The therapeutic effectiveness of acupuncture relies on both safety and stability, making these factors essential in acupuncture manipulation research. However, manual manipulation introduces unavoidable inaccuracies, which can impact the reliability of research findings. To address this challenge, a unique lifting and thrusting manipulation control cannula was designed in this study, offering flexible adjustment of movement amplitude. The cannula was created using 3D printing technology, and its effectiveness in maintaining stability was verified by recording the acupuncture needle's movement range with optical sensor technology. The study's results show that the control cannula significantly enhances the stability of acupuncture manipulation, reducing human error. This innovation suggests that the cannula could serve as a valuable auxiliary tool for ensuring both the precision and safety of acupuncture-related experimental research. Its adoption could also contribute to the standardization of acupuncture practices, ensuring more consistent and accurate research outcomes, which is essential for future advancements in acupuncture research and clinical applications.

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 °C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBIMFGE8 UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBIMFGE8 UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBIMFGE8 subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBIMFGE8 UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBIMFGE8 UC-MSCs.

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. However, because of the lack of sensitivity of hardware at longer wavelengths, absorption spectroscopy has typically been applied for wavelengths in the visible (VIS) and near-infrared (NIR) range from 400 to 1,000 nm. Hardware advancements in the short-wave infrared (SWIR) range have enabled investigators to explore wavelengths in the ~1,000 nm to 3,000 nm range in which fall characteristic absorption peaks for lipid, protein, and water. These molecules are difficult to visualize in the VIS-NIR and can provide label-free sources of biological contrast. Furthermore, lower SWIR absorption has been observed for melanin, the primary chromophore responsible for skin pigmentation. In vivo optical devices like clinically standard pulse oximeters have been found to have reduced accuracy in people with darkly pigmented skin, possibly because of the stronger melanin absorption in the VIS range. Thus, error associated with skin pigmentation could be reduced by using devices operating in the SWIR. Optical instrument design is facilitated by the understanding of the absorption properties of core tissue components from the VIS to the SWIR range. This article describes protocols and instrumentation for obtaining VIS-SWIR absorption spectra of common tissue absorbers: oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water, and lipid.

Glioblastoma (GBM) is described as a group of highly malignant primary brain tumors and stands as one of the most lethal malignancies. The genetic and cellular characteristics of GBM have been a focal point of ongoing research, revealing that it is a group of heterogeneous diseases with variations in RNA expression, DNA methylation, or cellular composition. Despite the wealth of molecular data available, the lack of transferable pre-clinic models has limited the application of this information to disease classification rather than treatment stratification. Transferring the patients' genetic information into clinical benefits and bridging the gap between detailed descriptions of GBM, genotype-phenotype associations, and treatment advancements remain significant challenges. In this context, we present an advanced human GBM organoid model, the Laboratory Engineered Glioblastoma Organoid (LEGO), and illustrate its use in studying the genotype-phenotype dependencies and screening potential drugs for GBM. Utilizing this model, we have identified lipid metabolism dysregulation as a critical milestone in GBM progression and discovered that the microsomal triglyceride transfer protein inhibitor Lomitapide shows promise as a potential treatment for GBM.

Dental ultrasonic scalers are commonly employed in periodontal treatment; however, their ability to roughen tooth surfaces is a worry since roughness may increase plaque production, a key cause of periodontal disease. This research studied the influence of a piezoelectric ultrasonic scaler on the roughness of two distinct flowable composite filling materials. To do this, 10 disc-shaped samples were generated from each of the two flowable composite materials. After standardized polishing, samples were submerged in water for 24 h before the first surface examination using electron microscopy and profilometry. The ultrasonic scaler was applied to a specified location of each sample for 60 s under water cooling and regulated force. Post-scaler surface parameters were again examined. Following the application of the scaler, both composite materials exhibited a notable increase in surface roughness, as determined by profilometry (p < 0.01). Additionally, the observed surface roughness was also qualitatively visualized with scanning electron microscopy. While initial roughness levels were comparable across the two composites (p = 0.143) after scaler application, no substantial discrepancy in surface texture was noticed between them (p = 0.684). The use of a high-power piezoelectric ultrasonic scaler on routinely used flowable composite restorations might generate considerable surface roughness, possibly leading to increased plaque accumulation. Nevertheless, it might be postulated that nanohybrid flowable composite materials having conventional monomer ingredients may demonstrate comparable surface alterations within the limitations of this experiment.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Clinical metaproteomics reveals host-microbiome interactions underlying diseases. However, challenges to this approach exist. In particular, the characterization of microbial proteins present in low abundance relative to host proteins is difficult. Other significant challenges are attributed to using very large protein sequence databases, which impedes sensitivity and accuracy during peptide and protein identification from mass spectrometry data in addition to retrieving taxonomy and functional annotations and performing statistical analysis. To address these problems, we present an integrated bioinformatics workflow for mass spectrometry-based metaproteomics that combines custom protein sequence database generation, peptide-spectrum match generation and verification, quantification, taxonomic and functional annotations, and statistical analysis. This workflow also offers characterization of human proteins (while prioritizing microbial proteins), thus offering insights into host-microbe dynamics in disease. The tools and workflow are deployed in the Galaxy ecosystem, enabling the development, optimization, and dissemination of these computational resources. We have applied this workflow for metaproteomic analysis of numerous clinical sample types, such as nasopharyngeal swabs and bronchoalveolar lavage fluid. Here, we demonstrate its utility via the analysis of residual fluid from cervical swabs. The complete workflow and accompanying training resources are accessible on the Galaxy Training Network to equip non-experts and experienced researchers with the necessary knowledge and tools to analyze their data.

