Selection Of Subpopulations Research Articles

Dipeptidyl peptidase-4 marks distinct subtypes of human adipose stromal/stem cells with different hepatocyte differentiation and immunoregulatory properties

BackgroundHuman adipose-derived stromal/stem cells (hASCs) play important roles in regenerative medicine and numerous inflammatory diseases. However, their cellular heterogeneity limits the effectiveness of treatment. Understanding the distinct subtypes of hASCs and their phenotypic implications will enable the selection of appropriate subpopulations for targeted approaches in regenerative medicine or inflammatory diseases.MethodshASC subtypes expressing dipeptidyl peptidase-4 (DPP4) were identified via fluorescence-activated cell sorting (FACS) analysis. DPP4 expression was knocked down in DPP4+ hASCs via DPP4 siRNA. The capacity for proliferation, hepatocyte differentiation, inflammatory factor secretion and T-cell functionality regulation of hASCs from DPP4−, DPP4+, and control siRNA-treated DPP4+ hASCs and DPP4 siRNA-treated DPP4+ hASCs were assessed.ResultsDPP4+ hASCs and control siRNA-treated DPP4+ hASCs presented a lower proliferative capacity but greater hepatocyte differentiation capacity than DPP4− hASCs and DPP4 siRNA-treated DPP4+ hASCs. Both DPP4+ hASCs and DPP4− hASCs secreted high levels of vascular endothelial growth factor-A (VEGF-A), monocyte chemoattractant protein-1 (MCP-1), and interleukin 6 (IL-6), whereas the levels of other factors, including matrix metalloproteinase (MMP)-1, eotaxin-3, fractalkine (FKN, CX3CL1), growth-related oncogene-alpha (GRO-alpha, CXCL1), monokine induced by interferon-gamma (MIG), macrophage inflammatory protein (MIP)-1beta, and macrophage colony-stimulating factor (M-CSF), were significantly greater in the supernatants of DPP4+ hASCs than in those of DPP4− hASCs. Exposure to hASC subtypes and their conditioned media triggered changes in the secreted cytokine profiles of T cells from healthy donors. The percentage of functional T cells that secreted factors such as MIP-1beta and IL-8 increased when these cells were cocultured with DPP4+ hASCs. The percentage of polyfunctional CD8+ T cells that secreted multiple factors, such as IL-17A, tumour necrosis factor alpha (TNF-α) and TNF-β, decreased when these cells were cocultured with supernatants derived from DPP4+ hASCs.ConclusionsDPP4 may regulate proliferation, hepatocyte differentiation, inflammatory cytokine secretion and T-cell functionality of hASCs. These data provide a key foundation for understanding the important role of hASC subpopulations in the regulation of T cells, which may be helpful for future immune activation studies and allow them to be customized for clinical application.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Protection Against Infectious Bronchitis Virus Vaccine Recombinants and Chicken-Selected Vaccine Subpopulations.

Outbreaks of infectious bronchitis (IB) continue to occur from novel variants of IB virus (IBV) emerging from selection of vaccine subpopulations and/or naturally occurring recombination events. S1 sequencing of Arkansas (Ark) -type viruses obtained from clinical cases in Alabama broilers and backyard chickens shows both Ark Delmarva Poultry Industry (ArkDPI) vaccine subpopulations as well as Ark vaccine viruses showing recombination with other IB vaccine viruses. IB Ark-type isolates AL5, most similar to an ArkDPI vaccine subpopulation selected in chickens, AL4, showing a cluster of three nonsynonymous changes from ArkDPI subpopulations selected in chickens, and AL9, showing recombination with Massachusetts (Mass) -type IBV, were examined for pathogenicity and ability to break through immunity elicited by vaccination with a commercial ArkDPI vaccine. Analysis of predicted S1 protein structures indicated the changes were in regions previously shown to comprise neutralizing epitopes. Thus, they were expected to contribute to immune escape and possibly virulence. Based on clinical signs, viral load, and histopathology, all three isolates caused disease in naïve chickens, although AL9 and AL5 viral loads in trachea were statistically significantly higher (30- and 40-fold) than AL4. S1 gene sequencing confirmed the stability of the relevant changes in the inoculated viruses in the chickens, although virus in some individual chickens exhibited additional S1 changes. A single amino acid deletion in the S1 NTD was identified in some individual chickens. The location of this deletion in the predicted structure of S1 suggested the possibility that it was a compensatory change for the reduced ability of AL4 to replicate in the trachea of naïve chickens. Chickens vaccinated with a commercial ArkDPI vaccine at day of hatch and challenged at 21 days of age showed that vaccination provided incomplete protection against challenge with these viruses. Moreover, based on viral RNA copy numbers in trachea, differences were detected in the ability of the vaccine to protect against these IBV isolates, with the vaccine protecting the most poorly against AL4. These results provide additional evidence supporting that IBV attenuated vaccines, especially ArkDPI vaccines, contribute to perpetuating the problem of IB in commercial chickens.

