Enlarged facial pores are a common aesthetic concern associated with aging and reduced dermal support. Hybrid Cooperative Complex (HCC), a stabilized hyaluronic acid formulation, has been proposed to improve skin quality through bioremodeling. This prospective observational pilot study evaluated changes in facial pore size after treatment with Hybrid Cooperative Complex (HCC) using standardized photography, clinical assessment, and validated rating scales. Ten healthy adult participants received two treatment sessions according to the Bio Aesthetic Points protocol, and outcomes were assessed at baseline and on Days 30, 60, 120, and 180. Visible improvement in facial pore size and skin texture was observed after treatment. Peak pore refinement was noted at Day 120 in most participants, while partial regression was observed in many cases by Day 180, although skin quality remained improved compared with baseline. HCC may be associated with visible improvement in facial pore size and skin quality in this pilot cohort. Changes in pore morphology may represent a practical clinical marker for estimating maintenance treatment timing, although larger controlled studies are needed.
In patients with a hip fracture, anaemia has been associated with increased transfusion requirements, poor functional outcomes, prolonged hospital stays and increased mortality. While anaemia in elderly patients with hip fractures has traditionally been attributed to bleeding during or after surgery, many of these patients are anaemic on hospital admission. Thus, detecting and managing anaemia in the perioperative, postoperative and, most significantly, the preoperative period is important to avoid the need for blood transfusions and to improve patient outcomes. The protocol for this clinical trial is designed to evaluate the efficacy and safety of both combined intravenous and topical tranexamic acid (TXA) therapy, or topical administration alone, assessing its effect on blood loss and the need for blood transfusions in elderly patients undergoing hip fracture surgery. This is a multicentre, double-blinded, randomised, placebo-controlled trial with a 1:1 allocation ratio. Patients of both sexes, aged ≥65 years, who are admitted to the emergency department and will undergo hip fracture surgery are eligible for enrolment. Eligible patients who provide their consent will be stratified according to the type of fracture (intracapsular and extracapsular) and whether or not they are suitable for intravenous TXA therapy, and they will then be randomly allocated to receive either TXA or a placebo. The primary outcome is the blood transfusion rate from patient admission to the emergency department until discharge, while the secondary outcomes include: the preoperative, perioperative and postoperative haemoglobin and haematocrit levels; the preoperative and postoperative occult and total blood loss; the mean length of hospital stay; and any adverse events assessed for up to 1 year after patient discharge. The study was approved by the Basque Country Ethics Committee (Ref.: 2021012) and the Spanish Agency for Medicines and Healthcare Products (Agencia Española de Medicamentos y Productos Sanitarios). All participants will provide their written informed consent prior to study inclusion. The trial's results, regardless of its outcomes, will be disseminated through presentations at scientific conferences and publication in peer-reviewed journals, and they will be made publicly available through the European Union Clinical Trials Register after the end of the clinical trial. EudraCT Number 2020-002144-23; EUCT Number 2024-519349-31-00.
Hidradenitis suppurativa (HS) is a chronic skin disease that greatly affects patients' health-related quality of life (HRQoL). Bimekizumab, a humanised monoclonal antibody that inhibits interleukin (IL)-17A and IL‑17F, has demonstrated efficacy in HS. This study aimed to assess the association between clinical response and patient-reported benefits on HRQoL, using pooled 48-week data from BE HEARD I&II, in moderate to severe HS. Patients received (initial/maintenance; treatment switch at Week 16) bimekizumab 320 mg every 2 weeks (Q2W)/Q2W, Q2W/every 4 weeks (Q4W), Q4W/Q4W or placebo/Q2W. Data are reported for patients randomised to bimekizumab from baseline (bimekizumab Total group) and Q2W/Q4W (Supplement). Patients were grouped into Week 16 HS Clinical Response (HiSCR) bands (HiSCR<50/50-<75/75-<90/90-100). Proportions of patients meeting pre-defined thresholds in HRQoL outcomes are reported at Weeks 16/48: meaningful improvements in HS QoL questionnaire (HiSQOL) total response and domain scores, no/mild impact of HS on HRQoL and Dermatology Life Quality Index (DLQI) scores 0/1 and 0-5. At Weeks 16/48, greater proportions of patients achieved HiSQOL total score responses with higher Week 16 HiSCR bands (HiSCR<50: 28.2%/35.9%; HiSCR50-< 75: 34.6%/37.2%; HiSCR75-< 90: 36.1%/47.5%; HiSCR90-100: 57.4%/66.2%). Similar patterns were observed across HiSQOL domains and in the proportions of patients who reported no/mild impact on HRQoL (HiSQOL