Methods in molecular biology (Clifton, N.J.)Cristovão De Jesus Vieira Teixeira, Kevin Bellande, Joop E M Vermeer
The study of plant organogenesis presents significant challenges due to the small number of cells involved in its initial stages. Observing the initial cell divisions becomes increasingly challenging when transitioning from Arabidopsis thaliana (Arabidopsis) to other species. Lateral root (LR) initiation is an essential process for enhancing a plant's ability to access water and nutrients. In most cases, LR formation starts in the pericycle of the parent root, giving rise to a new meristem. Understanding the mechanisms coordinating LR development is important for improving plant resilience to biotic and abiotic stresses. The prediction of the precise timing and location of LR initiation along the root axis remains challenging, even in Arabidopsis. This is magnified when attempting to observe LR development in crops. Brachypodium distachyon (Brachypodium) has emerged as a versatile model for cereal crops. However, studying Brachypodium LR development requires a revision of protocols and methodologies to be applied from seedling growth to root imaging. Here, we present protocols for seed preparation, in vitro growth, and tissue clearing for Brachypodium. Whereas ClearSee appeared to be unsuitable for rendering Brachypodium root tissues transparent after a reasonable incubation period, our modified DEEP-Clear method resulted in improved efficiency in tissue clearing and is compatible with major fluorescent proteins and dyes. Finally, we introduce a simple and straightforward approach to locally synchronize LR development. These tools and methodologies are crucial for advancing our knowledge of plant root system architecture, transitioning from a model plant to agronomically important species.
Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder
Deep learning has transformed medical image analysis, but progress in cancer and stem cell applications is often constrained by limited access to large, diverse, well-annotated imaging datasets. This bottleneck is especially acute for studies of tumor heterogeneity and cancer stem cell (CSC) biology, where rare phenotypes and dynamic cell-state transitions-frequently linked to stemness-associated transcriptional programs (e.g., OCT4, SOX2, NANOG)-benefit from high-quality imaging across many samples and conditions. At the same time, regulatory and practical barriers (patient privacy, acquisition cost, and uneven institutional data sharing) restrict dataset scale and reuse. Diffusion models offer a practical route to synthetic data expansion by generating high-fidelity synthetic images that retain salient radiologic and pathologic features. In this chapter, we present an end-to-end protocol for adapting latent diffusion (Stable Diffusion) to oncology imaging using DreamBooth fine-tuning with small numbers of representative images, coupled with text-to-image and image-to-image workflows to generate controlled variations across modalities and disease presentations (e.g., brain tumor MRI, breast cancer mammography/CESM). We also describe quantitative and qualitative evaluation strategies, including Fréchet Inception Distance (FID) benchmarking and expert review considerations, to assess realism and diversity. These methods enable cancer and stem cell biologists to augment training data for segmentation and classification, build shareable educational resources, and prototype analyses for rare tumors or stemness-enriched subtypes while potentially reducing reliance on direct sharing of patient images.
Methods in molecular biology (Clifton, N.J.)Benjamin L Kidder
Alkaline phosphatase staining is routinely used to evaluate the undifferentiated state of embryonic stem cells, yet quantitative interpretation of colony assays frequently depends on manual inspection and categorical scoring that introduce subjectivity and limit scalability. ColonyQuant provides a standardized, automated framework for objective analysis of alkaline phosphatase-stained colonies from conventional bright-field images. The software performs adaptive colony detection, per-colony intensity measurement, and extraction of eight geometric descriptors that collectively characterize colony size, compactness, symmetry, and boundary complexity. Feature tables generated by the workflow are structured to support statistical comparison across replicates and experimental conditions. Integrated analysis modules enable dimensionality reduction, supervised classification, and feature ranking, facilitating interpretation of phenotypic differences without requiring custom scripting. Visualization routines generate distribution plots, contour-density maps, multivariate embeddings, and representative shape mosaics to summarize population heterogeneity and morphological organization. Applied to pluripotent stem cell cultures subjected to chromatin perturbation, the platform detects coordinated changes in colony growth behavior and structural architecture that may not be evident through visual scoring alone. The protocol describes installation, configuration, batch image processing, quality control, hierarchical data aggregation, and downstream statistical analysis, providing a reproducible approach for transforming qualitative colony assays into quantitative, high-content phenotypic datasets suitable for stem cell research and screening applications.
Methods in molecular biology (Clifton, N.J.)H M Suraj, André Fleissner, Jan A L van Kan
Botrytis cinerea is a widely studied model organism for plant-fungal interactions. This chapter provides simple and easy protocols for live-cell imaging techniques and subcellular protein localization studies in B. cinerea in vitro. Using a simple microscopy setup, we describe methods for imaging spore germlings and hyphae and for studying protein dynamics in response to the antifungal compound α-tomatine. The protocols highlight the application of fluorescence microscopy to visualize sterol distribution and protein localization patterns, enabling deeper insights into fungal cell biology.
International journal of neural systemsZhihuan Yang, Xin Chang, Junxia Chen, Haonan Pei, Jiangyan Liao, Xianmei Luo, Hui He, Mingjun Duan, Roberto Rodriguez-Labrada, Sisi Jiang, Dezhong Yao, Cheng L…
The global signal (GS) represents the broad variations in neural activity throughout the brain. Recent research has identified changes in the GS of functional magnetic resonance imaging (fMRI) in schizophrenia, challenging the traditional view of GS as mere noise that is typically discarded during data preprocessing. However, there has been no comprehensive characteristic analysis of GS in schizophrenia. This study recruited 100 patients with schizophrenia and 113 healthy subjects to perform resting-state fMRI. The averaged gray matter fMRI signal is denoted as GS. The GS topography was constructed by calculating Pearson correlation (i.e. GSCORR) between the GS and time series of each gray matter voxel. Furthermore, the relevance between GS topography and the function network features was constructed according to graph theory. Finally, we implemented an integrated analytical framework combining independent component analysis and multiple linear regression to quantify the contributions of resting-state brain networks to the spatiotemporal characteristics of the GS. The GSCORR of schizophrenia decreased in the bilateral insula and exhibited a significant negative association with disease duration. The increased GSCORR in the thalamus and default mode network (DMN) showed a positive correlation with the scale scores. We found that global properties are represented by GS topography in schizophrenia. In addition, the linear characterization results of GS showed that the component contribution decreased at the primary sensory network but increased at the high-order associated network. Our results further demonstrated that GS contains brain features associated with schizophrenia, which would help to understand the neural mechanisms underlying the psychopathological symptoms of schizophrenia.
