Amino Acid Changes Research Articles

Discovery and computational exploration of SNPs in GNRHR gene and their influence on protein structure and function in Indian goat breeds

This study focuses on identifying functional single nucleotide polymorphisms (SNPs) within the gonadotropin-releasing hormone receptor (GNRHR) gene and conducting subsequent in-silico analysis of their effects on protein structure and function in two distinct South Indian goat breeds, namely Attapady Black (n = 120) and Malabari goats (n = 180), known for their divergent prolificacy traits. Utilizing a DNA pool sequencing assay, ten SNPs were uncovered in the study population: c.-1129T>G, c.-1069A>G, c.-978A>C, c.-605A>G, c.-33A>G, c.-29T>G, c.48G>A, c.75G>A, c.209T>G, and c.*212A>G. Notably, two polymorphisms, c.-1129T>G and c.-33A>G, were novel. Additionally, two polymorphisms, c.-33A>G and c.-978A>C, were exclusive to Malabari goats. Analysis of upstream variants revealed modifications to transcription factor and micro-RNA (miRNA) binding sites, suggesting potential alterations in GNRHR expression. Of particular significance was the non-synonymous exonic variant at c.209T>G locus, resulting in methionine to arginine substitution at the 70th position within the first intracellular loop of the receptor protein. This amino acid change may have implications for the functional dynamics of the receptor as GnRHR intracellular loops are involved in G protein coupling thereby facilitation of downstream signalling pathways. The identified SNPs and their in-silico impact analysis contribute to our understanding of the molecular mechanisms underlying reproductive traits in these goat populations, with implications for future breeding strategies and genomic selection programs.

The metastasis-promoting P1597L mutation in PlexinB1 enhances Ras activity

BackgroundMetastatic prostate cancer is a leading cause of cancer-related morbidity and mortality in men, yet the underlying molecular mechanisms are poorly understood. Plexins are transmembrane receptors for semaphorins with divergent roles in many forms of cancer. We recently found that a single clinically relevant specific amino acid change (Proline1597Leucine, (P1597L)), found in metastatic deposits of prostate cancer patients, converts PlexinB1 from a metastasis suppressor to a gene that drives prostate cancer metastasis in vivo. However, the mechanism by which PlexinB1(P1597L) promotes metastasis is not known.MethodsPull down assays using GST-RalGDS or -GSTRaf1-RBD were used to reveal the effect of mutant or wild-type PlexinB1 expression on Rap and Ras activity respectively. Protein–protein interactions were assessed in GST pulldown assays, Akt/ERK phosphorylation by immunoblotting and protein stability by treatment with cycloheximide. Rho/ROCK activity was monitored by measuring MLC2 phosphorylation and actin stress fiber formation. PlexinB1 function was measured using cell-collapse assays.ResultsWe show here that the single clinically relevant P1597L amino acid change converts PlexinB1 from a repressor of Ras to a Ras activator. The PlexinB1(P1597L) mutation inhibits the RapGAP activity of PlexinB1, promoting a significant increase in Ras activity. The P1597L mutation also blocks PlexinB1-mediated reduction in Rho/ROCK activity, restraining the decrease in MLC2 phosphorylation and actin stress fiber formation induced by overexpression of wild-type PlexinB1. PlexinB1(P1597L) has little effect on the interaction of PlexinB1 with small GTPases or receptor tyrosine kinases and does not inhibit PlexinB1-stimulated Akt or ERK phosphorylation. These results indicate that the mutation affects Rho signalling via the Rap/Ras pathway. The PlexinB1(P1597L) mutation inhibits morphological cell collapse induced by wild-type PlexinB1 expression, suggesting that the mutation induces a loss of an inhibitory tumour suppressor function.ConclusionThese results suggest that the clinically relevant P1597L mutation in PlexinB1 may transform PlexinB1 from a suppressor to a driver of metastasis in mouse models of prostate cancer by reducing the RapGAP activity of PlexinB1, leading to Ras activation. These findings highlight the PlexinB1-Rap-Ras pathway for therapeutic intervention in prostate cancer.

