Amino Acid Changes Research Articles

Changes in Physiological Indices, Amino Acids, and Volatile Compounds in Vitis vinifera L. cv. Pinot Noir under UV-B Radiation and Water Deficit Conditions.

UV-B radiation and water deficit can challenge Pinot noir growth and fruit quality. The aim of this work is to determine the effects of UV-B and water deficit on the physiological indices, amino acids, and volatile compounds of Pinot noir vine and fruit. The results showed that both individual and combined treatments caused a decrease in the leaf SPAD, with the largest amplitude being observed in the combined treatment. Water deficit also decreased the leaf water potential and increased the juice δ13C‱ at harvest, which was the opposite of the latter under UV-B radiation. Interestingly, most of the physiological indices under combined stresses did not show significant changes compared with that under no UV-B and the well-watered control treatment. Moreover, the concentrations of amino acids and volatile compounds in the berries were determined at harvest. The amino acid contents were significantly increased by the combined treatment, particularly proline (Pro), aspartate (Arg), alanine (Ala), and threonine (Thr). There were slight increases in volatile compounds. This research substantially contributed to improve our scientific understanding of UV-B and water deficit responses in an important commercial species. In addition, it highlighted some future research to produce high-quality wines with the anticipated specific characteristics.

Nontargeted metabolomics reveals sequential changes in amino acid and ferroptosis-related metabolism in Parkinson's disease.

Parkinson's disease (PD) is inseparable from metabolic disorders but lacks assessment of specific metabolite alteration. To explore the sequential metabolic changes in PD progression, we evenly divided 78 C57BL/6 mice (10weeks) into six groups (one control group and five experimental groups) and collected the hippocampus tissue of mice after treating with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and probenecid (twice a week) at five periods (1, 2, 3, 4, and 5weeks) for metabolome analysis. Our study identified 567 differentially abundant metabolites (DAMs) (total 4348 metabolites). Compared with controls, 145, 146, 171, 208, and 213 DAMs were obtained from the five experimental groups, respectively. Notably, 40 shared DAMs were present in five experimental groups, of which 22 shared DAMs formed a new metabolic network based on amino acid metabolism. Compared with group W3, 84 DAMs were identified in group W5, including 12 unique DAMs. DAMs in different stages of PD were significantly enriched in amino acid metabolism pathway, lipid metabolism pathway, and ferroptosis pathway. l-Glutamine, spermidine, and l-tryptophan were the key hubs in the whole metabolic process of PD. N-Formyl-l-methionine gradually increased in abundance with PD progression, whereas 5-methylcytosine gradually decreased. The study emphasized the sequential changes in DAMs in PD progression, stimulating subsequent studies.

Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.

Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus Mammarenavirus, family Arenaviridae and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.

Genetic and phenotypic presentation of Usher syndrome - a case report

Usher syndrome is a genetic, clinically heterogeneous condition characterized by sensorineural hearing loss, progressive retinal degeneration, and vestibular dysfunction. There are three phenotypically recognizable types of Usher syndrome. Individuals with Usher syndrome type 1 have no vestibular function and profound sensorineural hearing loss. Individuals with Usher syndrome type 2 have normal vestibular function and mild-to-severe hearing loss with visual impairment that is presented later in life. People with Usher syndrome type III experience hearing and vision loss beginning later in life. In this case report, we are reporting a 7-year-old boy consulted for progressive hearing loss and bilateral vision impairment, and fundus exam revealed mild bilateral retinal vessel attenuation and bone spicule deposits in both eyes. A molecular genetic test done by next-generation sequencing identified a homozygous pathogenic variant in the CDH23 gene (NM_022124.5:c.2255del variant coordinate with amino acid change of p.(Gly752Valfs*13)), confirming the diagnosis of autosomal recessive Usher syndrome type ID (USH1D). The patient had a remarkable improvement with visual and optical aids. Genetic counseling, including reproductive counseling, was provided to the parents. Clinical evaluation, visual hearing tests, and genetic workup confirmed Usher syndrome, which is a rare but dangerous cause of hearing loss and visual impairment that needs to be thoroughly evaluated by a multi-disciplinary team approach.

The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones.