Methods in molecular biology (Clifton, N.J.)David J Delgado Diaz, Katherine M Nelson, Vonetta L Edwards, Ian J Glomski, Filipa Ribeiro, Patrik M Bavoil, Alison K Criss, Jacques Ravel, Jason P Gleghorn, I…
Chlamydia trachomatis is an obligate intracellular bacterium that infects the columnar epithelium of the human endocervix. While conventional two-dimensional cell cultures and animal models have been instrumental in advancing our understanding of C. trachomatis biology, they are limited in capturing the multicellularity, architecture, and physiological microenvironment of the human cervix. This chapter describes the use of a three-dimensional (3D) microphysiologic model to study Chlamydia trachomatis infection. The model is inexpensively made without specialized equipment and is designed to recreate the epithelial-stromal interface. We outline procedures for coculturing cervical epithelial cells and fibroblasts, infecting epithelial cells with fluorescently labeled C. trachomatis, monitoring infection progression via fluorescent microscopy, and quantifying infectious progeny. The complete developmental cycle of C. trachomatis within this model provides a robust and accessible platform to investigate C. trachomatis-specific host-pathogen interactions, immune responses, and the influence of diverse physiological and environmental stimuli within a relevant cervical context.
Methods in molecular biology (Clifton, N.J.)Laura Gull, Mireia Sueca-Comes, Ana Pires
Here, we present an optimized whole-organoid immunofluorescence staining protocol that preserves morphology, achieves uniform antibody penetration, and enables deep-tissue imaging without the need for tissue clearing. Organoids are three-dimensional, self-organizing structures that replicate many of the cellular and architectural features of their tissue of origin, making them powerful preclinical models for studying development, disease, and therapeutic responses. Most organoid research is performed on formalin-fixed, paraffin-embedded (FFPE) samples, in which organoids are sectioned across the Z-planes. While this method facilitates conventional histological analysis, it limits spatially resolved visualization of protein expression and tissue organization. The inherent density and abundant extracellular matrix of organoids present substantial barriers to reagent penetration and imaging depth, further constraining the ability to capture their three-dimensional architecture in full. Our new workflow includes steps for fixation, permeabilization, and antibody incubation and has been validated on intestinal organoids using markers for different membrane-associated proteins. By providing high-quality staining through the full organoid depth without clearing, this protocol streamlines sample preparation, reduces processing artifacts, and allows more rapid, accurate 3D mapping of cell types and structures in organoid-based research.
The division of a mother cell into two daughter cells is a fundamental process in biology, involving the transfer of genomic information to the next generation of cells. In plant roots, cell divisions occur in the root apical meristem, a specialized tissue located at the plant's root tip. Cell proliferation rates in the root apical meristem shape root growth, thereby contributing to general development as well as the acclimatization to stressful environmental conditions through phenotypic plasticity. Here, we present a protocol for the microscopical analysis of cell proliferation in Arabidopsis thaliana root meristems by 5-ethynyl-2'-deoxyuridine (EdU) staining. EdU, a thymine-analog, is incorporated into newly synthesized DNA of proliferative cells during the S-phase of the cell cycle. Subsequently, EdU-containing nuclei can be labeled with a fluorophore and detected by confocal laser scanning microscopy. The combination of the EdU assay with cell wall staining enables the quantification of root meristem architecture parameters and cell division activity from microscopic images. Together, this can elucidate how root meristems respond to changes in environmental conditions or address fundamental questions of developmental plant biology.
Botrytis cinerea, a notorious pathogenic fungus, leads to postharvest decay in horticultural crops and substantial economic losses, while the pathogenicity of B. cinerea is closely related to its structural characteristics and cellular homeostasis. Among the currently available techniques, fluorescence and ultrastructural observations serve as crucial tools for investigating subcellular structures and cellular events during B. cinerea growth. These techniques enable the monitoring of cell structural integrity, such as variations in the cell membrane and cell wall, as well as intracellular changes, such as reactive oxygen species bursts and cell apoptosis. The results at cytological levels may not only facilitate close-up dissection of B. cinerea cells, but also provide important cues to uncover the inhibitory mechanism of exogenous treatments. This chapter introduces fluorescence staining and observation, as well as the preparation of ultrathin sections of B. cinerea conidia, aiming to provide methodological guidance for cytological examinations of B. cinerea.
The pathogenic bacterium Chlamydia replicates via an unusual developmental cycle that takes place within a membrane-bound inclusion inside an infected host cell. Immunofluorescence microscopy has revealed many details about the two specialized developmental forms and the recruitment of host organelles to the inclusion, but lacks the high resolution of electron microscopy (EM). In this chapter, we describe the use of an innovative microscopy method, called Tenfold Robust Expansion (TREx) microscopy, to visualize proteins in Chlamydia-infected cells at nanoscale resolution. We present a detailed protocol describing the steps of this expansion microscopy method, including immunostaining, anchoring, gelation, homogenization, expansion, and visualization with a standard confocal microscope.
Methods in molecular biology (Clifton, N.J.)Marcel Rühling, Thomas Rudel
The obligate intracellular bacterial pathogen Chlamydia trachomatis establishes a membrane-bound compartment called the inclusion, which serves as a replicative niche, within host cells. Sphingolipids, a major class of eukaryotic membrane lipids, are acquired from the host and incorporated into chlamydial membranes. Visualization of individual bacteria within inclusions or sub-bacterial structures by fluorescence microscopy remains challenging due to the limited resolution of conventional light microscopy. Expansion microscopy (ExM), a super-resolution imaging technique, enables nanoscale resolution on standard confocal microscopes and is an ideal tool to study Chlamydia infections. However, current ExM protocols are not compatible with sphingolipid visualization. Here, we present a strategy that enables ExM-based imaging of sphingolipids during C. trachomatis infection.
Methods in molecular biology (Clifton, N.J.)Jodi Bianca Callwood, Clarice Felix Gonzales, Dior Rose Kelley
Imaging has revolutionized phenotyping analysis by providing accurate documentation and records of observed phenotypes. Analyzing imaging data remains a major bottleneck in root phenotyping analysis. Advances in technology offer a plethora of analysis options at varying automation levels and price points, providing a reliable open-source pipeline for image analysis that delivers stable, reproducible results. Here, we discuss a maize seedling image analysis pipeline powered by open-source tools that enables rapid analysis of seedling root phenotypes. This image analysis pipeline uses Root Painter, an optional AI cleanup step, and RhizoVision Explorer to extract phenotypic data from maize seedlings grown on rolled towel assays, which are commonly used, enabling rapid analysis of these images.
Here, we present detailed protocols for histological staining and imaging of root cross sections of the fern Ceratopteris richardii, providing high-resolution visualization of cellular and tissue structures essential for understanding root development, nutrient and water uptake, and responses to environmental cues. This chapter describes the preparation of root samples, fixation, embedding, sectioning, and the application of specific histological stains that highlight key anatomical features, including the epidermis, cortex, endodermis, vascular tissues, and root cap. The protocol also covers imaging techniques using confocal microscopy to obtain clear, reproducible images suitable for quantitative and qualitative analyses. This standardized approach facilitates comparative studies of root anatomy and development, offering a valuable tool for plant developmental biology research while ensuring the preservation of tissue morphology.
