Specific Amino Acid Substitutions Research Articles

Prediction models for COVID-19 disease outcomes

ABSTRACT SARS-CoV-2 has caused over 6.9 million deaths and continues to produce lasting health consequences. COVID-19 manifests broadly from no symptoms to death. In a retrospective cross-sectional study, we developed personalized risk assessment models that predict clinical outcomes for individuals with COVID-19 and inform targeted interventions. We sequenced viruses from SARS-CoV-2-positive nasopharyngeal swab samples between July 2020 and July 2022 from 4450 individuals in Missouri and retrieved associated disease courses, clinical history, and urban-rural classification. We integrated this data to develop machine learning-based predictive models to predict hospitalization, ICU admission, and long COVID. The mean age was 38.3 years (standard deviation = 21.4) with 55.2% (N = 2453) females and 44.8% (N = 1994) males (not reported, N = 4). Our analyses revealed a comprehensive set of predictors for each outcome, encompassing human, environment, and virus genome-wide genetic markers. Immunosuppression, cardiovascular disease, older age, cardiac, gastrointestinal, and constitutional symptoms, rural residence, and specific amino acid substitutions were associated with hospitalization. ICU admission was associated with acute respiratory distress syndrome, ventilation, bacterial co-infection, rural residence, and non-wild type SARS-CoV-2 variants. Finally, long COVID was associated with hospital admission, ventilation, and female sex. Overall, we developed risk assessment models that offer the capability to identify patients with COVID-19 necessitating enhanced monitoring or early interventions. Of importance, we demonstrate the value of including key elements of virus, host, and environmental factors to predict patient outcomes, serving as a valuable platform in the field of personalized medicine with the potential for adaptation to other infectious diseases.

Rational design of structure‐based vaccines targeting misfolded alpha‐synuclein conformers of Parkinson's disease and related disorders

AbstractSynucleinopathies, including Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are neurodegenerative disorders caused by the accumulation of misfolded alpha‐synuclein protein. Developing effective vaccines against synucleinopathies is challenging due to the difficulty of stimulating an immune‐specific response against alpha‐synuclein without causing harmful autoimmune reactions, selectively targeting only pathological forms of alpha‐synuclein. Previous attempts using linear peptides and epitopes without control of the antigen structure failed in clinical trials. The immune system was unable to distinguish between native alpha‐synuclein and its amyloid form. The prion domain of the fungal HET‐s protein was selected as a scaffold to introduce select epitopes from the surface of alpha‐synuclein fibrils. Four vaccine candidates were generated by introducing specific amino acid substitutions onto the surface of the scaffold protein. The approach successfully mimicked the stacking of the parallel in‐register beta‐sheet structure seen in alpha‐synuclein fibrils. All vaccine candidates induced substantial levels of IgG antibodies that recognized pathological alpha‐synuclein fibrils derived from a synucleinopathy mouse model. Furthermore, the antisera recognized pathological alpha‐synuclein aggregates in brain lysates from patients who died from DLB, MSA, or PD, but did not recognize linear alpha‐synuclein peptides. Our approach, based on the rational design of vaccines using the structure of alpha‐synuclein amyloid fibrils and strict control over the exposed antigen structure used for immunization, as well as the ability to mimic aggregated alpha‐synuclein, provides a promising avenue toward developing effective vaccines against alpha‐synuclein fibrils.

Structure-function analysis of plant G-protein regulatory mechanisms identifies key Gα-RGS protein interactions

Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.

A pilot investigation of the association between HIV-1 Vpr amino acid sequence diversity and the tryptophan-kynurenine pathway as a potential mechanism for neurocognitive impairment

