Acid Variations Research Articles

A chromosome-level genome assembly of Solanum chilense, a tomato wild relative associated with resistance to salinity and drought.

Solanum chilense is a wild relative of tomato reported to exhibit resistance to biotic and abiotic stresses. There is potential to improve tomato cultivars via breeding with wild relatives, a process greatly accelerated by suitable genomic and genetic resources. In this study we generated a high-quality, chromosome-level, de novo assembly for the S. chilense accession LA1972 using a hybrid assembly strategy with ~180 Gbp of Illumina short reads and ~50 Gbp long PacBio reads. Further scaffolding was performed using Bionano optical maps and 10x Chromium reads. The resulting sequences were arranged into 12 pseudomolecules using Hi-C sequencing. This resulted in a 901 Mbp assembly, with a completeness of 95%, as determined by Benchmarking with Universal Single-Copy Orthologs (BUSCO). Sequencing of RNA from multiple tissues resulting in ~219 Gbp of reads was used to annotate the genome assembly with an RNA-Seq guided gene prediction, and for a de novo transcriptome assembly. This chromosome-level, high-quality reference genome for S. chilense accession LA1972 will support future breeding efforts for more sustainable tomato production. Gene sequences related to drought and salt resistance were compared between S. chilense and S. lycopersicum to identify amino acid variations with high potential for functional impact. These variants were subsequently analysed in 84 resequenced tomato lines across 12 different related species to explore the variant distributions. We identified a set of 7 putative impactful amino acid variants some of which may also impact on fruit development for example the ethylene-responsive transcription factor WIN1 and ethylene-insensitive protein 2. These variants could be tested for their ability to confer functional phenotypes to cultivars that have lost these variants.

Substrate Promiscuity of the Triceptide Maturase XncB Leads to Incorporation of Various Amino Acids and Detection of Oxygenated Products.

Triceptides are cyclophane-containing ribosomally synthesized and post-translationally modified peptides. The characteristic cross-links are formed between an aromatic ring to Cβ on three-residue Ω1X2X3 motifs (Ω1 = aromatic). Here, we explored the promiscuity of the XYE family triceptide maturase, XncB from Xenorhabdus nematophila DSM 3370. Single amino acid variants were coexpressed with XncB in vivo in Escherichia coli, and we show that a variety of amino acids can be incorporated into the Phe-Gly-Asn cyclophane. Aromatic amino acids at the X3 position were accepted by the enzyme but yielded hydroxylated, rather than the typical cyclophane, products. These studies show that oxygen can be inserted but diverges in the final product formed relative to daropeptide maturases. Finally, truncations of the leader peptide showed that it is necessary for complete modification by XncB.

Rationally introducing non-canonical amino acids to enhance catalytic activity of LmrR for Henry reaction

The use of enzymes to catalyze Henry reaction has advantages of mild reaction conditions and low contamination, but low enzyme activity of promiscuous catalysis limits its application. Here, rational design was first performed to identify the key amino acid residues in Henry reaction catalyzed by Lactococcal multidrug resistance Regulator (LmrR). Further, non-canonical amino acids were introduced into LmrR, successfully obtaining variants that enhanced the catalytic activity of LmrR. The best variant, V15CNF, showed a 184% increase in enzyme activity compared to the wild type, and was 1.92 times more effective than the optimal natural amino acid variant, V15F. Additionally, this variant had a broad substrate spectrum, capable of catalyzing reactions between various aromatic aldehydes and nitromethane, with product yielded ranging from 55 to 99%. This study improved enzymatic catalytic activity by enhancing affinity between the enzyme and substrates, while breaking limited types of natural amino acid residues by introducing non-canonical amino acids into the enzyme, providing strategies for molecular modifications.Graphical

A Pilot Investigation of the Association Between Vpr Amino Acid Substitutions and Peripheral Immune Marker Levels in People With Human Immunodeficiency Virus: Implications for Neurocognitive Impairment.

