Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes.

Most patients diagnosed with resected pancreatic adenocarcinoma (PDAC) survive less than 5 years, but a minor subset survives longer. Here, we dissect the role of the tumor microbiota and the immune system in influencing long-term survival. Using 16S rRNA gene sequencing, we analyzed the tumor microbiome composition in PDAC patients with short-term survival (STS) and long-term survival (LTS). We found higher alpha-diversity in the tumor microbiome of LTS patients and identified an intra-tumoral microbiome signature (Pseudoxanthomonas-Streptomyces-Saccharopolyspora-Bacillus clausii) highly predictive of long-term survivorship in both discovery and validation cohorts. Through human-into-mice fecal microbiota transplantation (FMT) experiments from STS, LTS, or control donors, we were able to differentially modulate the tumor microbiome and affect tumor growth as well as tumor immune infiltration. Our study demonstrates that PDAC microbiome composition, which cross-talks to the gut microbiome, influences the host immune response and natural history of the disease.

Pragmatic Trial of Hospitalization Rate in Chronic Kidney Disease.

BACKGROUND: Despite the availability of effective therapies for patients with chronic kidney disease, type 2 diabetes, and hypertension (the kidney-dysfunction triad), the results of large-scale trials examining the implementation of guideline-directed therapy to reduce the risk of death and complications in this population are lacking. METHODS: In this open-label, cluster-randomized trial, we assigned 11,182 patients with the kidney-dysfunction triad who were being treated at 141 primary care clinics either to receive an intervention that used a personalized algorithm (based on the patient's electronic health record [EHR]) to identify patients and practice facilitators to assist providers in delivering guideline-based interventions or to receive usual care. The primary outcome was hospitalization for any cause at 1 year. Secondary outcomes included emergency department visits, readmissions, cardiovascular events, dialysis, and death. RESULTS: We assigned 71 practices (enrolling 5690 patients) to the intervention group and 70 practices (enrolling 5492 patients) to the usual-care group. The hospitalization rate at 1 year was 20.7% (95% confidence interval [CI], 19.7 to 21.8) in the intervention group and 21.1% (95% CI, 20.1 to 22.2) in the usual-care group (between-group difference, 0.4 percentage points; P = 0.58). The risks of emergency department visits, readmissions, cardiovascular events, dialysis, or death from any cause were similar in the two groups. The risk of adverse events was also similar in the trial groups, except for acute kidney injury, which was observed in more patients in the intervention group (12.7% vs. 11.3%). CONCLUSIONS: In this pragmatic trial involving patients with the triad of chronic kidney disease, type 2 diabetes, and hypertension, the use of an EHR-based algorithm and practice facilitators embedded in primary care clinics did not translate into reduced hospitalization at 1 year. (Funded by the National Institutes of Health and others; ICD-Pieces ClinicalTrials.gov number, NCT02587936.).

Bile acid-induced IRF3 phosphorylation mediates cell death, inflammatory responses, and fibrosis in cholestasis-induced liver and kidney injury via regulation of ZBP1.

BACKGROUND AND AIMS: Cell death and inflammation play critical roles in chronic tissue damage caused by cholestatic liver injury leading to fibrosis and cirrhosis. Liver cirrhosis is often associated with kidney damage, which is a severe complication with poor prognosis. Interferon regulatory factor 3 (IRF3) is known to regulate apoptosis and inflammation, but its role in cholestasis remains obscure. In this study. APPROACH AND RESULTS: We discovered increased IRF3 phosphorylation in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. In the bile duct ligation model of obstructive cholestasis in mice, we found that tissue damage was associated with increased phosphorylated IRF3 (p-IRF3) in the liver and kidney. IRF3 knockout ( Irf3-/- ) mice showed significantly attenuated liver and kidney damage and fibrosis compared to wide-type mice after bile duct ligation. Cell-death pathways, including apoptosis, necroptosis, and pyroptosis, inflammasome activation, and inflammatory responses were significantly attenuated in Irf3-/- mice. Mechanistically, we show that bile acids induced p-IRF3 in vitro in hepatocytes. In vivo , activated IRF3 positively correlated with increased expression of its target gene, Z-DNA-Binding Protein-1 (ZBP1), in the liver and kidney. Importantly, we also found increased ZBP1 in the liver of patients with primary biliary cholangitis and primary sclerosing cholangitis. We discovered that ZBP1 interacted with receptor interacting protein 1 (RIP1), RIP3, and NLRP3, thereby revealing its potential role in the regulation of cell-death and inflammation pathways. In conclusion. CONCLUSIONS: Our data indicate that bile acid-induced p-IRF3 and the IRF3-ZBP1 axis play a central role in the pathogenesis of cholestatic liver and kidney injury.

