Quantitative Gene Expression Analysis Research Articles

Therapeutic application of nicotinamide: As a potential target for inhibiting fibrotic scar formation following spinal cord injury.

We aimed to confirm the inhibitory effect of nicotinamide on fibrotic scar formation following spinal cord injury in mice using functional metabolomics. We proposed a novel functional metabolomics strategy to establish correlations between gene expression changes and metabolic phenotypes using integrated multi-omics analysis. Through the integration of quantitative metabolites analysis and assessments of differential gene expression, we identified nicotinamide as a functional metabolite capable of inhibiting fibrotic scar formation and confirmed the effect invivo using a mouse model of spinal cord injury. Furthermore, to mimic fibrosis models invitro, primary mouse embryonic fibroblasts and spinal cord fibroblasts were stimulated by TGFβ, and the influence of nicotinamide on TGFβ-induced fibrosis-associated genes and its underlying mechanism were examined. Administration of nicotinamide led to a reduction in fibrotic lesion area and promoted functional rehabilitation following spinal cord injury. Nicotinamide effectively downregulated the expression of fibrosis genes, including Col1α1, Vimentin, Col4α1, Col1α2, Fn1, and Acta2, by repressing the TGFβ/SMADs pathway. Our functional metabolomics strategy identified nicotinamide as a metabolite with the potential to inhibit fibrotic scar formation following SCI by suppressing the TGFβ/SMADs signaling. This finding provides new therapeutic strategies and new ideas for clinical treatment.

Prediction and validation of putative candidate genes underlying drought‐tolerant QTLs through in silico and RT‐PCR approaches in rice (Oryza sativa)

AbstractIn the present study, the aim was to predict and validate putative candidate genes underlying drought‐tolerant quantitative trait loci (QTLs) in rice crop using in silico approaches and real‐time polymerase chain reaction (RT‐PCR). The genes underlying major drought‐tolerant QTLs which have been reported by data mining, sequence variation, gene ontology analysis, quantitative traits gene finder and gene expression analysis were subjected to RiceVarmap software to design primers, and only a few variants gave the SNP/InDel primers; thus, finally, 15 primers were ultimately selected, which were used in identification of differentially expressed genes (DEGs) among contrasting rice genotypes IR 64 and N 22 for drought tolerance trait using quantitative RT‐PCR studies by providing drought stress treatment during panicle initiation stage. In this investigation, we predicted 11 genes as candidate genes underlying drought‐tolerant QTLs. Out of these, only four QTLs were found responsible for the major effect in drought tolerance regions such as QTL‐Qsn‐4b, QTL‐rn7a, QTL‐Qtgw‐2a and QTL‐phc4.1 and 11 prioritized candidates were identified that expressed in leaf tissues. Only four primers belong to two QTLs, primer vg0712623096 from QTL‐rn7a (LOC_Os07g22450) located on chromosome‐7 encoding NAC domain‐containing protein and the primers vg0431750843(LOC_Os04g53310) encoding soluble starch synthase 3‐ chloroplast precursor, vg0432626757 (LOC_Os04g54850) encoding pectin acetylesterase domain‐containing protein and vg0433031562 (LOC_Os04g55520) encoding AP2 domain‐containing protein, from QTL‐Qsn‐4b, located on chromosome‐4 found to have higher differential expression in N 22 in comparison with IR 64 during drought stress as per quantitative RT‐PCR 2–ΔΔCt values. Considering the overall study, these four primers/genes were identified as candidate genes underlying genomic regions governing drought tolerance. Therefore, these putative candidate genes could be focussed for further functional analysis to exploit in rice breeding.

Biomass-derived carbon dots as fluorescent quantum probes to visualize and modulate inflammation