Single Cell RNA Sequencing Enables Identification of Mantle Cell Lymphoma Subclones with Drug-Resistant Profiles Already at Diagnosis and May be Used for Therapy Prognostication and Personalization

Mantle cell lymphoma (MCL) is a chronically relapsing B cell lymphoproliferative malignancy characterized by translocation t(11;14)(q13;q32), cyclin D1 overexpression and significant chromosomal instability. One of the main hypotheses of MCL relapses is a clonal evolution driven by therapy selection of subpopulations carrying adverse genetic and epigenetic aberrations. Our previous whole exome sequencing analysis of 25 MCL tumors showed that the majority of the detected single-nucleotide variants and copy number variants (CNV) were already detectable at diagnosis. To further analyze the subclonal structure of MCL at diagnosis and clonal evolution at relapse, we implemented single cell RNA sequencing analysis of five patients with MCL at diagnosis and at the first clinical relapse after failure of standard immunochemotherapy. All analyzed patients achieved at least partial remission. Fresh-frozen tumor cells were sorted to obtain Hoechst − CD45 + population which was processed using single cell 3′ protocol (10X Genomics). The libraries were paired-end sequenced on Novaseq 6000 (Illumina). The reads were aligned to GRCh38 human genome. The MCL cells were distinguished from the normal B cells based on of CCND1 expression and unsupervised clustering. CNVs were predicted using inferCNV package. These CNVs shared a high degree of similarity with the CNVs predicted using available exome sequencing data. The t-SNE representation of the data clearly showed unique positions for the malignant lymphocytes obtained from each of the five analyzed patients ( Figure 1a). In contrast, non-malignant cells (T cells, B cells and monocytes) have grouped together irrespective of the patient or compartment of origin ( Figure 1b). These data suggest two conclusions. First, each patient's malignant cells have unique genetic background. Second, the impact of the microenvironment does not override the impact of the genetic makeup of the non-malignant cells. The data thus suggest comparability of the malignant cells obtained from different compartments. Hierarchical clustering of the per-cell CNV profiles revealed subclonal heterogeneity of the MCL population in all analyzed compartments both at diagnosis and at relapse. In all analyzed patients the clustering identified several large subclones with significant differences (DG L) and at least one minor subclone (DG S) with significant similarities compared to the major clone detectable at relapse (R L). These data suggest that the majority of the diagnostic MCL cells (i.e., DG L clones) were indeed eliminated by therapy, which on clinical level corresponded to achieving remission. On the other hand, the data also suggest that MCL cells with CNV profiles highly similar to the major relapse clone (i.e., R L) were detectable already at diagnosis (i.e., DG S, Figure 1c). To gain a better insight into the biology of the DG S clones with potential drug-resistant phenotype, we analyzed transcriptome differences between DG S and DG L clones. For each patient we identified different sets of significantly upregulated genes in DG S compared to DG L. These included genes coding for important cell surface molecules ( MARCKS, CD52, CD79A, LTB), regulators of cell motility and cytoskeleton dynamics ( STMN1, SWAP70, CFL1, TUBB, TUBA1B, TMSB4X), transcription factor PRDM2, energy metabolism proteins ( MT-ND4, MT-CO1, ATP5MG), protein degradation proteins ( UBA52, PSMC4, UFD1, UBE2L3) and others. Among these genes, significant upregulation of TMSB4X, SWAP70, HLA-A, TUBB4B, RPS26, UBA52, PSMB2, SNRPG, ARPC1B, ATP5MG was detected in three out of five patients. Many of these genes code for oncogenes involved in cell motility and aggressiveness. To further characterize subclonal clusters in MCL population we used marker gene detection and subsequent gene set enrichment analysis, which identified cell adhesion molecules as major category upregulated in four out of five patients in clusters associated with relapse. Our data shows the applicability of the single cell genomic approaches not only to analyze subclonal heterogeneity for the study of disease biology, but also to identify drug-resistant subclones at diagnosis, predict response to various types of therapies thereby enabling therapy personalization. Acknowledgement: Supported by Grant Agency of the Czech Republic GA23-05474S and National Institute for Cancer Research (EXCELES) LX22NPO5102.

A mechanism-based pathway toward administering highly active N-phage cocktails.