Microbial competition within plant tissues affects invading pathogens' fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce's disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography-mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.

Genomic resistant determinants of multidrug-resistant Campylobacter spp. isolates in Peru

Antimicrobial resistant (AMR) Campylobacter is a global health threat; however, there is limited information on genomic determinants of resistance in low- and middle-income countries. We evaluated genomic determinants of AMR using a collection of whole genome sequenced Campylobacter jejuni and C. coli isolates from Iquitos, Peru. Campylobacter isolates from two paediatric cohort studies enriched with isolates that demonstrated resistance to ciprofloxacin and azithromycin were sequenced and mined for AMR determinants. The gyrA mutation leading to the Thr86Ile amino acid change was the only gyrA mutation associated with fluoroquinolone resistance identified. The A2075G mutation in 23S rRNA was present, but three other 23S rRNA mutations previously associated with macrolide resistance were not identified. A resistant-enhancing variant of the cmeABC efflux pump genotype (RE-cmeABC) was identified in 36.1% (35/97) of C. jejuni genomes and 17.9% (12/67) of C. coli genomes. Mutations identified in the CmeR-binding site, an inverted repeat sequence in the cmeABC promoter region that increases expression of the operon, were identified in 24/97 C. jejuni and 14/67 C. coli genomes. The presence of these variants, in addition to RE-cmeABC, was noted in 18 of the 24 C. jejuni and 9 of the 14 C. coli genomes. Both RE-cmeABC and mutations in the CmeR-binding site were strongly associated with the MDR phenotype in C. jejuni and C. coli. This is the first report of RE-cmeABC in Peru and suggests it is a major driver of resistance to the principal therapies used to treat human campylobacteriosis in this setting.

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.

Colistin heteroresistance in Citrobacter freundii clinical isolates from Republic of Korea

We investigated colistin heteroresistance in Citrobacter freundii isolates from Korean hospitals. Using population analysis profiling (PAP), we detected colistin heteroresistance in 31.3% of isolates. Among these, ST217 was the most prevalent clone (58.5%), particularly within colistin-heteroresistant isolates (80.0%). Interestingly, the second most common clone, ST248, was not found in heteroresistant isolates. We identified amino acid changes in PhoQ, PmrA, and PmrB, along with mRNA overexpression in pmrB and arnD. Colistin monotherapy showed no efficacy, but a combination of colistin and ciprofloxacin successfully eradicated all five isolates, even at 0.5 × minimum inhibitory concentrations. This study underscores the high prevalence of colistin heteroresistance in C. freundii isolates, limiting the effectiveness of colistin monotherapy. Combining colistin with ciprofloxacin may offer a viable treatment option for C. freundii infections.

Creation of the HIV-1 antisense gene asp coincided with the emergence of the pandemic group M and is associated with faster disease progression.

Despite being first identified more than three decades ago, the antisense gene asp of HIV-1 remains an enigma. asp is present uniquely in pandemic (group M) HIV-1 strains, and it is absent in all non-pandemic (out-of-M) HIV-1 strains and virtually all non-human primate lentiviruses. This suggests that the creation of asp may have contributed to HIV-1 fitness or worldwide spread. It also raises the question of which evolutionary processes were at play in the creation of asp. Here, we show that HIV-1 genomes containing an intact asp gene are associated with faster HIV-1 disease progression. Furthermore, we demonstrate that the creation of a full-length asp gene occurred via the evolution of codon usage in env overlapping asp on the opposite strand. This involved differential use of synonymous codons or conservative amino acid substitution in env that eliminated internal stop codons in asp, and redistribution of synonymous codons in env that minimized the likelihood of new premature stops arising in asp. Nevertheless, the creation of a full-length asp gene reduced the genetic diversity of env. The Luria-Delbruck fluctuation test suggests that the interrupted asp open reading frame (ORF) is the progenitor of the intact ORF, rather than a descendant under random genetic drift. Therefore, the existence of group-M isolates with a truncated asp ORF indicates an incomplete transition process. For the first time, our study links the presence of a full-length asp ORF to faster disease progression, thus warranting further investigation into the cellular processes and molecular mechanisms through which the ASP protein impacts HIV-1 replication, transmission, and pathogenesis.IMPORTANCEOverlapping genes engage in a tug-of-war, constraining each other's evolution. The creation of a new gene overlapping an existing one comes at an evolutionary cost. Thus, its conservation must be advantageous, or it will be lost, especially if the pre-existing gene is essential for the viability of the virus or cell. We found that the creation and conservation of the HIV-1 antisense gene asp occurred through differential use of synonymous codons or conservative amino acid substitutions within the overlapping gene, env. This process did not involve amino acid changes in ENV that benefited its function, but rather it constrained the evolution of ENV. Nonetheless, the creation of asp brought a net selective advantage to HIV-1 because asp is conserved especially among high-prevalence strains. The association between the presence of an intact asp gene and faster HIV-1 disease progression supports that conclusion and warrants further investigation.