Subtype-specific amino acid variations in viral proteins of human immunodeficiency virus type 1 (HIV-1) influence disease progression. Furthermore, Vpr sequence variation correlates with chronic inflammation, a central mechanism in HIV-1 (neuro)pathogenesis. Nevertheless, no clinical study has investigated the link between Vpr sequence variation and peripheral inflammation in people with HIV (PWH). The aim of this pilot study was to ascertain whether specific Vpr amino acid variants were associated with immune markers in PWH. We included a unique cohort of 48 treatment-naive South African PWH to determine the association between blood-derived Vpr sequence variation and peripheral immune marker levels using Sanger sequencing and enzyme-linked immunosorbent assay analysis, respectively. Our findings indicate that among the many neuropathogenic Vpr amino acid variants and immune markers examined, after applying Bonferroni corrections (P = .05/3) and adjusting for sex and locality, soluble urokinase plasminogen activator receptor (suPAR) was nearing significance for higher levels in participants with the G41 amino acid variant compared to those with the S41 variant (P = .035). Furthermore, amino acid variations at position 41 (between G41 and S41) exhibited a significant association with suPAR (adjusted R2 = 0.089, β = .386 [95% confidence interval, .125-3.251]; P = .035). These findings suggest that Vpr amino acid sequence variations might contribute to dysregulated inflammation, which could explain the observed association between specific Vpr variants and HIV-1 (neuro)pathogenesis found in prior research. These Vpr variants merit further investigation to fully understand their roles in HIV-1 pathogenesis and neuropathogenesis.

Effect of New Methods for Inhibiting Malolactic Fermentation on the Analytical and Sensory Parameters of Wines

This study focuses on the impact of new methods for inhibiting malolactic fermentation in white wines on their analytical and sensory properties. Enological preparations with different mechanisms of effect were tested: fumaric acid, chitosan, Estaan (a preparation based on tannin inhibition), medium-chain fatty acids (MCFAs), sulphur dioxide and a control variant. Malolactic fermentation (MLF) was also performed. The samples underwent analysis through HPLC (high-performance liquid chromatography) to determine the concentrations of malic and lactic acid, as well as biogenic amines. GC (gas chromatography) analysis was used to monitor volatile substances, alongside sensory evaluation. This study demonstrated a significant influence of individual enological preparations on the aromatic profile of the examined wines. The SO2 and MCFA variants exhibited the highest concentrations of volatile substances within the esters group, specifically isoamyl acetate, 1-hexyl acetate and phenylethyl acetate. Conversely, the fumaric acid and Estaan variants displayed the lowest concentrations of these esters. The most notable disparities were observed in acetoin concentration, with the MCFA variant exhibiting the lowest values. Additionally, the chitosan variant showed higher concentrations of putrescine and spermidine compared to the MCFA and fumaric acid variants, which presented the lowest levels.

From microbial heterogeneity to evolutionary insights: A strain-resolved metagenomic study of H2S-induced changes in anaerobic biofilms

The hidden layers of genomic diversity in microbiota of biotechnological interest have been only partially explored and a deeper investigation that overcome species level resolution is needed. CO2-fixating microbiota are prone to such evaluation as case study. A lab-scale trickle-bed reactor was employed to successfully achieve simultaneous biomethanation and desulfurization on artificial biogas and sulfur-rich biogas, and oxygen supplementation was also implemented. Under microaerophilic conditions, hydrogen sulfide removal efficiency of 81% and methane content of 95% were achieved. Methanobacterium sp. DTU45 emerged as predominant, and its metabolic function was tied to community-wide dynamics in sulfur catabolism. Genomic evolution was investigated in Gammaproteobacteria sp. DTU53, identified as the main contributor to microaerophilic desulfurization. Positive selection of variants in the hydrogen sulfide oxidation pathway was discovered and amino acid variants were localized on the sulfide entrance channel for sulfide:quinone oxidoreductase. Upon oxygen supplementation strain selection was the primary mechanism driving microbial adaptation, rather than a shift in species dominance. Selective pressure determined the emergence of new strains for example on Gammaproteobacteria sp. DTU53, providing in depth evidence of functional redundancy within the microbiome.