HIV infection compromises both the peripheral and central immune systems due to its pathogenic and neuropathogenic features. The mechanisms driving HIV-1 pathogenesis and neuropathogenesis involve a series of events, including metabolic dysregulation. Furthermore, HIV-subtype-specific variations, particularly alterations in the amino acid sequences of key viral proteins, are known to influence the severity of clinical outcomes in people living with HIV. However, the impact of amino acid sequence variations in specific viral proteins, such as Viral protein R (Vpr), on metabolites within the Tryptophan (Trp)-kynurenine (Kyn) pathway in people living with HIV remains unclear. Our research aimed to explore the relationship between variations in the Vpr amino acid sequence (specifically at positions 22, 41, 45, and 55, as these have been previously linked to neurocognitive function) and peripheral Trp-Kyn metabolites. Additionally, we sought to clarify the systems biology of Vpr sequence variation by examining the link between Trp-Kyn metabolism and peripheral inflammation, as a neuropathogenic mechanism. In this preliminary study, we analyzed a unique cohort of thirty-two (n = 32) South African cART naïve people living with HIV. We employed Sanger sequencing to ascertain blood-derived Vpr amino acid sequence variations and a targeted LC-MS/MS metabolomics platform to assess Trp-Kyn metabolites, such as Trp, Kyn, kynurenic acid (KA), and quinolinic acid (QUIN). Particle-enhanced turbidimetric assay and Enzyme-linked immunosorbent assays were used to measure immune markers, hsCRP, IL-6, suPAR, NGAL and sCD163. After applying Bonferroni corrections (p =.05/3) and adjusting for covariates (age and sex), only the Vpr G41 and A55 groups was nearing significance for higher levels of QUIN compared to the Vpr S41 and T55 groups, respectively (all p =.023). Multiple regression results revealed that Vpr amino acid variations at position 41 (adj R2 = 0.049, β = 0.505; p =.023), and 55 (adj R2 = 0.126, β = 0.444; p =.023) displayed significant associations with QUIN after adjusting for age and sex. Lastly, the higher QUIN levels observed in the Vpr G41 group were found to be correlated with suPAR (r =.588, p =.005). These results collectively underscore the importance of specific Vpr amino acid substitutions in influencing QUIN and inflammation (specifically suPAR levels), potentially contributing to our understanding of their roles in the pathogenesis and neuropathogenesis of HIV-1.

HIV-1 Vif protein sequence variations in South African people living with HIV and their influence on Vif-APOBEC3G interaction

PurposeDespite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein, which neutralizes the host protein APOBEC3G, has been implicated in differences in clinical outcomes among people living with HIV (PLHIV). Most studies on Vif sequence diversity have focused on subtype B, leaving gaps in understanding Vif variations in HIV-1C regions like South Africa. This study aimed to identify and compare Vif sequence diversity in a cohort of 51 South African PLHIV and other HIV-1C prevalent regions.MethodsSanger sequencing was used for Vif analysis in the cohort, and additional sequences were obtained from the Los Alamos database. Molecular modeling and docking techniques were employed to study the influence of subtype-specific variants on Vif-APOBEC3G binding affinity.ResultsThe findings showed distinct genetic variations between Vif sequences from India and Uganda, while South African sequences had wider distribution and closer relatedness to both. Specific amino acid substitutions in Vif were associated with geographic groups. Molecular modeling and docking analyses consistently identified specific residues (ARGR19, LYS26, TYR30, TYR44, and TRP79) as primary contributors to intermolecular contacts between Vif and APOBEC3G, essential for their interaction. The Indian Vif variant exhibited the highest predicted binding affinity to APOBEC3G among the studied groups.ConclusionsThese results provide insights into Vif sequence diversity in HIV-1C prevalent regions and shed light on differential pathogenesis observed in different geographical areas. The identified Vif amino acid residues warrant further investigation for their diagnostic, prognostic, and therapeutic potential.

Mutations in genes lpxL1, bamA, and pmrB impair the susceptibility of cystic fibrosis strains of Pseudomonas aeruginosa to murepavadin.

Murepavadin is a peptidomimetic exhibiting specific inhibitory activity against Pseudomonas species. In the present study, its in vitro activity was assessed on 230 cystic fibrosis (CF) strains of Pseudomonas aeruginosa isolated from 12 French hospitals, in comparison with 12 other antipseudomonal antibiotics. Although murepavadin is still in preclinical stage of development, 9.1% (n = 21) of strains had a minimum inhibitory concentration (MIC) >4 mg/L, a level at least 128-fold higher than the modal MIC value of the whole collection (≤0.06 mg/L). Whole-genome sequencing of these 21 strains along with more susceptible isogenic counterparts coexisting in the same patients revealed diverse mutations in genes involved in the synthesis (lpxL1 and lpxL2) or transport of lipopolysaccharides (bamA, lptD, and msbA), or encoding histidine kinases of two-component systems (pmrB and cbrA). Allelic replacement experiments with wild-type reference strain PAO1 confirmed that alteration of genes lpxL1, bamA, and/or pmrB can decrease the murepavadin susceptibility from 8- to 32-fold. Furthermore, we found that specific amino acid substitutions in histidine kinase PmrB (G188D, Q105P, and D45E) reduce the susceptibility of P. aeruginosa to murepavadin, colistin, and tobramycin, three antibiotics used or intended to be used (murepavadin) in aerosols to treat colonized CF patients. Whether colistin or tobramycin may select mutants resistant to murepavadin or the opposite needs to be addressed by clinical studies.