Exploring the effects of missense mutations on protein thermodynamics through structure-based approaches: findings from the CAGI6 challenges.

Missense mutations are known contributors to diverse genetic disorders, due to their subtle, single amino acid changes imparted on the resultant protein. Because of this, understanding the impact of these mutations on protein stability and function is crucial for unravelling disease mechanisms and developing targeted therapies. The Critical Assessment of Genome Interpretation (CAGI) provides a valuable platform for benchmarking state-of-the-art computational methods in predicting the impact of disease-related mutations on protein thermodynamics. Here we report the performance of our comprehensive platform of structure-based computational approaches to evaluate mutations impacting protein structure and function on 3 challenges from CAGI6: Calmodulin, MAPK1 and MAPK3. Our stability predictors have achieved correlations of up to 0.74 and AUCs of 1 when predicting changes in ΔΔG for MAPK1 and MAPK3, respectively, and AUC of up to 0.75 in the Calmodulin challenge. Overall, our study highlights the importance of structure-based approaches in understanding the effects of missense mutations on protein thermodynamics. The results obtained from the CAGI6 challenges contribute to the ongoing efforts to enhance our understanding of disease mechanisms and facilitate the development of personalised medicine approaches.

Impact of Sprouting Process on the Protein Quality of Yellow and Red Quinoa (Chenopodium quinoa).

The demand for plant-based proteins has increased remarkably over the last decade. Expanding the availability and variety of plant-based protein options has shown positive potential. This study aimed to investigate the qualitative and quantitative changes in amino acids of yellow and red quinoa seeds (YQ and RQ) during a 9-day germination period. The results showed that the germination process led to an increase in the total amino acids by 7.43% and 14.36% in the YQ and RQ, respectively. Both varieties exhibited significant (p < 0.05) increases in non-essential and essential amino acids, including lysine, phenylalanine, threonine, and tyrosine. The content of non-essential amino acids nearly reached the standard values found in chicken eggs. These results were likely attributed to the impact of the germination process in increasing enzymes activity and decreasing anti-nutrient content (e.g., saponins). A linear relationship between increased seeds' hydration and decreased saponins content was observed, indicating the effect of water absorption in changing the chemical composition of the plant. Both sprouts showed positive germination progression; however, the sprouted RQ showed a higher germination rate than the YQ (57.67% vs. 43.33%, respectively). Overall, this study demonstrates that germination is a promising technique for enhancing the nutritional value of quinoa seeds, delivering sprouted quinoa seeds as a highly recommended source of high-protein grains with notable functional properties.

Hao-Fountain syndrome: 32 novel patients reveal new insights into the clinical spectrum.

Hao-Fountain syndrome (HAFOUS, OMIM: #616863) is a neurodevelopmental disorder caused by pathogenic variants in the gene USP7 coding for USP7, a protein involved in several crucial cellular homeostatic mechanisms and the recently described MUST complex. The phenotype of HAFOUS is insufficiently understood, yet there is a great need to better understand the spectrum of disease, genotype-phenotype correlations, and disease trajectories. We now present a larger cohort of 32 additional individuals and provide further clinical information about six previously reported individuals. A questionnaire-based study was performed to characterize the phenotype of Hao-Fountain syndrome more clearly, to highlight new traits, and to better distinguish the disease from related neurodevelopmental disorders. In addition to confirming previously described features, we report hyperphagia and increased body weight in a subset of individuals. HAFOUS patients present an increased rate of birth complications, congenital anomalies, and abnormal pain thresholds. Speech impairment emerges as a potential hallmark of Hao-Fountain syndrome. Cognitive testing reports reveal borderline intellectual functioning on average, although some individuals score in the range of intellectual disability. Finally, we created a syndrome-specific severity score. This score neither indicates a sex- nor age-specific difference of clinical severity, yet highlights a more severe outcome when amino acid changes colocalize to the catalytic domain of the USP7 protein.