Bacteriophage (phage) therapy is being explored as a possible response to the antimicrobial resistance public health emergency. Administering a mixture of different phage types as a cocktail is one proposed strategy for therapeutic applications, but the optimal method for formulating phage cocktails remains a major challenge. Each phage strain has complex pharmacokinetic/pharmacodynamic (PK/PD) properties which depend on the nano-scale size, target-mediated, self-dosing nature of each phage strain, and rapid selection of resistant subpopulations. The objective of this study was to explore the pharmacodynamics (PD) of three unique and clinically relevant anti-Pseudomonas phages after simulation of dynamic dosing strategies. The Hollow Fiber Infection Model (HFIM) is an in vitro system that mimics in vivo pharmacokinetics (PK) with high fidelity, providing an opportunity to quantify phage and bacteria concentration profiles over clinical time scales with rich sampling. Exogenous monotherapy-bolus (producing max concentrations of Cmax = 7 log10 PFU/mL) regimens of phages LUZ19, PYO2, and E215 produced Pseudomonas aeruginosa nadirs of 0, 2.14, or 2.99 log10 CFU/mL after 6 h of treatment, respectively. Exogenous combination therapy bolus regimens (LUZ19 + PYO2 or LUZ19 + E215) resulted in bacterial reduction to <2 log10 CFU/mL. In contrast, monotherapy as a continuous infusion (producing a steady-state concentration of Css,avg = 2 log10PFU/mL) was less effective at reducing bacterial densities. Specifically, PYO2 failed to reduce bacterial density. Next, a mechanism-based mathematical model was developed to describe phage pharmacodynamics, phage-phage competition, and phage-dependent adaptive phage resistance. Monte Carlo simulations supported bolus dose regimens, predicting lower bacterial counts with bolus dosing as compared to prolonged phage infusions. Together, in vitro and in silico evaluation of the time course of phage pharmacodynamics will better guide optimal patterns of administration of individual phages as a cocktail.

An extension to a 2-in-1 adaptive design with biomarker subpopulation selection

In a recent article, Jin and Zhang (2022) proposed an adaptive 2-in-1 design which can expand an ongoing Phase 2 trial with multiple treatment or dose arms into a confirmatory Phase 3 trial with the selected arms based on interim data, and proved that the design can preserve the familywise Type I error rate under a mild assumption. The proposed adaptive design provides an efficient pathway to combine the treatment or dose selection stage and the confirmatory stage into one trial and can expedite drug development. Here we extend the adaptive 2-in-1 design by Jin and Zhang (2022) to an adaptive 2-in-1 design with biomarker subpopulation selection with a similar framework.

Molecular epidemiological analyses reveal extensive connectivity between Echinostoma revolutum (sensu stricto) populations across Eurasia and species richness of zoonotic echinostomatids in England.

Echinostoma revolutum (sensu stricto) is a widely distributed member of the Echinostomatidae, a cosmopolitan family of digenetic trematodes with complex life cycles involving a wide range of definitive hosts, particularly aquatic birds. Integrative taxonomic studies, notably those utilising nad1 barcoding, have been essential in discrimination of E. revolutum (s.s.) within the 'Echinostoma revolutum' species complex and investigation of its molecular diversity. No studies, however, have focussed on factors affecting population genetic structure and connectivity of E. revolutum (s.s.) in Eurasia. Here, we used morphology combined with nad1 and cox1 barcoding to determine the occurrence of E. revolutum (s.s.) and its lymnaeid hosts in England for the first time, in addition to other echinostomatid species Echinoparyphium aconiatum, Echinoparyphium recurvatum and Hypoderaeum conoideum. Analysis of genetic diversity in E. revolutum (s.s.) populations across Eurasia demonstrated haplotype sharing and gene flow, probably facilitated by migratory bird hosts. Neutrality and mismatch distribution analyses support possible recent demographic expansion of the Asian population of E. revolutum (s.s.) (nad1 sequences from Bangladesh and Thailand) and stability in European (nad1 sequences from this study, Iceland and continental Europe) and Eurasian (combined data sets from Europe and Asia) populations with evidence of sub-population structure and selection processes. This study provides new molecular evidence for a panmictic population of E. revolutum (s.s.) in Eurasia and phylogeographically expands the nad1 database for identification of echinostomatids.