A novel synthetic peptide analog enhanced antibacterial activity of the frog-derived skin peptide wuchuanin-A1

In recent years, there has been a growing focus on the development of novel antibacterial compounds for clinical applications, such as antimicrobial peptide (AMP). Among the developed AMP, wuchuanin-A1, a coil-shaped bioactive peptide derived from Odorrana wuchuanensis frog skin, has been reported to exhibit antibacterial, antifungal, and antioxidant activity, but there are limited studies on its potential as an antibacterial agent. Therefore, this study aims to molecularly modify the sequence of wuchuanin-A1 to enhance its antibacterial properties. The interaction of both the native and analog peptide with bacterial inner membranes was initially assessed using computational methods. Specific amino acid substitutions were then used to enhance the modified peptide’s antibacterial efficacy, followed by several preliminary tests to evaluate its activity. This study bridges the gap in exploring the potential of wuchuanin-A1 for antibacterial purposes, providing insights into the design of effective antimicrobial agents. Communicated by Ramaswamy H. Sarma

Distinct SF3B1 Allele HEAT Repeat Location Is Associated with Co-Occurring Mutation Patterns in MDS

Background: In 2022, the WHO classified a new subtype of myelodysplastic neoplasms (MDS) with low blast count and SF3B1 mutation called MDS- SF3B1. SF3B1 is the major subunit of the SF3B spliceosome complex and is responsible for recognizing 3' splice sites in pre-mRNA for processing into mature mRNA. Mutations in SF3B1 have also been observed in other myeloid neoplasms. While MDS- SF3B1 has been associated with a better survival outcome, allelic variants such as SF3B1 K666N are associated with higher-risk MDS, transformation to AML and decreased OS. Additionally, SF3B1 E592K is more likely to be co-mutated with RUNX1, with a worse prognosis. Thus, not all SF3B1 allelic variants should be treated the same. The IPSS-M model incorporates SF3B1 mutations with different weights depending on co-mutations (i.e. isolated del(5q) or BCOR, BCORL1, RUNX1, NRAS, STAG2, SRSF2 mutations) but did not find a difference based on SF3B1 hotspot mutation. However, these and other studies have focused on specific amino acid substitutions but have not taken into consideration the protein domain structure of SF3B1. Using a large cohort of SF3B1-mutant myeloid malignancies, we determined whether HEAT repeat domain location was associated with differences between SF3B1-mutant myeloid neoplasms when comparing co-mutations and clinical characteristics. Methods: Bone marrow, peripheral blood, or FFPE tissue samples from a cohort of 2,996 unique patients with a suspected myeloid neoplasm from 6/30/2020-5/30/2023 were sequenced using a DNA 297 gene myeloid panel. We also analyzed a separate cohort of 112 patients from Sylvester Comprehensive Cancer Center (SCCC) and the University of Texas Southwestern Medical Center (UTSW) from 06/01/2017-06/30/2023 to validate the findings and assess for associations with clinical parameters such as blast count, time to disease progression, and survival. Statistics were performed using Chi-Square. SF3B1 has 22 HEAT repeats with SF3B1 mutations occurring predominately in HEAT Domains 2-6. For this study, we used UniProt definitions of amino acids 569-603 (HEAT2), 604-641 (HEAT3), 643-677 (HEAT4), 680-718 (HEAT5), and 763-801 (HEAT6) as shown in Figure 1. Results: Out of 2,996 myeloid neoplasm patients with SF3B1 mutations, the SF3B1 allele with the highest prevalence was K700E, which falls into HEAT repeat number 5, followed by K666N/R/T and H662Q in HEAT repeat number 4, and R635C in HEAT repeat number 3. Mutations in HEAT repeat 2 and 6 were less common. Several genes were significantly co-mutated in different SF3B1 heat domains, including ASXL1 (11%, p-value =<0.00001), CUX1 (4.7%, p-value = <0.00001), DNMT3A (22.5%, p-value = <0.00001), EZH2 (4.3%, p-value = 0.07), JAK2 (11.2%, p-value= <0.00001), RUNX1 (7.8%, p-value = <0.00001), STAG2 (3.2%, p-value = <0.00001), and TET2 (28.2%, p-value = 0.003) shown in Table 1. The most common co-mutations within each HEAT domain were: HEAT2- ASXL1 (80.0%), RUNX1 (55%), and STAG2 (25%); HEAT3- TET2 (29.5%), DNMT3A (17.5%), and ASXL1 (12.6%); HEAT4- TET2 (24%), D NMT3A (15.2%), and JAK2 (16.9%); HEAT5- TET2 (31.4%) and DNMT3A (31%);and HEAT6- TET2 (28.9%), ASXL1 (15.8%), DMNT3A (14.5%), and EZH2(10.5%). In a separate cohort of 112 patients with clinical follow-up, 97 were diagnosed with MDS- SF3B1: 53 (55%) were male and the average age at diagnosis of 70 years (range: 27-89 years old). A total of 88 patients had 297 gene myeloid panel information, which revealed a similar HEAT repeat distribution: HEAT3 (19%), HEAT4 (27%), HEAT5 (45%), and HEAT6 (6%). The median OS of the entire cohort was 10.5 years (CI 95% 4.93-NE). With a median follow-up of 2 years; 75, 81, 63 and 80% of patients were alive in HEAT 3, 4, 5 and 6, respectively. Conclusion: Distinct SF3B1 alleles defined by HEAT repeat location reflect distinct co-mutation patterns and may play a role in the biology of myeloid disorders. The relatively most enriched co-mutation patterns included: ASXL1 and RUNX1 in HEAT2, JAK2 in HEAT4, and EZH2 in HEAT6. Despite numerical differences in survival larger multicenter patient cohorts are needed to further define how distinct SF3B1 allelic variants and their HEAT repeat location correlate with prognosis and outcome in MDS and other myeloid neoplasms. In addition, further research is need into whether distinct SF3B1 alleles result in differences in RNA splicing signatures that may influence interactions with co-occurring mutations and disease biology.