Quantum dots, which won the Nobel Prize in Chemistry, have recently gained significant attention in precision medicine due to their unique properties, such as size-tunable emission, high photostability, efficient light absorption, and vibrant luminescence. Consequently, there is a growing demand to identify new types of quantum dots from various sources and explore their potential applications as stimuli-responsive biosensors, biomolecular imaging probes, and targeted drug delivery agents. Biomass-waste-derived carbon quantum dots (CQDs) are an attractive alternative to conventional QDs, which often require expensive and toxic precursors, as they offer several merits in eco-friendly synthesis, preparation from renewable sources, and cost-effective production. In this study, we evaluated three CQDs derived from biomass waste for their potential application as non-toxic bioimaging agents in various cell lines, including human dermal fibroblasts, HeLa, cardiomyocytes, induced pluripotent stem cells, and an in-vivo medaka fish (Oryzias latipes) model. Confocal microscopic studies revealed that CQDs could assist in visualizing inflammatory processes in the cells, as they were taken up more by cells treated with tumor necrosis factor-α than untreated cells. In addition, our quantitative real-time PCR gene expression analysis has revealed that citric acid-based CQDs can potentially reduce inflammatory markers such as Interleukin-6. Our studies suggest that CQDs have potential as theragnostic agents, which can simultaneously identify and modulate inflammatory markers and may lead to targeted therapy for immune system-associated diseases.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.)

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Fine mapping and targeted genomic analyses of photoperiod‐sensitive gene (GB_PPD1) in Pima cotton (Gossypium barbadense L.)

AbstractCotton grown in the United States are day‐length insensitive annuals and are grown under long‐day summers. Photoperiod sensitivity, present in tropical wild and landraces endemic to the center of origin and diversity, is a major barrier for the introgression of tropical gene pool into the US cotton. Previously, we mapped the major photoperiod response locus Gb_Ppd1 on chromosome D06 of Pima cotton (Gossypium barbadense L.). In the current study, an F2 population of 2112 gametes was used to fine map the Gb_Ppd1 locus. Two sequence‐tagged‐site and nine kompetitive allele‐specific polymerase chain reaction (KASP) markers were developed and the Gb_Ppd1 locus was fine mapped to 1.1 cM region flanked by novel markers 17‐KASP‐8, 17‐KASP‐10, and 15‐PR‐10A. The closely linked markers identified 2.10 Mb region in the G. barbadense genome that contained 18 putative gene sequences. A candidate gene Gbar_D06G014560 showed high homology to VASCULAR PLANT ONE ZINC FINGER PROTEIN 1 involved in photoperiodism in Arabidopsis. We evaluated the diagnostic polymorphisms for the flanking markers on a diversity panel of 91 Pima accessions. These markers would be useful in marker‐assisted selection of photoperiod response in cotton breeding. Further, quantitative gene expression analysis indicated that the CONSTANS (CO)/FLOWERING LOCUS T (FT) system is conserved in the photoperiod response pathway in Pima cotton, while CO and FT are likely not the causal genes underlying flowering time at the Gb_Ppd1 locus. The Gb_Ppd1 gene may be an upstream regulatory sequence in the FT gene pathway that controls photoperiodism in photoperiod‐sensitive cotton by affecting the CO/FT interaction under long‐day‐length conditions.

The RNA sequencing results revealed the expression of different genes and signaling pathways during chemotherapy resistance in peripheral T-cell lymphoma

BackgroundPeripheral T-cell lymphoma (PTCL) is a subtype of non-Hodgkin's lymphoma that occurs primarily at extranodal sites and is commonly treated using chemotherapy and radiotherapy. PTCL is more malignant than other lymphoid tumors, resulting in a poor prognosis.The 5-year recurrence rate remains high, and there is a lack of standard treatment for patients with relapse-resistant disease. However, the molecular mechanisms underlying the resistance of peripheral T-cell lymphoma cells to chemotherapeutic drugs, as well as identifying strategies to overcome drug resistance remains unclear. In this study, we aimed to identify pivotal genes and signaling pathways associated with chemotherapy resistance in PTCL.MethodsIn this study, a total of 5 healthy controls and 7 clinical patients were enrolled; 4 patients were classified as chemotherapy sensitive, and 3 patients were classified as chemotherapy resistant. Peripheral blood samples were collected from each participant, and total RNA was extracted from the white blood cells. RNA sequencing was conducted on the Illumina HiSeq platform to obtain comprehensive gene expression profiles. Subsequently, the expression patterns of the DEGs associated with the most enriched signaling pathways, with a special focus on cancer-related genes, were validated using quantitative real-time polymerase chain reaction (qRT–PCR) in peripheral TCL patients.ResultsRNA sequencing (RNA-seq) analysis revealed 4063 differentially expressed genes (DEGs) in peripheral T-cell lymphoma specimens from patients with chemotherapy resistance, of which 1128 were upregulated and 2935 were downregulated. Subsequent quantitative gene expression analysis confirmed a differential expression pattern in all the libraries, with 9 downregulated genes and 10 upregulated genes validated through quantitative real-time PCR in 6 clinical specimens from patients with chemotherapy resistance. KEGG pathway analysis revealed significant alterations in several pathways, with 6 downregulated pathways and 9 upregulated pathways enriched in the DEGs. Notably, the TNF signaling pathway, which is extensively regulated, was among the pathways that exhibited significant changes. These findings suggest that DEGs and the TNF signaling pathway may play crucial roles in chemotherapy resistance in peripheral T-cell lymphoma.ConclusionOur study revealed that the expression of specific genes, including TNFRSF1B, TRADD2, and MAP3K7, may play an important role in chemotherapy resistance in peripheral T-cell lymphoma. Moreover, we identified the downregulation of the TNF signaling pathway, a crucial pathway involved in cell survival, death, and differentiation, as a potential contributor to the development of chemotherapy resistance in peripheral T-cell lymphoma. These findings provide valuable insights into the molecular mechanisms underlying chemotherapy resistance and highlight potential targets for overcoming treatment resistance in this challenging disease.

