Mutational Changes Research Articles

Development of a Reagent Kit for the Quantitative Determination of Hepatitis B Virus (HBV) DNA in Clinical Material by PCR with Hybridization-Fluorescence Detection

Relevance.Hepatitis B virus (HBV) is one of the most common viral infections affecting people worldwide and can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently 3% of the world's population are infected with hepatitis B virus and are at risk of developing life-threatening liver disease. Immunological and molecular biological methods of detection of HBV are currently used in laboratory diagnostics. The polymerase chain reaction (PCR) is currently the most sensitive method for the detection and quantification of HBV. HBV DNA quantification is widely used to monitor the antiviral treatment of HBV infection.Aim.To develop a real-time PCR kit for the quantification of HBV DNA.Materials and methods.A total of 200 plasma and serum samples positive and negative for HBV were used in the development. The performance of the developed kit was compared with the use of other commercially registered HBV diagnostic kits in Russia. Additionally, the nucleotide sequences of all existing virus genotypes analysed for the selection of primers using GeneBank system.Results and discussion.Comparison analysis of the results of quantitative determination by real-time PCR in 200 clinical serum and blood plasma samples showed that the diagnostic sensitivity of the developed kit was 100% and specificity 100%. The primers developed specific to the POL gene region. The kit is capable of detecting all types of virus genotypes.Conclusions.The developed reagent kit allows detection of hepatitis B virus and determination of its quantity within 70 minutes. In addition to a large number of genotypes and subgenotypes, the virus is characterized by mutational changes in the genome, which complicates its diagnosis and, as a consequence, the ongoing therapy with drugs. Conservative regions for primer and probe selection taken into account in the development, and the sequencing results obtained are applicable to all HBV genotypes. The reagent kit is designed to monitor HBV infected patients and will allow the analysis of different HBV viral loads.

Advanced Age in Humans and Mouse Models of Glioblastoma Show Decreased Survival from Extratumoral Influence.

Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan. Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy. Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice. This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM.

Remdesivir increases mtDNA copy number causing mild alterations to oxidative phosphorylation

SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it accumulates intracellularly. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA). These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated us to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu cells (35.26% increase ± 11.33%) and liver (100.27% increase ± 32.73%) upon treatment. However, these increases only resulted in mild changes to mitochondrial function. Surprisingly, skeletal muscle and heart were extremely resistant to RDV treatment, tissues that have preferentially been affected by other nucleoside analogues. Although our data suggest that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease.

347. LASER CAPTURE MICRODISSECTION AND WHOLE GENOME SEQUENCING OF ESOPHAGEAL ADENOCARCINOMA SAMPLES WITH MINIMAL TUMOUR

Abstract Background Esophageal adenocarcinoma (EAC) samples, particularly biopsies, can have low tumour cellularity, which limits their use for sequencing. Previous studies have focused on untreated resection specimens with high cellularity (i.e. TCGA requiring 60% cellularity on histology). Laser capture microdissection (LCM) is a method to enrich tumour cells from histological specimens and has been successfully used in other samples. We hypothesize that LCM can enrich for tumour from biopsies and resection specimens with very low cellularity. Methods Biopsy and resection EAC specimens were subjected to laser capture microdissection and whole genome sequencing (N = 49 biopsies and N = 15 resections) as part of the MOCHA trial (NCT04219137). LCM samples were reviewed by a board-certified pathologist to quantify tumour cellularity prior to LCM. Matched germline DNA was extracted from patient blood. Samples were processed using an in-house pipeline to call somatic single nucleotide variants (SNVs), insertions, deletions, copy number changes, and structural variants. Post LCM tumour cellularity was estimated from the WGS data. Results We had slightly higher WGS tumour cellularity for biopsies (66.2% +/− 16.8) and resections (71.3% +/− 18.0) than TCGA (58.1% +/− 17.5), while our histological cellularity was much lower than TCGA’s 60% cutoff at 32.2% +/− 24.5 for biopsies and 31.0% +/− 22.7 for resections. Overall, our LCM strategy led to an increase of 35.4 +/− 25.1% for cellularity by WGS compared to the histology. Finally, we observed a similar tumour ploidy and SNV composition as TCGA, which further verified that our tumour enrichment strategy was successful. Conclusion We have successfully applied an LCM strategy to enrich for tumour cells from low cellularity specimens. For example, we have successfully enriched for tumour cells from specimens with as low as 1% histological cellularity. We are currently exploring the results of this dataset including clinical correlations, changes in potential driver mutations from biopsy to resection, and comparing our results to treatment response.