Monkeypox: Can we count on the current smallpox immunization?

Two vaccines ACAM 2000 and JYNNEOS have obtained approval from the Food and Drug Administration as preventive measures against monkeypox, contributing significantly to the management of the monkeypox epidemic. Nonetheless, research has demonstrated that smallpox vaccination offers approximately 88.8% protection against monkeypox, while immunization with these vaccines generates relatively low levels of neutralizing antibodies. In this work, we performed a comprehensive comparison of antigens between the 2022–2023 monkeypox strains and the smallpox vaccine strains. Our analysis has revealed considerable amino acid changes in all 27 antigens, including core and envelope proteins. Amino acid substitutions within B cell epitopes were observed in 26 of these antigens, with at least half of the antigen substitutions occurring within B cell epitopes in 20 out of the 26 antigens analyzed. These findings may raise potential concerns regarding the efficacy of these vaccines.

Characterization of a Novel Mutation in the COPI Vesicle on Binding to Dilysine Motifs

Background/Objective: The heptameric COPI coatomer complex is involved in the formation of vesicles and the intracellular trafficking of proteins between the Golgi and Endoplasmic Reticulum as well as throughout the cytoplasm. Members of the COPI complex bind dilysine motifs found in the C-terminal domain of the cargo protein, particularly KKxx or KxKxx. We generated a point mutation in the WD40 domain of the COPI alpha subunit (⍺-COP). We hypothesized that the E269V mutant ⍺-COP would not co-immunoprecipitate (co-IP) COPI cargo proteins terminating with a dilysine domain of KxKxx (Nucleolin and Stasimon/Tmem41b), but would bind cargo proteins terminating in KKxx (FLAGSyntaxin17). We predicted that a mutation in the ⍺-COP C-terminus, which impairs interaction with ε–COP, would not affect its ability to co-IP dilysine-containing cargo.&#x0D; Experimental Design or Project Methods: HEK-293TT cells were transfected with Myctagged wild-type, E269V, and triple mutant (3X) ⍺-COP. The E269V ⍺-COP mutant has an amino acid change at position 269 from glutamic acid to valine. The triple ⍺-COP mutant has three amino acid changes that eliminate binding with the ε–COP COPI subunit. Transfected ⍺-COP was immunoprecipitated using magnetic anti-Myc beads. Endogenous Nucleolin was immunoprecipitated using magnetic Protein A beads conjugated to rabbit polyclonal anti- Nucleolin antibody. Western blots of inputs and immunoprecipitates of each experiment were conducted to determine the ability of ⍺-COP to co-IP C-terminal dilysine-containing proteins.&#x0D; Results: Endogenous Nucleolin and Stasimon co-immunoprecipitated with WT and 3X ⍺-COP, but not E269V ⍺-COP.&#x0D; Conclusions and Potential Impact: The inability of mutation E269V to co-IP dilysine proteins implies that the WD40 domain of the COPI ⍺-COP protein is required for binding to KxKxxterminating proteins, as typified by Nucleolin and Stasimon.The C-terminal 3X mutation shows that ε-COP is not necessary for dilysine recognition and implies that ⍺-COP directly binds to this KxKxx motif.