Methods in molecular biology (Clifton, N.J.)Huifang Ma, Zhicheng Ji
10× Visium is a widely used spatial transcriptomics platform that enables joint profiling of gene expression and the spatial locations of cells. However, the histology images generated by the 10× Visium platform often contain technical artifacts, including fiducial markers and background noise, which degrade image quality. Here, we describe how a computational method, Vispro, can be applied to process and enhance these images. The resulting high-quality images lead to improved performance across a range of downstream analyses.
Methods in molecular biology (Clifton, N.J.)Josie Blair, Prakash Sah, George Liechti
The incorporation and visualization of modified D-amino acids into the peptidoglycan (PG) of actively replicating microbes have made a tremendous impact on the field of bacterial physiology. Here, we describe the optimization of this technique for labeling PG in obligate, intracellular Chlamydia species as well as Chlamydia-like organisms. Fluorescent D-amino acids (FDAAs) are D-alanine analogs that readily incorporate into the stem peptides of living bacteria and can be visualized rapidly with a fluorescence microscope without the need for laborious postfixation procedures. By comparison, D-amino acid analogs (DAAAs) and D-amino acid dipeptides (DAADs) similarly incorporate into newly synthesized peptidoglycan but require postfixation steps to effectively visualize PG. These probes were foundational in demonstrating the presence of PG in Protochlamydia and Chlamydia species, and PG labeling is a convenient tool that can augment other fluorescence imaging approaches. Here, we provide a protocol for the incorporation of D-alanine analogs into the PG of intracellular bacteria and the subsequent steps enabling these amino acids to be visualized via fluorescence microscopy. We also describe the use of an analysis tool that enables the mapping of PG-labeled objects in three-dimensional space and a protocol for confirming that modified amino acids are incorporating into bacterial PG.
Methods in molecular biology (Clifton, N.J.)Jordan Wesolowski, Fabienne Paumet
Chlamydia subverts host cell pathways by secreting effector proteins into the membrane of its inclusion. These bacterial membrane proteins, called Incs, mediate interactions between the inclusion and the host cell. To study Inc protein dynamics in live cells, we have developed a methodology that relies on host cell expression of antibodies fused to fluorescent proteins, known as frankenbodies, which detect epitopes exposed to the cytosol. HeLa cells are first stably transfected with a plasmid encoding an mScarlet3-tagged anti-FLAG frankenbody. These cells are then infected with Chlamydia expressing a FLAG-tagged Inc protein, which is secreted into the inclusion membrane with the FLAG tag facing the cytosol. Recruitment of the fluorescent anti-FLAG frankenbody to the inclusion is monitored in real time using time-lapse confocal microscopy. This approach has been successfully employed to elucidate the dynamics of IncA on the inclusion membrane and can be expanded to simultaneously investigate multiple Incs, as well as Inc:Inc interactions, by using different tag-frankenbody combinations.
Methods in molecular biology (Clifton, N.J.)Hang Zhou, Irina A Okkelman, Amber De Kinder, Bert Devriendt, Max Nobis, Ruslan I Dmitriev
Apical-basal polarity is essential for gastrointestinal epithelium function, where the apical membrane mediates nutrient absorption, host-microbiota interactions, and pathogen defense. However, conventional intestinal organoid culture displays an "apical-in" topology, limiting functional research with respect to the apical membrane. F-actin labeling with fluorescent phalloidin, a well-established apical marker, is also incompatible with live imaging. To address these limitations, in this chapter we describe an extracellular matrix (ECM) removal protocol to achieve "apical-out" topology in porcine intestinal organoid model and further validated WGA and Nile Red live imaging tracers enabling discrimination between apical-out (AO) and basal-out (BO) organoids. Subsequently, using integrated computational pipelines (automatic nuclei segmentation and single-nucleus phasor-based lifetime analysis), we can quantify organoid proliferative states at single-nucleus resolution following BrdU and Hoechst 33342 staining. Using the analysis of Hoechst 33342 fluorescence lifetime, we demonstrate that both organoid topologies exhibit intrinsic proliferative heterogeneity, but overall proliferative capacity is topology-dependent: apical-out organoids displayed decreased proliferation, whereas basal-out organoids remain highly proliferative.
Methods in molecular biology (Clifton, N.J.)Rachel Weild, Richard D Hayward
This chapter describes methodological approaches for studying intracellular ion dynamics during Chlamydia trachomatis infection. It emphasizes the requirement for live-cell imaging to capture active and compartmentalized ion fluxes in infected cells precluded by fixed-cell techniques. Specifically, we present step-by-step protocols for labeling Chlamydia-infected cells with a fluorescent potassium ion (K+)-sensitive probe for performing live imaging with confocal microscopy or spinning disc confocal microscopy for higher resolution and procedures for image processing and quantification. We also describe time-lapse microscopy to monitor K+ dynamics over the extended time course of the infection. These advanced microscopy methods have the potential to visualize ion transport in real time at the host-pathogen interface.
Methods in molecular biology (Clifton, N.J.)Carine Alcon, Tou Cheu Xiong
Iron (Fe) is involved in numerous key physiological processes due to its transfer or gain electron capacity. However, in excess, Fe is toxic to cells. The understanding of mechanisms governing its homeostasis requires imaging techniques capable of localizing and quantifying Fe and its redox state in situ. The Perls-DAB histochemical staining is a widely used method for visualizing Fe at a cellular level. Other advanced approaches, such as micro X-ray fluorescence and X-ray absorption near-edge structure spectroscopy, also provide information on the spatial distribution and oxidation state of Fe. However, all of these methods require sample fixation before observing Fe localization, which can introduce biases in the results. Recent development in synthetic fluorescent probes now enables the real-time visualization of ferrous (Fe2+) and ferric (Fe3+) forms in living roots, offering a robust and user-friendly approach to study Fe redox distribution in planta. This live imaging approach provides access to Fe dynamics and enables semi-quantitative analyses, thereby opening new avenues for Fe homeostasis research. This chapter provides detailed protocols for applying these methods in Arabidopsis roots.
Methods in molecular biology (Clifton, N.J.)Jasim Basheer, Petra Marhava
Quantitative methods using imaging and spectroscopy offer valuable insights into cell biology and molecular biophysics by elucidating complex mechanisms across various cellular environments. Fluorescence correlation spectroscopy (FCS) is particularly advantageous for quantifying the dynamics of fluorescent particles in low-concentration samples. As a high-resolution spectroscopic method with single-molecule sensitivity, FCS is well-suited to investigating membrane proteins, molecular interactions, aggregation, and conformational changes in living systems, including plant cells. However, challenges such as background fluorescence and artifacts arising from biological sample properties require careful experimental design, thereby limiting the routine application of FCS in plant tissues. Recent advancements in technology, such as confocal optics and highly efficient photon detectors, have substantially enhanced FCS's sensitivity and expanded its capacity for single-molecule detection. These advancements now empower plant biologists to explore the dynamics of plant proteins under various conditions. This chapter focuses on methodologies in fluorescence autocorrelation spectroscopy (FACS/FCS) and their application to plant research, with a particular emphasis on plasma membrane proteins in Arabidopsis root cells.