Molecular evolution of wound healing-related genes during cetacean secondary aquatic adaptation.

The marine environment presents challenges for wound healing in cetaceans, despite their remarkable recovery abilities with minimal infections or complications. However, the molecular mechanism underlying this efficient wound healing remains underexplored. To better understand the molecular mechanisms behind wound healing in cetaceans, we investigated the evolutionary patterns of 37 wound healing-related genes in representative mammals. We found wound healing-related genes experience adaptive evolution in cetaceans: (1) Three extrinsic coagulation pathway-related genes-tissue factor (F3), coagulation factor VII (F7), and coagulation factor X (F10)-are subject to positive selection in cetaceans, which might promote efficient hemostasis after injury; positive selection in transforming growth factor-beta 2 (TGF-β2), transforming growth factor-beta 3 (TGF-β3), and platelet-derived growth factor D (PDGFD), which play immunological roles in wound healing, may help cetaceans enhance inflammatory response and tissue debridement. (2) Coagulation factor XII (F12) is the initiation factor in the intrinsic coagulation pathway. It had a premature stop codon mutation and was subjected to selective stress relaxation in cetaceans, suggesting that the early termination of F12 may help cetaceans avoid the risk of vascular blockage during diving. (3) Fibrinogen alpha chain (FGA) and FIII, which were detected to contain the specific amino acid substitutions in marine mammals, indicating similar evolutionary mechanisms might exist among marine mammals to maintain strong wound-healing ability. Thus, our research provides further impetus to study the evolution of the wound healing system in cetaceans and other marine mammals, extending knowledge of preventing coagulation disorder and atherosclerosis in humans.

Effects of tea, catechins and catechin derivatives on Omicron subvariants of SARS-CoV-2

The Omicron subvariants of SARS-CoV-2 have multiple mutations in the S-proteins and show high transmissibility. We previously reported that tea catechin (−)-epigallocatechin gallate (EGCG) and its derivatives including theaflavin-3,3’-di-O-digallate (TFDG) strongly inactivated the conventional SARS-CoV-2 by binding to the receptor binding domain (RBD) of the S-protein. Here we show that Omicron subvariants were effectively inactivated by green tea, Matcha, and black tea. EGCG and TFDG strongly suppressed infectivity of BA.1 and XE subvariants, while effect on BA.2.75 was weaker. Neutralization assay showed that EGCG and TFDG inhibited interaction between BA.1 RBD and ACE2. In silico analyses suggested that N460K, G446S and F490S mutations in RBDs crucially influenced the binding of EGCG/TFDG to the RBDs. Healthy volunteers consumed a candy containing green tea or black tea, and saliva collected from them immediately after the candy consumption significantly decreased BA.1 virus infectivity in vitro. These results indicate specific amino acid substitutions in RBDs that crucially influence the binding of EGCG/TFDG to the RBDs and different susceptibility of each Omicron subvariant to EGCG/TFDG. The study may suggest molecular basis for potential usefulness of these compounds in suppression of mutant viruses that could emerge in the future and cause next pandemic.

Prevalence and genomic analysis of t203-like G9 (G9-VI) rotaviruses circulating in children with gastroenteritis in Beijing, China