Abstract A030: DNA repair gene polymorphism(s): Association with relapse of oral squamous cell carcinoma

Abstract Differences in DNA repair capacity have been linked to the risk and prognosis of cancer. In specific types of cancer, certain variations known as Single Nucleotide Polymorphisms (SNPs) within DNA repair genes can influence the effectiveness of treatments. This phenomenon has been observed in patients with Oral Squamous Cell Carcinoma (OSCC), especially in those undergoing chemotherapy and radiation therapy. One such gene, Excision Repair Cross-Complementation 5 (ERCC5), contains a SNP (rs17655) G&amp;gt;C, leading to a missense variation and a change in amino acid from D to H. This variation has been noted in various cancer types. Hence, this study aimed to investigate the presence of this DNA repair gene polymorphism (rs17655) and its association with OSCC. To achieve this goal, blood samples were collected from OSCC patients exposed to chemotherapy and radiation therapy, as well as from OSCC patients not exposed to these treatments, following approval from the relevant institutional review committees. Informed consent was obtained from 100 patients, and an equal number of age and gender-matched controls. Genomic DNA was extracted using the salting-out method and analyzed for quality and quantity through agarose gel electrophoresis and nanodrop spectrophotometry. Primers were designed using online primer designing software. Genetic variations were identified using Tetra Primers-Amplifies Refractory Mutation System Polymerase Chain Reaction (t-ARMS PCR), and the amplified products were visualized via agarose gel electrophoresis. The genotypic frequency analysis of rs17655 revealed a higher prevalence of the homozygous mutant type (CC) in the blood samples of OSCC patients, both exposed and non-exposed to chemotherapy and radiation therapy. In contrast, the homozygous wild type (GG) was more prevalent among the control group. Statistical analysis, including a chi-square test (χ 2 = 36.76, p &amp;lt; 0.001) and odds ratio (10.27), demonstrated a significant association between the rs17655 polymorphism and oral squamous cell carcinoma, indicating an increased risk of OSCC due to this genetic variation. These genomic variants have the potential to serve as biomarkers for predicting clinical outcomes in OSCC patients undergoing chemotherapy and radiotherapy. Citation Format: Umaiya Abdali, Saima Saleem. DNA repair gene polymorphism(s): Association with relapse of oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A030.

Assessment of thimerosal effects in sublethal concentrations on zebrafish (Danio rerio) embryos exploring NMR-based metabolomics profile

Thimerosal, a preservative commonly used in the pharmaceutical and cosmetic industry, has raised concerns regarding its potentially toxic effects as an organic mercury compound. Within this context, using an NMR-based metabolomics profile and chemometric analysis, zebrafish embryos were used as an in vivo model to study the effects of thimerosal in metabolic profiles after exposure to sublethal concentrations of the mercury compound. The thimerosal concentrations of 40 and 80 nM were employed, corresponding to 40% and 80% of the LC50, respectively, for zebrafish embryos. The most significant alterations in the metabolic profile included changes in carbohydrates, amino acids, nucleotides, trimethylamine-N-oxide, ethanolamine, betaine, and ethanol. Furthermore, thimerosal exposure affects various metabolic pathways, impairing the nervous system, disrupting protein metabolism, and potentially causing oxidative damage. Therefore, adopting a metabolomics approach in this investigation provided insights into the potentially implicated metabolic pathways contributing to the deleterious effects of thimerosal in biological systems.

WGCCRR: a web-based tool for genome-wide screening of convergent indels and substitutions of amino acids.

Genome-wide analyses of proteincoding gene sequences are being employed to examine the genetic basis of adaptive evolution in many organismal groups. Previous studies have revealed that convergent/parallel adaptive evolution may be caused by convergent/parallel amino acid changes. Similarly, detailed analysis of lineage-specific amino acid changes has shown correlations with certain lineage-specific traits. However, experimental validation remains the ultimate measure of causality. With the increasing availability of genomic data, a streamlined tool for such analyses would facilitate and expedite the screening of genetic loci that hold potential for adaptive evolution, while alleviating the bioinformatic burden for experimental biologists. In this study, we present a user-friendly web-based tool called WGCCRR (Whole Genome Comparative Coding Region Read) designed to screen both convergent/parallel and lineage-specific amino acid changes on a genome-wide scale. Our tool allows users to replicate previous analyses with just a few clicks, and the exported results are straightforward to interpret. In addition, we have also included amino acid indels that are usually neglected in previous work. Our website provides an efficient platform for screening candidate loci for downstream experimental tests. The tool is available at: https://fishevo.xmu.edu.cn/.