Author Correction: Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury.

Change history: In Fig. 1j of this Letter, one data point was inadvertently omitted from the graph for the acute kidney injury (AKI), double knockout (-/-), S-nitrosothiol (SNO) condition at a nitrosylation level of 25.9 pmol mg-1 and the statistical significance given of P = 0.0221 was determined by Fisher's test instead of P = 0.0032 determined by Tukey's test (with normalization for test-day instrument baseline). Figure 1 and its Source Data have been corrected online.

Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury.

Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood1,2. Nitric oxide-based cellular signalling is generally mediated by protein S-nitrosylation, the oxidative modification of Cys residues to form S-nitrosothiols (SNOs). S-nitrosylation regulates proteins in all functional classes, and is controlled by enzymatic machinery that includes S-nitrosylases and denitrosylases, which add and remove SNO from proteins, respectively3,4. In Saccharomyces cerevisiae, the classic metabolic intermediate co-enzyme A (CoA) serves as an endogenous source of SNOs through its conjugation with nitric oxide to form S-nitroso-CoA (SNO-CoA), and S-nitrosylation of proteins by SNO-CoA is governed by its cognate denitrosylase, SNO-CoA reductase (SCoR)5. Mammals possess a functional homologue of yeast SCoR, an aldo-keto reductase family member (AKR1A1)5 with an unknown physiological role. Here we report that the SNO-CoA-AKR1A1 system is highly expressed in renal proximal tubules, where it transduces the activity of eNOS in reprogramming intermediary metabolism, thereby protecting kidneys against acute kidney injury. Specifically, deletion of Akr1a1 in mice to reduce SCoR activity increased protein S-nitrosylation, protected against acute kidney injury and improved survival, whereas this protection was lost when Enos (also known as Nos3) was also deleted. Metabolic profiling coupled with unbiased mass spectrometry-based SNO-protein identification revealed that protection by the SNO-CoA-SCoR system is mediated by inhibitory S-nitrosylation of pyruvate kinase M2 (PKM2) through a novel locus of regulation, thereby balancing fuel utilization (through glycolysis) with redox protection (through the pentose phosphate shunt). Targeted deletion of PKM2 from mouse proximal tubules recapitulated precisely the protective and mechanistic effects of S-nitrosylation in Akr1a1-/- mice, whereas Cys-mutant PKM2, which is refractory to S-nitrosylation, negated SNO-CoA bioactivity. Our results identify a physiological function of the SNO-CoA-SCoR system in mammals, describe new regulation of renal metabolism and of PKM2 in differentiated tissues, and offer a novel perspective on kidney injury with therapeutic implications.

Functional hierarchy of PCNA-interacting motifs in DNA processing enzymes.

Numerous eukaryotic DNA processing enzymes, such as DNA polymerases and ligases, bind the processivity factor PCNA, which acts as a platform to recruit and regulate the binding of enzymes to their DNA substrate. Multiple PCNA-interacting motifs (PIPs) are present in these enzymes, but their individual structural and functional role has been a matter of debate. Recent cryo-EM reconstructions of high-fidelity DNA polymerase Pol δ (Pol δ), translesion synthesis DNA polymerase κ (Pol κ) and Ligase 1 (Lig1) bound to a DNA substrate and PCNA demonstrate that the critical interaction with PCNA involves the internal PIP proximal to the catalytic domain. The ancillary PIPs, located in long disordered regions, are instead invisible in the reconstructions, and appear to function as flexible tethers when the enzymes fall off the DNA. In this review, we discuss the recent structural advancements and propose a functional hierarchy for the PIPs in Pol δ, Pol κ, and Lig1.

