Although breathing dynamics and mechanical strain play critical roles in alveolar biology, their contributions to the pathogenesis of respiratory diseases are often overlooked. To address this gap, we developed an organoid model in which alveolar epithelial-fibroblast organoids are subjected to controlled mechanical strain using a cell-stretching bioreactor. This platform replicates the spatial organization and multicellular complexity of alveolar tissue and enables investigation of how cell-cell communication and mechanotransduction influence tissue structure and function in health and disease. In this protocol, we describe methods for generating and culturing epithelial-fibroblast organoids using A549 alveolar epithelial and MRC-5 lung fibroblast cell lines in a basement membrane extracellular matrix hydrogel. Organoids are then non-enzymatically isolated and embedded in 3D collagen gels in specialized plates in the bioreactor. We outline procedures for applying equibiaxial strain to mimic pathological breathing patterns seen in respiratory exacerbations. Finally, we present a standard characterization workflow, including immunostaining for cell-specific markers and immunoassays to profile immune mediator release. Together, these analyses support the assessment of organoid structure and function. This protocol provides a user-friendly, reproducible, and adaptable platform for studying alveolar biology under mechanical stress, with easily modifiable parameters to meet diverse experimental goals and advance research into lung disease mechanisms.

Tooth discoloration is a common esthetic concern that may influence psychosocial development and peer interactions during childhood. This in vitro study aimed to compare the discoloration potential of fruit juices frequently consumed by children-black mulberry, organic pomegranate, and organic orange-and to evaluate whether brushing with fluoridated or non-fluoridated toothpastes reduces these changes in primary teeth. Eighty sound primary teeth extracted due to trauma, orthodontic indications, or natural exfoliation and free of caries, restorations, enamel defects, or pre-existing discoloration were included. Samples were divided into four immersion groups (n = 20): saline (control), black mulberry juice, organic pomegranate juice, and organic orange juice. Each group was further divided into brushed and unbrushed subgroups, with brushed specimens treated using either fluoridated or non-fluoridated toothpaste (n = 10). All teeth were immersed for 28 days, and color measurements were obtained at baseline, day 7, and day 28 using a dental spectrophotometer according to the CIELab system. All fruit juices produced clinically perceptible discoloration (ΔE > 3.3), and each juice caused higher staining than the saline control. Black mulberry and pomegranate produced the greatest total discoloration, while orange juice resulted in markedly lower ΔE values. Brushing consistently reduced discoloration across all groups; however, no significant difference was found between fluoridated and non-fluoridated toothpastes, indicating that the mechanical action of brushing-rather than fluoride content-was primarily responsible for stain reduction. These findings reinforce the susceptibility of primary enamel to pigment-rich beverages and highlight the importance of effective brushing habits in minimizing extrinsic discoloration in children. Clinically, these findings suggest that despite the high staining potential of these fruit juices, strict adherence to daily brushing routines can effectively mitigate esthetic degradation in primary teeth, regardless of the toothpaste's fluoride content.

Understanding the healthy human virosphere (the viral component of the microbiome) requires accurate measurements of viral community composition across a diverse range of viral types. Building on prior experience with soil viral community ecology methods, here we demonstrate a series of laboratory approaches for enriching and extracting DNA from extracellular DNA viruses in human stool samples. A working primary protocol is presented, along with options for deviations at different steps. The general approach involves adding a liquid buffer (default: protein-enhanced phosphate buffered saline, PPBS) to facilitate removal of free viral particles from the stool matrix, centrifugation to separate the liquid fraction containing viral particles, filtration (default: 0.2 µm pore size) to remove most cells, concentration of viral particles (default: ultracentrifugation), removal of free nucleic acids with nucleases prior to virion lysis, and then DNA extraction for sequencing. Alternative techniques, including different buffers, filter sizes, and concentration methods, are also noted. Overall, multiple options for generating high-quality viromic DNA for sequencing are offered. Rather than tailoring the approach to specific equipment and resources, the protocol's flexibility should make it broadly applicable across labs with varying standard molecular biology equipment.