Methods in molecular biology (Clifton, N.J.)Michele Cervellera, Hang Zhou, Bram van den Broek, Kees Jalink, Ruslan I Dmitriev
Intracellular calcium signaling plays a crucial role in intestinal epithelial function. In this chapter, we describe a method to monitor calcium dynamics in live pig small intestinal organoids using stably expressed genetically encoded FRET-based biosensor in combination with intensity-based ratiometric and fluorescence lifetime imaging (FLIM) microscopy readouts. The calcium biosensor Twitch-2B was introduced into organoids by electroporation. Despite the modest lifetime contrast, the biosensor provides a highly reliable ratiometric signal and is compatible with both conventional fluorescence and FLIM-based imaging platform. Following electroporation, organoids are allowed to form and subsequently were selected to establish stable biosensor-expressing lines. This is followed by the live microscopy of heterogeneous and cell-specific calcium responses upon pharmacological stimulation. The presented protocol offers a versatile approach for studies of calcium signaling in relevant 3D cell models, such as organoids and tissues-on-a-chip.
International braz j urol : official journal of the Brazilian Society of UrologyMarcos Tobias-Machado, Ricardo C Brianson, John Eder Gamarra Bravo, Alcedir Raiser, Eliney Faria
INTRODUCTION: Horseshoe kidney is an uncommon congenital fusion anomaly that can make renal tumor surgery especially challenging because of altered rotation, limited mobility, variable vascular supply, and an unpredictable collecting system (1-7). This video presents a robot-assisted partial nephrectomy for a high-complexity renal tumor in this setting. CASE PRESENTATION: A 33-year-old man, with ECOG 0 and no relevant comorbidities, was diagnosed with a 7.5-cm solid renal mass in the central posterior portion of the left moiety of a horseshoe kidney. The lesion had a RENAL score of 10p. Contrast-enhanced computed tomography and three-dimensional reconstruction were used to understand the relationship between the tumor, aberrant vessels, renal hilum, and collecting system, supporting the decision to attempt nephron-sparing surgery (5, 8). Surgical technique and results: The procedure was performed through a transperitoneal robotic approach with the patient in right lateral decubitus using the Da Vinci Si platform. Port placement followed a standard renal robotic configuration, with a paramedian supraumbilical camera port, three robotic working ports along a craniocaudal lateral axis, a caudal fourth-arm port, and two medial assistant ports for suction, exposure, and support during renorrhaphy. After exposure of the horseshoe kidney and left hilar dissection, two arterial branches and one renal vein were identified. Tumor excision was performed under vascular control, with 20 minutes of warm ischemia and no collecting system opening, followed by two-layer absorbable renorrhaphy with adjunctive hemostatic agents. The operative time was 150 minutes. No transfusion, conversion, drain placement, or relevant immediate complication occurred. The urinary catheter was removed after 24 hours, and the patient was discharged 72 hours after surgery. Pathology showed clear cell renal cell carcinoma, Fuhrman grade 3, pT2N0M0, with negative surgical margins. During 12 months of oncologic follow-up, renal function remained stable and semiannual imaging showed no evidence of recurrence. Contemporary video reports have also emphasized the feasibility of advanced robotic renal surgery and complex partial nephrectomy strategies in selected patients (9, 10). CONCLUSION: In a carefully selected patient, robot-assisted partial nephrectomy supported by three-dimensional planning was feasible for a complex renal tumor in a horseshoe kidney, with negative surgical margins, preserved renal function, and no recurrence during 12 months of follow-up.
Methods in molecular biology (Clifton, N.J.)Hans Motte, Joris Jourquin, Kenzo Vereecken, Wouter Smet, Tom Beeckman, Jan Verwaeren
Root hairs play pivotal roles in nutrient and water acquisition and in plant-microbe interactions. Consequently, understanding the mechanisms underlying root hair development and their regulatory pathways is an important aspect of plant physiology research. Quantifying root hairs and root hair length is often essential in such studies, but is labor-intensive and prone to subjectivity. The availability of straightforward tools for automated root hair measurements is limited, and existing options are often tailored for specific images or do not measure individual root hairs. To address this, we developed RootHairML, a flexible and simple Python-based machine learning tool designed for efficient quantification of root hair lengths. Based on labeled images and pixel features, RootHairML trains a Random Forest model to enable the detection of root hairs in new images. It provides measurements of individual root hair lengths per image and generates annotated images showing all detected root hairs, allowing for manual verification and adjustments. Here, we describe and showcase the use of RootHairML. Overall, RootHairML offers a valuable tool for root hair analysis, enabling researchers to increase data collection and enhance the reproducibility of root hair studies.
Methods in molecular biology (Clifton, N.J.)Lana H Jachmann, Barbara S Sixt
Chlamydia trachomatis, an obligate intracellular pathogen responsible for trachoma and widespread urogenital infections, replicates exclusively within the confines of a membrane-bound vacuole known as the inclusion. This compartment shields the bacterium from host cellular immune detection; thus, clarifying how inclusion integrity is maintained and protected from vacuole-destabilizing host attacks may identify new therapeutic strategies. To enable such insights, we recently developed split-GFP-based microscopic reporters that can detect early forms of membrane damage and track individual bacteria released from compromised inclusions. These reporters rely on infecting GFP1-10-expressing cells with C. trachomatis strains engineered to express either a GFP11-tagged outer membrane protein, exposing GFP11 on the bacterial surface, or a GFP11-tagged inclusion membrane protein, exposing GFP11 to the inclusion lumen. Damage to the inclusion allows GFP1-10 to access the bacteria and inner surface of the inclusion membrane, leading to GFP reconstitution and fluorescence at either cytosol-exposed bacteria or damaged membrane sites. Here, we provide detailed protocols for applying these reporter systems, including the generation of the required bacterial strains, development of a GFP1-10-expressing cell line, and execution of a representative infection experiment.
