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.
The Global Initiative for Asthma recommends patients with asthma uncontrolled on medium-dose inhaled corticosteroid (ICS) plus long-acting β2-agonist (LABA) therapy to first escalate their ICS dose before considering add-on biologic therapy despite acknowledging that high-dose ICS provides little additional benefit for most patients and carries an increased risk of adverse effects. Dupilumab, an IL-4Rα antagonist that inhibits signaling of IL-4 and IL-13, has demonstrated efficacy in patients with uncontrolled moderate-to-severe asthma and type 2 inflammation. The AIM4: Next Step study will evaluate whether earlier intervention with dupilumab provides clinical benefit compared to escalation to high-dose ICS/LABA. AIM4: Next Step (NCT06572228) is a randomized, double-blind, active-controlled study enrolling approximately 250 adolescents and adults with type 2 inflammatory asthma uncontrolled with medium-dose ICS/LABA treatment. Patients will be randomized 1:1 to receive dupilumab 300 mg every 2 weeks plus medium-dose ICS/LABA or placebo plus high-dose ICS/LABA. The primary end point is the annualized rate of severe asthma exacerbations over 52 weeks. Key secondary end points include change from baseline in prebronchodilator forced expiratory volume in 1 second at week 12, annualized systemic corticosteroid exposure for exacerbations, and asthma control at week 12 based on 5-item Asthma Control Questionnaire. AIM4: Next Step is the first study designed to address an important gap in asthma management by directly comparing ICS dose escalation versus early biologic intervention with dupilumab in patients with uncontrolled type 2 inflammatory asthma. Findings may inform future treatment strategies for patients whose asthma remains uncontrolled despite ICS/LABA.
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Relying solely on MRI scans may prove insufficient for obtaining a complete view of a patient's supraspinatus health status comprising both objective and subjective disability level. On the other hand, while Patient Reported Outcome Measures (PROM) capture patients' personal experiences, they may not provide objective data.This study assessed the correlation level between preoperative MRI radiomics features and pre-and post-operative PROM scores in patients diagnosed with supraspinatus lesions. 44 patients who underwent arthroscopic repair for full-thickness Rotator Cuff Tears (RCT) of any grade between January 2019 and January 2022 in a single surgical centre were enrolled in the study. All coronal T2-weighted MRI scans were evaluated independently by two orthopedic surgeons blinded to the patients' clinical information, and the radiomics features were extracted. The strength of linear and non-linear relationships were assessed respectively through Fisher's statistical test applied to linear regression models, namely F-statistics, and through a model-free AI-based Mutual Information estimator. Together, these methods provided a comprehensive feature selection framework, with overlapping results reinforcing feature relevance and differences revealing complementary patterns in the radiomics-PROM relationship. Besides, they allowed us to construct an integrated ranking of radiomic features based on their strength of association with clinical outcomes, from which we selected those ranking in the top 10 most important of at least three PROMs.The radiomics features most frequently related to the patient's health condition were Maxi- mum2DDiameterColumn, Flatness, LeastAxisLength, and features gathered from the Gray Level Co-occurrences Matrix (GLCM), like Correlation, Maximum Correlation Coefficient (MCC), and Informational Measure of Correlation 2 (Imc2).Identifying crucial visual features pertaining to the patient's health status highlighted the potential for radiomics-guided personalized medicine in rotator cuff surgery. Incorporating both linear and non-linear analyses adds depth to the findings, establishing a strong groundwork for future advancements in the field.