Microfluidic devices offer precise control over solution mixing and gradient generation, essential for cell-based assays in cryobiology and biomedical research. However, traditional fabrication methods are time-consuming, costly, and require specialized expertise, which limits accessibility. To address these challenges, we developed a cost-effective, reliable, and fully 3D-printed microfluidic device workflow to facilitate rapid and inexpensive prototyping using a consumer-grade printer and biocompatible plastic resins. Here, we demonstrate this workflow with a fluidic mixing device capable of generating programmable concentration gradients and solution combinations. Commercial mixing devices cost more than $300 each and cannot be customized. By utilizing affordable resin materials and an innovative open-channel design sealed with transparent adhesive tape, we overcame common fabrication issues such as channel clogging, enabling rapid and reproducible fabrication of complex microfluidic architectures, all at a materials cost of less than $5. Here we demonstrate this workflow, integrating dual-syringe pumps to create linear osmotic gradients, ranging from iso-osmotic (~300 mOsm/kg) to hyperosmotic (~9,000 mOsm/kg) conditions, followed by a return to isotonicity over defined intervals. To ensure automation and reproducibility, we developed an open-source Python-based software tool that precisely regulates syringe pump activation, flow rates, and gradient timing. The device's performance was validated through continuous osmometric measurements, which confirmed both the linearity and accuracy of gradient generation, and colorimetric measurements to confirm mixing efficacy. This accessible and cost-effective microfluidic platform significantly improves the reproducibility of osmotic exposure studies and shows potential for various biomedical applications, including drug screening and precise chemical modulation.

Perianal refractory wounds refer to long-term non-healing wounds of the skin and soft tissues surrounding the anus, often resulting from surgery or complications of chronic diseases. Current treatment mainly focuses on maintaining an open wound environment and allowing healing to follow its natural course. Due to the specific anatomical location, susceptibility to secondary infection, and the local wound environment, healing often requires more than 4 weeks. Vacuum-sealed drainage (VSD), combined with counter-current irrigation technology, has been widely adopted and demonstrated significant efficacy in specialties such as burn and plastic surgery, orthopedics, and general surgery. This non-invasive negative-pressure technology offers new approaches for treating refractory perianal wounds, promoting active intervention in the healing process, accelerating healing speed, improving healing quality, and reducing patient discomfort. This article describes the management and application details of this technology for refractory perianal wounds, including methods of outer-film application at different positions, differences in negative-pressure settings based on wound distance from the anal margin, and strategies to avoid secondary contamination. This methodology may facilitate broader clinical adoption of VSD for perianal refractory wounds and presents new therapeutic opportunities for managing such challenging non-healing wounds.

Laparoscopic anatomic liver resection is the optimal treatment for intrahepatic bile duct stones confined to a liver segment or lobe. However, recurrent inflammation often obscures the first hepatic hilum, making traditional dissection approaches challenging. The "Landmark Vein approach," using a major hepatic vein as a real-time intraoperative guide for the parenchymal transection plane, offers a technical strategy to facilitate precise anatomic resection in this context. This technical note demonstrates the feasibility of this approach in a patient with complex hepatolithiasis undergoing a right hemihepatectomy. We detail the critical steps: preoperative 3D reconstruction for venous mapping, intraoperative ultrasound for confirmation, and a caudal-to-cephalad parenchymal transection meticulously guided along the middle hepatic vein. This report aims to provide a structured description of the technique, highlighting its potential utility in managing hepatolithiasis with distorted hilar anatomy. While the approach may contribute to improved surgical outcomes, further studies with larger cohorts are needed to validate its broader applicability and impact.

Early embryogenesis requires an exponential decrease in cell size, while the embryo remains constant in size at the earliest stages of development. The functional importance and the mechanisms by which cell size impacts embryo development are still largely understudied. The Xenopus early embryoprovides a unique system to address these major gaps. Compared with other model organisms, the Xenopus early embryo is extremely large and contains a cell-size gradient, from small cells at the animal pole to large cells at the vegetal pole, across the entire embryo at stages around the mid-blastula transition. Here, we describe a protocol for dissociating single cells from Xenopus early embryos and sorting them by size using cell strainers with varying mesh pore sizes. As determined by imaging and size measurements, the sorted cells are within the expected size range and can be used for live-cell analysis or sequencing, or fixed for imaging. This system will shed new light on revealing size-dependent regulatory mechanisms during early development.