Multiple Lines of Evidence Support 199 SARS-CoV-2 Positively Selected Amino Acid Sites.

SARS-CoV-2 amino acid variants that contribute to an increased transmissibility or to host immune system escape are likely to increase in frequency due to positive selection and may be identified using different methods, such as codeML, FEL, FUBAR, and MEME. Nevertheless, when using different methods, the results do not always agree. The sampling scheme used in different studies may partially explain the differences that are found, but there is also the possibility that some of the identified positively selected amino acid sites are false positives. This is especially important in the context of very large-scale projects where hundreds of analyses have been performed for the same protein-coding gene. To account for these issues, in this work, we have identified positively selected amino acid sites in SARS-CoV-2 and 15 other coronavirus species, using both codeML and FUBAR, and compared the location of such sites in the different species. Moreover, we also compared our results to those that are available in the COV2Var database and the frequency of the 10 most frequent variants and predicted protein location to identify those sites that are supported by multiple lines of evidence. Amino acid changes observed at these sites should always be of concern. The information reported for SARS-CoV-2 can also be used to identify variants of concern in other coronaviruses.

An integrated QTL mapping and transcriptome sequencing provides further molecular insights and candidate genes for stem strength in rapeseed (Brassica napus L.).

We detected the major QTL- qSR.A07, which regulated stem strength and was fine-mapped to 490 kb. BnaA07G0302800ZS and BnaA07G0305700ZS as the candidate functional genes were identified at qSR.A07 locus. The stem's mechanical properties reflect its ability to resist lodging. In rapeseed (Brassica napus L.), although stem lodging negatively affects yield and generates harvesting difficulties, the molecular regulation of stem strength remains elusive. Hence, this study aimed to unravel the main loci and molecular mechanisms governing rapeseed stem strength. A mapping population consisting of 267 RILs (recombinant inbred lines) was developed from the crossed between ZS11 (high stem strength) and 4D122 (low stem strength), and two mechanical properties of stems including stem breaking strength and stem rind penetrometer resistance were phenotyped in four different environments. Four pleiotropic QTLs that were stable in at least two environments were detected. qSR.A07, the major one, was fine-mapped to a 490kb interval between markers SA7-2711 and SA7-2760 on chromosome 7. It displayed epistatic interaction with qRPR.A09-2. Comparative transcriptome sequencing and analysis unveiled methionine/S-adenosylmethionine cycle (Met/SAM cycle), cytoskeleton organization, sulfur metabolism and phenylpropanoid biosynthesis as the main pathways associated with high stem strength. Further, we identified two candidate genes, BnaA07G0302800ZS and BnaA07G0305700ZS, at qSR.A07 locus. Gene sequence alignment identified a number of InDels, SNPs and amino acid variants in sequences of these genes between ZS11 and 4D122. Finally, based on these genetic variants, we developed three SNP markers of these genes to facilitate future genetic selection and functional studies. These findings offer important genetic resources for the molecular-assisted breeding of novel rapeseed stem lodging-resistant varieties.

Realization of digital twin for dynamic control towardsample variation of ion exchange chromatography in antibody separation.