Our previous surveillance revealed that t203-like G9 (tentatively designated subtype G9-VI) rotaviruses re-emerged in 2010 in Beijing and rapidly prevailed over the G9-III subtype (the most common G9 subtype globally) and previously predominant G genotypes over the following two years. G9-VI belongs to the VP7 evolutionary lineage VI, which includes unusual and sporadic human rotaviruses from China (t203) and Japan. To obtain insight into the epidemiology, evolution, and transmission advantages of G9-VI rotavirus, we performed follow-up surveillance (2014-2017) and whole-genome analysis of 12 representative G9 strains. The results showed that the G9 genotype was predominant (77.4%), with a marked increase in prevalence (previously 43.5%). Within the G9 genotype, subtype G9-VI accounted for the majority (98.3%) of cases. The most prevalent P-genotype was P[8] (93.7%), within which subtype P[8]b was rare (0.7%). Phylogenetically, the G9-VI subtype strains in this study clustered closely with contemporary emerging human rotaviruses from many other countries in VP7 lineage VI, indicating that this subtype is capable of spreading globally. These currently emerging G9-VI rotaviruses formed a distinct monophyletic subcluster when compared to early G9-VI rotaviruses. Furthermore, four specific amino acid substitutions and synonymous codon substitutions were observed in the VP7 genes between the current G9-VI and globally common G9-III rotaviruses. The remaining nine genes of all of the analyzed representative G9 strains, whether G9-VI or G9-III, combined with the P[8]a, P[8]b, or P[6] genotype and exhibited the same Wa-like backbone constellation.

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract.

The lower female reproductive tract is notoriously dominated by Lactobacillus species, among which Lactobacillus crispatus emerges for its protective and health-promoting activities. Although previous comparative genome analyses highlighted genetic and phenotypic diversity within the L. crispatus species, most studies have focused on the presence/absence of accessory genes. Here, we investigated the variation at the single nucleotide level within protein-encoding genes shared across a human-derived L. crispatus strain selection, which includes 200 currently available human-derived L. crispatus genomes as well as 41 chromosome sequences of such taxon that have been decoded in the framework of this study. Such data clearly pointed out the presence of intra-species micro-diversities that could have evolutionary significance contributing to phenotypical diversification by affecting protein domains. Specifically, two single nucleotide variations in the type II pullulanase gene sequence led to specific amino acid substitutions, possibly explaining the substantial differences in the growth performances and competition abilities observed in a multi-strain bioreactor culture simulating the vaginal environment. Accordingly, L. crispatus strains display different growth performances, suggesting that the colonisation and stable persistence in the female reproductive tract between the members of this taxon is highly variable.

Organotypic hippocampal culture model reveals differential responses to highly similar Zika virus isolates

IntroductionZika virus (ZIKV) caused an outbreak in Brazil, in 2015, being associated to microcephaly. ZIKV has a strong neurotropism leading to death of infected cells in different brain regions, including the hippocampus, a major site for neurogenesis. The neuronal populations of the brain are affected differently by ZIKV from Asian and African ancestral lineages. However, it remains to be investigated whether subtle variations in the ZIKV genome can impact hippocampus infection dynamics and host response.ObjectiveThis study evaluated how two Brazilian ZIKV isolates, PE243 and SPH2015, that differ in two specific missense amino acid substitutions, one in the NS1 protein and the other in the NS4A protein, affect the hippocampal phenotype and transcriptome.MethodsOrganotypic hippocampal cultures (OHC) from infant Wistar rats were infected with PE243 or SPH2015 and analyzed in time series using immunofluorescence, confocal microscopy, RNA-Seq and RT-qPCR.ResultsUnique patterns of infection and changes in neuronal density in the OHC were observed for PE243 and SPH2015 between 8 and 48 h post infection (p.i.). Phenotypic analysis of microglia indicated that SPH2015 has a greater capacity for immune evasion. Transcriptome analysis of OHC at 16 h p.i. disclosed 32 and 113 differentially expressed genes (DEGs) in response to infection with PE243 and SPH2015, respectively. Functional enrichment analysis suggested that infection with SPH2015 activates mostly astrocytes rather than microglia. PE243 downregulated biological process of proliferation of brain cells and upregulated those associated with neuron death, while SPH2015 downregulated processes related to neuronal development. Both isolates downregulated cognitive and behavioral development processes. Ten genes were similarly regulated by both isolates. They are putative biomarkers of early hippocampus response to ZIKV infection. At 5, 7, and 10 days p.i., neuronal density of infected OHC remained below controls, and mature neurons of infected OHC showed an increase in the epigenetic mark H3K4me3, which is associated to a transcriptionally active state. This feature is more prominent in response to SPH2015.ConclusionSubtle genetic diversity of the ZIKV affects the dynamics of viral dissemination in the hippocampus and host response in the early stages of infection, which may lead to different long-term effects in neuronal population.