Evaluating the Cysteine-Rich and Catalytic Subdomains of Human Tyrosinase and OCA1-Related Mutants Using 1 μs Molecular Dynamics Simulation.

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a 'molten globule' state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain's rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism.

Transcription factor expression levels and environmental signals constrain transcription factor innovation.

Evolutionary innovation of transcription factors frequently drives phenotypic diversification and adaptation to environmental change. Transcription factors can gain or lose connections to target genes, resulting in novel regulatory responses and phenotypes. However the frequency of functional adaptation varies between different regulators, even when they are closely related. To identify factors influencing propensity for innovation, we utilise a Pseudomonas fluorescens SBW25 strain rendered incapable of flagellar mediated motility in soft-agar plates via deletion of the flagellar master regulator (fleQ). This bacterium can evolve to rescue flagellar motility via gene regulatory network rewiring of an alternative transcription factor to rescue activity of FleQ. Previously, we have identified two members (out of 22) of the RpoN-dependent enhancer binding protein (RpoN-EBP) family of transcription factors (NtrC and PFLU1132) that are capable of innovating in this way. These two transcription factors rescue motility repeatably and reliably in a strict hierarchy – with NtrC the only route in a ∆fleQ background, and PFLU1132 the only route in a ∆fleQ∆ntrC background. However, why other members in the same transcription factor family have not been observed to rescue flagellar activity is unclear. Previous work shows that protein homology cannot explain this pattern within the protein family (RpoN-EBPs), and mutations in strains that rescued motility suggested high levels of transcription factor expression and activation drive innovation. We predict that mutations that increase expression of the transcription factor are vital to unlock evolutionary potential for innovation. Here, we construct titratable expression mutant lines for 11 of the RpoN-EBPs in P. fluorescens . We show that in five additional RpoN-EBPs (FleR, HbcR, GcsR, DctD, AauR and PFLU2209), high expression levels result in different mutations conferring motility rescue, suggesting alternative rewiring pathways. Our results indicate that expression levels (and not protein homology) of RpoN-EBPs are a key constraining factor in determining evolutionary potential for innovation. This suggests that transcription factors that can achieve high expression through few mutational changes, or transcription factors that are active in the selective environment, are more likely to innovate and contribute to adaptive gene regulatory network evolution.

Baseline mutations and ctDNA dynamics as prognostic and predictive factors in ER-positive/HER2-negative metastatic breast cancer patients.