Development and Implementation of a Novel CAPS Assay Reveals High Prevalence of a Boscalid Resistance Marker and Its Co-Occurrence with an Azole Resistance Marker in Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs) are frequently used against powdery mildew (PM) fungi, such as Erysiphe necator, the causal agent of grapevine PM. Fungicide resistance, however, hinders effective control. DNA-based monitoring facilitates the recognition of resistance. We aimed (i) to adapt an effective method to detect a widespread genetic marker of resistance to boscalid, a commonly used SDHI, and (ii) to study the co-occurrence of the marker with a marker of resistance to demethylase inhibitor (DMI) fungicides. Sequencing of the sdhB gene identified a nonsynonymous substitution, denoted as sdhB-A794G, leading to an amino acid change (H242R) in the sdhB protein. In vitro fungicide resistance tests showed that E. necator isolates carrying sdhB-A794G were resistant to boscalid. We adopted a cleaved amplified polymorphic sequence-based method and screened more than 500 field samples collected from five Hungarian wine regions in two consecutive years. The sdhB-A794G marker was detected in all wine regions and in both years, altogether in 61.7% of samples, including 20.5% in which both sdhB-A794G and the wild-type were present. The frequency of sdhB-A794G was higher in SDHI-treated vineyards than in vineyards without any SDHI application. A significant difference in the presence of the marker was detected among wine regions; its prevalence ranged from none to 100%. We identified significant co-occurrence of sdhB-A794G with the CYP51-A495T (Y136F) mutation of the CYP51 gene, a known marker of resistance to DMIs. The monitoring of fungicide resistance is fundamental for the successful control of E. necator. Our rapid, cost-effective diagnostic method will support decision-making and fungicide resistance monitoring and management.

SARS-CoV-2 JN.1 variant evasion of IGHV3-53/3-66 B cell germlines.

The severe acute respiratory syndrome coronavirus 2 variant JN.1 recently emerged as the dominant variant despite having only one amino acid change on the spike (S) protein receptor binding domain (RBD) compared with the ancestral BA.2.86, which never represented more than 5% of global variants. To define at the molecular level the JN.1 ability to spread globally, we interrogated a panel of 899 neutralizing human monoclonal antibodies. Our data show that the single leucine-455-to-serine mutation in the JN.1 spike protein RBD unleashed the global spread of JN.1, likely occurring by elimination of more than 70% of the neutralizing antibodies mediated by IGHV3-53/3-66 germlines. However, the resilience of class 3 antibodies with low neutralization potency but strong Fc functions may explain the absence of JN.1 severe disease.

Regulation of ClC-K/barttin by endocytosis influences distal convoluted tubule hyperplasia.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

Nitrite and sulfide stress affects physiological and metabolic functions of gills in crab Eriocheir sinensis

Nitrite and sulfide are environmental factors that affect crabs survival and immunity. Gills are important respiratory and immune organs of Chinese mitten crabs Eriocheir sinensis, and both nitrite and sulfide can affect the physiological function stability of gill tissues. The present study investigated the acute toxicity of 10 mg/L nitrite and 5 mg/L sulfide, single and in combination, on the morphological, immune, osmoregulatory, and metabolic functions of the gill tissues of Chinese mitten crab. Both single and combined stressors were observed to disrupt the structural integrity of gill tissues. Oxidative stress-related indicators, including MDA, H2O2, and ·O2− content, exhibited an increase in all stress groups. The activities of antioxidant enzymes such as CAT and SOD and the expression levels of antioxidant genes fluctuated to varying degrees in all stress groups. The expression levels of osmotic regulating genes such as aqp, vatp, and nhe increased, as did the expression levels of lipid metabolism-related genes such as pparγ, rxr, fabp1, gyk, and dgat1. Furthermore, nitrite and sulfide stress affected the functions of metabolic pathways related to arachidonic acid, linoleic acid, D-amino acids, phenylalanine, and other substances in Chinese mitten crabs gill tissues. Additionally, the study identified 18 potential biomarkers related to stress responses. The nitrite and sulfide stressors disrupt the structure of crab gill tissues, cause osmoregulation disturbances, immune dysfunction, induce amino acid and lipid metabolic changes in gill tissue.

Genetic structure of apical membrane antigen-1 in Plasmodium falciparum isolates from Pakistan.

Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.