A gated group sequential design for seamless Phase II/III trial with subpopulation selection

BackgroundDue to the high cost and high failure rate of Phase III trials where a classical group sequential design (GSD) is usually used, seamless Phase II/III designs are more and more popular to improve trial efficiency. A potential attraction of Phase II/III design is to allow a randomized proof-of-concept stage prior to committing to the full cost of a Phase III trial. Population selection during the trial allows a trial to adapt and focus investment where it is most likely to provide patient benefit. Previous methods have been developed for this problem when there is a single primary endpoint and two possible populations.MethodsTo find the population that potentially benefits with one or two primary endpoints (e.g., progression free survival (PFS), overall survival (OS)), we propose a gated group sequential design for a seamless Phase II/III trial design with adaptive population selection.ResultsThe investigated design controls the familywise error rate and allows multiple interim analyses to enable early stopping for efficacy or futility. Simulations and an illustrative example suggest that the proposed gated group sequential design has more power and requires less time and resources compared to the group sequential design and adaptive design.ConclusionsCombining the group sequential design and adaptive design, the gated group sequential design has more power and higher efficiency while controlling for the familywise error rate. It has the potential to save drug development cost and more quickly fulfill unmet medical needs.

Evolution of avian infectious bronchitis vaccine viruses reisolated from vaccinated broilers

Genetic and phenotypic diversity among infectious bronchitis virus (IBVs) is a key element of the epidemiology of avian infectious bronchitis. Virtually, all studies have focused on the evolution of wild viruses. Live-attenuated vaccine strains deserve a special attention. Two strains of IBV, 2T/17 and 16T/17, detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in samples from H120 vaccinated chickens showing respiratory signs, were genetically analysed targeting the S1 gene. The total RNA was extracted by the classical Trizol method. The whole S1 gene was amplified in an end-point RT-PCR using a specific primer pair, before being sequenced. The obtained sequences were compared to those of vaccine strains and reference viruses and finally subjected to a phylogenetic analysis. The two isolates turned out to be vaccine strains as evidenced by the monophyletic cluster they formed with the H120 vaccine strain (100% bootstrap). Also, they shared with this vaccine strain 99.60% and 99.47% of nucleotides (nt) for strain 2T/17 and 16T/17 respectively. The sequence alignment revealed, for each of the two strains, one non-synonymous nt substitution outside the hypervariable regions: C56T and T39G leading to two amino acid (aa) substitutions: Alv19Val and Cys13Trp, respectively for the strain 2T/17 and 16T/17. The second hypervariable region of strain 2T/17 displayed 2 nt substitutions at positions 345 and 347, resulting in an aa substitution Lys116Thr. As for 16T/17 strain, it harboured two non-synonymous nt substitutions (T353G and T380G), leading to two aa substitutions: Val118Gly and Met127Arg. The chromatogram analysis revealed minor and major peaks at all positions where nt changes were observed. Nucleotides associated with minor peaks matched with those observed in the sequence of the H120 vaccine strain at the same positions, indicating subpopulation selection. Only the substitution T353G results in a mutation as evidenced by the absence of minor peak at this position. Viral subpopulations contained in IBV live-attenuated vaccines are susceptible to be in vivo selected and emerge as persistent pathotype with reversion to virulence. This draws attention to the need of monitoring the evolution of IBV vaccine strains.

Pharmacodynamics of colistin resistance in carbapenemase-producing Klebsiella pneumoniae: the double-edged sword of heteroresistance and adaptive resistance.

Introduction. The presence of heteroresistant subpopulations and the development of resistance during drug exposure (adaptive resistance) limits colistin's efficacy against carbapenemase-producing Klebsiella pneumoniae (CP-Kp) isolates.Hypothesis/Gap statement. The pharmacokinetic/pharmacodynamic (PK/PD) characteristics of both types of colistin resistance against CP-Kp are unknown.Aim. We therefore studied the PK/PD characteristics of colistin resistance in an in vitro PK/PD model simulating clinical colistin exposures.Methods. Two K. pneumoniae clinical isolates, one non-CP-Kp and one CP-Kp, with colistin MICs of 0.5-1 mg l-1 at a final inoculum of 107 c.f.u. ml-1 were used in an in vitro PK/PD dialysis/diffusion closed model simulating 4.5 MU q12h and 3 MU q8h clinical dosing regimens. Heteroresistant (HRS, bacteria with stable high-level resistance present before drug exposure) and adaptive resistant (ARS, bacteria with reversible low-level resistance emerging after drug exposure) subpopulations were measured and optimal PK/PD targets for reducing both ARS and HRS were determined. Cumulative fractional response (CFR) was calculated with Monte Carlo simulation for 9 MU q24h, 4.5 MU q12h and 3 MU q8h clinical dosing regimens.Results. A 2-5 log10c.f.u. ml-1 decrease of the total bacterial population was observed within the first 2 h of exposure, followed by regrowth at 12 h. Colistin exposure was positively and negatively correlated with HRS and ARS 24-0 h c.f.u. ml-1 changes, respectively. An optimal PK/PD (~0.5log10 increase) target of 35 fAUC/MIC (the ratio of the area under the unbound concentration-time curve to the MIC) was found for reducing both HRS and ARS of high-level resistance (MIC >16 mg l-1). The 4.5 MU q12h regimen had slightly higher CFR (74 %) compared to the other dosing regimens.Conclusions. High colistin exposures reduced high-level adaptive resistance at the expense of selection of heteroresistant subpopulations.