Methods in molecular biology (Clifton, N.J.)Julie Ducla, Iwona Bernacka-Wojcik, Eleni Stavrinidou, Peter Marhavý
The capillary-based Organic Electronic Ion Pump (cOEIP) is an emerging bioelectronic delivery tool that enables precise and electronically controlled delivery of charged biomolecules to biological targets without inducing bulk fluid flow. Traditional treatment methods often induce mechanical disturbances or secondary signaling that obscure the direct physiological effects of the delivered compounds. In contrast, the iontronic devices enable precise, flow-free ionic delivery, allowing the isolation of responses specifically attributable to the applied biomolecule. Here, we present a detailed protocol for integrating the cOEIP with live confocal imaging of Arabidopsis thaliana roots, enabling spatiotemporally resolved studies of root physiology under controlled ionic delivery. As a model application, we demonstrate the localized delivery of protons (H⁺) to the root microenvironment, providing a framework for investigating pH-dependent signaling processes. The protocol includes step-by-step instructions for device assembly, preparation, calibration, and experimental setup, accompanied by representative images illustrating the mounting and operational procedures. Importantly, this approach can be adapted for the delivery of a wide range of charged biomolecules, expanding the toolkit for dynamic in vivo studies of plant-environment interactions.
Methods in molecular biology (Clifton, N.J.)Shivam Patel, Paloma Ordóñez-Morán
Human intestinal organoids (HIOs) recapitulate the architecture and cell diversity of intestinal epithelium, hence providing a system for modeling processes like regeneration and tumorigenesis. Here, we describe a detailed whole-mount immunostaining protocol for HIOs to visualize proliferative cell population. Briefly, this protocol includes preparation of HIOs, fixation and permeabilization of tissue, blocking of non-specific binding, antibody crosslinking, and visualization using a confocal microscope. Additionally, the protocol is broadly adaptable for investigating different antibodies, enabling the exploration of other cell signaling processes in HIO models. By eliminating embedding and sectioning steps, this method is both time-efficient and preserves spatial tissue architecture.
Pakistan journal of pharmaceutical sciencesZhongshu Han, Ping Zhu
BACKGROUND: Diabetes retinopathy (DR) is the main blinding complication of type 2 diabetes (T2DM). Glycated hemoglobin (HbA1c) can reflect long-term blood glucose control and optical coherence tomography (OCT) can evaluate retinal microvessels. The effects of metformin and insulin regimens on microvascular structure are not yet clear. OBJECTIVES: To explore the relationship between HbA1c levels and retinal microvascular changes in T2DM patients receiving metformin and/or insulin and to evaluate the impact of different hypoglycemic regimens on DR progression. METHODS: This retrospective study included T2DM patients with non‑proliferative DR treated between January 2023 and December 2024. After propensity score matching (PSM), 31 patients per group received metformin monotherapy (M), insulin‑based therapy (Ins), or combined therapy (M‑Ins). HbA1c, HbA1c variability (HbA1c‑SD), DR grade, central macular thickness (CMT), retinal nerve fiber layer (RNFL) thickness and superficial/deep capillary plexus (SCP/DCP) density were recorded at baseline, 1‑ and 2‑year follow‑up. Multiple linear regression and Cox regression were used. RESULTS: After 2 years, HbA1c and HbA1c‑SD decreased significantly in all groups (p<0.05), with greater reduction in the M‑Ins group. DR progression was documented in 23 patients (24.7%), with rates of 16.1%, 38.7% and 19.4% in the M, Ins and M-Ins groups, respectively (p>0.05). OCT showed reduced CMT/RNFL and increased SCP/DCP density in all groups (p<0.05), with better improvement in M‑Ins group (p<0.05). HbA1c correlated positively with CMT/RNFL and negatively with SCP/DCP (p<0.05). Cox regression indicated that higher HbA1c-SD was an independent risk factor for DR progression (HR = 3.216, 95% CI: 1.011-10.230, p= 0.048), while metformin use was an independent protective factor (HR=0.694, 95% CI: 0.451-0.840, p=0.001). CONCLUSION: Metformin plus insulin was associated with greater HbA1c reduction, decreased glycemic variability and increased retinal vessel density and may thus slow DR progression. Both HbA1c-SD and SCP density were correlated with DR progression risk, supporting the combined use of glycemic variability and OCT parameters for clinical evaluation and treatment guidance.
Neuropsychopharmacology reportsNaoki Obata, Kenshi Terajima, Gen Miura, Yuta Muratake, Shun Irie, Yuichiro Watanabe
BACKGROUND: Neurosyphilis may present with a variety of clinical symptoms and neuroimaging findings and is thus likely to be overlooked. We report a case of neurosyphilis presenting with dementia, limbic encephalitis, and normal pressure hydrocephalus. CASE PRESENTATION: A 47-year-old man had gradually experienced cognitive decline for over a year. He was admitted to a psychiatric hospital. Brain computed tomography imaging showed ventricular enlargement. Serological tests for syphilis were positive. He was transferred to our hospital, where he exhibited gait disturbance, cognitive impairment, and urinary incontinence. Brain magnetic resonance imaging showed high signal intensity in both medial temporal lobes. Treponemal cerebrospinal fluid testing was positive. He was treated with intravenous benzylpenicillin for 36 days and two courses of intravenous methylprednisolone pulse therapy. His psychomotor agitation required physical restraint for 37 days and treatment with risperidone. Although his gait disturbance and urinary incontinence improved, no clinically meaningful change in cognitive impairment was observed. The high signal intensity on repeat imaging was attenuated. He was transferred back to the psychiatric hospital 3 months after admission. CONCLUSION: This case of neurosyphilis was characterized by early-onset dementia with limbic encephalitis and normal pressure hydrocephalus. Psychiatrists should be aware of the possibility of neurosyphilis in such cases.
Journal of foot and ankle researchKazuki Kanazawa, Shunsuke Akiho, Masahiko Sakai, Ken Ichikawa, Soshiro Goshima, Ryohei Haraguchi, Takuaki Yamamoto
BACKGROUND: Hallux valgus is a frequent forefoot deformity in older adults and has been linked to impaired balance, gait dysfunction, and increased fall risk. However, its prevalence among patients with femoral neck fracture and its association with postoperative functional outcomes remain unclear. METHODS: This single-center retrospective study included 235 patients aged ≥ 65 years with available radiographic data who underwent surgery for femoral neck fracture between 2022 and 2024. Hallux valgus was defined on weight-bearing radiographs as a hallux valgus angle (HVA) ≥ 20°. Functional outcomes were evaluated using the Functional Independence Measure (FIM) at 3 months postoperatively. Multivariable linear regression analysis was performed to determine whether hallux valgus was independently associated with motor FIM scores after adjustment for age, sex, surgical procedure, and cognitive function. RESULTS: Among the 235 patients with radiographic data, 178 (76%) were women, with a median age of 85 (77-90) years; 86% of patients underwent bipolar hemiarthroplasty. Hallux valgus was present in 55% (130/235), including 20% (47/235) with moderate-to-severe deformity (HVA ≥ 30°). Patients with hallux valgus had significantly lower motor FIM scores at 3 months than those without the deformity (p = 0.044), whereas cognitive and total FIM scores did not differ. In multivariable analysis, hallux valgus (HVA ≥ 20°) was independently associated with lower motor FIM scores (B = -5.92, 95% CI -10.95 to -0.89; p = 0.021). CONCLUSIONS: Hallux valgus is highly prevalent in patients with femoral neck fracture and is independently associated with lower postoperative motor function. Assessment of hallux valgus may provide additional insight into postoperative functional prognosis in this population.