Heightened incidence of adverse events associated with a live attenuated varicella vaccine strain that lacks critical genetic polymorphisms in open reading frame 62

ObjectivesThis study aimed to identify the specific vaccine strain associated with herpes zoster (HZ) in children following a series of diagnosed cases and to explore whether differences in single nucleotide polymorphisms (SNPs) among various vaccine strains are linked to an increased incidence of herpes zoster after vaccination. MethodsFrom February 2021 to March 2024, children <12 years old suspected of vaccine-related varicella-like rash or HZ were included. Varicella zoster virus DNA isolated from the patients were sequenced to differentiate vaccine type versus wild-type. 3D protein structures of pORF62 were simulated using open reading frame 62 sequences extracted from whole genome sequencing of vOka, MAV/06, Oka/SK vaccines, and pOka reference. ResultsA total of 27 children with a median age of 2.1 (interquartile range, 1.5–3.4) years old presented with vaccine-related varicella-like rash (n = 4/27, 14.8%) or HZ (n = 23/27, 85.2%). One patient with varicella-like rash and 34.8% (n = 8/23) with HZ had disseminated skin involvement. All were immunized with the Oka/SK strain varicella vaccine. Genotyping showed 88.2% (n = 15/17) had SNPs specific to the Oka/SK strain, and two had SNPs considered pOka type contained within the Oka/SK vaccine. Despite accumulations of SNPs in ORF 62 of Oka/SK, the translated amino acid sequence and 3D protein structure were identical to wild-type pOka's pORF62. In vOKA and MAV/06, changes in amino acids occurred at two positions, S628G and R958G, within pORF62. The predicted 3D protein structure of vOka and MAV/06's pORF62 showed that the α helical structure within region I undergoes conformational change, potentially increasing difficulties in interactions with infection-related proteins and thereby decreasing virulence. pORF62 in pOka and Oka/SK exhibited more stable structure complex of the α helical structure. DiscussionLack of structural alternations in region I of pORF62 due to the absence of critical genetic polymorphisms in open reading frame 62 could be associated with the heightened incidence of adverse events.

Structure-based design of a Plasmodium vivax Duffy-binding protein immunogen focuses the antibody response to functional epitopes.

The Duffy-binding protein (DBP) is a promising antigen for a malaria vaccine that would protect against clinical symptoms caused by Plasmodium vivax infection. Region II of DBP (DBP-II) contains the receptor-binding domain that engages host red blood cells, but DBP-II vaccines elicit many non-neutralizing antibodies that bind distal to the receptor-binding surface. Here, we engineered a truncated DBP-II immunogen that focuses the immune response to the receptor-binding surface. This immunogen contains the receptor-binding subdomain S1S2 and lacks the immunodominant subdomain S3. Structure-based computational design of S1S2 identified combinatorial amino acid changes that stabilized the isolated S1S2 without perturbing neutralizing epitopes. This immunogen elicited DBP-II-specific antibodies in immunized mice that were significantly enriched for blocking activity compared to the native DBP-II antigen. This generalizable design process successfully stabilized an integral core fragment of a protein and focused the immune response to desired epitopes to create a promising new antigen for malaria vaccine development.

Geographic Distribution of Rabies Virus and Genomic Sequence Alignment of Wild and Vaccine Strains, Kenya.

Rabies, a viral disease that causes lethal encephalitis, kills ≈59,000 persons worldwide annually, despite availability of effective countermeasures. Rabies is endemic in Kenya and is mainly transmitted to humans through bites from rabid domestic dogs. We analyzed 164 brain stems collected from rabid animals in western and eastern Kenya and evaluated the phylogenetic relationships of rabies virus (RABV) from the 2 regions. We also analyzed RABV genomes for potential amino acid changes in the vaccine antigenic sites of nucleoprotein and glycoprotein compared with RABV vaccine strains commonly used in Kenya. We found that RABV genomes from eastern Kenya overwhelmingly clustered with the Africa-1b subclade and RABV from western Kenya clustered with Africa-1a. We noted minimal amino acid variances between the wild and vaccine virus strains. These data confirm minimal viral migration between the 2 regions and that rabies endemicity is the result of limited vaccine coverage rather than limited efficacy.