Abstract B021: Pan-RAS IMM-1-104 activity in humanized 3D tumor models is independent of specific amino acid substitution

Abstract Introduction: Novel dual-MEK inhibitor IMM-1-104 is under clinical investigation for use in RAS-addicted solid tumors. Approved KRAS G12C inhibitors are available but cover a limited subset of patients. For example, the KRAS G12C substitution occurs in only 1-3% of pancreatic cancers. We assessed response to IMM-1-104 across RAS mutant preclinical models to determine whether a preference was observed for mutation position, or for specific amino acid substitutions. Experimental Procedures: Response to IMM-1-104 was measured in a humanized 3D tumor growth assay across 133 tumor models. Sixty-nine of these models have a reported RAS mutation, and all models are being mutationally profiled by whole exome sequencing, with the majority (~75%) completed to date. The RAS-mutant panel spans 11 tissue types and includes a subset of 30 confirmed KRAS G12 mutated cell lines drawn from three major indications: 12 pancreatic, 11 lung, and 7 colorectal cancer models. Based on the 3D assay, cell lines were classified into sensitive, intermediate, and resistant to IMM-1-104. The distribution of responses was then assessed across mutation position and amino acid substitutions. Summary of New Data: In the full dataset across 69 RAS-mutant models, at least one model displayed response to IMM-1-104 (sensitive or intermediate) for each observed mutation in K/N/HRAS. That is, no particular mutation position or amino acid substitution was exclusively found to be resistant. Association of response with amino acid identity was further evaluated in a subset of lines for the most frequently altered residue in KRAS, G12. A distribution of responses was observed for each amino acid substitution. The G12 substitutions having at least four cell lines each total across the three indications included G12C (8 lines), D (5 lines), R (4 lines), and V (11 lines). Across cell lines for each of these substitutions, multiple response categories were observed. In each case, half or more lines fell into the intermediate category with the rest falling into one or both of the other response categories. For example, out of the 8 KRAS G12C lines, 6 showed intermediate response, 1 showed resistance, and 1 showed sensitivity. Examining these distributions together, no significant statistical relationship was seen between the amino acid substitution and response categories by Fisher’s exact test. Conclusions: Across 69 RAS-mutated cell lines, each mutation position or amino acid substitution was associated with sensitive or intermediate response in at least one line. Looking at the most commonly mutated position in KRAS, across 30 KRAS G12 mutated cell lines from three cancer indications, no significant preference was observed with respect to response to IMM-1-104 for a particular amino acid at G12. These observations suggest potential relevance of IMM-1-104 to a broad RAS-driven patient population. Citation Format: Sarah Kolitz, Praveen Nair, Mai Johnson, Jason Funt, Peter J. King, Kevin D. Fowler, Anna Travesa, Frank Wang, John Brothers II, Amy Axel, Scott Barrett, Benjamin J. Zeskind, Brett Hall. Pan-RAS IMM-1-104 activity in humanized 3D tumor models is independent of specific amino acid substitution [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B021.

The Roles of Fibroblast Growth Factor Receptor 3 (FGFR3) in the Spectrum of Skeletal Dysplasia

The fibroblast growth factor receptor 3 (FGFR3) gene mutations were identified to be involved in the pathogenesis of most chondropathies. The FGFR3 gene encodes the FGFR3 receptor and is involved in the regulation of bone growth by limiting the ossification of long bones. Mutations of the FGFR3 gene result in abnormal cell proliferation and improper cartilage development. We aim to provide an overview of the roles of FGFR3 in skeletal dysplasia by highlighting the pathogenesis, clinical variants of skeletal dysplasia, diagnosis, and their management.
 Achondroplasia is the most common form of chondropathies, occurring in approximately 1 in 20,000-30,000 live births, and it is the most common form of genetic dwarfism. In over 80% of cases of achondroplasia, the mutation is sporadic, and only 20% are inherited autosomal dominant. Achondroplasia results from a point mutation in the gene encoding the transmembrane portion of FGFR3. Two viable base substitutions are identified in achondroplasia, including a point mutation of guanine substituted for adenine (c.1138G>A); this is identified in approximately 98% of the affected individuals, and transversion of guanine to cytosine (c.1138G>C).
 Hypochondroplasia is a milder form of chondropathies with an incidence between 1 in 33,000 and 1 in 47,000 live births. Missense mutations of FGFR3 (p.Asn540Lys) are isolated in tyrosine kinase domain I occurring in approximately 60% of cases, and missense mutation of FGFR3 (p.Lys650Asn) identified in the tyrosine kinase domain II of FGFR3. Thanatophoric dysplasia is the most lethal form of chondropathies, with neonatal fetal death secondary to pulmonary hypoplasia. In thanatophoric dysplasia, there is Lys650Met substitution in FGFR3 (Type I) with impairment of endochondral bone growth and a pathogenic variant of p.Lys650Glu substitution in FGFR3 (Type II). In addition, specific amino acid substitution in the FGFR3 gene (G380R) was identified to be associated with severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN).
 The diagnosis of achondroplasias is routinely made from clinical presentations and radiological findings. FGFR3 molecular genetic testing is performed in children with atypical presentations. In addition, a pre-implantation genetic diagnosis should be available for parents pursuing in-vitro fertilization and embryo implantation procedures. Management of chondropathies includes symptomatic treatment with drugs, surgical intervention, and lifelong follow-up care. Different pharmacological options have been used, including those that directly block FGFR3 activation or regulate signalling pathways controlling chondrocyte proliferation and differentiation.