Evaluation and normalization of a set of reliable reference genes for quantitative sgk-1 gene expression analysis in Caenorhabditis elegans-focused cancer research

Multiple signaling pathways have been discovered to play a role in aging and longevity, including the insulin/IGF-1 signaling system, AMPK pathway, TOR signaling, JNK pathway, and germline signaling. Mammalian serum and glucocorticoid-inducible kinase 1 (sgk-1), which has been associated with various disorders including hypertension, obesity, and tumor growth, limits survival in C. elegans by reducing DAF-16/FoxO activity while suppressing FoxO3 activity in human cell culture. C. elegans provides significant protection for a number of genes associated with human cancer. The best known of these are the lin-35/pRb (mammalian ortholog pRb) and CEP-1 (mammalian ortholog p53) genes. Therefore, in this study, we aimed to investigate the expression analyzes of sgk-1, which is overexpressed in many types of mammalian cancer, in mutant lin-35 and to demonstrate the validation of reference genes in wild-type N2 and mutant lin-35 for C. elegans-focused cancer research. To develop functional genomic studies in C. elegans, we evaluated the expression stability of five candidate reference genes (act-1, ama-1, cdc-42, pmp-3, iscu-1) by quantitative real-time PCR using five algorithms (geNorm, NormFinder, Delta Ct method, BestKeeper, RefFinder) in N2 and lin-35 worms. According to our findings, act-1 and cdc-42 were effective in accurately normalizing the levels of gene expression in N2 and lin-35. act-1 and cdc-42 also displayed the most consistent expression patterns, therefore they were utilized to standardize expression level of sgk-1. Furthermore, our results clearly showed that sgk-1 was upregulated in lin-35 worms compared to N2 worms. Our results highlight the importance of definitive validation using mostly expressed reference genes.

Multi-tissue transcriptome analysis to identify candidate genes associated with weight regulation in Hanwoo cattle.

While genetic markers related to meat production traits have been identified in many other cattle breeds, research on weight in Hanwoo cattle (Korean native cattle) is still insufficient. In this study, we performed expression quantitative trait loci (eQTL) analysis and differential gene expression analysis to detect candidate genes influencing the weight characteristics of 32 castrated Hanwoo cattle across 22 tissues and, we identified variants that affect gene expression levels. In total, we identified a total of 3,298 differentially expressed genes, among which we discovered key genes such as UBD, RGS2, FASN, and SCD that have functions related to adipogenesis, body weight, obesity, and lipid metabolism. Gene-set enrichment analysis revealed that candidate genes in adipose tissue are involved in metabolic pathways linked to obesity-related traits, adipose metabolism, and lipid metabolism. Additionally, we found that decreased expression of TRIM31 contributes to weight gain which can be explained by the associated candidate cis-eQTL genotypes for TRIM31 and their effect on differential gene expression between the lower and higher weight groups. Our findings revealed candidate genes associated with the weight of Hanwoo cattle and perhaps can provide comprehensive insights into the association of weight with various tissues beyond adipose tissue and muscle, indicating the potential for expanding the focus of livestock trait research.