Mechanism and threshold of environmental stressors on seagrass in high-turbidity estuary: case of Zostera japonica in Yellow River Estuary, China

Zostera japonica (Z. japonica), the most widely distributed seagrass species in temperate estuaries, has experienced a dramatic decline of nearly 75% over the past decade. While previous research has investigated the adaptation of seagrass individuals and populations to single stress factors, the molecular mechanisms underlying the interaction of multiple stressors remain poorly understood. This study conducted laboratory experiments to examine the response of Z. japonica at different life stages to environmental pressures, specifically salinity and turbidity, as indicated by changes in free amino acids (FAAs). The results demonstrate that Z. japonica exhibits stronger adaptability to high salinity environments but displays weaker adaptability to freshwater conditions. Through single stress experiments, the salinity and turbidity thresholds for FAA homeostatic disturbance in Z. japonica were determined at seedling, juvenile, and mature stages. As Z. japonica matures, its metabolic pathways expand and diversify, allowing the regulation of key FAAs to enhance stress resistance. Turbidity stress exerts a more pronounced negative impact on the cellular homeostasis of Z. japonica compared to salinity stress, and when turbidity levels exceed 150 NTU, they significantly intensify the negative effects of salinity stress on the seagrass. Furthermore, under strong salinity-turbidity interactions, the concentration of key FAAs generally decreases by 20-30%, indicating inhibition of growth and development in Z. japonica. These findings have important implications for the conservation of intertidal seagrass beds and estuarine ecosystems in the face of multiple human activities and environmental stressors. The study provides valuable insights into the molecular mechanisms underlying Z. japonica’s adaptations to salinity and turbidity stress, contributing to the development of targeted strategies to mitigate the impacts of environmental pressures on seagrass populations and promote the resilience of these critical marine ecosystems.

The effect of biofertilizers on nickel accumulation, nitrogen metabolism and amino acid profile of corn (Zea mays L.) exposed to nickel stress.

The issue of heavy metal pollution such as nickel poses a significant environmental concern, exerting detrimental effects on the growth and viability of plant life. Plants have various mechanisms to effectively manage heavy metal stress, including the ability to modify their amino acid type and content. This adaptive response allows plants to mitigate the detrimental effects caused by excessive heavy metal accumulation. The aim of this study was to investigate the effect of biofertilizers on nickel accumulation, nitrogen metabolism and amino acid profile of corn (Zea maysL.) cv. 'PL438' exposed to Ni stress. After disinfecting and soaking in water for 24 h, corn seeds were primed with bacterial biofertilizers (T2: NPK + FZ), fungal biofertilizers (T3: Arbuscular mycorrhizal fungi (AMF) + Trichoderma (T)), or a combination of them (T4: NPK + FZ + AMF + T) and were cultured by the hydroponic method in completely controlled conditions. Then, they were simultaneously exposed to nickel chloride at various rates (0, 75, or 150 µM) at the three-leaf stage. They were harvested two weeks later and were subjected to the measurement of Ni content, nitrate and nitrite content, nitrate reductase activity, and amino acid profile by high-performance liquid chromatography. The results showed that the application of Ni at higher rates increased Ni, nitrate, and nitrite contents and nitrate reductase activity. The study of Ni accumulation and TF revealed that Ni accumulated in the roots to a greater extent than in the shoots and TF was < 1 in all treatments. The shoot amino acid profile showed that the treatment of Ni+2 increased som amino acids such as aspartic acid, asparagine, serine, histidine, and glycine versus the control, whereas T4Ni+2 increased aspartic acid, glutamic acid, threonine and arginine. The change in amino acids in Ni-treated plants may play a key role in their adaptation to Ni stress. The findings indicate that biofertilizers played a crucial role in mitigating the negative impacts of Ni on corn plants through alterations in amino acid composition and decreased absorption and translocation of Ni.