Digital twin (DT) is a virtual and digital representation of physical objects or processes. In this paper, this concept is applied to dynamic control of the collection window in the ion exchange chromatography (IEC) towardsample variations. A possible structure of a feedforward model-based control DT system was proposed. Initially, a precise IEC mechanistic model was established through experiments, model fitting, and validation. The average root mean square error (RMSE) of fitting and validation was 8.1% and 7.4%, respectively. Then a model-based gradient optimization was performed, resulting in a 70.0% yield with a remarkable 11.2% increase. Subsequently, the DT was established by systematically integrating the model, chromatography system, online high-performance liquid chromatography, and a server computer. The DT was validated under varying load conditions. The results demonstrated that the DT could offer an accurate control with acidic variants proportion and yield difference of less than 2% compared to the offline analysis. The embedding mechanistic model also showed a positive predictive performance with an average RMSE of 11.7% during theDT test under >10% sample variation. Practical scenario tests indicated that tightening the control target could further enhance the DT robustness, achieving over 98% success rate with an average yield of 72.7%. The results demonstrated that the constructed DT could accurately mimic real-world situations and perform an automated and flexible pooling in IEC. Additionally, a detailed methodology for applying DT was summarized.

Uncovering the fragmentation and separation characteristics of sophorolipid biosurfactants with LC-MS-ESI.

This article describes the process and challenges in identifying different structures of eco-friendly biosurfactants, providing a novel database to compare results.

Countercurrent preferential precipitation of acidic variants from monoclonal antibody pools

The process of countercurrent multistage precipitation (CnMP) was developed for reduction of the acidic variant (av) content in monoclonal antibody (mAb) pools. The process was performed in polyethylene glycol (PEG) solutions of low ionic strength, at which av was the most prone to precipitate. To design the process, a mathematical model was formulated, which consisted of steady-state mass balance equations and underlying thermodynamic dependencies. The model was solved for different combinations of the process variables, including the protein concentration in the feed material and the PEG concentration in subsequent precipitation stages. The model solution was projected onto two-dimensional planes, where the performance indicators, such as the separation yield and the av reduction level, were correlated with the process variables. CnMP overperformed crosscurrent multistage precipitation (CsMP) and ion exchange chromatography (IEX) used in previous studies; in the comparison with CsMP, the 3-stage CnMP process allowed up to 28% increase in yield and up to 25% increase in the av reduction level, and in the comparison with IEX, up to 37% increase in yield and up 12.5% increase in the av reduction level. The yield benefit of CnMP depended on the composition of the feed material and the demand for the av reduction. The concept was experimentally verified; the 2- and 3-stage CnMP processes were performed using the input process variables provided by the model solution. The performance indicators calculated by the model and experimentally measured were in a good agreement, which indicated both feasibility and predictability of CnMP.

The adaptive potential of North American subtype H7N2 avian influenza viruses to mammals

Introduction. H7 subtype avian influenza viruses causing severe epizootics among birds are phylogenetically different in the Eastern and Western hemispheres. Numerous human infections caused by these viruses in the Eastern hemisphere indicate that H7 viruses can overcome the interspecies barrier and pose a potential threat of a new pandemic.The H7N2 viruses with deletion of amino acids 221–228 (H3 numbering) in hemagglutinin (HA) had been circulating among poultry in the Western Hemisphere during 1996–2006, and had once again been detected in 2016 in an animal shelter, where they caused cat diseases.
 The objective of this study is to elucidate the mechanism of adaptation to mammals of North American H7N2 influenza viruses with deletion in HA.
 Materials and methods. The A/chicken/New Jersey/294598-12/2004 (H7N2) virus was adapted to mice by the lung passages. Complete genomes of original and mouse-adapted viruses were analyzed. The receptor specificity and thermostability of viruses, HA activation pH and virulence for mice were determined.
 Results. The non-pathogenic H7N2 avian influenza virus became pathogenic after 10 passages in mice. Amino acid substitutions occurred in five viral proteins: one in PB2 (E627K), NA (K127N), NEP (E14Q), four in HA and six in NS1. Mutations in HA slightly changed receptor specificity but increased the pH of HA activation by 0.4 units. The NS1 protein undergone the greatest changes in the positions (N73T, S114G, K118R, G171A, F214L and G224R), where amino acid polymorphisms were observed in the original virus, but only minor amino acid variants have been preserved in the mouse adapted variant.
 Conclusion. The results show that H7N2 viruses have the potential to adapt to mammals. The increase in virulence is most likely due to the adaptive E627K mutation in PB2 and possibly in HA.