Silicon and arbuscular mycorrhizal fungi alleviate chromium toxicity in Brassica rapa by regulating Cr uptake, antioxidant defense expression, the glyoxalase system, and secondary metabolites

The potential contribution of silicon (Si) (300 mg kg−1 potash silica) or arbuscular mycorrhizal fungi (AMF) (Rhizophagus irregularis) to reduce chromium toxicity (Cr; 0 and 300 mg kg−1) in Brassica rapa was examined in this work. Under Cr stress, Si and AMF were used separately and in combination (no Si, or AMF, Si, AMF, and Si + AMF). Brassica rapa growth, colonization, photosynthesis, and physio-biochemical characteristics decreased under Cr stress. Oxidative stress was a side effect of Cr stress and was associated with high levels of methylglyoxal (MG), hydrogen peroxide (H2O2), lipid peroxidation (MDA), and maximum lipoxygenase activity (LOX). On the other hand, quantitative real-time PCR analyses of gene expression showed that under Cr stress, the expression of genes for secondary metabolites and antioxidant enzymes was higher than that under the control. The co-application of Si and AMF activated the plant defense system by improving the antioxidative enzymes activities, the potassium citrate and glutathione pool, the glyoxalase system, metabolites, and genes encoding these enzymes under Cr stress. Under the influence of Cr stress, oxidative stress was reduced by the coordinated control of the antioxidant and glyoxalase systems. However, the restricted Cr uptake and root and shoot accumulation of Si and AMF co-applied to only Cr-stressed plants was more significant. In summary, Si and AMF applied together successfully counteract the deleterious effects of Cr stress and restore growth and physio-biochemical characteristics. As a result, the beneficial effects of the combined Si and AMF application may be attributed to mycorrhizae-mediated enhanced Si absorption and metal resistance.

FLAgellum Member 8 modulates extravascular distribution of African trypanosomes.

In the mammalian host, the biology of tissue-dwelling Trypanosoma brucei parasites is not completely understood, especially the mechanisms involved in their extravascular colonization. The trypanosome flagellum is an essential organelle in multiple aspects of the parasites' development. The flagellar protein termed FLAgellar Member 8 (FLAM8) acts as a docking platform for a pool of cyclic AMP response protein 3 (CARP3) that is involved in signaling. FLAM8 exhibits a stage-specific distribution suggesting specific functions in the mammalian and vector stages of the parasite. Analyses of knockdown and knockout trypanosomes in their mammalian forms demonstrated that FLAM8 is not essential in vitro for survival, growth, motility and stumpy differentiation. Functional investigations in experimental infections showed that FLAM8-deprived trypanosomes can establish and maintain an infection in the blood circulation and differentiate into insect transmissible forms. However, quantitative bioluminescence imaging and gene expression analysis revealed that FLAM8-null parasites exhibit a significantly impaired dissemination in the extravascular compartment, that is restored by the addition of a single rescue copy of FLAM8. In vitro trans-endothelial migration assays revealed significant defects in trypanosomes lacking FLAM8. FLAM8 is the first flagellar component shown to modulate T. brucei distribution in the host tissues, possibly through sensing functions, contributing to the maintenance of extravascular parasite populations in mammalian anatomical niches, especially in the skin.

Aquaporin 1 overexpression may enhance glioma tumorigenesis by interacting with the transcriptional regulation networks of Foxo4, Maz, and E2F families

BackgroundThe glioblastoma has served as a valuable experimental model system for investigating the growth and invasive properties of glioblastoma. Aquaporin-1 (AQP1) in facilitating cell migration and potentially contributing to tumor progression. In this study, we analyzed the role of AQP1 overexpression in glioblastoma and elucidated the main mechanisms involved.MethodsAQP1 overexpression recombinant vector was introduced into C6 rat glioma cells to construct an AQP1 overexpression C6 cell line, and its effect on cell viability and migration ability was detected by MTT and Transwell. RNA was extracted by Trizol method for gene sequencing and transcriptomics analysis, and the differentially expressed genes (DEGs) were enriched for up- and downregulated genes by Principal component analysis (PCA), and the molecular mechanism of AQP1 overexpression was analyzed in comparison with the control group using the NCBI GEO database. Statistical analysis was performed using Mann-Whitney paired two tailed t test.ResultsThe cell viability of AQP1-transfected cell lines increased by 23% and the mean distance traveled increased by 67% compared with the control group. Quantitative analysis of gene expression showed that there were 12,121 genes with an average transcripts per million (TPM) value greater than 1. DEGs accounted for 13% of the genes expressed, with the highest correlation with upregulated genes being FOXO4 and MAZ, and the highest with downregulated genes being E2F TFs.ConclusionsAQP1 may be implicated in glioma formation by interacting with the transcriptional regulation networks involving the FOXO4, MAZ, and E2F1/2. These findings shed light on the potential significance of AQP1 in glioma pathogenesis and warrant further investigations to unravel the underlying molecular mechanisms.

