 4 weeks old followed by progressive hearing loss starting at high frequencies, while heterozygotes show largely normal thresholds until 6 months of age, when signs of worse thresholds are detected. In contrast, Slc26a5tm1Cre homozygotes have stable but raised thresholds across all frequencies tested, 3 to 42 kHz, at least from 4 to 8 weeks old, while heterozygotes have raised thresholds at high frequencies. Distortion product otoacoustic emissions and cochlear microphonics show deficits similar to auditory brainstem responses in both mutants, suggesting that the origin of hearing impairment is in the outer hair cells. Endocochlear potentials are normal in the two mutants. Scanning electron microscopy revealed normal development of hair cells in Ocmtm1e homozygotes but scattered outer hair cell loss even at 4 weeks old when thresholds appeared normal, indicating that there is not a direct relationship between numbers of outer hair cells present and auditory thresholds.

Structural Basis of Main Proteases of Coronavirus Bound to Bofutrelvir.

Globally, the continuous spread and evolution of SARS-CoV-2, along with its variants, profoundly impact human well-being, health, security, and the growth of socio-economic. In the field of development of drugs against COVID-19, the main protease (Mpro) is a critical target as it plays a core role in the lifecycle of SARS-CoV-2. Bofutrelvir acts as a potent inhibitor of SARS-CoV-2 Mpro, demonstrating high efficacy and broad-spectrum antiviral activity. Compared to therapies that require pharmacokinetic boosters, such as ritonavir, the monotherapy approach of Bofutrelvir reduces the risk of potential drug interactions, making it suitable for a wider patient population. However, further studies on the potency and mechanism of inhibition of Bofutrelvir against the Mpro of COVID-19 and its variants, together with other coronaviruses, are needed to prepare for the possibility of a possible re-emerging threat from an analogous virus in the future. Here, we reveal the effective inhibition of Bofutrelvir against the Mpro of SARS-CoV-2, SARS-CoV, and HCoV-229E through FRET and crystallographic analysis. Furthermore, the inhibitory mechanisms of Bofutrelvir against two SARS-CoV-2 Mpro mutants (G15S and K90R) were also elucidated through FRET and crystallographic studies. Through detailed analysis and comparison of these crystal structures, we identified crucial structural determinants of inhibition and elucidated the binding mode of Bofutrelvir to Mpros from different coronaviruses. These findings are hopeful to accelerate the development of safer and more potent inhibitors against the Mpro of coronavirus, and to provide important references for the prevention and treatment of similar viruses that may emerge in the future.

ROS production by cytochrome bc1: Its mechanism as inferred from the effects of heme b cofactor mutants.

Cytochrome bc1 is one of the enzymes of electron transport chain responsible for generation of reactive oxygen species (ROS). While ROS are considered to be products of side reactions of quinol oxidation site (Qo), molecular aspects of their generation remain unclear. One of them concerns significance of hemes b (bL and bH) redox potentials (Em) and properties on ROS generation by Qo. Here we addressed this question by examining ROS production in mutants of bacterial cytochrome bc1 that replaced one of the His ligand of either heme bL or bH with Lys or Asn. We observed that severe slowing down of electron flow by the Asn mutants induces similar effects on ROS production as inhibition by antimycin in the native cytochrome bc1 (WT). An increase in the Em of hemes b (either bL or bH) in Lys mutants does not exert major effect on the ROS production level, compared to WT. The experimental data were analyzed in the frame of a dynamic model to conclude that the observed ROS rates and levels reflect a combinatory effect of two factors: probability of heme bL being in the reduced state and probability of electron transfer from heme bL towards Qo. A significant contribution from short-circuits maintains the ROS levels at ~15 % in all tested forms. Overall, ROS production by cytochrome bc1 shows remarkably low susceptibility to changes in the Em of heme b cofactors, leaving significance of tuning the Em of hemes b as factor limiting superoxide production an open question.

Olfactory deficit and gastrointestinal dysfunction precede motor abnormalities in alpha-Synuclein G51D knock-in mice.

Parkinson's disease (PD) is typically a sporadic late-onset disorder, which has made it difficult to model in mice. Several transgenic mouse models bearing mutations in SNCA, which encodes alpha-Synuclein (α-Syn), have been made, but these lines do not express SNCA in a physiologically accurate spatiotemporal pattern, which limits the ability of the mice to recapitulate the features of human PD. Here, we generated knock-in mice bearing the G51D SNCA mutation. After establishing that their motor symptoms begin at 9 mo of age, we then sought earlier pathologies. We assessed the phosphorylation at Serine 129 of α-Syn in different tissues and detected phospho-α-Syn in the olfactory bulb and enteric nervous system at 3 mo of age. Olfactory deficit and impaired gut transit followed at 6 mo, preceding motor symptoms. The SncaG51D mice thus parallel the progression of human PD and will enable us to study PD pathogenesis and test future therapies.

A prioritization tool for cilia-associated genes and their in vivo resources unveil new avenues for ciliopathy research.

Defects in ciliary signaling or mutations in proteins that localize to primary cilia lead to a class of human diseases known as ciliopathies. About 10% of mammalian genes encode cilia-associated proteins and a major gap in the cilia research field is prioritizing which genes to study and finding the in vivo vertebrate mutant alleles and reagents available for their study. Here we present a unified resource listing the cilia-associated human genes cross-referenced to available mouse and zebrafish mutant alleles, their associated phenotypes as well as expression data in kidney and functional data for vertebrate Hedgehog signaling. This resource empowers researchers to easily sort and filter genes based on their own expertise and priorities, cross-reference with newly-generated -omics datasets, and quickly find in vivo resources and phenotypes associated with a gene of interest.