Stability of ranibizumab during continuous delivery from the Port Delivery Platform

The Port Delivery System with ranibizumab (PDS) is an innovative intraocular drug delivery system that has the potential to reduce treatment burden in patients with retinovascular diseases. The Port Delivery Platform (PD-P) implant is a permanent, indwelling device that can be refilled in situ through a self-sealing septum and is designed to continuously deliver ranibizumab by passive diffusion through a porous titanium release control element. We present results for the studies carried out to characterize the stability of ranibizumab for use with the PD-P. Simulated administration, in vitro release studies, and modeling studies were performed to evaluate the compatibility of ranibizumab with the PD-P administration components, and degradation and photostability in the implant. Simulated administration studies demonstrated that ranibizumab was highly compatible with the PD-P administration components (initial fill and refill needles) and commercially available administration components (syringe, transfer needle, syringe closure). Subsequent simulated in vitro release studies examining continuous delivery for up to 12 months in phosphate buffered saline, a surrogate for human vitreous, showed that the primary degradation products of ranibizumab were acidic variants. The presence of these variants increased over time and potency remained high. The stability attributes of ranibizumab were consistent across multiple implant refill-exchanges. Despite some degradation within the implant, the absolute mass of variants released daily from the implant was low due to the continuous release mechanism of the implant. Simulated light exposure within the implant resulted in small increases in the relative amount of ranibizumab degradants compared with those seen over 6 months.

Unveiling the effect of CaCl2 on amyloid β aggregation via supercritical angle Raman and fluorescence spectroscopy and microscopy.

Amyloid β aggregation is an important factor in Alzheimer's disease. Since calcium homeostasis plays an important role in amyloid β aggregation, it is crucial to study the interaction between calcium ions and amyloid β directly at the surface of the lipid membrane. With supercritical angle techniques, the signal of interest at the surface is easily separated from the bulk solution, making them a powerful tool for aggregation study. In this work, the influence of calcium ions on amyloid β aggregation over different aggregation time periods is investigated with supercritical angle Raman and fluorescence spectroscopy and microscopy. Note that calcium ions have a larger influence on amyloid β1-42 than on the 40 amino acid variant. We found that a small layer of calcium ions significantly protects the lipid membrane against the protein insertion process.

Isotretinoin Use and Liver Enzymes Changes: A Single-Center Study in Saudi Arabia.

Introduction Isotretinoin is a bioactive retinoic acid variant that is taken orally to treat moderate to severe acne vulgaris. One of the adverse effects of isotretinoin is elevated liver enzymes. This study estimated the prevalence of liver enzyme changes during isotretinoin use among dermatology clinic patients in Al-Ahsa, Kingdom of Saudi Arabia. Methods This study was a retrospective analysis that reviewed the medical data of 97 patients with acne at the King Faisal University Polyclinic who were taking systemic isotretinoin. It determined the baseline, second, and last readings of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Among the 97 patients, 67 (69.1%) were female and 30 (30.9%) were male. Results Of the patients,41 (50.6%) weighed 51-70 kg, and 45 (46.4%) were 21-23 years old. The age of acne onset was 20 years or younger. Most patients had a starting isotretinoin dose of 10-20 mg and an ending dose of 30-40 mg over six months. Ninety (92.8%) patients had not used isotretinoin in the past. Before treatment, AST was elevated in three (3.1%) patients, and ALT was elevated in two (2.1%) patients. In the last readings, AST was elevated in eight (8.2%) patients, and ALT was elevated in four (4.1%) patients. Conclusion The result of this study indicates that the incidence of high levels of AST and ALT with oral isotretinoin was low. So frequent laboratory monitoring is not recommended since the elevation was not associated with any morbidity and carries financial and emotional burdens. An exception is patients with higher body weight, males, and those whose acne started at age 16-19, in whom frequent monitoring may be considered for AST more than ALT.