Prognostic and predictive biomarkers to CDK4/6 inhibitors are lacking. Circulating tumour DNA (ctDNA) can be used to profile these patients and dynamic changes in ctDNA could be an early predictor of treatment efficacy. Here, we conducted plasma ctDNA profiling in patients from the PEARL trial comparing palbociclib+fulvestrant versus capecitabine to investigate associations between baseline genomic landscape and on-treatment ctDNA dynamics with treatment efficacy. Correlative blood samples were collected at baseline (C1D1) and prior to treatment (C1D15). Plasma ctDNA was sequenced with a custom error-corrected capture panel, with both univariate and multivariate Cox models used for treatment efficacy associations. A pre-specified methodology measuring ctDNA changes in clonal mutations between C1D1 and C1D15 was used for the on-treatment ctDNA dynamic model. 201 patients were profiled at baseline, with ctDNA detection associated with worse PFS/OS. Detectable TP53 mutation showed worse PFS and OS in both treatment arms, even after restricting population to baseline ctDNA detection. ESR1 mutations were associated with worse OS overall, which was lost when restricting population to baseline ctDNA detection. PIK3CA mutations confer worse OS only to patients on the palbociclib+fulvestrant treatment arm. ctDNA dynamics analysis (n=120) showed higher ctDNA suppression in the capecitabine arm. Patients without ctDNA suppression showed worse PFS in both treatment arms. We show impaired survival irrespective of endocrine or chemotherapy-based treatments for patients with HR+/HER2- MBC harbouring plasma TP53 mutations. Early ctDNA suppression may provide treatment efficacy predictions. Further validation to fully demonstrate clinical utility of ctDNA dynamics is warranted.

Mechanistic insights into the allosteric inactivation mechanism of ZAP-70 induced by the hot spot W165C mutation

Zeta chain-associated protein kinase 70 (ZAP-70) is a non-receptor tyrosine kinase that interacts with the activated T-cell receptor to transduce downstream signals, and thus plays an important role in the adaptive immune system. The biphosphorylated immunotyrosine-based activation motifs (ITAM-Y2P) binds to the N-SH2 and C-SH2 domains of ZAP-70 to promote the activation of ZAP-70. The present study explores molecular mechanisms of allosteric inactivation of ZAP-70 induced by the hot spot W165C mutation through atomically detailed molecular dynamics simulation approaches. We report microsecond-length simulations of two states of the tandem SH2 domains of ZAP-70 in complex with the ITAM-Y2P motif, including the wild-type and W165C mutant. Extensive analysis of local flexibility and dynamical correlated motions show that W165C mutation changes coupled motions of protein domains and community networks. The binding affinities of the ITAM-Y2P motif to the wild-type and W165C mutant of ZAP-70 are predicted using binding free energy calculations. The results suggest that the driving force to decrease the binding affinity in the W165C mutant derives from the difference in the protein-protein electrostatic interactions. Moreover, the per-residue free energy decomposition unravels that the contributions from residues in the phosphorylated Tyr315 (pY315) binding site, in particular pY315 of ITAM-Y2P, and Arg43, Tyr240 of ZAP-70, are the key determinants for the loss of binding affinity. This study may insights into our understanding of the pathological mechanism of ZAP-70. Communicated by Ramaswamy H. Sarma

The Molecular and Genetic Interactions between Obesity and Breast Cancer Risk.

Breast cancer (BC) is considered the leading cause of death among females worldwide. Various risk factors contribute to BC development, such as age, genetics, reproductive factors, obesity, alcohol intake, and lifestyle. Obesity is considered to be a pandemic health problem globally, affecting millions of people worldwide. Obesity has been associated with a high risk of BC development. Determining the impact of obesity on BC development risk in women by demonstrating the molecular and genetic association in pre- and post-menopause females and risk to BC initiation is crucial in order to improve the diagnosis and prognosis of BC disease. In epidemiological studies, BC in premenopausal women was shown to be protective in a certain pattern. These altered effects between the two phases could be due to various physiological changes, such as estrogen/progesterone fluctuating levels. In addition, the relationship between BC risk and obesity is indicated by different molecular alterations as metabolic pathways and genetic mutation or epigenetic DNA changes supporting a strong connection between obesity and BC risk. However, these molecular and genetic alteration remain incompletely understood. The aim of this review is to highlight and elucidate the different molecular mechanisms and genetic changes occurring in obese women and their association with BC risk and development.

Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs.

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNAleu for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, Δ(ΔG‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with 32Pα-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Parallel genetic adaptation of Bacillus subtilis to different plant species.