total score ≤14). At Weeks 16/48, greater proportions of patients who achieved Week 16 HiSCR75-<90/HiSCR90-100 reported DLQI 0/1 (HiSCR75-< 90: 19.1%/27.3%; HiSCR90-100: 34.8%/43.3%) compared with patients who achieved HiSCR<50/HiSCR50-<75 (HiSCR<50: 16.1%/19.6%; HiSCR50-<75: 12.9%/17.2%). Similar trends were observed in the proportions of patients reporting DLQI scores of 0-5. Across HiSCR bands, HiSQOL/DLQI outcomes were maintained or improved from Weeks 16-48. For patients with HS, achievement of greater lesion reduction with bimekizumab (at Week 16) translated into greater clinically meaningful improvements in HRQoL over the longer term. ClinicalTrials.gov identifiers, NCT04242446; NCT04242498. Hidradenitis suppurativa (HS) is a long-term skin condition that causes painful sores, known as lesions, and negatively impacts patients’ quality of life. Bimekizumab is a drug that can treat the symptoms of HS, leading to what is known as ‘clinical response’. This study evaluated whether improvements in clinical response with bimekizumab enhanced quality of life for patients living with HS. We collected data from two clinical trials (BE HEARD I and II). Patients received bimekizumab or a treatment that did not contain any medication (a ‘placebo’). Patients were grouped into categories based on their clinical response level after 16 weeks of treatment. Clinical response was measured by the reduction in the number of lesions each patient had after 16 weeks compared with the start of the trial. Patients completed questionnaires that asked how their HS affected their day-to-day lives; their answers were measured over 48 weeks of treatment. Out of 1,014 patients, 868 received bimekizumab from the start of the trials. Patients with higher clinical response after 16 weeks generally showed greater improvements in their quality of life over 48 weeks of bimekizumab treatment. Patients with the highest clinical response levels showed particularly large improvements in their quality of life. These results suggest that, for patients with HS, achieving a higher level of clinical response with bimekizumab treatment leads to greater improvements in quality of life, highlighting the importance of aiming for high clinical response levels.
Single-cell RNA sequencing (scRNA-seq) resolves cell types and molecular phenotypes within heterogeneous specimens but typically requires fresh, high-quality single-cell suspensions processed immediately to preserve transcriptional profiles. This constraint complicates samples with long preparation times and prevents collection at remote sites lacking single-cell instrumentation. Several commercial assays now enable preservation at the point of collection through fixation or cryopreservation, allowing processing to occur months later. The Association of Biomolecular Research Facilities' DNA Sequencing and Genomics and Bioinformatics Research Groups undertook a cross-platform, multisite study to assess the performance and reproducibility of three such platforms: 10x Genomics FLEX, Parse Biosciences Evercode WT v2, and Honeycomb Bio HIVE. Total leukocytes and peripheral blood mononuclear cells (PBMCs) were isolated from a single healthy individual, with EasySep reagent used for red blood cell depletion of the leukocyte fraction. Cells were characterized by a 21-color flow cytometry panel to provide a reference, and the remaining material was fixed or cryopreserved according to each platform's protocol. Preserved leukocyte samples were prepared in parallel by two technicians ("A" and "B" replicates) and distributed to multiple ABRF member core facilities for downstream processing, while fresh leukocytes processed with the 10x 3' v3.1 (3pGEX) chemistry served as a reference. Libraries were sequenced at a central site, and performance was evaluated across standard scRNA-seq quality control metrics, gene and transcript detection sensitivity, cell-type discovery and annotation, differential expression, and correlation analyses. Data from all platforms integrated effectively and produced concordant results for cell-type annotation and relative abundance, with cell-type proportions broadly consistent with the flow cytometry reference. However, platform-specific expression signatures were evident for a subset of genes, and cross-site reproducibility varied between methods, with the FLEX workflow showing greater susceptibility to technical variation introduced during on-site sample processing. Preservation-based methods (FLEX and HIVE) showed better retention of fragile granulocyte populations than fresh samples processed with the 10x 3pGEX workflow. Improvements to preservation methods are changing how single-cell research is conducted by decoupling sample collection from downstream processing. Our investigation into the performance and reproducibility of each platform provides a resource to help investigators and core facilities select the most appropriate single-cell preservation workflow given their sample type, cell populations of interest, sample collection logistics, and laboratory infrastructure constraints.