Complete genome sequence analysis of Reticuloendotheliosis virus integrated in nonhomologous Avipoxvirus

Integration of nucleic acid sequences of Reticuloendotheliosis virus (REV) in Avipoxvirus（APV） has become commonplace. In this study, 4 strains of suspected Fowlpox virus (FPV) and 1 strain of suspected Pigeonpox virus (PPV) collected in Taiyuan, Shanxi Province were cultured in chicken embryos, and the 4b core protein gene was amplified by PCR, and the identity and genome similarity were determined by sequence analysis. The sequences between the end of ORF201 and the beginning of ORF203 of FPV and PPV were then amplified, sequenced, and subjected to sequence comparison to determine genome similarity. The results showed that the isolates were 4 strains of FPV and 1 strain of PPV. The 4 isolated strains of FPV belong to type A1 virus, with 100 % identity to each other and to the FWPV-09-Jilin strain isolated in Jilin, China, and the lowest identity to the type B2 virus TNPV5/NZL/2009, which is only 74 %. PPV belongs to type A2 virus, and its identity with local strain of fowlpox virus was 90.1 %, with the highest identity of 100 % with PPLH and ROPI/W370/ON/2012 and ow_2017_3 strains, which also belong to type A2 pigeonpox virus, and the lowest identity of 73.7 % with TNPV5/NZL/2009, a type B2 virus. The complete genome of REV sequences integrated into FPV and PPV were amplified, and 5 REV nucleic acid sequences were obtained after sequencing and concatenation, with lengths ranging from 7942 to 8005 bp. The identity analysis results indicate that it has high identity with isolates from Northeast China, Guangdong, and Guangxi regions in China. Based on its gp90 protein gene, the REV integrated into the poxvirus belong to type III, with the highest identity of 99.9% with strains such as APC-566 and CY1111, and the lowest identity with REV-Anhui1, at 95.4 %. The length of the pol gene varies among different strains of REV, and its encoded amino acid changes significantly after position 675, with deletions and alterations. This study indicates that all fowlpox viruses isolated in Taiyuan, Shanxi Province have integrated the entire REV gene sequence, with high identity between them. At the same time, it indicates that the pigeonpox virus isolate has also integrated the entire REV gene sequence, and has the highest identity with the integrated REV gene sequence in fowlpox virus.

Hypoxic-Ischemic Insult Alters Polyamine and Neurotransmitter Abundance in the Specific Neonatal Rat Brain Subregions.

Neonatal hypoxic-ischemic (HI) brain insult is a major cause of neonatal mortality and morbidity. To assess the underlying pathological mechanisms, we mapped the spatiotemporal changes in polyamine, amino acid, and neurotransmitter levels, following HI insult (by the Rice-Vannucci method) in the brains of seven-day-old rat pups. Matrix-assisted laser desorption/ionization mass spectrometry imaging of chemically modified small-molecule metabolites by 4-(anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide revealed critical HI-related metabolomic changes of 22 metabolites in 14 rat brain subregions, much earlier than light microscopy detected signs of neuronal damage. For the first time, we demonstrated excessive polyamine oxidation and accumulation of 3-aminopropanal in HI neonatal brains, which was later accompanied by neuronal apoptosis enhanced by increases in glycine and norepinephrine in critically affected brain regions. Specifically, putrescine, cadaverine, and 3-aminopropanal increased significantly as early as 12 h postinsult, mainly in motor and somatosensory cortex, hippocampus, and midbrain, followed by an increase in norepinephrine 24 h postinsult, which was predominant in the caudate putamen, the region most vulnerable to HI. The decrease of γ-aminobutyric acid (GABA) and the continuous dysregulation of the GABAergic system together with low taurine levels up to 36 h sustained progressive neurodegenerative cellular processes. The molecular alterations presented here at the subregional rat brain level provided unprecedented insight into early metabolomic changes in HI-insulted neonatal brains, which may further aid in the identification of novel therapeutic targets for the treatment of neonatal HI encephalopathy.

Combined mediterranean diet-based sustainable healthy diet and multicomponent training intervention impact on plasma biomarkers and metabolome in older adults