The purpose of this retrospective case series was to find the Substantial Clinical Benefit (SCB), Minimal Clinically Important Difference (MCID) and Patient Acceptable Symptomatic State (PASS) of Simple Shoulder Test (SST) score in patients who underwent a rotator cuff repair. The SCB, defined as substantial improvement; the PASS, defined as acceptable symptom state; and the MCID, defined as minimal meaningful change, were calculated for the SST, a 12-point functional score. 57 patients (mean age 64 ± 11 years, 29 men and 28 women) who underwent rotator cuff repair in Campus Bio-Medico hospital in Rome were enrolled. Patients were selected from the existing surgical waiting list. Exclusion criteria encompassed inflammatory joint disease, prior surgical interventions involving the shoulder in question, labral pathology, degenerative osteoarthritis affecting the glenohumeral joint, symptomatic arthritis concerning the acromioclavicular joint, presence of rotator cuff arthropathy, and incapability to fill out the designated questionnaires. To calculate the SCB, PASS and MCID values of SST score, the patients completed anchor questions at final evaluation, 6 months post-operatively. The anchor questions for the SCB and PASS were: "How do you feel after the surgical procedure you have undergone?" and "Would you say that, overall, your physical health is at least satisfying?", respectively. The SCB thresholds were found comparing the "no improvement" group with the "substantially improved" group. The PASS thresholds were computed comparing satisfied and unsatisfied patients. An improvement was found in the SST score from baseline to post-operative 6-months follow-up, from 3.4 ± 2.7 to 8.6 ± 2.7 (p < 0.001). The MCID cut-off calculated using the Receiver Operating Characteristic (ROC) method was 4.5. The SCB thresholds found were 4.5 with the ROC method and 5.4 with the Mean Change (MC) approach. The PASS cut-off assessed with the ROC method was 9.5 and 11 the with 75th percentile approach. The SST score represents a valid Patient-Reported Outcome Measures (PROMs) score for patients undergoing rotator cuff repair. Patients who have at least a 5.4-point variation in the SST score from pre- operative to final 6-month post-operative follow-up recognize a substantial clinically important change in self-assessed outcome.As per the PASS of the SST score, patients who have at least 9.5-point value in the SST score at final evaluation experience an acceptable state of symptoms. The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Campus BioMedico University of Rome (COSMO study, Protocol number 78/18 OSS ComEt CBM, 16/10/18).
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.
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.
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.
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%.
A general electrosynthetic strategy for the preparation and direct functionalization of 2-oxa-bicyclo[2.1.1]hexanes (i.e. 2-oxa-BCHs) from hydroxymethyl-substituted bicyclo[1.1.0]butanes is reported. The method relies on the anodic generation of electrophilic heteroatom-centred species, including TEMPO-derived oxy-cations, halogen radicals, and thiyl radicals, rendering C(4) functionalized 2-oxa-BCHs selectively in good to excellent yields. The protocol operates under mild conditions, avoids stoichiometric oxidants, and displays broad substrate scope (41 examples). The synthetic utility of the resulting scaffolds is further demonstrated through late-stage functionalization, bio-conjugation, and telescoped synthesis from commercially available precursors. Mechanistic studies (cyclovoltammetry as well as DFT computations) support a pathway involving anodic oxidation for the formation of the electrophilic trigger followed by C-C bond capture and transannular intramolecular cyclization. Overall, this work establishes electrosynthesis as a powerful platform for the modular construction and diversification of 2-oxa-BCHs with potential relevance in drug discovery and expanding the chemical space of benzene bioisosteres.
Peptidoglycan (PG), a network of glycan strands crosslinked by short peptides, is an essential and bacterial-specific structure that determines cell shape and protects cells from lysis. Understanding how bacteria assemble, maintain, and modify their PG not only addresses fundamental questions in cell biology but also provides a basis for developing strategies to treat bacterial infections. Although several in vitro methods, such as zymography, Remazol Brilliant Blue (RBB) assay, and LC-MS analyses, are available to quantify the activities of PG-modification enzymes, these approaches are not readily applicable in vivo. Here, we describe a single-particle tracking photo-activated localization microscopy (sptPALM)-based method to quantify the binding of enzymes to PG in vivo, which serves as a proxy for their enzymatic activities. Because the PG meshwork is relatively immobile, fluorescently tagged enzymes that transiently or stably bind it exhibit reduced mobility, reflected by lower diffusion coefficients. This approach provides sensitive, quantitative, and real-time insights into enzyme behavior in vivo under diverse physiological conditions or genetic backgrounds. The protocol is particularly valuable for investigating PG-modification enzymes that are essential or functionally redundant, which are often difficult to analyze using traditional genetic methods. Key features • Builds upon the method developed by Fu et al. [1] for single-particle tracking with high spatial and temporal resolution. • Streamlined imaging and data processing workflow. • Automatic analysis on large datasets with minimum human bias. • Reveals the regulatory relationship between PG-modification enzymes, bypassing complex genetic experiments.