BACKGROUND: Elderly patients with cervical radiculopathy present therapeutic challenges owing to comorbidities and medication-related risks. Long-term pharmacotherapy and surgical interventions are often suboptimal, necessitating evaluation of optimized pulsed radiofrequency strategies under image guidance. OBJECTIVES: This superiority trial compared the efficacy and safety of ultrasound-guided cervical nerve root high-voltage pulsed radiofrequency (HVP-PRF) versus conventional pulsed radiofrequency (C-PRF) for pain management in elderly patients with cervical radiculopathy. METHODS: This single-center, parallel-group, assessor-blinded randomized controlled trial enrolled patients aged 60-85 years with cervical radiculopathy, randomly assigned (1:1) to HVP-PRF (70 V) or C-PRF (45 V). Procedures were performed under ultrasound guidance with sensory/motor stimulation confirmation and temperature ≤42°C. The primary outcome was change in upper-limb radiating pain on the Numeric Rating Scale (ΔNRS) from baseline to 3 months. Secondary outcomes included Neck Disability Index (NDI), neck pain NRS, Patient Global Impression of Change, responder rates, rescue analgesia use, and adverse events. Follow-up occurred at 1 week, 1, and 3 months. RESULTS: A total of 104 patients were randomized and 101 received treatment. At 3 months, HVP-PRF demonstrated significantly greater radiating pain improvement versus C-PRF (adjusted mean difference 1.24, 95% CI 0.46-2.02, P=0.002). Functional improvement (NDI) was superior in the HVP-PRF group at 3 months (AMD 6.47, 95% CI 2.11-10.83, P=0.004). Responder rates (≥50% pain reduction) were higher with HVP-PRF at 3 months (68.75% vs. 42.22%, OR 3.01, P=0.011) and 6 months (65.22% vs. 43.18%, OR 2.52, P=0.035). Rescue analgesic use was lower in the HVP-PRF group during 1-3 months intervals (both P<0.05). Adverse event rates were comparable (27.45% vs. 32.00%). CONCLUSION: Under ultrasound visualization and electrical stimulation-based target confirmation with temperature control ≤42°C, HVP-PRF provided greater and more durable relief of upper limb radiating pain compared with C-PRF in elderly patients with cervical radiculopathy, with a comparable safety profile.
Rapid communications in mass spectrometry : RCMGuang Xu, Shengfeng Gan, Bo Guo, Li Yang
RATIONALE: Mass spectrometry imaging (MSI) generates high-dimensional spatial-spectral data that requires efficient computational methods for tissue classification and candidate biomarker feature extraction. Deep learning offers a promising approach, yet the interpretability of model predictions and identification of biologically relevant spectral features remain challenging. METHODS: A comprehensive computational pipeline was developed for automatic tissue layer classification of a public mouse urinary bladder MSI dataset. Building upon prior work that compared manual tissue layer labels with those automatically generated via spectral preprocessing, t-SNE, and hierarchical clustering, in this study we separately use each type of class label to train convolutional neural networks (CNNs) for supervised classification. Gradient-weighted Class Activation Mapping (Grad-CAM) and SHapley Additive exPlanations (SHAP) were employed to compute layer-specific summed importance scores to evaluate each mass spectral feature and extract class-discriminative features. RESULTS: On the mouse urinary bladder MSI dataset, both manual labels and cluster-derived labels (t-SNE + hierarchical clustering) enabled the CNN model to achieve training accuracies exceeding 0.9 for classifying three tissue layers. Interpretability methods successfully identified discriminative m/z features, including known lipids such as SM(34:1) (m/z 741.54) and PC(34:1) (m/z 798.54), consistent with previously reported biological markers. Compared to the intensity values, the importance scores of the top class-discriminative features generated by both interpretability methods exhibited a sharper contrast and superior ability to delineate tissue-layer-specific distributions in their ion images. Furthermore, evaluation on an independent colorectal cancer dataset yielded a test accuracy of 0.758, suggesting that cross-patient generalizability varied across different data sources. CONCLUSIONS: This study presented an effective deep learning framework for accurate and interpretable tissue classification in MSI data. The CNN modeling and deep learning interpretability provided a robust approach for both automated segmentation and biological discovery, facilitating the identification of spatially resolved metabolic features in tissue sections.
Pain practice : the official journal of World Institute of PainHaneen Sabet, Ahmed Samir, Basant Lashin, Abdallah Abbas, Shrouk Ramadan, Mohamed El-Moslemani, Ahmed F Younis, Obai Yousef, Rovan Ahmed Rouby, Alaa Abd-Elsayed
OBJECTIVE: To evaluate the effectiveness and safety of proximal and distal ultrasound-guided greater occipital nerve block (US-GONB) in patients with migraine. METHODS: PubMed, Scopus, Web of Science, and the Cochrane Library were searched up to July 2025. Studies assessing US-GONB in migraine were included. Data were pooled using a random-effects model and expressed as mean difference (MD) with 95% confidence intervals (CI). Subgroup analyses were conducted based on the injection site. RESULTS: Twelve studies (658 patients) were included. US-GONB significantly reduced pain intensity (MD = -3.48 points, 95% CI [-3.84, -3.11]), monthly headache frequency (MD = -9.12 days, 95% CI [-11.65, -6.58]), headache duration (MD = -18.98 h, 95% CI [-26.86, -11.11]), and monthly analgesic use (MD = -10.01, 95% CI [-12.91, -7.11]). No significant subgroup difference was observed in pain intensity between injection sites. For monthly headache frequency, a significant subgroup difference was observed, with the proximal subgroup demonstrating numerically larger reductions than the distal subgroup (-10.83 vs. -6.36 days; p = 0.006). Adverse events were generally mild and transient: loss of pinprick sensation occurred in 80% of proximal vs. 100% of distal injections, dizziness in 22.7% vs. 3.9%, moderate-to-severe migraine attacks in 20.1% vs. 12.9%, and cerebellar-like symptoms in 12.5% vs. 0%, respectively. Vasovagal syncope (9.1%) and local pain (8.1%) were reported only in proximal injections. CONCLUSIONS: US-GONB appears to be a safe and effective intervention for migraine, significantly improving pain and headache burden. Proximal injections may be associated with greater reductions in headache frequency; however, these findings were based on indirect comparisons. Further comparative trials are warranted to optimize anatomical targeting.