Background and AimsHealthy dietary patterns and exercise practices have been associated with improved metabolic and inflammatory profiles. However, studies regarding the combined effect of these interventions on plasma biomarkers and metabolome in older adults are sparser. The primary aim of this study was to investigate the impact of a combined Mediterranean Diet-based Sustainable Healthy Diet (SHD) and Multicomponent Training (MT) intervention on the plasma biomarkers and metabolome and how dietary intake and exercise could modulate these effects. MethodsSHD intervention included a weekly supply of Mediterranean Diet-based SHD food and four nutrition sessions involving a Mediterranean-Diet culinary workshop, and the exercise program included 50-minute MT group sessions, held three times a week, lasting both 12 weeks. Plasma biomarkers were obtained through standard biochemical analysis. A proton (1H) nuclear magnetic resonance (NMR) spectroscopy-based metabolomics approach was used to study the metabolome in blood plasma. Repeated measures ANOVA were performed and adjusted for confounders. ResultsSHD+MT intervention significantly decreased HDL-C and calcium. SHD+MT showed some changes in common with the SHD and MT group, namely a significant decrease in citrate levels (p=0.009 for SHD+MT; p=0.037 for SHDT) and an increase in pyruvate (p<0.001 for MT and SHD+MT). The SHD+MT group also revealed specific changes in the levels of some amino acids (decrease in alanine, glutamine and lysine: p=0.026; p<0.001; p=0.038, respectively). Formate (p=0.025) and unsaturated lipids (p=0.011) increase are consistent with changes in energy, amino acid and lipoprotein metabolism. ConclusionOur data show that a combined lifestyle intervention program, including a Mediterranean Diet-based SHD and MT, could modulate biomarker and metabolome and there seems to be a metabolic path associated to these interventions in older adults. Due to its wide-ranging relevance, it is pertinent to assess to what extent combined SHD and MT can contribute to better clinical profiles.

Structure-based humanization of a therapeutic antibody for multiple myeloma

The optimal efficacy of xenogeneically generated proteins intended for application in humans requires that their own antigenicity be minimized. This necessary adaptation of antibodies to a humanized version poses challenges since modifications even distant from the binding sites can greatly influence antigen recognition and this is the primary feature that must be maintained during all modifications. Current strategies often rely on grafting and/or randomization/selection to arrive at a humanized variant retaining the binding properties of the original molecule. However, in terms of speed and efficiency, rationally directed approaches can be superior, provided the requisite structural information is available. We present here a humanization procedure based on the high-resolution X-ray structure of a chimaeric IgG against a marker for multiple myeloma. Based on in silico modelling of humanizing amino acid substitutions identified from sequence alignments, we devised a straightforward cloning procedure to rapidly evaluate the proposed sequence changes. Careful inspection of the structure allowed the identification of a potentially problematic amino acid change that indeed disrupted antigen binding. Subsequent optimization of the antigen binding loop sequences resulted in substantial recovery of binding affinity lost in the completely humanized antibody. X-ray structures of the humanized and optimized variants demonstrate that the antigen binding mode is preserved, with surprisingly few direct contacts to antibody atoms. These results underline the importance of structural information for the efficient optimization of protein therapeutics.Key messagesStructure-based humanization of an IgG against BCMA, a marker for Multiple Myeloma.Identification of problematic mutations and unexpected modification sites.Structures of the modified IgG-antigen complexes verified predictions.Provision of humanized high-affinity IgGs against BCMA for therapeutic applications.

Untargeted metabolomics reveals the mechanism of amantadine toxicity on Laminaria japonica.

The antiviral agent amantadine is frequently detected in seawater and marine organisms. Because of increasing concentrations, amantadine has become a contaminant of emerging concern. This compound has toxic effects on the brown algae Laminaria japonica. The effects of amantadine on the biological processes of L. japonica and the corresponding toxic mechanisms remain unclear. In this study, amantadine toxicity on L. japonica was investigated using histopathological and physiological characteristics combined with metabolomics analysis. Changes in metabolites were determined by untargeted metabolomics after exposure to 107ng/L amantadine for 72h. The catalase activity in the exposure group slightly increased, whereas the superoxide dismutase activity greatly decreased. An increase in the malondialdehyde concentration was observed after amantadine exposure, which suggested that lipid peroxidation and cell damage occurred. Metabolomics analysis showed that there were 406 differentially expressed metabolites after amantadine exposure. These were mainly phospholipids, amino acids, purines, and their derivatives. Inhibition of the glycerophospholipid metabolism affected the lipid bilayer and cell structure, which was aligned with changes in histological observation. Changes in amino acids led to perturbation of protein synthesis and induced oxidative stress through interference with glutathione metabolism and tyrosine metabolism. Amantadine also interfered with energy metabolism in L. japonica by disturbing the tricarboxylic acid cycle and purine metabolism. The results of this study provide new insights into the mechanism of amantadine toxicity on L. japonica.

