clone selection

Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TIL) is effective in melanoma patients, although long-term responses seem restricted to patients who have complete remissions. Many patients develop secondary resistance to TIL-ACT but the involved mechanisms are unclear. Here, we describe a case of secondary resistance to TIL-ACT likely due to intratumoral heterogeneity and selection of a resistant tumor cell clone by the transferred T cells. To our knowledge, this is the first case of clonal selection of a pre-existing non-dominant tumor cell clone and it demonstrates a mechanism involved in secondary resistance to TIL-ACT that could potentially change current clinical practice, because it advocates for T-cell collection from multiple tumor sites and analysis of tumor heterogeneity before the treatment with TIL-ACT...

The GATA transcription factors (TFs) have been extensively studied for its regulatory role in various biological processes in many plant species. The functional and molecular mechanism of GATA TFs in regulating tolerance to abiotic stress has not yet been studied in the common bean. This study analyzed the functional identity of the GATA gene family in the P. vulgaris genome under different abiotic and phytohormonal stress. The GATA gene family was systematically investigated in the P. vulgaris genome, and 31 PvGATA TFs were identified...

Chemoresistance is one of the main reasons for poor prognosis of pancreatic cancer. The effects of mesothelin (MSLN) on chemoresistance in pancreatic cancer are still unclear. We aim to investigate potential roles of MSLN in chemoresistance and its relationship with proliferation, epithelial-mesenchymal transition (EMT) and cancer stemness of pancreatic cancer cells. Human pancreatic cancer cell lines ASPC-1 and Mia PaCa-2 with high and low expression of MSLN, respectively, were selected. The ASPC-1 with MSLN knockout (KO) and Mia PaCa-2 of MSLN overexpression (OE) were generated...

BACKGROUND: Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations...

Fruit length is a crucial agronomic trait of snake gourd (Trichosanthes anguina L); however, genes associated with fruit length have not been characterised. In this study, F2 snake gourd populations were generated by crossing the inbred lines, S1 and S2 (fruit lengths: 110 and 20 cm, respectively). Subsequently, bulk segregant analysis, sequencing, and fine-mapping were performed on the F2 population to identify target genes. Our findings suggest that the fruit length of snake gourd is regulated by a major-effect regulatory gene...

Molecular cloning facilitates the assembly of heterologous DNA fragments with vectors, resulting in the generation of plasmids that can steadily replicate in host cells. To efficiently and accurately screen out the expected plasmid candidates, various methods, such as blue-white screening, have been developed for visualization. However, these methods typically require additional genetic manipulations and costs. To simplify the process of visualized molecular cloning, here we report Rainbow Screening, a method that combines Gibson Assembly with chromoproteins to distinguish Escherichia coli (E...

Biofilms inhabit a range of environments, such as dental plaques or soil micropores, often characterized by noneven surfaces. However, the impact of surface irregularities on the population dynamics of biofilms remains elusive, as most experiments are conducted on flat surfaces. Here, we show that the shape of the surface on which a biofilm grows influences genetic drift and selection within the biofilm. We culture E scherichia coli biofilms in microwells with a corrugated bottom surface and observe the emergence of clonal sectors whose size corresponds to that of the corrugations, despite no physical barrier separating different areas of the biofilm...

A novel β-galactosidase (TsGal48) from Thermus scotoductus was cloned, and the enzyme was biochemically characterized. TsGal48 catalyzed the synthesis of lacto- N -neotetraose (LNnT) from lactose via the transglycosylation reaction with a maximal yield of 20%, which is the highest yield for the synthesis of LNnT so far. To further improve the yield of LNnT, TsGal48 was successfully engineered by directed evolution and site-saturation mutagenesis. A mutated β-galactosidase (mTsGal48) was selected and characterized...

BACKGROUND: The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein...

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library...

Drought stress is a crucial environmental factor that limits plant growth, development, and productivity. Autophagy of misfolded proteins can help alleviate the damage caused in plants experiencing drought. However, the mechanism of autophagy-mediated drought tolerance in plants remains largely unknown. Here, we cloned the gene for a maize (Zea mays) selective autophagy receptor, NEXT TO BRCA1 GENE 1 (ZmNBR1), and identified its role in the response to drought stress. We observed that drought stress increased the accumulation of autophagosomes...

The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species...

Cell line generation of mammalian cells is a time-consuming and labor-intensive process, especially because of challenges in clone selection after transfection. Antibiotics are common selection agents for mammalian cells due to their simplicity of use. However, the optimal antibiotic concentration must be determined with a kill curve experiment before clone selection starts. The traditional kill curve experiments are resource-intensive and time-consuming due to necessary sampling and offline analysis effort...

OBJECTIVES: This study aimed to determine the association of E. coli microbiological factors with 30-day mortality in BSI patients presenting with a dysregulated response to infection (i.e., sepsis or septic shock). METHODS: Whole genome sequencing was performed on 224 E. coli isolates of patients with sepsis/septic shock, from 22 Spanish hospitals. Phylogroup, sequence type, virulence, antibiotic resistance and pathogenicity islands were assessed. A multivariable model for 30-day mortality including clinical and epidemiological variables was built, to which microbiological variables were hierarchically added...

The assembly of tissue-damaging membrane attack complexes (MACs; C5b-9) is a major mechanism by which excessive complement activation causes diseases. We previously developed a mouse anti-human C6 monoclonal antibody (mAb) 1C9 that selectively inhibits the assembly of MACs in human and non-human primates. In this project, we found that 1C9 also cross-reacted with rat and guinea pig C6, and determined its binding domains on C6 using different truncated C6 proteins. We then humanized the anti-C6 mAb by molecular modeling and complementarity-determining region grafting...

OBJECTIVES: To determine the frequencies and clonal distributions of putative genetic determinants of resistance to antimicrobials applied for treatment of Clostridioides difficile infection (CDI), as documented in the genomic record. METHODS: We scanned 26 557 C. difficile genome sequences publicly available from the EnteroBase platform for plasmids, point mutations and gene truncations previously reported to reduce susceptibility to vancomycin, fidaxomicin or metronidazole, respectively...

We find evidence of selection for local adaptation and extensive genotype-by-environment interaction in the potato National Chip Processing Trial (NCPT). We present a novel method for dissecting the interplay between selection, local adaptation and environmental response in plant breeding schemes. Balancing local adaptation and the desire for widely adapted cultivars is challenging for plant breeders and makes genotype-by-environment interactions (GxE) an important target of selection. Selecting for GxE requires plant breeders to evaluate plants across multiple environments...

BACKGROUND: Carbapenem resistance is epidemic worldwide, these last resort antimicrobials are listed in the WHO 'watch group' with higher resistance potential. During the years 2017-18 Pakistan Antimicrobial Resistance Surveillance System reported an increase in carbapenem resistance. However, a comprehensive information on prevalence and molecular epidemiology of carbapenem resistance in Pakistan is not available. This systematic review and meta-analysis is aimed to report the current carbapenem resistance situation in Pakistan and its treatment options...

Viruses are obligate parasites that completely rely on host cells for survival and replication. Razi Bovine Kidney (RBK) cell line was introduced and developed by the Razi Vaccine and Serum Research Institute. It has been successfully established as a continuous cell line over successive passages. As demonstrated in this experimental study, the RBK cells have shown suitable sensitivity to certain viruses, including Bovine Herpesvirus-1 (BoHV-1) virus. In the present research, the RBK cell line characteristics were analyzed using molecular and karyotype methods and growth specifications...

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39...