This study aimed to evaluate an animation-based caregiver-patient interaction model for postoperative pain and anxiety management in preschool children with congenital heart disease (CHD). This randomized controlled trial included 70 preschoolers undergoing CHD surgery under general anesthesia, who were enrolled in the Cardiac Critical Care Unit at Beijing Children's Hospital (June-October 2024). Participants were stratified into intervention (n=35) and control (n=35) groups. The control group received standard postoperative care, while the intervention group additionally received a structured animation-based program involving preoperative personalized animation selection, post-extubation viewing (≤30 min/session, 3-6 times/day) with nurse-guided interaction, and parental involvement. Pain (WongBaker FACES scale) and anxiety (mYPAS) were assessed at 1 h/6 h postextubation, 24 h/48 h postsurgery, during OR transport, and 24 h postsurgery.Pain was assessed at four time points: 1 h/6 h post-extubation and 24 h/48 h post-surgery. At 1 h post-extubation, there was no significant difference in pain scores between the intervention and control groups (median 9 vs 8, p= 0.462). Pain scores were significantly lower in the intervention group than in controls at 6 h post-extubation (median 6 vs 9), 24 h post-operative (3 vs 6), and 48 h post-operative (2 vs 4); all p < 0.001. Anxiety scores (m-YPAS) were also significantly lower in the intervention group, both on transport to the OR (median 76 vs 80) and at 24 h after surgery (60 vs 80), p < 0.001. Friedman tests demonstrated significant within-group changes over time in anxiety levels (intervention p < 0.001), confirming that the intervention group experienced substantially reduced anxiety at these two key perioperative time points. The interactive animation model significantly reduces postoperative pain and perioperative anxiety in preschool patients with CHD, offering a safe non-pharmacological adjuvant for pain management.

This meta-analysis evaluated whether adding Buyang Huanwu Decoction (BYHWD) to standard surgery improves spinal-cord nerve recovery after traumatic spinal-cord injury (SCI). Following PRISMA and PROSPERO protocol CRD42024612859, we systematically searched PubMed, Cochrane, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang and VIP to January 2025 for randomized controlled trials (RCTs) comparing surgery plus usual care with the same regimen plus BYHWD. Data were pooled with RevMan 5.3. Nine Chinese RCTs (614 patients, 306 BYHWD, 308 control) were included. Relative to controls, BYHWD significantly raised post-operative American Spinal Injury Association(ASIA) sensory score (MD 7.51; 95% CI 5.08-9.93; P <0.01), ASIA motor score (MD 7.65; 5.81-9.49; P <0.01), somatosensory evoked potential amplitude (MD 0.37 µV; 0.18-0.55; P <0.01), motor evoked potential amplitude (MD 0.43 µV; 0.19-0.68; P <0.01) and the proportion of patients achieving ≥1 ASIA grade improvement (RR 1.23; 1.09-1.38; P <0.01). Heterogeneity was low for most outcomes. No serious herb-related adverse events were reported. Adjunctive BYHWD appears to accelerate short-term neurological recovery after surgical decompression for SCI. However, all trials were Chinese, most lacked allocation concealment or blinding, follow-up was brief and outcome sets varied. Large-scale, rigorously blinded, multicentre RCTs with long-term functional endpoints are needed before routine clinical adoption.

This study utilized a multi-omics and computational biology framework to investigate the therapeutic potential of the Traditional Chinese Medicine (TCM) formula Buti Huatan Tang (BTHTT) against chronic obstructive pulmonary disease (COPD). Significant physiological improvements were observed in a rat model following BTHTT intervention. Histological analysis showed a reversal of lung pathological damage, while biochemical assays, and transcriptomics confirmed the normalization of IL-1β and IL-1R2 levels. Additionally, metabolic profiling revealed that BTHTT corrected disruptions in T3 and T4 thyroid hormone levels. A negative correlation was observed between the IL-1β/IL-1R2 axis and these thyroid hormones, indicating that their regulation is associated with the formula's therapeutic effect. Beyond direct measurements, machine learning algorithms identified ten COPD signature genes from clinical databases. Pathway enrichment analysis suggests that BTHTT may act through cytokine-cytokine-receptor interactions and thyroid hormone synthesis pathways. Furthermore, while 283 components were identified in vivo, compounds such as tanshinone IIA and cryptotanshinone are currently considered candidate active substances. Their role as primary drivers is supported by a model in which they stably bind to IL-1R2; this inference is based on molecular docking and molecular dynamics (MD) simulations rather than direct experimental isolation. Overall, the data support a model in which BTHTT exerts a multi-target effect on COPD by modulating inflammation and metabolic homeostasis. This integrated approach provides a refined scientific basis for the clinical application of BTHTT and highlights specific pathways for future experimental validation.