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism.

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5'-, but not 3'-ends of biotinylated DNA.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Nucleotide excision repair (NER) removes bulky DNA lesions and is thereby crucial in maintaining transcription and genomic integrity. Here, the authors show a dual function for the XPG nuclease that is critical for finding and excising the damage. During the separation of the damage-containing strand from the undamaged strand, XPG stimulates TFIIH dependent dsDNA unwinding 10 fold. In return, when TFIIH stalls at the damage it stimulates XPG nuclease activity 700 fold. Remarkably, this mutually exclusive coordination requires a bubble longer than 15 nucleotides. This study addressees why a bubble of a certain size is needed to facilitate NER and why XPG is recruited at the beginning of NER when its endonucleolytic activity is required at the very end.

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing.

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme. Using novel HelQ mutant proteins we identify that inhibition of Pol δ is independent of DNA binding, and maps to a 70 amino acid intrinsically disordered region of HelQ. Pol δ and its POLD3 subunit robustly stimulated DNA single-strand annealing by HelQ, and POLD3 and HelQ interact physically via the intrinsically disordered HelQ region. This data, and inability of HelQ to inhibit DNA synthesis by the POLD1 catalytic subunit of Pol δ, reveal a mechanism for limiting DNA synthesis and promoting DNA strand annealing during human DNA break repair, which centres on POLD3.

Motors, switches, and contacts in the replisome.

Replisomes are the protein assemblies that replicate DNA. They function as molecular motors to catalyze template-mediated polymerization of nucleotides, unwinding of DNA, the synthesis of RNA primers, and the assembly of proteins on DNA. The replisome of bacteriophage T7 contains a minimum of proteins, thus facilitating its study. This review describes the molecular motors and coordination of their activities, with emphasis on the T7 replisome. Nucleotide selection, movement of the polymerase, binding of the processivity factor, unwinding of DNA, and RNA primer synthesis all require conformational changes and protein contacts. Lagging-strand synthesis is mediated via a replication loop whose formation and resolution is dictated by switches to yield Okazaki fragments of discrete size. Both strands are synthesized at identical rates, controlled by a molecular brake that halts leading-strand synthesis during primer synthesis. The helicase serves as a reservoir for polymerases that can initiate DNA synthesis at the replication fork. We comment on the differences in other systems where applicable.

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed. DESIGN: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites. RESULTS: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.

Mitochondrial genetic variation and risk of chronic kidney disease and acute kidney injury in UK Biobank participants.

Experimental models suggest an important role for mitochondrial dysfunction in the pathogenesis of chronic kidney disease (CKD) and acute kidney injury (AKI), but little is known regarding the impact of common mitochondrial genetic variation on kidney health. We sought to evaluate associations of inherited mitochondrial DNA (mtDNA) variation with risk of CKD and AKI in a large population-based cohort. We categorized UK Biobank participants who self-identified as white into eight distinct mtDNA haplotypes, which were previously identified based on their associations with phenotypes associated with mitochondrial DNA copy number, a measure of mitochondrial function. We used linear and logistic regression models to evaluate associations of these mtDNA haplotypes with estimated glomerular filtration rate by serum creatinine and cystatin C (eGFRCr-CysC, N = 362,802), prevalent (N = 416 cases) and incident (N = 405 cases) end-stage kidney disease (ESKD), AKI defined by diagnostic codes (N = 14,170 cases), and urine albumin/creatinine ratio (ACR, N = 114,662). The mean age was 57 ± 8 years and the mean eGFR was 90 ± 14 ml/min/1.73 m2. MtDNA haplotype was significantly associated with eGFR (p = 2.8E-12), but not with prevalent ESKD (p = 5.9E-2), incident ESKD (p = 0.93), AKI (p = 0.26), or urine ACR (p = 0.54). The association of mtDNA haplotype with eGFR remained significant after adjustment for diabetes mellitus and hypertension (p = 1.2E-10). When compared to the reference haplotype, mtDNA haplotypes I (ß = 0.402, standard error (SE) = 0.111; p = 2.7E-4), IV (ß = 0.430, SE = 0.073; p = 4.2E-9), and V (ß = 0.233, SE = 0.050; p = 2.7E-6) were each associated with higher eGFR. Among self-identified white UK Biobank participants, mtDNA haplotype was associated with eGFR, but not with ESKD, AKI or albuminuria.