Developmental scienceMegan M Hare, Yanbin Niu, Charles H Zeanah, Kathryn L Humphreys
Early functional connectivity within the brain's reward circuitry may provide insight into the developmental origins of individual differences in pleasure and motivation. This study examined resting-state functional connectivity between two key reward-related regions, the nucleus accumbens (NAc) and ventral tegmental area (VTA), in 94 one-month-old infants (58% male; M = 4.84 weeks, SD = 0.86) and its association with later reward-related temperament. Infants completed a resting-state functional magnetic resonance imaging scan during natural sleep with a mean of 11.09 (SD = 2.78) min of retained low-motion data. Caregivers reported their infants' temperament at age six months, including high- and low-intensity pleasure as indicators of individual differences relevant to reward sensitivity. Positive resting-state functional connectivity between the NAc and VTA was present in early infancy (Cohen's d = 0.44, 95% CI [0.22, 0.65]), p < 0.001). Less positive NAc-VTA connectivity was associated with greater high-intensity pleasure at six months (β = -0.32, 95% CI [-0.54, -0.10], p = 0.004), indicating that infants with stronger connectivity between these regions exhibited less enjoyment of highly stimulating, energetic activities. NAc-VTA connectivity was not associated with low-intensity pleasure (β = -0.01, 95% CI [-0.24, 0.21], p = 0.908). These findings suggest that early variation in mesolimbic connectivity may serve as a marker of emerging individual differences in reward responsiveness, with implications for understanding affective development in infancy.
Annals of human biologyErin Marie Williams-Hatala, Allison J Rutledge, Michael L Collyer, Noah Bettinger, Lucas Tavares Naief, Karyne N Rabey, Angel Zeininger, Daniel Schmitt, Caley …
INTRODUCTION: Many methods for evaluating enthesis rugosity are qualitative or fail to capture the entire entheseal surface. Here we present a new 3D method quantifying rugosity across the entire entheseal surface and test it by measuring and comparing the rugosity of the opponens pollicis enthesis to a non-enthesis region on the same bone in a sample of great apes. METHODS: To quantify rugosity, the enthesis area is isolated on high-resolution 3D models and deviations are measured between a best-fit surface and the 3D point cloud representing the enthesis. RESULTS: We demonstrate that this method quantifies entheseal rugosity and is resilient to interobserver differences in defining entheseal boundaries. It is also sufficiently sensitive to detect intergeneri differences. In Gorilla, Homo, and Pan the magnitude of entheseal rugosity is significantly greater than non-entheseal rugosity. In Pongo, there are no significant differences between the two regions or between the entheseal region of Pongo and the non-entheseal regions of the other genera, indicating a unique pollical metacarpal morphology among great apes. DISCUSSION: The results support the validity and repeatability of the method for measuring rugosity. The observed pattern of enthesis rugosity reaffirms a complex relationship among entheses, muscle anatomy, and behaviour.
Annals of medicineHong-Yu Long, Yong-Mei Wu, Xue Li, Bai-Qing Chen
BACKGROUND: To compare the predictive performance of computed tomography (CT) body composition indices, anthropometric indices, and laboratory indices for gallstones occurrence, and to construct machine-learning models to improve performance. METHODS: The dual-center retrospective cohort enrolled patients who underwent initial abdominal CT between January 2017 and January 2023, had no gallstones detected, and completed at least 3 years of follow-up. The data analysis was performed in April 2026. They were divided into gallstone group and non‑gallstone group by follow‑up findings. A deep-learning tool, Body and Organ Analysis (BOA), was used to quantify fat, muscle, and bone at the level of the third lumbar vertebra. The area under the receiver operating characteristic curve (AUC) of these indices was compared with that of anthropometric and laboratory indices. Predictive models were developed in the training cohort. Model performance was evaluated using fivefold cross-validation and an independent test cohort. RESULTS: 1,944 patients were evaluated, including 1,437 in the training cohort (Center 1; median age, 63 years [25th-75th percentile, 55-72]; 699 males) and 507 in the test cohort (Center 2; median age, 63 years [55-71]; 266 males). In univariate analysis, neutrophil-to-lymphocyte ratio (NLR) showed the highest AUC (0.627, 95% confidence interval [CI]: 0.586-0.668). The extreme trees (ET) model performed best, with a test-set AUC of 0.772 (95% CI: 0.714-0.822). SHapley Additive exPlanations (SHAP) analysis identified the area ratio of subcutaneous to total fat as the most important feature. CONCLUSIONS: NLR was the best single predictor but had limited standalone utility. Among models integrating the three indicator categories, the ET performed best.
Gut microbesAnna Voulgari-Kokota, Els Janson, Ineke Heikamp de Jong, Jan Knol, Ruurd van Elburg, Niek E van der Aa, Lisa M Hortensius, Jeroen Dudink, Caroline G M de Theij…
Preterm birth, a major cause of brain injury, is often linked to dysregulated gut microbiome development. This association underscores microbial metabolic function as a modifiable target to support neurodevelopment. In this secondary analysis of data derived from a randomized controlled trial (Trial Registration: ISRCTN96620855), we tested whether daily nutritional supplementation with Bifidobacterium breve M-16V, short- and long-chain oligosaccharides, and L-glutamine could steer the gut microbiome of very and extremely preterm infants toward communities that support brain maturation. The gut microbiome was profiled with longitudinal shotgun metagenomics at nine time points during the intervention, which started at 48-72 h after birth and continued until 36 weeks postmenstrual age. Additionally, MRI scans were conducted when infants reached term-equivalent age to evaluate brain maturation. Supplementation promoted the early establishment of Bifidobacterium-rich communities in the test group, with enhanced capacity for amino acid biosynthesis and pyruvate fermentation towards acetate and lactate production. Integration of microbiome data with brain developmental markers post hoc showed that the same functions were markedly reduced in infants with delayed white-matter myelination. By integrating microbiome functional capacity profiling and evaluation of brain maturation via MRI, this study demonstrated that early microbial modulation could influence brain development, positioning the preterm gut microbiome as a clinically actionable target.
International journal of hyperthermia : the official journal of European Society for Hyperthermic Oncology, North American Hyperthermia GroupChenchen Bing, Teija Sainio, Ari Partanen, Simon Köttgen, Roberto Blanco Sequeiros
INTRODUCTION: Non-perfused volume (NPV) is a key imaging biomarker for treatment efficacy in MR -guided high-intensity focused ultrasound (MR-HIFU). In uterine fibroids therapies, the NPV ratio is strongly associated with clinical outcomes and long-term efficacy. We developed and clinically evaluated a deep learning-based automatic NPV segmentation model to facilitate standardized and efficient post-treatment assessment. MATERIALS AND METHODS: Contrast-enhanced T1-weighted MR images from 79 patients treated with MR-HIFU were retrospectively analyzed, with NPVs manually delineated as the reference. A 2D U-Net convolutional neural network was optimized using a Tversky loss function (α = 0.7) to prioritize volumetric robustness. Performance was evaluated using similarity metrics (Dice coefficient [DC], 95th percentile Hausdorff distance [HD95]) and clinically relevant measures, including Bland-Altman analysis of volumetric quantification error, precision, recall, slice-level missing rate, and processing time. RESULTS: In the independent test cohort (n = 12), the model achieved a median DC of 0.900 (IQR: 0.841-0.920) and a median HD95 of 0.42 (IQR: 0.32-0.60) mm. Median absolute NPV quantification error was 4.3 mL (IQR: 2.6-8.4 mL), corresponding to a median relative error of 2.3% (IQR: -2.4% - 10.5%). Precision and recall were 0.909 (IQR: 0.843-0.940) and 0.912 (IQR: 0.881-0.931), respectively, with 2.8% of ground truth-positive slices missed. Average processing time per patient (∼384 slices) was 1-2 min. CONCLUSION: The proposed 2D U-Net provides accurate and time-efficient automatic NPV segmentation. Quantitative and clinically oriented evaluation demonstrates reliable volumetric assessment with minimal slice-level omission, supporting integration into routine MR-HIFU treatment workflows.