Plant growth-promoting rhizobacteria benefit plants by stimulating their growth or protecting them against phytopathogens. Rhizobacteria must colonize and persist on plant roots to exert their benefits. However, little is known regarding the processes by which rhizobacteria adapt to different plant species, or behave under alternating host plant regimes. Here, we used experimental evolution and whole-population whole-genome sequencing to analyse how Bacillus subtilis evolves on Arabidopsis thaliana and tomato seedlings, and under an alternating host plant regime, in a static hydroponic setup. We observed parallel evolution across multiple levels of biological organization in all conditions, which was greatest for the two heterogeneous, multi-resource, spatially structured environments at the genetic level. Species-specific adaptation at the genetic level was also observed, possibly caused by the selection stress imposed by different host plants. Furthermore, a trade-off between motility and biofilm development was supported by mutational changes in motility- and biofilm-related genes. Finally, we identified several condition-specific and common targeted genes in different environments by comparing three different B. subtilis biofilm adaptation settings. The results demonstrate a common evolutionary pattern when B. subtilis is adapting to the plant rhizosphere in similar conditions, and reveal differences in genetic mechanisms between different host plants. These findings will likely support strain improvements for sustainable agriculture.

Apo state pore opening as functional basis of increased EAAT anion channel activity in episodic ataxia 6.

SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Mutational signature dynamics shaping the evolution of oesophageal adenocarcinoma

A variety of mutational processes drive cancer development, but their dynamics across the entire disease spectrum from pre-cancerous to advanced neoplasia are poorly understood. We explore the mutagenic processes shaping oesophageal adenocarcinoma tumorigenesis in 997 instances comprising distinct stages of this malignancy, from Barrett Oesophagus to primary tumours and advanced metastatic disease. The mutational landscape is dominated by the C[T > C/G]T substitution enriched signatures SBS17a/b, which are linked with TP53 mutations, increased proliferation, genomic instability and disease progression. The APOBEC mutagenesis signature is a weak but persistent signal amplified in primary tumours. We also identify prevalent alterations in DNA damage repair pathways, with homologous recombination, base and nucleotide excision repair and translesion synthesis mutated in up to 50% of the cohort, and surprisingly uncoupled from transcriptional activity. Among these, the presence of base excision repair deficiencies show remarkably poor prognosis in the cohort. In this work, we provide insights on the mutational aetiology and changes enabling the transition from pre-neoplastic to advanced oesophageal adenocarcinoma.

Clonal Myeloid Dysplasia Following CAR T-Cell Therapy: Chicken or the Egg?

Multiple myeloma (MM) is characterized by recurrent relapses. Consequently, patients receive multiple therapy lines, including alkylating agents and immune modulators, which have been associated with secondary malignancies such as myelodysplastic syndrome (MDS). Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T cell (CART) therapy is efficacious in patients with relapsed/refractory (R/R) MM. However, the long-term complications, particularly MDS, are not well understood. Whether CART therapy causes or promotes MDS has not been thoroughly investigated. In this study, we explored the causal relationship between MDS and CART therapy. We retrospectively examined the prevalence of MDS-related morphological and mutational changes before and after administration of CART therapy in five patients. Among them, four developed MDS after CART therapy, while one had pre-existing MDS prior to CART. None of the four patients who developed post-CART MDS showed morphological MDS changes prior to CART therapy. However, all four patients exhibited molecular alterations associated with MDS in their pre-CART as well as post-CART therapy bone marrow. No new mutations were observed. Our findings provide initial evidence suggesting that anti-BCMA CART therapy in MM may promote expansion of pre-existing MDS clones rather than causing development of new clones.

Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Comparison of Attenuated and Virulent Strains of African Swine Fever Virus Genotype I and Serogroup 2.