Severe Combined Immunodeficiency from a Homozygous DNA Ligase 1 Mutant with Reduced Catalytic Activity but Increased Ligation Fidelity.

A cell's ability to survive and to evade cancer is contingent on its ability to retain genomic integrity, which can be seriously compromised when nucleic acid phosphodiester bonds are disrupted. DNA Ligase 1 (LIG1) plays a key role in genome maintenance by sealing single-stranded nicks that are produced during DNA replication and repair. Autosomal recessive mutations in a limited number of individuals have been previously described for this gene. Here we report a homozygous LIG1 mutation (p.A624T), affecting a universally conserved residue, in a patient presenting with leukopenia, neutropenia, lymphopenia, pan-hypogammaglobulinemia, and diminished in vitro response to mitogen stimulation. Patient fibroblasts expressed normal levels of LIG1 protein but exhibited impaired growth, poor viability, high baseline levels of gamma-H2AX foci, and an enhanced susceptibility to DNA-damaging agents. The mutation reduced LIG1 activity by lowering its affinity for magnesium 2.5-fold. Remarkably, it also increased LIG1 fidelity > 50-fold against 3' end 8-Oxoguanine mismatches, exhibiting a marked reduction in its ability to process such nicks. This is expected to yield increased ss- and dsDNA breaks. Molecular dynamic simulations, and Residue Interaction Network studies, predicted an allosteric effect for this mutation on the protein loops associated with the LIG1 high-fidelity magnesium, as well as on DNA binding within the adenylation domain. These dual alterations of suppressed activity and enhanced fidelity, arising from a single mutation, underscore the mechanistic picture of how a LIG1 defect can lead to severe immunological disease.

Refining the phenotypic spectrum of CCDC88A-related PEHO-like syndrome.

Progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) and PEHO-like syndromes are very rare infantile disorders characterized by profound intellectual disability, hypotonia, convulsions, optic, and progressive brain atrophy. Many causative genes for PEHO and PEHO-like syndromes have been identified including CCDC88A. So far, only five patients from two unrelated families with biallelic CCDC88A variants have been reported in the literature. Herein, we describe a new family from Egypt with a lethal epileptic encephalopathy. Our patient was the youngest child born to a highly consanguineous couple and had a family history of five deceased sibs with the same condition. She presented with postnatal microcephaly, poor visual responsiveness, and epilepsy. Her brain MRI showed abnormal cortical gyration with failure of opercularization of the insula, hypogenesis of corpus callosum, colpocephaly, reduced white matter, hypoplastic vermis, and brain stem. Whole exome sequencing identified a new homozygous frameshift variant in CCDC88A gene (c.1795_1798delACAA, p.Thr599ValfsTer4). Our study presents the third reported family with this extremely rare disorder. We also reviewed all described cases to better refine the phenotypic spectrum associated with biallelic loss of function variants in the CCDC88A gene.

Thiamine for Renal Protection in Septic Shock (TRPSS): A Randomized, Placebo-controlled, Clinical Trial.