Herein, we report a redox-neutral photoredox-catalyzed anti-Markovnikov intermolecular hydroamidation of alkenes, achieved through the strategic integration of photoredox iron catalysis and thiol catalysis. Amidyl radicals are generated from readily accessible α-amido-oxy acids via photoredox iron-catalyzed decarboxylation under mild reaction conditions. This catalytic system displays an excellent substrate scope and broad functional group tolerance. The synthetic utility of this protocol is highlighted by its facile scalability and successful application in the late-stage functionalization of alkenes derived from biologically active molecules.
In current genomic research, molecular dating is challenged by both imperfect substitution modeling and analysis efficiency, as genome-scale datasets often exhibit substantial rate heterogeneity and complex patterns of sequence evolution, which can make divergence-time estimation sensitive to modeling assumptions and computational settings. Meanwhile, commonly used molecular dating workflows remain operationally demanding; preparing correctly formatted inputs, implementing model settings, configuring fossil calibrations, and performing basic diagnostics and visualization frequently require multiple tools and extensive manual steps, resulting in high hands-on time and avoidable operational errors. To facilitate the practical implementation of molecular dating analyses and lower the operational barrier for users, this protocol describes a GUI-based workflow in PhyloSuite v2 for molecular dating analysis. Using a dataset of fish nuclear genomes as an example, the tutorial covers multi-format data import, visual configuration of fossil calibrations, automatic selection and implementation of substitution models, automation of complex analytical procedures, and assessment of Markov chain Monte Carlo (MCMC) convergence, along with data visualization. Through this protocol, users can quickly master the full workflow-from input preparation and molecular dating to MCMC sample statistical assessment and timetree visualization-thus significantly enhancing the efficiency of molecular dating analysis and result verification. Key features • Guides users through a complete molecular dating workflow in PhyloSuite v2, from input preparation to final output. • Specifies required inputs, intermediate files, and final outputs for molecular dating analyses. • Provides a practical visual procedure for setting and checking fossil calibrations before analysis. • Helps users configure reproducible runs and interpret diagnostic outputs to assess convergence and dating performance.
The α-synuclein seed amplification assay (synSAA) is a biomarker test for synucleinopathies. Different synSAA conditions have different properties, and some of these conditions enable differentiation between diseases associated with Lewy bodies versus those associated with glial inclusions. Direct cross-assay and inter-site synSAA comparisons evaluating these features remain limited. Two synSAA conditions (identified here as sodium phosphate buffer (SPB) and piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) SAA conditions) were tested in a controlled 2 × 2 design, each performed at two independent laboratories. Cerebrospinal fluid (CSF) from 60 participants was analyzed, comprising 29 multiple system atrophy (MSA) samples and 31 additional CSF samples including 16 Parkinson's disease/dementia with Lewy bodies (PD/DLB) cases previously screened using SPB-SAA conditions, and 15 neurology ward controls, which were used as analytical controls. Inter-site concordance was high for SPB-SAA (100%) and PIPES-SAA (95%, 0.913 Fleiss' kappa). Compared to clinical diagnosis, sensitivity for detecting synSAA+ MSA cases was 75.0% for PIPES-SAA_A and 81.4% for PIPES-SAA_C, with specificities of 76.9% and 92.3%, respectively. SPB-SAA did not detect MSA at either of the two sites and showed 100% specificity. Inter-assay concordance was high for PD/DLB (88%) and controls (86%), but low for MSA (31%), reflecting the different sensitivity in MSA cases. The overall inter-assay concordance yielded a Fleiss' kappa of 0.23. Both synSAA conditions exhibit high reproducibility and replicability across laboratories yet differ in their diagnostic profiles: PIPES-SAA conditions enable detection of synuclein seeds in MSA, while SPB-SAA conditions showed high specificity for PD/DLB but not detect synuclein seeds in MSA. These findings support context-dependent assay selection and underscore the need for larger multicenter validation of analytical tools intended for clinical use.