Analysis of Polycyclic Aromatic Hydrocarbons via GC-MS/MS and Heterocyclic Amines via UPLC-MS/MS in Crispy Pork Spareribs for Studying Their Formation during Frying.

This study aims to explore the effects of frying conditions on the formation of HAs and PAHs in crispy pork spareribs, a popular meat commodity sold on Taiwan's market. Raw pork spareribs were marinated, coated with sweet potato powder, and fried in soybean oil and palm oil at 190 °C/6 min or 150 °C/12 min, followed by an analysis of HAs and PAHs via QuEChERS coupled with UPLC-MS/MS and GC-MS/MS, respectively. Both HAs and PAHs in pork spareribs during frying followed a temperature- and time-dependent rise. A total of 7 HAs (20.34-25.97 μg/kg) and 12 PAHs (67.69-85.10 μg/kg) were detected in pork spareribs fried in soybean oil and palm oil at 150 °C/12 min or 190 °C/6 min, with palm oil producing a higher level of total HAs and a lower level of total PAHs than soybean oil. The content changes of amino acid, reducing sugar, and creatinine played a vital role in affecting HA formation, while the degree of oil unsaturation and the contents of precursors including benzaldehyde, 2-cyclohexene-1-one, and trans,trans-2,4-decadienal showed a crucial role in affecting PAH formation. The principal component analysis revealed that HAs and PAHs were formed by different mechanisms, with the latter being more liable to formation in pork spareribs during frying, while the two-factorial analysis indicated that the interaction between oil type and frying condition was insignificant for HAs and PAHs generated in crispy pork spareribs. Both CcdP (22.67-32.78 μg/kg) and Pyr (16.70-22.36 μg/kg) dominated in PAH formation, while Harman (14.46-17.91 μg/kg) and Norharman (3.41-4.55 μg/kg) dominated in HA formation in crispy pork spareribs during frying. The outcome of this study forms a basis for learning both the variety and content of HAs and PAHs generated during the frying of pork spareribs and the optimum frying condition to minimize their formation.

Phylogenetic conservation of Trop-2 across species-rodent and primate genomics model anti-Trop-2 therapy for pre-clinical benchmarks.

A phylogenetic conservation analysis of Trop-2 across vertebrate species showed a high degree of sequence conservation, permitting to explore multiple models as pre-clinical benchmarks. Sequence divergence and incomplete conservation of expression patterns were observed in mouse and rat. Primate Trop-2 sequences were found to be 95%-100% identical to the human sequence. Comparative three-dimension primate Trop-2 structures were obtained with AlphaFold and homology modeling. This revealed high structure conservation of Trop-2 (0.66 ProMod3 GMQE, 0.80-0.86 ± 0.05 QMEANDisCo scores), with conservative amino acid changes at variant sites. Primate TACSTD2/TROP2 cDNAs were cloned and transfectants for individual ORF were shown to be efficiently recognized by humanized anti-Trop-2 monoclonal antibodies (Hu2G10, Hu2EF). Immunohistochemistry analysis of Macaca mulatta (rhesus monkey) tissues showed Trop-2 expression patterns that closely followed those in human tissues. This led us to test Trop-2 targeting in vivo in Macaca fascicularis (cynomolgus monkey). Intravenously injected Hu2G10 and Hu2EF were well tolerated from 5 to 10mg/kg. Neither neurological, respiratory, digestive, urinary symptoms, nor biochemical or hematological toxicities were detected during 28-day observation. Blood serum pharmacokinetic (PK) studies were conducted utilizing anti-idiotypic antibodies in capture-ELISA assays. Hu2G10 (t1/2 = 6.5days) and Hu2EF (t1/2 = 5.5days) were stable in plasma, and were detectable in the circulation up to 3weeks after the infusion. These findings validate primates as reliable models for Hu2G10 and Hu2EF toxicity and PK, and support the use of these antibodies as next-generation anti-Trop-2 immunotherapy tools.

The artificial amino acid change in the sialic acid-binding domain of the hemagglutinin neuraminidase of newcastle disease virus increases its specificity to HCT 116 colorectal cancer cells and tumor suppression effect

BackgroundOncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects.MethodsSialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells.ResultsS519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence.ConclusionThese results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.