Identification and Functional Characterization of Two Major Loci Associated with Resistance against Brown Planthoppers (Nilaparvata lugens (Stål)) Derived from Oryza nivara.

The brown planthopper (BPH) is a highly destructive pest of rice, causing significant economic losses in various regions of South and Southeast Asia. Researchers have made promising strides in developing resistance against BPH in rice. Introgression line RPBio4918-230S, derived from Oryza nivara, has shown consistent resistance to BPH at both the seedling and adult stages of rice plants. Segregation analysis has revealed that this resistance is governed by two recessive loci, known as bph39(t) and bph40(t), contributing to 21% and 22% of the phenotypic variance, respectively. We later mapped the genes using a backcross population derived from a cross between Swarna and RPBio4918-230S. We identified specific marker loci, namely RM8213, RM5953, and R4M17, on chromosome 4, flanking the bph39(t) and bph40(t) loci. Furthermore, quantitative expression analysis of candidate genes situated between the RM8213 and R4M17 markers was conducted. It was observed that eight genes exhibited up-regulation in RPBio4918-230S and down-regulation in Swarna after BPH infestation. One gene of particular interest, a serine/threonine-protein kinase receptor (STPKR), showed significant up-regulation in RPBio4918-230S. In-depth sequencing of the susceptible and resistant alleles of STPKR from Swarna and RPBio4918-230S, respectively, revealed numerous single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel) mutations, both in the coding and regulatory regions of the gene. Notably, six of these mutations resulted in amino acid substitutions in the coding region of STPKR (R5K, I38L, S120N, T319A, T320S, and F348S) when compared to Swarna and the reference sequence of Nipponbare. Further validation of these mutations in a set of highly resistant and susceptible backcross inbred lines confirmed the candidacy of the STPKR gene with respect to BPH resistance controlled by bph39(t) and bph40(t). Functional markers specific for STPKR have been developed and validated and can be used for accelerated transfer of the resistant locus to elite rice cultivars.

Comprehensive analyses of immune tumor microenvironment in papillary renal cell carcinoma

BackgroundPapillary renal cell carcinoma (pRCC) is the most common non-clear cell RCC, and associated with poor outcomes in the metastatic setting. In this study, we aimed to comprehensively evaluate the...

Expression of defense responsive genes in tripartite interaction of cucumber mosaic virus and plant growth promoting rhizobacteria in ridge gourd (Luffa acutangula (L.) Roxb)

The mosaic caused by cucumber mosaic virus (CMV) on ridge gourd (Luffa acutangula (L.) Roxb) is an important disease. An attempt was made to use a consortium of plant growth promoting rhizobacteria to manage the disease with an aim to reduce the usage of pesticide to control its vector. Eleven isolates of Bacillus spp. were selected based on their plant gowth promotion ability on ridge gourd cv. COH1. Among these, Bacillus pumilus (BSC 4), B. subtilis (Bbv 57) and B. subtilis (BST 8) had shown their potentiality in reducing the severity of CMV disease under artificial conditions. The compatibility test showed that these isolates were compatible to each other. A Bacillus consortium was prepared and evaluated for its efficacy against CMV on ridge gourd and the results showed a reduction of 49.98 % in disease severity over control. The quantitative gene expression analysis revealed that the defense genes viz., peroxidase, polyphenol oxidase, catalase and super oxide dismutase were upregulated in consortium treated ridge gourd plants. The upregulation of these genes indicated the activation of defense pathways which witnessed the induction of systemic resistance in Bacillus consortium treated plants. All gene expressions were reached its peak nearly two to three fold in the consortium treated plants except catalase, its peak expression was found in salicylic acid treated plants which served as a positive control. Correspondingly, the quantitative enzymatic analysis of these defense enzymes at different time intervals was also reflected in similar manner. The elevated level of defense enzymes in plant growth promoting rhizobacteria consortium treated plants was the evident for the onset of systemic resistance.

Transcriptome analysis of Apis mellifera antennae reveals molecular divergence underlying the division of labour in worker bees.