Germ cell tumors (GCTs) are highly curable, yet a subset of patients with metastatic disease experience early death (ED) soon after starting first-line chemotherapy. These patients are underrepresented in trials, and risk factors remain unclear. We performed a retrospective multicenter cohort study including adults ( ≥ 18 years) with metastatic GCT who died within 3 months of completing their last cycle of first-line chemotherapy. Primary outcomes were cause and timing of death; secondary endpoints included clinical predictors of acute respiratory failure (ARF) and very early death ( ≤ 30 days). Among 102 patients (1.7% of treated cases), 69.6% had non-seminoma, 83.3% testicular primaries, and 67.6% poor-risk disease. Median time to death was 28 days (range, 2-179). Leading causes were ARF (34.1%), disease progression (16.7%), septic shock (15.7%), hemorrhage (12.7%), and cardiovascular events (4.0%). ARF correlated with > 50% lung involvement, dyspnoea, and haemoptysis, but not choriocarcinoma histology or bleomycin use. Mostly, ED (51%) was associated with liver metastases, massive lung involvement, β-hCG > 50,000 mIU/mL, ECOG 2-3, elevated neutrophil/lymphocyte ratio, and need for intensive care (all P < 0.05). Early death in metastatic GCT, though rare, remains a critical clinical issue. Early identification, adapted induction regimens, and optimized supportive care may help prevent avoidable mortality.
Definitive radiotherapy (RT) for prostate cancer (PC) with dose intensification and/or focal boosting has excellent oncologic outcomes, but many patients experience adverse events. Dose escalation to the whole prostate improves outcomes at the expense of increased late adverse events. Intraprostatic recurrence after definitive RT typically occurs at the site of the primary tumor, suggesting that dose to the site of the dominant lesion is an important predictor of future failure. The efficacy and safety of tumor-focused RT compared to that of standard RT for definitive treatment of localized PC has not been assessed. RadTARGET (RAdiation Dose TAiloRing Guided by Enhanced Targeting; NCT06990542) is a phase II randomized trial that aims to demonstrate superior safety of image-guided, tumor-focused RT compared to standard RT for acute genitourinary (GU) or gastrointestinal (GI) in the setting of definitive RT for intermediate- and high-risk PC. The study intervention is image-guided, tumor-focused RT with dose intensification of cancer visible on imaging and dose de-intensification to remaining prostate. Patients will be randomized to two arms: those who receive standard RT dose and those that receive tumor-focused RT. The study population will be patients with intermediate- or high-risk PC planning to undergo definitive RT with or without systemic therapy. The primary endpoint to compare between randomized arms is acute GU or GI grade ≥2 adverse events. Participant and study duration are 5 years and 8 years, respectively. RadTARGET will compare the efficacy and safety of tumor-focused RT to that of standard RT for definitive treatment of localized PC. We hypothesize that the tumor-focused approach will substantially reduce adverse events after prostate RT while retaining high efficacy. If this hypothesis is confirmed, we will conclude that a phase III randomized control trial is warranted to formally establish oncologic non-inferiority compared to the current standard of whole-gland dose escalation.
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Non-invasive technologies for observing internal skin structures without damaging the surface have advanced in recent years. However, an integrated understanding of internal structures together with the morphology of surface "microrelief"-the surface pattern consisting of furrows and ridges (i.e., sulci cutis and cristae cutis)-remains limited. We elucidated the relationship between internal skin structure and microrelief by combining line-field confocal optical coherence tomography (LC-OCT) with a proprietary AI system. We applied a semantic segmentation-based image AI system to LC-OCT data from 11 adults. The system extracted the coordinates of sulci cutis, stratum corneum thickness, viable epidermal thickness, and dermal-epidermal junction (DEJ) topography. The analysis revealed that the skin surface and DEJ in sulci cutis were nearly parallel. The DEJ undulation index in cristae cutis correlated with epidermal thickness, stratum corneum multilayering rate, and ceramide subclass parameters. We verified the relationship between the skin surface and the DEJ structure by incorporating positional information of the microrelief. LC-OCT combined with AI-based analysis provides a non-invasive approach to link age-related changes in DEJ structure with alterations in microrelief and related biomarkers.