The conventional kinesin-1 is a plus-end-directed microtubule-dependent motor protein with distinct motor head, stalk, and tail domains. Along with the motor head, which binds and walks along microtubules in an adenosine 5'-triphosphate (ATP) dependent manner, kinesin also contains a C-terminal microtubule binding tail. Motor-driven collective motility is well characterized using in vitro gliding assays, which show uninterrupted, smooth trajectories of transport. However, gliding assays driven by the full-length Drosophila kinesin-1 with both head and tail resulted in the emergence of spontaneous spatial microtubule patterns and stop-and-go motion. This was reproduced by an equimolar ratio of the active head and passive tail. Here, we describe the detailed protocol to reconstitute these microtubule gliding assays using multiple motor types: the full-length kinesin-1, the motor head or tail, mixtures of both head and tail, and a rigor mutant of the kinesin. We provide details of the approach taken to acquire the image time-series, to then quantify the spatial patterns that result from these motor combinations. Our approach provides a framework to systematically characterize the spatiotemporal effects of molecular motor-driven collective microtubule transport. Key features • This protocol highlights the cloning and expression of two major Drosophila kinesin-1 constructs: the microtubule binding tail and isoleucine-alanine-lysine (IAK)-deleted kinesin-1 full length. • This protocol describes a typical gliding assay setup for the kinesin motor domain, alone as well as in combination with the kinesin tail. • We present a systematic framework for typical gliding assays, including experimental acquisition as well as quantitative analysis of microtubule collective transport. • This protocol gives a quantitative metric for microtubule spatial patterns, which enables systematic analysis of microtubule curvature, both in vitro and in vivo.
Age-associated dysregulation of the gut microbiota is a hallmark of aging and has been linked to multiple age-related diseases, yet upstream host factors driving these changes remain incompletely defined. Extensive bidirectional crosstalk between gut microbiota and mucosal immunity has been described. Aging is accompanied by a progressive decline in immune function, collectively termed aging-associated immune remodeling (AAIR). AAIR encompasses widespread compositional and functional changes that impair an effective response to pathogens, vaccines, and tissue damage. We examined whether AAIR is an upstream host factor influencing the composition of the microbiome upon aging. Hallmarks of AAIR were also present in the ileal lamina propria, including reduced naïve CD4+ and CD8+ T cell populations and expansion of memory and regulatory T cell subsets. To test whether mucosal AAIR reflects intrinsic aging of the hematopoietic system, we used an HSC transplantation model where young RAG1-/- recipients develop an adaptive immune system derived exclusively from either young or aged donor HSC in an otherwise young host environment. Recipients of aged HSCs recapitulated key features of mucosal AAIR, particularly loss of naïve T cells, demonstrating that AAIR in the ileal LP is driven at least in part by aged HSCs. Shotgun metagenomic sequencing of fecal samples revealed that ileal AAIR is associated with alterations in gut microbiota. In detail, there was a reduced abundance of taxa associated with the vitamin B6 (VB6) biosynthesis and salvage pathways. Accordingly, VB6 levels in serum were reduced in mice with aged immune systems. Our findings link AAIR to reduced microbial VB6 pathway abundance and lower systemic VB6 availability, suggesting that immune aging shapes the functional output of the microbiome in ways that diminish its VB6 biosynthetic capacity. This postulates an immune-microbiome-VB6 association that warrants further investigations for therapeutic strategies to increase VB6 levels upon aging. Video Abstract.