The olfactory system plays a fundamental role in mediating insect behaviour. Worker bees exhibit an age-dependent division of labour, performing discrete sets of behaviours throughout their lifespan. The behavioural states of bees rely on their sense of the environment and chemical communication via their olfactory system, the antennae. However, the olfactory adaptation mechanism of worker bees during their behavioural development remains unclear. In this study, we conducted a comprehensive and quantitative analysis of antennal gene expression in the Apis mellifera of newly emerged workers, nurses, foragers and defenders using RNA-seq. We found that the antenna tissues of honey bees continued developing after transformation from newly emerged workers to adults. Additionally, we identified differentially expressed genes associated with bee development and division of labour. We validated that major royal jelly protein genes are highly and specifically expressed in nurse honey bee workers. Furthermore, we identified and validated significant alternative splicing events correlated with the development and division of labour. These findings provide a comprehensive transcriptome profile and a new perspective on the molecular mechanisms that may underlie the worker honey bee division of labour.

Comparative Analysis of Phospholipase D (PLD) Gene Family in Camelina sativa and Brassica napus and Its Responses in Camelina Seedlings under Salt Stress

Phospholipases are among the important elements involved in lipid-dependent cell signaling that lead to the induction of downstream pathways. In the current study, phospholipases D (PLDs) gene family was characterized and compared in two important oilseed crops, Brassica napus and Camelina sativa. The results revealed that PLD has 33 members in Camelina sativa (CsPLD) and 41 members in Brassica napus (BnPLD). All studied PLDs showed a negative GRAVY value, indicating that PLDs are probably hydrophilic proteins. Phylogenetic analysis classified PLDs into five main subfamilies, including gamma, delta, beta, alpha, and zeta. According to evolution analysis, a different evolution process was observed between CsPLD and BnPLD. In addition, the results disclosed that most of the PLD genes have been segmentally duplicated under purifying selection. Cis-regulatory elements related to ABA and auxin responsiveness were found more in the upstream region of CsPLDs, while elements linked with MeJA responsiveness were observed more in the promoter region of BnPLDs. Analysis of the expression data showed that PLD alpha genes have a wide expression in most tissues. Quantitative expression analysis (qPCR) of CsPLD genes under salt stress, 200 mM of NaCl, was conducted in different time series. The results revealed that the CsPLD genes are involved in the response to salinity stress and their expression levels enhance with increasing salinity stress period. The outcomes of this research will be useful for future molecular works related to lipid signaling in oilseed plants.

Investigating the impact of pedoclimatic conditions on the oenological performance of two red cultivars grown throughout southern Italy.

The cultivated grapevine, Vitis vinifera subsp. vinifera, possesses a rich biodiversity with numerous varieties. Each variety adapts differently to varying pedoclimatic conditions, which greatly influence the terroir expression of wine regions. These conditions impact vine growth, physiology, and berry composition, ultimately shaping the unique characteristics and typicity of the wines produced. Nowadays, the potential of the different adaptation capacities of grape varieties has not yet been thoroughly investigated. We addressed this issue by studying two grape varieties, Aglianico and Cabernet Sauvignon, in two different pedoclimatic conditions of Southern Italy. We evaluated and compared the effect of different pedoclimatic conditions on plant physiology, the microbial quality of grapes using Next-Generation Sequencing (NGS) technology, the expression trends of key genes in ripe berries and the concentration of phenolic compounds in grapes and wines by HPLC-MS, HPLC-DAD, NMR and spectrophotometric analyses. Metabolomic and microbiome data were integrated with quantitative gene expression analyses to examine varietal differences and plasticity of genes involved in important oenological pathways. The data collected showed that the phenotypic response of studied grapes in terms of vigor, production, and fruit quality is strongly influenced by the pedoclimatic conditions and, in particular, by soil physical properties. Furthermore, Aglianico grape variety was more influenced than the Cabernet Sauvignon by environmental conditions. In conclusion, the obtained findings not only reinforce the terroir concept and our comprehension of grape's ability to adapt to climate variations but can also have implications for the future usage of grape genetic resources.

Evaluation of reference genes for quantitative analysis of gene expression in Lippia alba under abiotic stress

Evaluation of reference genes for quantitative analysis of gene expression in Lippia alba under abiotic stress

A phase 1 trial of umbilical cord blood–derived tumor-reactive PD-L1+ natural killer cells engineered to express soluble IL-15 (TRACK-NK) in patients with non–small-cell lung cancer (NSCLC) refractory to PD-1/PD-L1 inhibitors.