Garadacimab (monoclonal antibody inhibiting activated factor XII [FXIIa]) is approved for long-term prophylaxis (LTP) against hereditary angioedema (HAE) attacks. Due to the novelty of FXIIa inhibition and the lifelong need for HAE LTP, long-term evaluation of garadacimab is important. Pooling data from multiple studies can provide valuable insights into treatment effects over longer durations. We report the integrated summary of safety (ISS) and efficacy (ISE) of garadacimab LTP across the HAE clinical development program. The ISS comprised data from phase 2 (13-week placebo-controlled period, 75/200/600 mg monthly or 400 mg every 2 weeks; ≥ 44-week open-label period, 200/600 mg monthly; NCT03712228), pivotal phase 3 (6 months; 200 mg monthly; NCT04656418), and ≥ 12-month phase 3 open-label extension (OLE) studies (200 mg monthly; NCT04739059). The ISE comprised data from phase 3 studies. Endpoints included treatment-emergent adverse events (TEAEs), serious adverse events (SAEs), adverse events of special interest (AESIs) per protocol, and efficacy. At data cutoff (June 15, 2024), median garadacimab exposure (ISS: any dose, n = 172) was 2.5 (range 0.2-5.5) years. Overall rates of TEAEs and garadacimab-related TEAEs were 2.8/patient-year and 0.2/patient-year, respectively. No deaths occurred; four TEAEs led to treatment discontinuation. Eleven patients experienced SAEs (0 garadacimab-related). No garadacimab-related AESIs were reported; one unrelated AESI was observed with garadacimab 600 mg (epistaxis; mild; resolved). The most common garadacimab-related TEAEs were mild/moderate injection-site reactions. In the ISE (median exposure 2.5 [range 0.3-3.2] years), the mean (95% confidence interval) monthly attack rate was 0.2 (0.1, 0.2) with garadacimab (n = 164), corresponding to a 94.9% (93.1, 96.7) reduction from run-in (3.5 [3.2, 3.9]). These integrated data, representing the longest garadacimab exposure reported to date, confirm the favorable long-term safety profile of garadacimab (exposure ≤ 5.5 years), and durable efficacy with sustained protection against HAE attacks (exposure ≤ 3.2 years). ClinicalTrials.gov identifiers, NCT03712228, NCT04656418, NCT04739059.
Benzazepinoindole architectures have gained increasing prominence in contemporary synthetic and medicinal chemistry owing to their structural complexity and biological relevance. However, efficient and selective approaches to benzazepinoindole frameworks remain limited and often require harsh conditions or multistep sequences. In this study, we describe an ion-pair-catalyzed strategy for the synthesis of benzazepinoindole derivatives that circumvents the limitations of traditional Brønsted acid activation. The protocol proceeds through an indole C3-alkylation followed by a Ph3C+[B(C6F5)4]- catalyzed Pictet-Spengler cyclization using α-keto esters and isatin, enabling enhanced electrophilic activation, and improved control over reactivity under mild conditions. Notably, all products feature a quaternary stereogenic carbon center. This method provides efficient access to structurally complex benzazepinoindole frameworks.
The research study is dedicated towards synthesis of novel spiro[indoline-3,2'-pyrrolidines] incorporating a urea functionality. Crucially, the study seeks to elucidate their anticipated multi-targeted mechanism of action, moving towards the final objective of producing multi-functional antineoplastic agents. 1,3-Dipolar cycloaddition was used to construct the final compounds, the spiro-analogs 15a-m. 2-Propen-1-ones 12a-j provided a crucial part of the structure (the urea group). Isatins 13a,b and sarcosine 14, reacted together during the process to form in situ the necessary dipole, azomethine ylide. This synthetic protocol produced the targeted agents 15a-m with high yields. Biological evaluation identified compound 15l as a highly potent agent against the HCT116 colon cancer line, outperforming sunitinib and 5-fluorouracil by 3.7-fold and 7.9-fold, respectively. It is also identified with efficacy against MCF7 (breast cancer) close to that of 5-fluorouracil. Additionally, compound 15h exhibited exceptional potency against A431 skin squamous carcinoma surpassing 5-fluorouracil by 9.1-fold. Mechanistic studies confirmed the dual-action of rational design. Compound 15h demonstrated VEGFR-2 inhibition comparable to sorafenib, while 15g emerged as a dual MDM2 inhibitor and p53 activator, significantly outperforming doxorubicin. The correlation between MDM2 inhibition and p53 activation suggests that the disruption of the MDM2-p53 protein-protein interaction, alongside VEGFR-2 inhibition, drives the antiproliferative effects. Furthermore, compounds 15g, 15h, 15j, and 15k induced apoptosis through the upregulation of caspase-3 and BAX and the downregulation of Bcl-2. Cell cycle (flow cytometry) studies of 15h and 15j confirmed G0/G1 phase arrest. Moreover, the Annexin V-FITC/PI dual staining assay evidenced the apoptotic potential of compounds 15h and 15j. CAM assays supported the anti-angiogenic potential of 15g, 15h, 15j, and 15k. Finally, molecular docking (PDB ID: 5LAW) and dynamic simulations validated the binding stability and mechanism within the MDM2 pocket.