Rationale: Kidney injury is common and associated with worse outcomes in patients with septic shock. Mitochondrial resuscitation with thiamine (vitamin B1) may attenuate septic kidney injury. Objectives: To assess whether thiamine supplementation attenuates kidney injury in septic shock. Methods: The TRPSS (Thiamine for Renal Protection in Septic Shock) trial was a multicenter, randomized, placebo-controlled trial of thiamine versus placebo in septic shock. The primary outcome was change in serum creatinine between enrollment and 72 hours after enrollment. Measurements and Main Results: Eighty-eight patients were enrolled (42 patients received the intervention, and 46 received placebo). There was no significant between-groups difference in creatinine at 72 hours (mean difference, -0.57 mg/dl; 95% confidence interval, -1.18, 0.04; P = 0.07). There was no difference in receipt of kidney replacement therapy (14.3% vs. 21.7%, P = 0.34), acute kidney injury (as defined by stage 3 of the Kidney Disease: Improving Global Outcomes acute kidney injury scale; 54.7% vs. 73.9%, P = 0.07), or mortality (35.7% vs. 54.3%, P = 0.14) between the thiamine and placebo groups. Patients who received thiamine had more ICU-free days (median [interquartile range]: 22.5 [0.0-25.0] vs. 0.0 [0.0-23.0], P < 0.01). In the thiamine-deficient cohort (27.4% of patients), there was no difference in rates of kidney failure (57.1% thiamine vs. 81.5% placebo) or in-hospital mortality (28.6% vs. 68.8%) between groups. Conclusions: In the TRPSS trial, there was no statistically significant difference in the primary outcome of change in creatinine over time. Patients who received thiamine had more ICU-free days, but there was no difference in other secondary outcomes. Clinical trial registered with www.clinicaltrials.gov (NCT03550794).

Development of autoantibody signatures for common cancers.

A humoral immune response in the form of autoantibodies to tumor antigens occurs early during tumor development. Identification of antigens that induce an autoantibody response restricted to a cancer type has the potential to yield markers useful for early detection. A multitude of strategies are currently available for the discovery of tumor antigens directed autoantibodies in circulation. Each approach has advantages and limitations for comprehensive discovery of antigenic epitopes. Herein, we review established and novel strategies and methodologies and highlight potential cancer applications.

Design, synthesis, molecular modelling and biological evaluation of novel 6-amino-5-cyano-2-thiopyrimidine derivatives as potent anticancer agents against leukemia and apoptotic inducers.

Herein, a novel series of 6-amino-5-cyano-2-thiopyrimidines and condensed pyrimidines analogues were prepared. All the synthesized compounds (1a-c, 2a-c, 3a-c, 4a-r and 5a-c) were evaluated for in vitro anticancer activity by the National Cancer Institute (NCI; MD, USA) against 60 cell lines. Compound 1c showed promising anticancer activity and was selected for the five-dose testing. Results demonstrated that compound 1c possessed broad spectrum anti-cancer activity against the nine cancerous subpanels tested with selectivity ratio ranging from 0.7 to 39 at the GI50 level with high selectivity towards leukaemia. Mechanistic studies showed that Compound 1c showed comparable activity to Duvelisib against PI3Kδ (IC50 = 0.0034 and 0.0025 µM, respectively) and arrested cell cycle at the S phase and displayed significant increase in the early and late apoptosis in HL60 and leukaemia SR cells. The necrosis percentage showed a significant increase from 1.13% to 3.41% in compound 1c treated HL60 cells as well as from 1.51% to 4.72% in compound 1c treated leukaemia SR cells. Also, compound 1c triggered apoptosis by activating caspase 3, Bax, P53 and suppressing Bcl2. Moreover, 1c revealed a good safety profile against human normal lung fibroblast cell line (WI-38 cells). Molecular analysis of Duvelisib and compound 1c in PI3K was performed. Finally, these results suggest that 2-thiopyrimidine derivative 1c might serve as a model for designing novel anticancer drugs in the future.

Whiteness and greenness assessments of a sensitive HPLC method with fluorimetric detection for dapagliflozin quantitation in human plasma: Application to a healthy human volunteer.