Genome-wide polymorphism and genic selection in feral and domesticated lineages of Cannabis sativa.

A comprehensive understanding of the degree to which genomic variation is maintained by selection versus drift and gene flow is lacking in many important species such as Cannabis sativa (C. sativa), one of the oldest known crops to be cultivated by humans worldwide. We generated whole genome resequencing data across diverse samples of feralized (escaped domesticated lineages) and domesticated lineages of C. sativa. We performed analyses to examine population structure, and genome wide scans for FST, balancing selection, and positive selection. Our analyses identified evidence for sub-population structure and further support the Asian origin hypothesis of this species. Feral plants sourced from the U.S. exhibited broad regions on chromosomes 4 and 10 with high F̄ST which may indicate chromosomal inversions maintained at high frequency in this sub-population. Both our balancing and positive selection analyses identified loci that may reflect differential selection for traits favored by natural selection and artificial selection in feral versus domesticated sub-populations. In the U.S. feral sub-population, we found six loci related to stress response under balancing selection and one gene involved in disease resistance under positive selection, suggesting local adaptation to new climates and biotic interactions. In the marijuana sub-population, we identified the gene SMALLER TRICHOMES WITH VARIABLE BRANCHES 2 to be under positive selection which suggests artificial selection for increased tetrahydrocannabinol yield. Overall, the data generated, and results obtained from our study help to form a better understanding of the evolutionary history in C. sativa.

Sympatric Recombination in Zoonotic Cryptosporidium Leads to Emergence of Populations with Modified Host Preference.

Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.

Recombination landscape divergence between populations is marked by larger low-recombining regions in domesticated rye.

The genomic landscape of recombination plays an essential role in evolution. Patterns of recombination are highly variable along chromosomes, between sexes, individuals, populations, and species. In many eukaryotes, recombination rates are elevated in sub-telomeric regions and drastically reduced near centromeres, resulting in large low-recombining (LR) regions. The processes of recombination are influenced by genetic factors, such as different alleles of genes involved in meiosis and chromatin structure, as well as external environmental stimuli like temperature and overall stress. In this work, we focused on the genomic landscapes of recombination in a collection of 916 rye (Secale cereale) individuals. By analyzing population structure among individuals of different domestication status and geographic origin, we detected high levels of admixture, reflecting the reproductive biology of a self-incompatible, wind-pollinating grass species. We then analyzed patterns of recombination in overlapping subpopulations, which revealed substantial variation in the physical size of LR regions, with a tendency for larger LR regions in domesticated subpopulations. Genome-wide association scans (GWAS) for LR region size revealed a major quantitative-trait-locus (QTL) at which, among 18 annotated genes, an ortholog of histone H4 acetyltransferase ESA1 was located. Rye individuals belonging to domesticated subpopulations showed increased synaptonemal complex length, but no difference in crossover frequency, indicating that only the recombination landscape is different. Furthermore, the genomic region harboring rye ScESA1 showed moderate patterns of selection in domesticated subpopulations, suggesting that larger LR regions were indirectly selected during domestication to achieve more homogeneous populations for agricultural use.

Rational selection of virologic and immunological biomarkers and therapeutic endpoints in the clinical trials of new drugs treating chronic hepatitis B

Hepatitis B virus (HBV) infection remains to be the major cause of chronic liver diseases in China. Since the nucleos(t)ide analogues and pegylated interferon-alpha do not directly target the covalently closed circular DNA (cccDNA) in the nuclei of HBV-infected hepatocytes, those standard-of-care medications cannot efficiently cure the infected hepatocytes and rarely achieve the functional cure of chronic hepatitis B (CHB). Therefore, new antiviral drugs targeting distinct steps of HBV replication and immunotherapeutics reinvigorating antiviral immune responses are urgently needed for the functional cure of CHB. Based on the extensive discussion of the biological and clinical significance of new virologic biomarkers and distinct mechanism of drug candidates currently in clinical development, we propose that the selection of virologic and immunological biomarkers for evaluation of therapeutic efficacy as well as setting the therapeutic endpoints in the clinical trials should be based on the mode of action of investigational drugs. In addition, due to the complexity of CHB pathogenesis, selection of specific subpopulation of CHB patients for the clinical trials of drugs with a specific mode of action should also be considered.

Hyper-Methylated Hub Genes of T-Cell Receptor Signaling Predict a Poor Clinical Outcome in Lung Adenocarcinoma.