Opening a 60-year time capsule: sequences of historical poliovirus cold variants shed a new light on a contemporary strain.

Polioviruses (PVs) are positive strand RNA viruses responsible for poliomyelitis. Many PVs have been isolated and phenotypically characterized in the 1940s-50s for the purpose of identifying attenuated strains that could be used as vaccine strains. Among these historical PVs, only few are genetically characterized. We report here the sequencing of four PV strains stored for more than 60 years in a sealed box. These PVs are cold variants that were selected by Albert Sabin based on their capacity to multiply at relatively low temperatures. Inoculation of permissive cells at 25°C showed that two of the four historical virus stocks still contained infectious particles. Both viruses reached titres that were higher at 25°C than at 37°C, thus demonstrating that they were genuine cold variants. We obtained sequences that span virtually all the genome for three out of the four strains; a short sequence that partly covers the 5' untranslated region was recovered for the last one. Unexpectedly, the genome of one historical cold variant (which derives from PV-3 Glenn) displayed a very high nucleotide identity (above 95%) with that of a PV strain (PV-3 strain WIV14) sampled in China in 2014 and then classified as a highly evolved vaccine-derived PV. Our analyses made this hypothesis very unlikely and strongly suggested that Glenn and WIV14 shared a very recent common ancestor with one another. Some strains used to produce the inactivated polio vaccine were also very close to Glenn and WIV14 in the capsid-encoding region, but they had not been sequenced beyond the capsid. We therefore sequenced one of these strains, Saukett A, which was available in our collection. Saukett A and WIV14 featured an identity higher than 99% at the nucleotide level. This work provides original data on cold variants that were produced and studied decades ago. It also highlights that sequences of historical PV strains could be crucial to reliably characterize contemporary PVs in case of release from a natural reservoir or from a facility, which is of highest importance for the PV eradication program.

Maize mutant screens: from classical methods to new CRISPR-based approaches.

Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings.

Screening of pathogenicity-deficient Penicillium italicum mutants established by Agrobacterium tumefaciens-mediated transformation.

Blue mold, caused by Penicillium italicum, is one of the main postharvest diseases of citrus fruits during storage and marketing. The pathogenic mechanism remains largely unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of P. italicum PI5 was established via Agrobacterium tumefaciens-mediated transformation (ATMT). The system yielded 200-250 transformants per million conidia, and the transformants were genetically stable after five generations of successive subcultures on hygromycin-free media. 2700 transformants were obtained to generate a T-DNA insertion library of P. italicum. Only a few of the 200 randomly selected mutants exhibited significantly weakened virulence on citrus fruits, with two mutants displaying attenuated sporulation. The T-DNA in the two mutants existed as a single copy. Moreover, the mutant genes PiBla (PITC_048370) and PiFTF1 (PITC_077280) identified may be involved in conidia production by regulating expressions of the key regulatory components for conidiogenesis. These results demonstrated that the ATMT system is useful to obtain mutants of P. italicum for further investigation of the molecular mechanisms of pathogenicity and the obtained two pathogenesis-related genes might be novel loci associated with pathogenesis and conidia production.

Kinetics of Secoisolariciresinol Glucosyltransferase LuUGT74S1 and Its Mutants.

The lignan secoisolariciresinol (SECO) diglucoside (SDG) is a phytoestrogen with diverse effects. LuUGT74S1 glucosylates SECO to SDG, whereby only small amounts of the monoglucoside SMG are formed intermediately, which exhibit increased activity. To identify critical amino acids that are important for enzymatic activity and the SMG/SDG ratio, 3D structural modeling and docking, as well as site-directed mutation studies, were performed. Enzyme assays with ten mutants revealed that four of them had identical kinetic data to LuUGT74S1, while three showed reduced and one increased catalytic efficiency kcat/Km. S82F and E189L substitutions resulted in the complete absence of activity. A17 and Q136 are crucial for the conversion of SMG to SDG as A17S and Q136F mutants exhibited the highest SMG/SDG ratios of 0.7 and 0.4. Kinetic analyses show that diglucosylation is an essentially irreversible reaction, while monoglycosylation is kinetically favored. The results lay the foundation for the biotechnological production of SMG.

Exchanges in the 'a' determinant of the hepatitis B virus surface antigen revisited.

The hepatitis B virus surface antigen's (HBsAg) 'a' determinant comprises a sequence of amino acid residues located in the major hydrophilic region of the S protein, whose exchanges are closely associated with compromising the antigenicity and immunogenicity of that antigen. The HBsAg is generally present in the bloodstream of individuals with acute or chronic hepatitis B virus (HBV) infection. It is classically known as the HBV infection marker, and is therefore the first marker to be investigated in the laboratory in the clinical hypothesis of infection by this agent. One of the factors that compromises the HBsAg detection in the bloodstream by the assays adopted in serological screening in both clinical contexts is the loss of S protein antigenicity. This can occur due to mutations that emerge in the HBV genome regions that encode the S protein, especially for its immunodominant region - the 'a' determinant. These mutations can induce exchanges of amino acid residues in the S protein's primary structure, altering its tertiary structure and the antigenic conformation, which may not be recognized by anti-HBs antibodies, compromising the infection diagnosis. In addition, these exchanges can render ineffective the anti-HBs antibodies action acquired by vaccination, compromise the effectiveness of the chronically HBV infected patient's treatment, and also the HBsAg immunogenicity, by promoting its retention within the cell. In this review, the residues exchange that alter the S protein's structure is revisited, as well as the mechanisms that lead to the HBsAg antigenicity loss, and the clinical, laboratory and epidemiological consequences of this phenomenon.

CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.