Herein, we report organocatalyst-controlled deuteration of enones at the α'-position or at both the α- and α'-positions under mild reaction conditions using D2O as the deuterium source. The key to the success of this protocol is the appropriate selection of the catalytic system. A quinine-derived primary amine-benzoic acid catalytic composite deuterated the α'-position of enones with high selectivity. On the other hand, the (1S,2S)-1,2-diphenylethane-1,2-diamine-benzoic acid catalytic system promotes deuteration at both the α- and α'-positions of enones. In addition, this flexible protocol also features a broad substrate scope (>48 examples), including drug molecules and natural products, mild reaction conditions, and facile scalability. A working mechanistic pathway is also proposed.
Dysregulated microRNA (miR) expression has emerged as a potential prognostic tool in head and neck squamous cell carcinoma (HNSCC), but the clinical value of miR-34a remains unclear. This systematic review, meta-analysis, and trial sequential analysis (TSA) evaluated the association between tumor tissue miR-34a expression and survival outcomes in HNSCC. Following a protocol registered in PROSPERO (n. CRD420251238772), PubMed/MEDLINE, Scopus, ScienceDirect, CENTRAL, Google Scholar, and grey literature sources were searched for studies reporting overall survival (OS) or disease-free survival (DFS) stratified by miR-34a expression in HNSCC or its subsites. Hazard ratios (HRs) were extracted directly or reconstructed from Kaplan-Meier (KM) curves using the Tierney method, supported by a dedicated Python application (KM2HR). Four retrospective studies, corresponding to six study/site-specific cohorts and 318 patients, met the inclusion criteria. For OS (four cohorts), the fixed-effects model yielded a pooled HR of 2.25 (95% CI 1.48-3.41) for low versus high miR-34a expression, indicating worse survival in the low-expression group. However, the random-effects model attenuated the association (HR 1.32, 95% CI 0.32-5.54), with substantial heterogeneity (I2 ≈ 77%). For DFS (two studies), the fixed-effects model suggested poorer outcomes with low miR-34a (HR 2.92, 95% CI 1.24-6.88), whereas the random-effects model reversed the direction of effect with extremely wide confidence intervals (HR 0.19, 95% CI ≈ 0.00-129.34; I2 = 91%). TSA for OS (accrued information size 225 patients; estimated power ≈66%) crossed the monitoring boundary but did not reach the a priori information size, supporting only a tentative signal. A bioinformatic exploration of the TCGA HNSCC cohort (n = 522) showed a non-significant trend towards worse OS with low miR-34a (HR 1.24, 95% CI 0.93-1.65) and was excluded from pooling. Overall, low tumor miR-34a expression appears to be associated with poorer OS, but the evidence is limited by retrospective design, small sample size, and marked heterogeneity. miR-34a is a promising biomarker for prognostic stratification in HNSCC, yet larger, prospective, site-specific studies with standardized assays, pre-defined cut-offs, and appropriate adjustment for HPV status and clinical covariates are required before clinical implementation can be recommended.
Non-invasive biomarkers are essential for monitoring transplant health, particularly for detecting acute rejection. Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising biomarker in solid organ transplantation, but optimized methodology and comprehensive evaluation in kidney transplantation (KTx), especially in Japan, are currently limited. We developed a customized approach for dd-cfDNA quantification and evaluated its utility as a biomarker in KTx. In this prospective longitudinal study, 322 blood samples were collected from 39 KTx recipients, mostly within the first year and up to 5 years post-transplant. Quantification was performed using a modified targeted sequencing protocol using a 1,000-SNP probe panel tailored to East Asian genetic backgrounds. The resulting dd-cfDNA values were correlated with clinical parameters and histological findings. General longitudinal dynamics of dd-cfDNA in KTx patients were comprehensively described. Elevated dd-cfDNA was consistently associated with acute rejection and graft injuries. In particular, dd-cfDNA significantly distinguished rejection from non-rejection episodes (p = 1.9 × 10- 4) and showed moderate consistency with serum markers and biopsy findings. Noticeable surges of dd-cfDNA were reported in antibody-mediated rejection and various forms of kidney injury. Distinct from conventional serum markers, dd-cfDNA exhibited a significant inverse correlation with immunosuppressant trough levels (Spearman's rho = - 0.25, p = 8 × 10- 3). Our study demonstrated that dd-cfDNA is a robust, non-invasive biomarker for detecting allograft rejection and injury in KTx and is superior for monitoring patients' immunosuppressive response. We also present a scalable, cost-effective sequencing-based methodology for quantifying dd-cfDNA, suitable for clinical application.