Mechanisms of Primaquine Induced Hemolysis in a Novel Humanized Murine Model of Mediterranean G6PD Deficiency

Background: Approximately half a billion individuals are genetically deficient in glucose 6-phosphate dehydrogenase (G6PD) due to amino acid variants generally thought to decrease enzyme half-life. Decreased G6PD activity limits the antioxidant capacity of red blood cells (RBCs). As such, certain drugs that induce oxidative stress can cause life-threatening hemolysis in G6PD deficient (G6DPd) individuals. Standard regiments of the only approved drugs that can cure liver phase Plasmodium vivax ( p. vivax) (i.e. primaquine and tafenoquine) are contraindicated in G6PDd patients due to risks of hemolysis. Effective treatment of G6PDd individuals infected with p. vivax requires a prolonged course of primaquine (8 weeks), leading to non-adherence, and subsequent reduced efficacy and increased risk of transmission. Despite decades of research and large drug screening programs, no non-hemolytic compound has been identified that can eradicate p. vivax. Lack of progress in this area has likely been hampered by an incomplete mechanistic understanding of primaquine induced hemolysis, in part due to the lack of an animal model that recapitulates G6PD instability. Methods: Recombinant non-deficient human hG6PD(ND) or the deficient Mediterranean variant (hG6PD(MED)) were expressed, purified, and subjected to thermal proteome profiling in combination with TMT10-assisted quantitative cross-linking proteomics. Mice were generated in which murine genomic G6PD is replaced with either genomic human hG6PD(ND) or hG6PD(MED). Hemolysis in humans is caused by primaquine metabolites (e.g. primaquine-5,6-orthoquinone (5,6-POQ)) and not primaquine itself. As such, RBCs from both strains were exposed to 5,6-POQ and then analyzed by 1) high resolution metabolomics and proteomics, 2) electron paramagnetic resonance (EPR) to measure superoxide, 3) methemoglobin (MetHb) formation, 4) light and electron microscopy, 5) in vitro hemolysis, and 6) in vivo hemolysis (clearance) after infusion into animals. In vivo pulse chase biotinylation allowed visualization and identification of younger (1-6 days) RBCs vs. older (7-55 day) RBCs. Results: Recombinant hG6PD(MED) enzyme had a 2.6-fold lower specific activity compared to hG6PD(ND). Thermal gradient crosslinking proteomics identified specific amino acid spacing (less than 26 angstroms) that was higher in hG6PD(ND) [82-95 and 91-432] vs. higher in hG6PD(MED) [89-205, 97-429, and 429-497]. Analysis of mice demonstrated that hG6PD(MED) RBCs had 5% of G6PD activity as compared to hG6PD(ND) RBCs, with an RBC age dependent decrease in G6PD activity in hG6PD(MED) RBCs. Exposure to 5,6-POQ caused increased superoxide generation, increased MetHb formation, alterations to morphology, in vitro hemolysis, and in vivo hemolysis of hG6PD(MED) RBCs compared to hG6PD(ND) RBCs. However, younger hG6PD(MED) RBCs with higher G6PD activity were highly resistant to 5,6-POQ induced hemolysis (both in vivo and in vitro). All experiments were carried out a minimum of 3 times and all stated findings were statistically significant (p<0.05). Proteomic and metabolomic analysis (with combined samples from multiple experiments) demonstrated oxidation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in a block in glycolysis that increases dihydroxyacetone phosphate (DHAP) leading to cytotoxic methylglyoxal that broadly oxidizes protein lysines to carboxymethyllysine (CML) - the first step in advanced glycation end products (AGEs) known to facilitate RBC clearance. Discussion: We identify specific conformational changes caused by the hG6PD(MED) variant resulting in decreased specific activity and half-life, and confirm that G6PD activity decreases with RBC age, in a humanized mouse model. The resistance of young hG6PD(MED) RBCs to 5,6-POQ has immediate practical ramifications, in that it supports the rationale of clinical studies currently proposing new dose escalation primaquine regimens to induce low level early hemolysis, leading to reticulocytosis, and decreasing the mean age of circulating RBCs such that increased parricidal doses of primaquine can then be tolerated in G6PDd patients. The elucidation of the metabolic and proteomic lesion (under conditions confirmed to cause 5,6-POQ induced hemolysis of hG6PD(MED) but not hG6PD(ND) RBCs) provides a mechanistic landscape to guide development of non-hemolytic drugs to treat p. vivax.