Molecular Evolution of Human Parainfluenza Virus Type 2 Based on Hemagglutinin-Neuraminidase Gene.

To understand the molecular evolution of human parainfluenza virus type 2 (HPIV2), 21 Hemagglutinin-Neuraminidase (HN) gene sequences covering seven Chinese provinces in 2011 and 2017 to 2021 were combined with 90 published HN sequences worldwide for phylogenetic analysis. The result showed that global HPIV2 could be classified into two distinct clusters (I and II), five lineages (IA to IIE), and four sublineages (IB1 and 2, and IIE1 and 2). The minimum genetic distances between different clusters and lineages were 0.049 and 0.014, respectively. In the last decade, one lineage (IID) and three sublineages (IB1, IB2, and IIE1) have been cocirculating in China, with the sublineages IB2 and IIE1 dominating, while sublineages IB1 and IIE1 are dominant globally. In addition, the spread of HPIV2 had relative spatial clustering, and sublineage IB2 has only been detected in China thus far. The overall evolution rate of HPIV2 was relatively low, on the order of 10-4 substitutions/site/year, except for sublineage IB2 at 10-3 substitutions/site/year. Furthermore, human-animal transmission was observed, suggesting that the HPIV2 might have jumped out of animal reservoirs in approximately 1922, the predicted time of a common ancestor. The entire HN protein was under purifying/negative selection, and the specific amino acid changes and two novel N-glycosylation sites (N316 and N517) in sublineages IB1, IB2, and IIE1 were mostly located in the globular head region of the HN protein. In this study, preliminary evolutionary characteristics of HPIV2 based on the HN gene were obtained, increasing the recognition of the evolution and adaptation of HPIV2. IMPORTANCE The phylogenetic analysis showed that global HPIV2 could be classified into two distinct clusters (I and II) and five lineages (IA to IIE) with at least 0.049 and 0.014 genetic distances between clusters and lineages, respectively. Furthermore, lineages IB and IIE could be further divided into two sublineages (IB1-2 and IIE1-2). All China sequences belong to one lineage and three sublineages (IB1, IB2, IID, and IIE1), among which sublineages IB2 and IIE1 are predominant and cocirculating in China, while sublineages IB1 and IIE1 are dominant globally. The overall evolution rate of HPIV2 is on the order of 10-4 substitutions/site/year, with the highest rate of 2.18 × 10-3 for sublineage IB2. The entire HN protein is under purifying/negative selection, and the specific amino acid substitutions and two novel N-glycosylation sites (N316 and N517) in sublineages IB1, IB2, and IIE1 are mostly located in the globular head region of the HN protein.

Identification of the NA+/K+-ATPase α-Isoforms in Six Species of Poison Dart Frogs and their Sensitivity to Cardiotonic Steroids

Cardiotonic steroids (CTS) are a group of compounds known to be toxic due to their ability to inhibit the Na+/K+-ATPase (NKA), which is essential to maintain the balance of ions in animal cells. An evolutionary strategy of molecular adaptation to avoid self-intoxication acquired by CTS defended organisms and their predators is the structural modification of their NKA where specific amino acid substitutions confer resistant phenotypes. Several lineages of poison dart frogs (Dendrobatidae) are well known to sequester a wide variety of lipophilic alkaloids from their arthropod diet, however there is no evidence of CTS-sequestration or dietary exposure. Interestingly this study identified the presence of α-NKA isoforms (α1 and α2) with amino acid substitutions indicative of CTS-resistant phenotypes in skeletal muscle transcriptomes obtained from six species of dendrobatids: Phyllobates aurotaenia, Oophaga anchicayensis, Epipedobates boulengeri, Andinobates bombetes, Andinobates minutus, and Leucostethus brachistriatus, collected in the Valle del Cauca (Colombia). P. aurotaenia, A. minutus, and E. boulengeri presented two variants for α1-NKA, with one of them having these substitutions. In contrast, O. anchicayensis and A. bombetes have only one α1-NKA isoform with an amino acid sequence indicative of CTS susceptibility and an α2-NKA with one substitution that could confer a reduced affinity for CTS. The α1 and α2 isoforms of L. brachistriatus do not contain substitutions imparting CTS resistance. Our findings indicate that poison dart frogs express α-NKA isoforms with different affinities for CTS and the pattern of this expression might be influenced by factors related to evolutionary, physiological, ecological, and geographical burdens.

Multiple amino acid substitutions in penicillin-binding protein-1A confer amoxicillin resistance in refractory Helicobacter pylori infection.