A sustainable and efficient protocol for the synthesis of imidazole-2-thione derivatives is herein reported. The method relies on the reaction of imidazolium salts with sodium sulfide (Na2S) in water at room temperature under visible-light irradiation. In this transformation, cobalt phthalocyanine (CoPc) serves as an inexpensive and robust catalyst, while ambient air acts as a clean and environmentally benign terminal oxidant. Operational simplicity, straightforward work-up, and excellent product yields highlight the practicality of this environmentally friendly methodology, making it an attractive approach for the green synthesis of imidazole-2-thiones.
The interest in therapeutic applications of tetrahydrocannabivarin (THCV) recently increased. For this reason, we validated an online extraction liquid chromatography- tandem mass spectrometry (LC-MS/MS) method to investigate the formation of urinary metabolites and understand potential cross-reactivity of THCV metabolites in cannabinoid immunoassays. Urine samples were obtained after oral administration of Δ8-THCV to healthy participants. The protocol was approved by the Advarra Institutional Review Board (Pro00059879; approved December 20, 2021) and the trial was registered on clinicaltrials.gov (NCT05210634). Urine samples were collected pre-dose and pooled 0-8 hours post-dose. Urine samples were extracted using a simple one-step protein precipitation procedure and the extracts analyzed using online trapping LC-MS/MS in positive multiple reaction monitoring mode. All compounds passed validation criteria in urine. In the clinical samples, 11-nor-9-carboxy-Δ8-THCV (Δ8-THCV-COOH) was the main metabolite detected before and after incubation with glucuronidases. Of the urine pooled 0-8 hours post-dose, 70 out of 80 were reported positive by a cannabinoid immunoassay targeting Δ9-THC-COOH, despite being negative for Δ9-THC-COOH and positive mainly for Δ8-THCV-COOH in the LC-MS/MS analysis. The major metabolites of Δ8-THCV in urine were Δ8-THCV-COOH, 11-hydroxy-Δ8-THCV and Δ9-THCV-COOH that were extensively glucuronidated and cross-react with immunoassay routinely used for toxicology testing resulting in false positive results for Δ9-THC exposure.
Changes in sperm motility can serve as an early indicator of reproductive effects caused by environmental chemicals or genetic perturbations. However, sperm motility is highly sensitive to external factors such as osmolarity, ionic composition, and the timing of measurement after activation, making it challenging to obtain consistent and reproducible measurements. Here, we present a standardized protocol for assessing sperm motility in Japanese medaka (Oryzias latipes) using a sperm motility analysis system (SMAS), an application for computer-aided sperm motility analysis (CASA). This protocol details the procedures for sperm collection, activation, and quantitative motility assessment, with particular focus on changes in the percentage of motile sperm post activation and the effects of sperm cryopreservation. We demonstrate time-dependent declines in sperm motility and velocity, and highlight the importance of early post-activation measurements to accurately capture peak motility. Notably, cryopreservation significantly accelerated the decline in sperm motility rate without affecting the initial proportion of motile sperm. To enable reliable comparisons among experimental groups, we recommend standardizing the initiation time after sperm activation by using CASA, and show that measurements should be initiated within 1 min after activation to obtain consistent and reliable data. This standardized SMAS-based protocol provides a robust and reproducible framework for sperm motility analysis in medaka and will be valuable not only for studies in reproductive biology, toxicology, and environmental risk assessment but also for applied research, such as breeding of aquacultural fishes.
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.