Regional Genetic Heterogeneity Among Patients with Pyruvate Kinase Deficiency

Background: Pyruvate kinase (PK) deficiency is a rare, congenital hemolytic anemia caused by homozygous or compound heterozygous mutations in the PKLR gene, with a prevalence of 3.2-8.5 individuals per million in Western populations. Over 350 pathogenic variants in the PKLR gene have been reported, with the majority being missense substitutions. Although their frequency differs across regions and populations, the geographic distribution of PKLR variants has not yet been fully investigated. Aim: To evaluate the PKLR genotype distribution by region among patients (pts) with PK deficiency in the merged real-world database of the Peak Registry (NCT03481738) and PK Deficiency Natural History Study (NHS; NCT02053480). Methods: The NHS was a global, longitudinal, observational study that enrolled pts with PK deficiency from 2014-2017 (study completed May 2020). The Peak Registry is an ongoing global registry which was initiated in 2018 to build on the NHS with expanded pt recruitment and geographic reach, broader data collection, and longer pt follow-up. Comparable data from these two studies were integrated to form a merged database providing enlarged sample size and prolonged follow-up from pts who participated in both studies. This analysis included pts with available genotype data from the NHS and Peak Registry, including those who were siblings; a large Amish cohort of pts carrying the homozygous p.R479H variant was excluded to avoid reporting bias. Genotype classifications were aligned between the two studies. Data were extracted from the merged database of NHS and Peak (data cutoff date: 13May2022) and summarized descriptively by region. Regions were defined as: Northern (N) Europe (Denmark, Ireland, Netherlands, UK); Central (C) Europe (Czech Republic, Germany, Switzerland); Southern (S) Europe (France, Italy, Spain, Portugal); Asia (S Korea, Thailand, Turkey); N America (Canada, US). Results: 362 pts were included in the analysis (N Europe, N=59; S Europe, N=62; C Europe, N=51; Asia, N=23; N America, N=167), 97 (27%) of whom were siblings. Most pts (72%) had compound heterozygous PKLR variants (Table). Homozygous mutations were most common among pts from Asia (15/23; 65%) and were least common among pts from S Europe (8/62; 13%). Almost half of pts from N Europe (49%) had a compound heterozygous missense/missense (M/M) genotype, while a similar proportion from S Europe (44%) had a compound heterozygous missense/non-missense (M/NM) genotype. A total of 724 PKLR variants were identified, including 197 unique variants, 26 of which were newly identified. Most were missense mutations (76%), followed by nonsense (10%), splice site (7%), and frameshift (5%) mutations. The majority of mutated alleles in the cohort were single nucleotide variants (90%), followed by small deletions (4%), large deletions (4%), and insertions (2%). The amino acid variant with the highest observed frequency was p.Arg510Gln, both overall (158/724; 22%), and in N Europe (31/118; 26%), C Europe (27/102; 26%), and N America (92/334; 28%), but it was not observed in Asia (0/46; 0%). The second most common variant overall, p.Arg486Trp (67/724; 9%), was the most frequent in S Europe (31/124; 25%). When stratified by genotype, p.Arg510Gln was the most common missense mutation in homozygous pts in each region (40-67%) except in Asia. In Asia, p.Thr157Ala, p.Lys541Asn, and p.Arg194Pro were the most common mutations overall (4/46; 9%), and also the most common missense mutations in homozygous pts (4/28; 14%). One hundred and seventy-four unique variants were observed in compound heterozygotes. In S Europe (27/102; 26%) and Asia (2/10; 20%), p.Arg486Trp was the most frequently observed missense mutation in compound heterozygote pts. Overall and in all regions except for Asia (where it was absent), p.Glu241Ter was the most common non-missense mutation in compound heterozygote pts (32/254; 13%); this was p.Gly540Ter (3/6; 50%) in Asia. Conclusions: This analysis, which used the largest real-world data source for pts with PK deficiency, confirms the genetic heterogeneity of the disease, showing that the mutation frequency in PKLR varies substantially based on geographic distribution and that the identification of novel genetic variants is relatively common in PK deficiency.