TPS2665 Background: The mechanism by which patients with PD-L1 – tumors can respond favorably to anti-PD-L1 monoclonal antibody therapy has been shown to involve the PD-L1+ NK cell in the tumor microenvironment (TME). PD-L1 is induced on the cell surface when NK cells recognize tumors, leading to enhanced NK-cell function without exhaustion. The PD-L1 + NK cell is capable of trafficking to the lung TME, recognizing and killing tumor cells as well as inducing activation of endogenous T cells via potent cytokine secretion, all without the expression of a chimeric antigen receptor (CAR). TRACK-NK cells are PD-L1+ NK cells derived from cord blood (CB) and engineered to express soluble IL-15 (sIL-15). The TRACK-NK cells express high levels of tumor reactive receptors that recognize "tumor stress" including DNAM-1, NKp30 and NKG2D. TRACK-NK cells induced significant reduction in tumor volume in mice injected with human A549 NSCLC cells compared to mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation. Survival was also prolonged in the H460 NSCLC model compared to vehicle alone. We hypothesized that TRACK-NK cells can be safely administered, and will provide meaningful clinical benefit in patients with NSCLC. Methods: NCT05334329 is a first-in-human, phase 1 dose finding study of allogeneic, off-the shelf frozen and thawed TRACK-NK cells in patients with NSCLC progressing on treatment with PD-1/PD-L1 inhibitors. TRACK-NK cells (CB NK cells engineered with retroviral transduction to express sIL-15 and ex vivo expanded and activated to express PD-L1) are administered in four weekly intravenous infusions per cycle following lymphocyte depletion (Fludarabine/Cyclophosphamide x 3 days) in the outpatient setting. Patients not experiencing disease progression (per RECIST v1.1) following cycle 1 may receive a second cycle. Primary endpoints include: 1. Toxicity/adverse events as per NCI-CTCA version 5.0, and ASTCT Consensus Grading for Cytokine Release Syndrome (CRS) and Neurotoxicity; and 2. Magnitude/duration of TRACK-NK cell persistence in peripheral blood via droplet digital PCR. We are employing a Utility Based-Bayesian Optimal Interval design to direct optimal dose determination. Dose limiting toxicity is defined as any ≥ grade 3 non-hematologic toxicity; any grade 3 CRS that does not resolve to ≤ grade 1 within 7 days; any ≥ grade 4 CRS; any ≥ grade 2 neurotoxicity that does not resolve to grade 1 within 72 hours; any ≥ grade 3 autoimmune, hypersensitivity, and acute graft-versus-host reactions. Correlatives also include cytokines levels, quantity and activation of CD4 and CD8 cells in peripheral blood, and NK and T cell trafficking and TME changes in paired tumor biopsies using quantitative immunofluorescence and gene expression analysis. Clinical trial information: NCT05334329 .

Understanding of arthrofibrosis: New explorative insights into extracellular matrix remodeling of synovial fibroblasts.

Arthrofibrosis following total knee arthroplasty is a fibroproliferative joint disorder marked by dysregulated biosynthesis of extracellular matrix proteins, such as collagens and proteoglycans. The underlying cellular events remain incompletely understood. Myofibroblasts are highly contractile matrix-producing cells characterized by increased alpha-smooth muscle actin expression and xylosyltransferase-I (XT-I) secretion. Human XT-I has been identified as a key mediator of arthrofibrotic remodeling. Primary fibroblasts from patients with arthrofibrosis provide a useful in vitro model to identify and characterize disease regulators and potential therapeutic targets. This study aims at characterizing primary synovial fibroblasts from arthrofibrotic tissues (AFib) regarding their molecular and cellular phenotype by utilizing myofibroblast cell culture models. Compared to synovial control fibroblasts (CF), AFib are marked by enhanced cell contractility and a higher XT secretion rate, demonstrating an increased fibroblast-to-myofibroblast transition rate during arthrofibrosis. Histochemical assays and quantitative gene expression analysis confirmed higher collagen and proteoglycan expression and accumulation in AFib compared to CF. Furthermore, fibrosis-based gene expression profiling identified novel modifier genes in the context of arthrofibrosis remodeling. In summary, this study revealed a unique profibrotic phenotype in AFib that resembles some traits of other fibroproliferative diseases and can be used for the future development of therapeutic interventions.