The development of nitrogen- and sulfur-containing heterocycles remains a central challenge in modern synthetic chemistry owing to their structural diversity and biological relevance. Herein, we report the synthesis of the ionic liquid (IL) 1-dodecyl-1-methylpiperidinium chloride ([C12mpip][Cl]) and, for the first time, its application as an efficient catalyst in an unprecedented divergent one-pot cyclization. The protocol enables the cyclization of 6-aminobenzothiazole with diverse aryl or heteroaryl aldehydes and 1,3-dimethylbarbituric acid in ethanol as a green solvent under reflux conditions. Apart from ortho-substituted and NH-unsubstituted heteroaryl aldehydes, the reaction proceeds with high regio- and diastereoselectivity to afford spiro[pyrimidine-5,8'-thiazolo[5,4-f]quinoline]triones 4(a-l) in yields of up to 94% and diastereomeric ratios exceeding >99:1 (syn:anti). Notably, the reaction outcome is governed by the aldehyde substitution pattern, enabling controlled access to structurally distinct fused heterocycles under identical conditions. The method constructs two new stereogenic centers and multiple σ-bonds in a single operation and is readily scalable to the gram scale with consistently high yields. All structures were confirmed by spectroscopic techniques and single-crystal X-ray analysis. Antiproliferative evaluation across six solid tumor cell lines identified compound 4l as a lead candidate, displaying uniform single-digit micromolar GI50 values (1.22-7.40 μM) and potent activity against patient-derived glioblastoma (GBM6) cells (GI50 = 2.40 μM). Compound 5b also exhibited consistent low-micromolar growth inhibition across multiple cancer cell lines. The operational simplicity, scalability, and promising biological profile facilitated gram-scale synthesis with yields of up to 94%.
Herein, a sequential catalytic system combining asymmetric aminocatalysis with iodine catalysis is introduced. This newly catalytic system promotes a Michael/iodization/nucleophilic substitution reaction to provide enantioenriched novel spirodihydrobenzofuran-pyrazolones from o-hydroxy enones and pyrazolones. The desired products are realized in moderate to good yields with good diastereoselectivities and high enantioselectivities. This one-pot method features the formation of contiguous stereocenters, of which one is the spiro-quaternary stereocenter, engagement of less reactive electrophiles, mild reaction conditions, and scalability. Importantly, the reported protocol provides a mechanistically distinct pathway to construct enantioenriched spirodihydrobenzofuran-pyrazolones.
Pseudoalteromonas haloplanktis TAC125 is a psychrophilic marine bacterium widely used to study cold adaptation and increasingly exploited as a non-conventional platform for biotechnological applications. The strain harbors the endogenous megaplasmid pMEGA (64.7 kb), whose presence may limit its exploitation as a cell factory, making its elimination advantageous to strain engineering. Traditional plasmid-curing approaches based on chemical and physical agents are often inefficient and unsuitable for stable endogenous replicons, such as pMEGA. Here, we describe a targeted protocol for pMEGA curing in P. haloplanktis TAC125 that combines homologous recombination with paired-termini antisense RNA (PTasRNA) gene silencing. First, a selectable marker cassette is inserted into pMEGA by homologous recombination using a suicide vector, enabling selective discrimination between plasmid-positive and plasmid-cured bacteria. Next, PTasRNA gene silencing technology is applied to target a gene essential for the replication of pMEGA, thereby transiently interfering with its replication and promoting its loss. This approach provides a specific method to cure a highly stable endogenous megaplasmid in a psychrophilic non-conventional bacterium, enabling improved functional studies and strain optimization, establishing a broadly applicable framework for targeted curing across diverse bacterial systems. Key features • Enables targeted curing of stable endogenous plasmids lacking selectable markers. • Combines replication silencing and homologous recombination for targeted plasmid elimination without permanent chromosomal modification. • Adaptable framework for non-model bacteria with limited genetic toolkits, such as marine psychrophiles.