Enhancing Selectivity of Protein Biopharmaceuticals in Ion Exchange Chromatography through Addition of Organic Modifiers

Charge heterogeneity among therapeutic monoclonal antibodies (mAbs) is considered an important critical quality attribute and requires careful characterization to ensure safe and efficacious drug products. The charge heterogeneity among mAbs is the result of chemical and enzymatic post-translational modifications and leads to the formation of acidic and basic variants that can be characterized using cation exchange chromatography (CEX). Recently, the use of mass spectrometry-compatible salt-mediated pH gradients has gained increased attention to elute the proteins from the charged stationary phase material. However, with the increasing antibody product complexity, more and more selectivity is required. Therefore, in this study, we set out to improve the selectivity by using a solvent-enriched mobile phase composition for the analysis of a variety of mAbs and bispecific antibody products. It was found that the addition of the solvents to the mobile phase appeared to modify the hydrate shell surrounding the protein and alter the retention behavior of the studied proteins. Therefore, this work demonstrates that the use of solvent-enriched mobile phase composition could be an attractive additional method parameter during method development in CEX.

Microscopic mechanisms of shear strength variation in acid- and alkali-contaminated loess

Microscopic mechanisms of shear strength variation in acid- and alkali-contaminated loess

A Survey of HBV Core/Pre-Core Mutations in Iraqi Patients with Chronic Hepatitis

Objectives: The present study was conducted to assess the pattern of HBV Core/Pre-Core mutations and HBV genotype in Iraqi patients with chronic hepatitis B virus (HBV) infection. Methods: In the current cross-sectional study in an Iraqi province, we evaluated 134 patients diagnosed with HBV hepatitis. We used PCR and, subsequently, Sanger sequencing to assess HBV Core/Pre-Core mutations. Sanger sequencing reads were further used for phylogenetic analysis and multiple sequence alignment. A phylogenetic tree was generated according to the neighbor-joining method. Results: The current study revealed that 58 (45%) of the patients were male, and 72 (55%) of them were female. The mean age of the patients was 36 ± 12.7 years, and the mean duration of infection was 5.2 ± 4.8 years. The results revealed 21 nucleic acid alterations in the samples analyzed. The generated phylogenetic tree divided samples into two genotypes. Pre-core/Core mutations were significantly associated with the treatment received (P = 0.0.001) but not with laboratory parameters. Most samples were matched with the genotype D clade, while only four samples were positioned adjacent to the genotype E clade. Direct nucleic acid translation disclosed five nucleic acid variants (73T>G, 347T>G, 364A>G, 365T>C, and 366A>G) on the core protein. Conclusions: This study has detected 21 nucleotide variants and 5 amino acid alterations within the coding sequences of the C gene. This study revealed that genotype D represents the primary genotype for the identified viral infections. The current study highlights the importance of these mutations evaluation for future, more comprehensive studies.