Background Immune checkpoint inhibitors (ICIs) emerge as the first-line treatment of lung adenocarcinoma (LUAD); selection of subpopulations acquiring clinical benefit is required. Associations between epigenetic modulation of tumor microenvironment (TME) and clinical outcome are far from clear. We focused on immune-related genes closely regulated by DNA methylation to identify the potential clinical outcome indicators. Methods We systematically calculated immunophenotype score (IMpS) and classified immunophenotypes based on seven TME features in three independent cohorts. The overlapping of differential expressed genes and methylated probes targeted genes was regarded as genes closely regulated by DNA methylation. Then, probe/gene pairs which highly correlated with each other and IMpS were identified and named as immune-related probe/gene pairs (mIMg). Prognostic mIMg were selected and verified in seven independent validation cohorts. Results Three immune phenotypes were clustered, and similar results were obtained in the three independent training cohorts. C2 displayed as an immunologically hot phenotype, whereas C3 corresponded with immunologically cold phenotype. Average methylation level was decreased from C2 to C3 (C2 > C1 > C3). Similarly, ICIs nonresponders showed global hypo-methylation compared with responders. Genes in mIMg were mainly enriched, especially in T-cell receptor activation, and repressed in noninflamed TME by hyper-methylation. Among mIMg, low expression and hyper-methylation of CD247, LCK, and PSTPIP1 were risk factors of overall survival (OS). ICIs nonresponders were more likely to be hyper-methylated in the three genes. By integrating with the oncogenes status, we demonstrated that EGFR wt and SRGN overexpressed patients were associated with chronic inflammation and immune evasion, showing an immunologically hot phenotype, which might lead to the short OS but derive clinical benefit from ICIs. Conclusions This study identifies hyper-methylation and concurrent repression of CD247, LCK, PSTPIP1 as immune negative indicators and risk factors for prognosis in LUAD. Moreover, EGFR/SRGN axis may participate in immune modification to influence ICIs response and clinical outcome in LUAD.

Signatures of selection in Nelore cattle revealed by whole-genome sequencing data.

Nelore cattle breed was farmed worldwide due to its economic importance in the beef market and adaptation to the tropics. In Brazil, purebred Nelore animals (PO) receive a certificate from the breeders' association based on the animal's genealogy and morphological characterization. The top 20 to 30% of the superior animals are eligible to receive the Special Certificate of Identification and Production (CEIP), meaning animals from this category were selected and evaluated in a breeding program to improve economically important traits. We used whole-genome sequencing and approaches based on haplotype differentiation and allelic differentiation to detect regions of selection signatures in Nelore cattle by comparing animals from PO and CEIP categories. From a total of 150 animals, a hierarchical clustering analysis was performed to choose the more unrelated animals from each category (16 PO and 40 CEIP). The hapFLK statistic was performed, and extensions of hapFLK values were investigated considering continuous regions with significant q-values. The Weir and Cockerham's Fst estimator (wcFst) was computed using the GPAT++ software library. The total of 82,326 SNPs with hapFLK values passed the FDR control (q-value<0.05), and 718 segments were target as signatures of selection. A total of 1713 highly differentiated genomic regions were identified based on the segmentFst approach. The signatures of selection were spread across the genome. Annotation of overlapping selection signature regions between the two methods revealed 118 genes in common. A variant located within the 3' region of the BOLA-DRB3 gene was found as a promising candidate polymorphism. Within genomic regions that deserves attention, we found genes previously associated with adaptation to tropical environments (HELB), growth and navel size (HMGA2), fat deposition and domestication (IRAK3), and feed efficiency and postmortem carcass traits (GABRG3). The genes BOLA-DQA2, BOLA-DQB, BOLA-DQA5, BOLA-DQA1, BOLA-DRB3, ENSBTAG00000038397 on chromosome 23 are part of the Bovine Major Histocompatibility Complex (MHC) Class II gene family, representing good candidates for immune response and adaptation to tropical conditions. The BoLA family genes and the interaction of ROBO1 with SLIT genes appeared in the enrichment results. Genomic regions located in intronic regions were also identified and might play a regulatory role in traits under selection in PO and CEIP subpopulations. The regions here identified contribute to our knowledge regarding genes and variants that have an important role in complex traits selected in this breed.

Therapy-Related Transcriptional Subtypes in Matched Primary and Recurrent Head and Neck Cancer.