Volume electron microscopy based on serial sectioning allows for three-dimensional (3D) visualization and analysis of the internal structures of tissues, cells, and organelles. One such technique, focused ion beam (FIB) scanning electron microscopy (SEM), has the advantages of nanoscale sectioning and high z-resolution, but the disadvantage of limited volume processing. Because of this limitation, targeting localized objects by FIB-SEM is difficult. Here, we developed a FIB-SEM observation workflow that enables the analysis of the filiform apparatus of synergid cells enclosed in the Arabidopsis ovule. In this protocol, plant samples are stained, embedded, trimmed, and carbon-coated while maintaining their orientation within the tissue. Then, sequential observations are performed using Cut & See function of FIB-SEM, followed by image processing for 3D reconstruction. Utilization of multi-scanning and image cropping from high-resolution data helps to identify localized targets within plant tissue. The filiform apparatus, which is an invaginated cell wall structure of the synergid cells, shows distinct contrast in each image, allowing for segmentation using brightness-based binarization. Such segmentation avoids the need to manually trace complex structures and facilitates 3D reconstruction by volume electron microscopy. Key features • Sampling and trimming of the resin block enable directionally loading in FIB-SEM. • Multi-scanning by FIB-SEM and target extraction by image processing software enable 3D reconstruction of local areas within the sample block. • Binarization using distinctive brightness of cellular structures enables segmentation without manual tracing of complex structures such as the filiform apparatus cell wall.
Evaluate safety, efficacy, and response markers to atezolizumab, an immune checkpoint inhibitor, when combined with neoadjuvant chemotherapy (NACT) for primary epithelial ovarian, tubal, and peritoneal cancer (EOC). Eligible patients had high-grade stage III/IV EOC planned for NACT and interval cytoreductive surgery (ICS). After pretreatment biopsy, patients receivedweekly paclitaxel (80 mg/m2) with carboplatin (AUC6) and atezolizumab (1200mg) every 3 weeks for 3 cycles before ICS, and 3 cycles +/- bevacizumab post-ICS, followed by maintenance atezolizumab +/- bevacizumab or a PARPinhibitor. The primary endpoint was frequency of delay in ICS due to atezolizumab-related toxicities. Frequency/severity of adverse events (AE), response prior to ICS, and immune translational parameters were assessed. Negative results of IMagyn050 led to early termination; 18 of 40 planned patients enrolled: median age 69 years (range 46-87); majority were white (78%), had Stage III disease (72%), high-grade serous histology (100%), and no BRCAmutation (72%). Fifteen patients underwent ICS; 3 stopped protocol therapy prior to cycle 3 for non-atezolizumab-related reasons. Thromboembolism led to delay of ICS in one patient. Optimal cytoreduction was achieved in 13 (86%) patients. Treatment-related AEs were as expected. Partial response occurred in 9 (60%) and stable disease in 6 (40%). Patients with response versus stable disease had elevated post-treatment CXCL10, TNFα, and IL10. Immune compartments showed increased inflammatory markers, while tumor showed increased inflammatory and suppressive markers. NACT with atezolizumab was feasible and did not delay ICS. Translational parameters showed heterogeneous changes reflecting both immune activation and adaptive immune resistance.
Securing a sustainable global food supply for the future necessitates a paradigm shift towards technologies that amplify biological potential while minimizing environmental harm, a goal that can be achieved through the controlled generation of reactive oxygen and nitrogen species (RONS) by non‑thermal plasma. This interdisciplinary review synthesizes current knowledge on plasma‑based approaches, a rapidly advancing interdisciplinary frontier converging physics, chemistry, and biology, to fundamentally enhance plant nutrient acquisition and performance. We elucidate how reactive oxygen and nitrogen species (RONS), generated by non-thermal plasma (NTP) or delivered via plasma-activated water (PAW), act as potent biochemical signals and physical agents. These trigger a cascade of responses from seed coat etching and hormonal modulation to profound root system remodeling that collectively boost the plant's innate nutrient foraging capacity. We critically evaluate evidence for improved uptake of essential macro- and micronutrients linked to increased biomass and yield across diverse species. Practical applications in seed priming, soil amendment, and foliar sprays are explored as pathways to reduce dependency on conventional fertilizers. However, to transition this promise into reality, we discuss the critical hurdles of scalability, protocol standardization, and regulatory frameworks. We conclude that plasma technology represents more than an incremental improvement; it is a transformative tool that can inform choices for sustainable agricultural intensification. By unlocking plant physiological potential, it offers a tangible pathway to align agricultural productivity with the urgent goals of planetary health, making it a pivotal area of research for biological sciences.