African swine fever (ASF) is a contagious disease of pigs caused by the ASF virus (ASFV). The main problem in the field of ASF control is the lack of vaccines. Attempts to obtain vaccines by attenuating the ASFV on cultured cell lines led to the production of attenuated viruses, some of which provided protection against infection with a homologous virus. Here we report on the biological and genomic features of the attenuated Congo-a (KK262) virus compared to its virulent homologue Congo-v (K49). Our results showed differences in in vivo replication and virulence of Congo-a. However, the attenuation of the K49 virus did not affect its ability to replicate in vitro in the primary culture of pig macrophages. Complete genome sequencing of the attenuated KK262 strain revealed an 8,8 kb deletion in the left variable region of the genome compared to the virulent homologue K49. This deletion concerned five genes of MGF360 and three genes of MGF505. In addition, three inserts in the B602L gene, genetic changes in intergenic regions and missense mutations in eight genes were detected. The data obtained contribute to a better understanding of ASFV attenuation and identification of potential virulence genes for further development of effective vaccines.

Tumor heterogeneity and treatment response assessment using next generation imaging and liquid biopsy in patients with metastatic castration resistant prostate cancer receiving abiraterone (ANGELA): A single-center prospective observational trial.

5057 Background: Metastatic castration resistant prostate cancer (mCRPC) is a disease of intra-patient tumor heterogeneity which poses obstacles in treatment and response evaluation. With the development of next generation imaging (NGI) and liquid biopsy, precise assessment of heterogeneity has entered a new era. In this study, we used longitudinal NGI (68Ga-PSMA and 18F-FDG PET/CT) and circulating tumor DNA (ctDNA) testing to assess tumor heterogeneity and treatment response in mCRPC patients receiving abiraterone. Methods: ANGELA is a single-center prospective observational trial (NCT05188911). We recruited mCRPC patients who received abiraterone as initial hormonal treatment. Eligible patients should have a life expectancy >12 months. CtDNA testing and NGI were performed at baseline and after 12 weeks of abiraterone (Week 13). Conventional imaging and PSA testing were performed at Week 1 and every 12 weeks. Primary end-point of this study is intra-patient lesion heterogeneity evaluated by 3 dimensions: (1) Imaging heterogeneity: defined as at least 1 lesion with FDG+/PSMA- phenotype at baseline and/or Week 13; (2) Genomic heterogeneity: quantified by ctDNA fraction increase and mutational changes per gene between baseline and Week 13; (3) Response heterogeneity: defined as at least 1 new tumoral lesion with either PSMA or FDG abnormal uptake at Week 13. Secondary end-points are correlations of heterogeneity with conventional-evaluated progression-free survival (PFS) and 2-year overall survival (OS). Results: Between May 20, 2020 and Feb 02, 2021, we included 33 eligible patients in our study. FDG+/PSMA- tumoral lesion was observed in 7/33 (21.2%) and 8/33 (24.2%) of patients at baseline and Week 13, respectively. Notably, 5 of 8 patients with imaging heterogeneity at Week 13 had no FDG+/PSMA- lesion at baseline. For genomic heterogeneity, we detected ctDNA fraction increase in 4/33 patients (12.1%). In addition, we found new gene mutations detected in Week 13 ctDNA testing in 5/33 patients (15.2%), 2 of 5 had ctDNA fraction increase. For response heterogeneity, we observed 15/33 patients (45.5%) patients developed at least 1 new tumoral lesion (FDG+ or PSMA+) at Week 13. We correlated heterogeneity factors with prognosis, and found that detection of new mutated genes (P=0.005 and P<0.001), new PSMA+ lesion (P<0.001 and P<0.001), and new FDG+ lesion (P=0.008 and P<0.0001) at Week 13 were significant predictors of shorter PFS and decreased 2-year OS. Detailed biomarker study for response analysis is still in process. Conclusions: Intra-patient tumor heterogeneity was common among mCRPC patients receiving abiraterone. Combining liquid biopsy with NGI can monitor changes in tumor heterogeneity and indicate early combination therapy. Clinical trial information: NCT05188911 .