The evident ecological impact of human actions, like air pollution, global warming, and ozone depletion, underscores the need for environmentally friendly approaches across various domains, including analytical chemistry. This study aimed to establish a validated, eco-friendly, and sustainable approach utilizing a fluorescence detector coupled with high-performance liquid chromatography for quantifying the antihyperglycemic agent dapagliflozin (DAPA), in human plasma. This method employed a C18 Microsorb MV (4.5 × 250 mm, 5 µm [particle size]) column at 40°C, with 40:60% v/v isocratic elution of acetonitrile and (0.1%) orthophosphoric acid as the mobile phase at 1 mL/min flow rate. DAPA and the internal standard demonstrated their greatest response by performing excitation at 225 nm (λex) and recording chromatograms at an emission wavelength (λem) equal to 305 nm. The presented approach demonstrated high linearity between 50 and 2000 ng/mL and full adherence to the guidelines of the US Food and Drug Administration regarding the validation of bioanalytical methods. The described technique was effectively used for quantification of DAPA in human plasma samples from a healthy male participant who received a tablet of 10 mg DAPA. Analytical Eco-Scale, Analytical GREEnness metric, and the recently created ChlorTox Scale were utilized for greenness assessment. Additionally, the "Red, Green, and Blue 12" model was used in whiteness evaluation.

Circulating microRNA Panel for Prediction of Recurrence and Survival in Early-Stage Lung Adenocarcinoma.

Early-stage lung adenocarcinoma (LUAD) patients remain at substantial risk for recurrence and disease-related death, highlighting the unmet need of biomarkers for the assessment and identification of those in an early stage who would likely benefit from adjuvant chemotherapy. To identify circulating miRNAs useful for predicting recurrence in early-stage LUAD, we performed miRNA microarray analysis with pools of pretreatment plasma samples from patients with stage I LUAD who developed recurrence or remained recurrence-free during the follow-up period. Subsequent validation in 85 patients with stage I LUAD resulted in the development of a circulating miRNA panel comprising miR-23a-3p, miR-320c, and miR-125b-5p and yielding an area under the curve (AUC) of 0.776 in predicting recurrence. Furthermore, the three-miRNA panel yielded an AUC of 0.804, with a sensitivity of 45.8% at 95% specificity in the independent test set of 57 stage I and II LUAD patients. The miRNA panel score was a significant and independent factor for predicting disease-free survival (p < 0.001, hazard ratio [HR] = 1.64, 95% confidence interval [CI] = 1.51-4.22) and overall survival (p = 0.001, HR = 1.51, 95% CI = 1.17-1.94). This circulating miRNA panel is a useful noninvasive tool to stratify early-stage LUAD patients and determine an appropriate treatment plan with maximal efficacy.

Epithelial SOX9 drives progression and metastases of gastric adenocarcinoma by promoting immunosuppressive tumour microenvironment.

OBJECTIVE: Many cancers engage embryonic genes for rapid growth and evading the immune system. SOX9 has been upregulated in many tumours, yet the role of SOX9 in mediating immunosuppressive tumour microenvironment is unclear. Here, we aim to dissect the role of SOX9-mediated cancer stemness attributes and immunosuppressive microenvironment in advanced gastric adenocarcinoma (GAC) for novel therapeutic discoveries. METHODS: Bulk RNAseq/scRNA-seq, patient-derived cells/models and extensive functional studies were used to identify the expression and functions of SOX9 and its target genes in vitro and in vivo. Immune responses were studied in PBMCs or CD45+ immune cells cocultured with tumour cells with SOX9high or knockout and the KP-Luc2 syngeneic models were used for efficacy of combinations. RESULTS: SOX9 is one of the most upregulated SOX genes in GAC and highly expressed in primary and metastatic tissues and associated with poor prognosis. Depletion of SOX9 in patient-derived GAC cells significantly decreased cancer stemness attributes, tumour formation and metastases and consistently increased CD8+ T cell responses when cocultured with PBMCs/CD45+ cells from GAC patients. RNA sequencing identified the leukaemia inhibitory factor (LIF) as the top secreted molecule regulated by SOX9 in tumour cells and was enriched in malignant ascites and mediated SOX9-induced M2 macrophage repolarisation and inhibited T cell function. CONCLUSION: Epithelial SOX9 is critical in suppressing CD8+ T cell responses and modified macrophage function in GAC through the paracrine LIF factor. Cotargeting LIF/LIFR and CSF1R has great potential in targeting SOX9-mediated cancer stemness, T cell immunosuppression and metastases suggesting the novel combination therapy against advanced GAC.