Exploring Variation in Ovine KRTAP19-5 and Its Effect on Fine Wool Fibre Curvature in Chinese Tan Sheep.

Sheep's wool is known to have unique biological, physical and chemical properties. The fibre primarily consists of proteins, but these have amino acid sequence variation, and at the phenotypic level wool fibre varies considerably. This can affect its utility and value. Unravelling the genetic factors that underpin the protein and phenotypic variability is crucial if we are to contemplate improving wool quality. Accordingly, this study investigates the high glycine and tyrosine content keratin-associated protein 19-5 gene (KRTAP19-5) in sheep. PCR-single strand confirmation polymorphism analysis, coupled with DNA sequencing of a region spanning whole coding sequence, revealed six sequence variants containing seven single nucleotide polymorphisms (SNPs). Five of the SNPs were located within the coding region, with four leading to amino acid changes if expressed. In 247 Chinese Tan sheep derived from 10 sire-lines, and renowned for their distinct 'spring-like' crimped wool at up to approximately 35 days after birth, one of the variants was found to be associated with decreased curvature of the fine wool fibres in the fleece. No associations were detected with other fibre traits or with variation in the heterotypic hair fibres of the Tan sheep. While these findings may be useful for developing gene markers to alter mean wool fibre curvature and improve sheep breeding, many other genes and environmental factors are known to contribute to variation in fibre traits.

Biosynthesis of a clickable pyoverdine via in vivo enzyme engineering of an adenylation domain

The engineering of non ribosomal peptide synthetases (NRPS) for new substrate specificity is a potent strategy to incorporate non-canonical amino acids into peptide sequences, thereby creating peptide diversity and broadening applications. The non-ribosomal peptide pyoverdine is the primary siderophore produced by Pseudomonas aeruginosa and holds biomedical promise in diagnosis, bio-imaging and antibiotic vectorization. We engineered the adenylation domain of PvdD, the terminal NRPS in pyoverdine biosynthesis, to accept a functionalized amino acid. Guided by molecular modeling, we rationally designed mutants of P. aeruginosa with mutations at two positions in the active site. A single amino acid change results in the successful incorporation of an azido-l-homoalanine leading to the synthesis of a new pyoverdine analog, functionalized with an azide function. We further demonstrated that copper free click chemistry is efficient on the functionalized pyoverdine and that the conjugated siderophore retains the iron chelation properties and its capacity to be recognized and transported by P. aeruginosa. The production of clickable pyoverdine holds substantial biotechnological significance, paving the way for numerous downstream applications.

Prevalent fast evolution of genes involved in heterochromatin functions.

Heterochromatin is a gene-poor and repeat-rich genomic compartment universally found in eukaryotes. Despite its low transcriptional activity, heterochromatin plays important roles in maintaining genome stability, organizing chromosomes, and suppressing transposable elements (TEs). Given the importance of these functions, it is expected that the genes involved in heterochromatin regulation would be highly conserved. Yet, a handful of these genes were found to evolve rapidly. To investigate whether these previous findings are anecdotal or general to genes modulating heterochromatin, we compile an exhaustive list of 106 candidate genes involved in heterochromatin functions and investigate their evolution over short and long evolutionary time scales in Drosophila. Our analyses find that these genes exhibit significantly more frequent evolutionary changes, both in the forms of amino acid substitutions and gene copy number change, when compared to genes involved in Polycomb-based repressive chromatin. While positive selection drives amino acid changes within both structured domains with diverse functions and intrinsically disordered regions (IDRs), purifying selection may have maintained the proportions of IDRs of these proteins. Together with the observed negative associations between evolutionary rates of these genes and genomic TE abundance, we propose an evolutionary model where the fast evolution of genes involved in heterochromatin functions is an inevitable outcome of the unique functional roles of heterochromatin, while the rapid evolution of TEs may be an effect rather than cause. Our study provides an important global view of the evolution of genes involved in this critical cellular domain and provides insights into the factors driving the distinctive evolution of heterochromatin.