Pulsed antibiotic treatments of gnotobiotic mice manifest in complex bacterial community dynamics and resistance effects

Bacteria can evolve to withstand a wide range of antibiotics (ABs) by using various resistance mechanisms. How ABs affect the ecology of the gut microbiome is still poorly understood. We investigated strain-specific responses and evolution during repeated AB perturbations by three clinically relevant ABs, using gnotobiotic mice colonized with a synthetic bacterial community (oligo-mouse-microbiota). Over 80days, we observed resilience effects at the strain and community levels, and we found that they were correlated with modulations of the estimated growth rate and levels of prophage induction as determined from metagenomics data. Moreover, we tracked mutational changes in the bacterial populations, and this uncovered clonal expansion and contraction of haplotypes and selection of putative AB resistance-conferring SNPs. We functionally verified these mutations via reisolation of clones with increased minimum inhibitory concentration (MIC) of ciprofloxacin and tetracycline from evolved communities. This demonstrates that host-associated microbial communities employ various mechanisms to respond to selective pressures that maintain community stability.

An optimized thermodynamics integration protocol for identifying beneficial mutations in antibody design.

Accurate identification of beneficial mutations is central to antibody design. Many knowledge-based (KB) computational approaches have been developed to predict beneficial mutations, but their accuracy leaves room for improvement. Thermodynamic integration (TI) is an alchemical free energy algorithm that offers an alternative technique for identifying beneficial mutations, but its performance has not been evaluated. In this study, we developed an efficient TI protocol with high accuracy for predicting binding free energy changes of antibody mutations. The improved TI method outperforms KB methods at identifying both beneficial and deleterious mutations. We observed that KB methods have higher accuracies in predicting deleterious mutations than beneficial mutations. A pipeline using KB methods to efficiently exclude deleterious mutations and TI to accurately identify beneficial mutations was developed for high-throughput mutation scanning. The pipeline was applied to optimize the binding affinity of a broadly sarbecovirus neutralizing antibody 10-40 against the circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant. Three identified beneficial mutations show strong synergy and improve both binding affinity and neutralization potency of antibody 10-40. Molecular dynamics simulation revealed that the three mutations improve the binding affinity of antibody 10-40 through the stabilization of an altered binding mode with increased polar and hydrophobic interactions. Above all, this study presents an accurate and efficient TI-based approach for optimizing antibodies and other biomolecules.

Newly Characterized Porcine Epidemic Diarrhea Virus GII Subtype Strain

Diarrhea outbreaks in piglets on pig farms are commonly attributed to porcine epidemic diarrhea virus (PEDV) infection. This research analyzed the S gene prevalence variation and recombination patterns in PEDV GII strains. Throughout the previous two years, 172 clinical samples of piglet diarrhea have been collected, from which 24 PEDV isolates have been isolated. Analysis of the evolutionary relationships among all 24 S genes revealed that 21 were most closely related to strains within the GII-a subgroup. The 2 isolates grouped into one clade with the GII-b subgroup. According to the mutation analysis of the amino acids (aa) that encode the S protein, 43 of the common aa in strains of the GII subtype were found to have undergone a change in polarity or charge, and 36 of these aa had a mutation frequency of more than 90%. Three different aa mutation sites were identified as exclusive to GII-a subtype strains. The genomes of three PEDV isolates were sequenced, and the resulting range in genome length was 28,035−28,041 nt. The results of recombination analysis showed that the SD1 isolate is a novel strain recombinant from the foreign S-INDEL strain and a domestic GII subtype strain. Based on the findings, the PEDV GII-a strain has been the most circulating strain in several parts of China during the previous two years. Our study reveals for the first time the unique change of aa mutations in the S protein of the GII-a subtype strain and the new characteristics of the recombination of foreign strains and domestic GII subtype strains, indicating that it is crucial to monitor the epidemic dynamics of PEDV promptly to prevent and control the occurrence of PED effectively.