Journal of obstetrics and gynaecology : the journal of the Institute of Obstetrics and GynaecologyKübra Kurt Bilirer, Hale Özer Çaltek, Barış Boza, Selvi Aydın Şenel, Hamdullah Pekkolay, Mehmet Gümüştaş
BACKGROUND: Foetal situs abnormalities, including situs inversus totalis (SIT), right atrial isomerism (RAI) and left atrial isomerism (LAI), are associated with substantial variation in cardiac anatomy and postnatal outcomes. This study aimed to compare prenatal characteristics, associated abnormalities, perinatal outcomes, postnatal management and survival among foetuses with SIT, RAI and LAI. METHODS: This retrospective cohort study included 73 foetuses prenatally diagnosed with SIT (n = 12), RAI (n = 24) or LAI (n = 37) at a tertiary foetal cardiology and perinatology centre between 2021 and 2025. Prenatal cardiac and extracardiac findings, pregnancy and neonatal outcomes, postnatal surgical management pathways, and survival were evaluated. Survival was assessed using Kaplan-Meier's analysis, and factors associated with 1-year mortality were examined using Cox proportional hazards regression. RESULTS: Cardiac phenotypes differed significantly among groups. Interrupted inferior vena cava (IVC) occurred predominantly in LAI, whereas aortic-IVC juxtaposition, double-outlet right ventricle, anomalous pulmonary venous return and unbalanced atrioventricular septal defect were more frequent in RAI. Arrhythmias occurred exclusively in LAI. Postnatal surgical management pathways differed significantly among groups (p = 0.001), with univentricular pathway most frequent in RAI. Survival distributions did not differ significantly among SIT, RAI and LAI (log-rank p = 0.193). Exploratory multivariable analysis showed an association between right ventricular outflow tract obstruction (RVOTO) and increased 1-year mortality (HR 4.79, 95% CI 1.51-15.10; p = 0.008). CONCLUSIONS: Prenatally diagnosed SIT, RAI and LAI demonstrate distinct cardiac phenotypes and postnatal management requirements. Prognosis appears to depend not only on the laterality phenotype but also on specific cardiac morphology, with RVOTO emerging as an important prognostic marker. Detailed segmental foetal echocardiography may improve prenatal risk stratification, counselling and postnatal management planning.
European journal of psychotraumatologyLaijun Bu, Zhen Wang
Objective: Childhood peer abuse is a developmentally salient form of interpersonal adversity, yet its role in shaping links between peripheral biological stress markers and brain structure remains poorly understood. The present cross-sectional study examined whether retrospectively reported childhood peer verbal and physical abuse moderated associations of systemic inflammatory burden and hair cortisol with subcortical brain volume.Methods: Participants from a community sample completed the Maltreatment and Abuse Chronology of Exposure scale and structural magnetic resonance imaging. Inflammatory burden was indexed by a composite of standardised IL-6, IL-8, TNF-alpha, and C-reactive protein values (n = 292), and hair cortisol was available in a subsample (n = 172). Bilateral amygdala, hippocampus, putamen, and caudate volumes were extracted from FreeSurfer segmentation. Linear regression models adjusted for gender, age, parental education, and estimated total intracranial volume; false discovery rate correction was applied across the four ROI tests within each marker-by-moderator family.Results: Peer verbal abuse moderated associations of inflammatory burden with bilateral amygdala volume (β = -.095, pFDR = .032) and bilateral putamen volume (β = -.115, pFDR = .017). Peer physical abuse moderated the association between hair cortisol and bilateral amygdala volume (β = .227, pFDR = .035). Caudate models did not survive correction. Core findings remained significant after sensitivity adjustment for broader childhood adversity and current depression/anxiety symptoms.Conclusions: Associations differed across brain regions, biological markers, and peer-abuse subtypes. These findings identify peer adversity as an important interpersonal context for understanding stress-related brain differences.
The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal ObstetriciansMeng Hou, Weihong Wang, Meili Pei, Lu Zeng, Fanfan Gao
OBJECTIVE: Fetal foramen ovale restriction (FO-R) is an abnormal intracardiac flow disorder that may affect fetal cardiac hemodynamics and perinatal management. This retrospective cohort study aimed to characterize the echocardiographic features, associated abnormalities, and perinatal outcomes of pregnancies complicated by fetal FO-R, providing clinical evidence for obstetric decision-making. METHODS: A total of 283 fetuses diagnosed with FO-R by fetal echocardiography were enrolled. The diagnostic criteria were defined as a foramen ovale (FO) diameter <3 mm and a Doppler flow velocity >40 cm/s. Maternal clinical data, fetal echocardiographic findings, delivery details, and neonatal follow-up outcomes were collected and analyzed systematically. RESULTS: The incidence of FO-R was 1.88% among all fetal echocardiographic examinations. The median gestational age at diagnosis was 35 + 4 weeks, with 11.7% of cases diagnosed before 32 weeks of gestation. Coexisting pregnancy complications or fetal abnormalities were observed in 51.6% of cases, while 41.2% presented with isolated FO-R. Right heart enlargement was the most common associated cardiac finding (74.6%). The cesarean delivery rate was 64.7%, with fetal distress as the leading indication (24.0%). Multivariate analysis showed that FO diameter, timing of diagnosis, and presence of right heart enlargement did not significantly influence the delivery mode (all p > 0.05). Among 240 neonates with complete follow-up data, 27.1% had postnatal cardiac abnormalities, 93.8% of which resolved spontaneously within one year. Only 1.7% of neonates had persistent congenital heart defects. CONCLUSION: Fetal FO-R is mostly diagnosed in the third trimester and is frequently associated with right heart enlargement. Importantly, FO-R does not independently require cesarean delivery or preterm birth. Neonatal outcomes of fetuses with FO-R are generally favorable, with a high rate of spontaneous resolution of postnatal cardiac findings, which supports conservative obstetric management for most cases.