Amoxicillin resistance in Helicobacter pylori is mainly associated with mutations in penicillin-binding protein-1A (PBP-1A). However, the specific amino acid substitutions in PBP-1A that confer amoxicillin resistance in H. pylori remain to be investigated. This study aimed to investigate the molecular mechanism underlying amoxicillin resistance in patients with refractory H. pylori infection. Esophagogastroduodenoscopy (EGD) was performed in patients with persistent H. pylori infection after at least two courses of H. pylori eradication therapy between January-2018 to March-2021. Refractory H. pylori was cultured from the gastric biopsy specimens. Antibiotic susceptibility testing was conducted to determine the minimum inhibitory concentrations (MICs). Sequence analysis of pbp-1A was performed for amoxicillin-resistant strains. Thirty-nine successfully cultured isolates were classified as refractory H. pylori isolates, and seventeen isolates were resistant to amoxicillin (MIC > 0.125mg/L). Sequence analysis of resistant strains showed multiple mutations in the C-terminal region of PBP-1A that conferred amoxicillin resistance in H. pylori. However, the number of PBP-1A mutations did not correlate with the high MICs of amoxicillin-resistant isolates. Notably, some amino acid substitutions were identified in all Taiwanese isolates with history of eradication failure but not in published amoxicillin-susceptible strains, suggesting that the mutations may play a role in conferring antibiotic resistance to these strains. Our results show that amoxicillin resistance in refractory H. pylori is highly correlated with numerous PBP-1A mutations that are strain specific. Continuous improvements in diagnostic tools, particularly molecular analysis approaches, can help to optimize current antimicrobial regimens.

Evolutionary analysis of p38 stress-activated kinases in unicellular relatives of animals suggests an ancestral function in osmotic stress.

p38 kinases are key elements of the cellular stress response in animals. They mediate the cell response to a multitude of stress stimuli, from osmotic shock to inflammation and oncogenes. However, it is unknown how such diversity of function in stress evolved in this kinase subfamily. Here, we show that the p38 kinase was already present in a common ancestor of animals and fungi. Later, in animals, it diversified into three JNK kinases and four p38 kinases. Moreover, we identified a fifth p38 paralog in fishes and amphibians. Our analysis shows that each p38 paralog has specific amino acid substitutions around the hinge point, a region between the N-terminal and C-terminal protein domains. We showed that this region can be used to distinguish between individual paralogs and predict their specificity. Finally, we showed that the response to hyperosmotic stress in Capsaspora owczarzaki, a close unicellular relative of animals, follows a phosphorylation-dephosphorylation pattern typical of p38 kinases. At the same time, Capsaspora's cells upregulate the expression of GPD1 protein resembling an osmotic stress response in yeasts. Overall, our results show that the ancestral p38 stress pathway originated in the root of opisthokonts, most likely as a cell's reaction to salinity change in the environment. In animals, the pathway became more complex and incorporated more stimuli and downstream targets due to the p38 sequence evolution in the docking and substrate binding sites around the hinge region. This study improves our understanding of p38 evolution and opens new perspectives for p38 research.

Rubbing Salt in the Wound: Molecular Evolutionary Analysis of Pain-Related Genes Reveals the Pain Adaptation of Cetaceans in Seawater.

Pain, usually caused by a strong or disruptive stimulus, is an unpleasant sensation that serves as a warning to organisms. To adapt to extreme environments, some terrestrial animals have evolved to be inherently insensitive to pain. Cetaceans are known as supposedly indifferent to pain from soft tissue injury representatives of marine mammals. However, the molecular mechanisms that explain how cetaceans are adapted to pain in response to seawater environment remain unclear. Here, we performed a molecular evolutionary analysis of pain-related genes in selected representatives of cetaceans. ASIC4 gene was identified to be pseudogenized in all odontocetes (toothed whales) except from Physeter macrocephalus (sperm whales), and relaxed selection of this gene was detected in toothed whales with pseudogenized ASIC4. In addition, positive selection was detected in pain perception (i.e., ASIC3, ANO1, CCK, and SCN9A) and analgesia (i.e., ASIC3, ANO1, CCK, and SCN9A) genes among the examined cetaceans. In this study, potential convergent amino acid substitutions within predicted proteins were found among the examined cetaceans and other terrestrial mammals, inhabiting extreme environments (e.g., V441I of TRPV1 in cetaceans and naked mole rats). Moreover, specific amino acid substitutions within predicted sequences of several proteins were found in the studied representatives of cetaceans (e.g., F56L and D163A of ASIC3, E88G of GRK2, and F159L of OPRD1). Most of the substitutions were located within important functional domains of proteins, affecting their protein functions. The above evidence suggests that cetaceans might have undergone adaptive molecular evolution in pain-related genes through different evolutionary patterns to adapt to pain, resulting in greater sensitivity to pain and more effective analgesia. This study could have implications for diagnosis and treatment of human pain.