The genetic relatedness between primary and recurrent head and neck squamous cell carcinomas (HNSCC) reflects the extent of heterogeneity and therapy-driven selection of tumor subpopulations. Yet, current treatment of recurrent HNSCC ignores the molecular characteristics of therapy-resistant tumor populations. From 150 tumors, 74 primary HNSCCs were RNA sequenced and 38 matched primary/recurrent tumor pairs were both whole-exome and RNA sequenced. Transcriptome analysis determined the predominant classical (CL), basal (BA), and inflamed-mesenchymal (IMS) transcriptional subtypes according to an established classification. Genomic alterations and clonal compositions of tumors were evaluated from whole-exome data. Although CL and IMS subtypes were more common in primary HNSCC with low recurrence rates, the BA subtype was more prevalent and stable in recurrent tumors. The BA subtype was associated with a transcriptional signature of partial epithelial-to-mesenchymal transition (p-EMT) and early recurrence. In 44% of matched cases, the dominant subtype changed from primary to recurrent tumors, preferably from IMS to BA or CL. Expression analysis of prognostic gene sets identified upregulation of hypoxia, p-emt, and radiotherapy resistance signatures and downregulation of tumor inflammation in recurrences compared with index tumors. A relevant subset of primary/recurrent tumor pairs presented no evidence for a common clonal origin. Our study showed a high degree of genetic and transcriptional heterogeneity between primary/recurrent tumors, suggesting therapy-related selection of a transcriptional subtype with characteristics unfavorable for therapy. We conclude that therapy decisions should be based on genetic and transcriptional characteristics of recurrences rather than primary tumors to enable optimally tailored treatment strategies.

Deregulation of AKT–mTOR Signaling Contributes to Chemoradiation Resistance in Lung Squamous Cell Carcinoma

Abstract Lung squamous cell carcinoma (LUSC) accounts for one of three of non–small cell lung carcinoma (NSCLC) and 30% of LUSC patients present with locally advanced, unresectable/medically inoperable disease, who are commonly treated with definitive chemoradiation. However, disease relapse in the radiation fields occurs in one of three cases. We aim to explore the underlying molecular mechanisms of chemoradiation resistance of LUSC. Patient-derived xenograft (PDX) models of LUSC were established in immunodeficient mice, followed by treatment with cisplatin in combination with clinically relevant courses of ionizing radiation (20, 30, and 40 Gy). The recurrent tumors were extracted for functional proteomics using reverse phase protein analysis (RPPA). We found that phospho-AKT-S473, phospho-AKT-T308, phospho-S6-S235/6, and phospho-GSK3β-S9 were upregulated in the chemoradiation-resistant 20 Gy + cisplatin and 40 Gy + cisplatin tumors compared with those in the control tumors. Ingenuity pathway analysis of the RPPA data revealed that AKT–mTOR signaling was the most activated signaling pathway in the chemoradiation-resistant tumors. Similarly, elevated AKT–mTOR signaling was observed in stable 40 Gy and 60 Gy resistant HARA cell lines compared with the parental cell line. Accordingly, pharmacologic inhibition of mTOR kinase by Torin2 significantly sensitized LUSC cell lines to ionizing radiation. In conclusion, using chemoradiation-resistant PDX models coupled with RPPA proteomics analysis, we revealed that deregulation of AKT–mTOR signaling may contribute to the chemoradiation resistance of LUSC. Implications: Clonal selection of subpopulations with high AKT–mTOR signaling in heterogeneous tumors may contribute to relapse of LUSC after chemoradiation. mTOR kinase inhibitors may be promising radiosensitizing agents in upfront treatment to prevent acquired resistance.

Fish out of water: Genomic insights into persistence of rainbowfish populations in the desert.

How populations of aquatic fauna persist in extreme desert environments is an enigma. Individuals often breed and disperse during favorable conditions. Theory predicts that adaptive capacity should be low in small populations, such as in desert fishes. We integrated satellite-derived surface water data and population genomic diversity from 20,294 single-nucleotide polymorphisms across 344 individuals to understand metapopulation persistence of the desert rainbowfish (Melanotaenia splendida tatei) in central Australia. Desert rainbowfish showed very small effective population sizes, especially at peripheral populations, and low connectivity between river catchments. Yet, there was no evidence of population-level inbreeding and a signal of possible adaptive divergence associated with aridity was detected. Candidate genes for local adaptation included functions related to environmental cues and stressful conditions. Eco-evolutionary modeling showed that positive selection in refugial subpopulations combined with connectivity during flood periods can enable retention of adaptive diversity. Our study suggests that adaptive variation can be maintained in small populations and integrate with neutral metapopulation processes to allow persistence in the desert.

Effects of BCL11A Shmir-Induced Post-Transcriptional Silencing on Distributions of HbF in Single-RBCs and Reticulocytes

NH and EE equally contributed. ADW and PB co-signed.The expression of fetal hemoglobin (HbF) is one of the main targets of sickle cell disease treatment, as it inhibits the polymerization of hemoglobin S. The hypothesis of an inhibitory threshold of HbF per red blood cell (RBC) has been suggested, 1 although not well defined, as the overall percentage of HbF does not reflect the heterogeneous distribution of HbF per cell. Likewise, the qualitative analysis of RBCs containing HbF, called F cells, is neither reproducible nor clinically interpretable, due to low expression. 2 We have developed a technique for measuring the amount of HbF per cell, to determine thresholds of HbF expression per RBC correlated with clinical and biological effects. 2 Among genes controlling its expression, BCL11A has a major repressive effect on the expression of gamma globin/HbF during the fetal to adult hemoglobin switch. Post-transcriptional silencing of BCL11A, using lentivirus expression of a shRNA embedded in a microRNA architecture (shmiR) to re-activate γ-globin expression, is safe and demonstrates high levels of %HbF in a pilot clinical study (NCT 03282656). 3 Here, we show the quantitative measurement of HbF per RBC and reticulocyte.Methods: During patient follow-up, HbF quantification per single cell RBC was performed using a fluorescent HbF antibody. 2 Addition of an anti-CD71 fluorescent antibody allowed selection of reticulocyte sub-populations for determining their HbF content. Fold-increase in percentage of RBC versus percentage of reticulocyte were calculated. Kinetics of HbF/RBC and HbF/Reticulocyte were modeled using mixed effects polynomial linear regression to account for the correlation between repeated data over time.Results: With a median follow-up of 15 months [12-20] after gene transfer, figure 1 shows the mathematical modeling of single-RBC HbF measurement representing RBC percentage containing at least 2, 4, 6, 8 and 10 pg of HbF. Percentage of RBC above each threshold was higher compared to 14 hydroxyurea treated patients for 6 months. Figure 2 shows fold increase between reticulocytes and RBCs with same thresholds of HbF/cell. For low thresholds, RBCs were found in same percentage as reticulocytes whereas RBCs containing increasing levels of HbF were found in higher percentage than reticulocytes, until 6pg/cell showing a clear selective advantage for red cells with a threshold ≥ 6pg/cell of HbF. Figure 3 shows different kinetics of HbF increase according to two different transduction strategies with 2 enhancers in patients 2-4 compared to one enhancer in patients 6-8.Conclusion: BCL11A down-regulation in six clinical trial subjects was associated with an in vivo selection process RBCs with ≥ 6pg HbF per cell attained with different engraftment kinetics, depending on transduction processes, and ultimately stable high level and broadly distributed HbF.1 Steinberg MH, Chui DH, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: a glass half full? Blood. 2014 Jan 23;123(4):481-5.2 Hebert N, Rakotoson MG, Bodivit G, et al. Individual red blood cell fetal hemoglobin quantification allows to determine protective thresholds in sickle cell disease. Am. J. Hematol.3 Esrick EB, Lehmann LE, Biffi A, et al. Post-Transcriptional Genetic Silencing of BCL11A to Treat Sickle Cell Disease. N. Engl. J. Med. 2021;384(3):205-215. [Display omitted] DisclosuresEsrick: bluebird bio: Consultancy. Audureau: GBT: Honoraria. Higgins: Sebia, Inc.: Honoraria; Danaher Diagnostics: Consultancy. Williams: BioMarin: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Advisory Board; Geneception: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Emerging Therapy Solutions: Membership on an entity's Board of Directors or advisory committees, Other: Chief Scientific Chair; Beam Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Scientific Advisory Board; Alerion Biosciences: Other: Co-founder (now licensed to Avro Bio, potential for future milestones/royalties); Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Steering Committee, Novartis ETB115E2201 (eltrombopag in aplastic anemia). Advisory fees donated to NAPAAC.; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Membership on a safety advisory board (SAB): SAB position ended 05/20/2021. Co-founder , Patents & Royalties: Potential for future royalty/milestone income, X-SCID. Provided GMP vector for clinical trial, Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees, Other: Insertion Site Analysis Advisory Board, Patents & Royalties: BCH licensed certain IP relevant to hemoglobinopathies to bluebird bio. The current license includes the potential for future royalty/milestone income. Bluebird has indicated they will not pursue this as a clinical program and BCH is negotiating return of, Research Funding. Bartolucci: AGIOS: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Lecture fees, Steering committee, Research Funding; Jazz Pharma: Other: Lecture fees; Emmaus: Consultancy; Addmedica: Consultancy, Other: Lecture fees, Research Funding; INNOVHEM: Other: Co-founder; Hemanext: Consultancy; GBT: Consultancy; Bluebird: Consultancy, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy; Fabre Foundation: Research Funding.