A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of Alzheimer's disease (AD) is presented here. To begin, fresh human neuro progenitor ReN cells expressing β-amyloid precursor protein (APP) containing familial Alzheimer's disease (FAD) or naïve ReN cells are grown in thin (1:100) Matrigel-coated tissue culture plates. After the cells reach confluency, these are electroporated with expression plasmids encoding red fluorescence protein (RFP)-conjugated mitochondria-binding sequence of AKAP1(34-63) (Mito-RFP) that detects mitochondria or constitutive MAM stabilizers MAM 1X or MAM 9X that stabilize tight (6 nm ± 1 nm gap width) or loose (24 nm ± 3 nm gap width) MAMs, respectively. After 16-24 h, the cells are harvested and enriched by a fluorescence-activated cell sorter (FACS). An equal number of FACS-enriched cells are seeded in the 3-dimensional matrix (1:1 Matrigel) and allowed to differentiate into mature neurons for 10 days. Live cell images of the 10-day differentiated cells expressing the RFP-conjugated MAM stabilizers are captured under a fluorescent microscope equipped with a live-cell imaging culture chamber maintaining the CO2 (5%), temperature (37 °C), and humidity (~90%). Toward this end, we performed live-cell imaging and kymographic analyses to measure the motility of free mitochondria labeled with Mito-RFP or ER-bound mitochondria of tight or loose gap widths stabilized by MAM 1X or MAM 9X, respectively, in the most extended neuronal process of each ReN GA neuron which is at least 500 nm long, considering these as axons.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity via continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

Zebrafish scales offer a variety of advantages for use in standard laboratories for teaching and research purposes. Scales are easily collected without the need for euthanasia, regenerate within a couple of weeks, and are translucent and small, allowing them to be viewed using a standard microscope. Zebrafish scales are especially useful in educational environments, as they provide a unique opportunity for students to engage in hands-on learning experiences, particularly in understanding cellular dynamics and in vitro culturing methods. The main objective of this protocol is to describe a method for collecting and maintaining zebrafish scales in culture for use in a variety of biological studies using basic laboratory equipment. Additionally, the protocol details their use in understanding bone homeostasis by examining the activity of bone cells involved in bone resorption and deposition. It also includes additional protocols for general techniques, such as the visualization of nuclei and apoptotic cells. The in vitro culturing protocol produces reliable results with minimal reagents and equipment. This article discusses the benefits of using in vitro cultures of zebrafish scales to foster scientific inquiry and outlines the resources needed to support their integration into educational settings.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

Cardiovascular disease (CVD) is the leading cause of death in the United States. Damage in the cardiovascular system can be due to environmental exposure, trauma, drug toxicity, or numerous other factors. As a result, cardiac tissue and vasculature undergo structural changes and display diminished function. The damage and the resulting remodeling can be detected and quantified with ultrasound (US) imaging at the organ level and mass spectrometry imaging (MSI) at the molecular level. This manuscript describes an innovative methodology for studying murine cardiac pathophysiology, coupling in vivo four-dimensional (4D) ultrasound imaging and analysis with ex vivo matrix-assisted laser desorption/ionization (MADLI) MSI of the heart. 4D ultrasound can provide dynamic volumetric measurements, including radial displacement, surface area strain, and longitudinal strain throughout an entire cardiac cycle. In the vasculature, MSI and ultrasound are used to assess vessel wall compositions, hemodynamics, and vessel wall dynamics. The methodology can be tailored to study a myriad of CV diseases by adjusting functional metrics of interest and/or varying MALDI MSI protocol to target specific molecules. MALDI MSI can be used to study lipids, small metabolites, peptides, and glycans. This protocol outlines the use of MALDI MSI for untargeted lipidomic analysis and the use of ultrasound imaging for cardiovascular hemodynamics and biomechanics.

The objective of this study was to investigate the cardioprotective effects of Munziq on abnormal body fluid myocardial ischemia-reperfusion injury (MIRI) and its underlying mechanism.Normal rats and rats with abnormal body fluid (ABF) were pre-treated with Munziq for 21 days. Following this, MIRI models were established. Histopathological changes and myocardial ultrastructure changes were observed by Hematoxylin and Eosin (HE)staining and transmission electron microscopy to observe pathological manifestations of myocardial injury. Serum CK-MB, cTn-T, and ICAM-1 levels were detected by Enzyme-Linked Immunosorbent Assay (ELISA) to observe myocardial injury-related markers. The levels of IL-1β, IL-6, and TNF-α in serum and myocardial tissue were also detected by ELISA to observe the anti-inflammatory effect. The expression levels of NF-κB signaling pathway-related proteins NIK, IKKα, Pikα, and p65 were detected by Western blot analysis. The results showed that myocardial injury in the ABF MIRI group was more severe compared to the control MIRI group. Munziq pretreatment has the potential to mitigate the pathological changes induced by ischemia-reperfusion injury and could protect cardiac function. Protein levels of the NF-κB pathway and downstream effectors IL-1β, IL-6, and TNF-α were significantly up-regulated in the MIRI group while down-regulated in the Munziq group. Interestingly, there was more activation of the NF-κB signaling pathway and higher levels of downstream inflammatory cytokines in the ABF MIRI group. The results suggest that MIRI was more severe in ABF. Munziq has cardioprotective effects in ischemia and reperfusion injury. This protective effect may be acted by suppressing the NF-κB signaling pathway.

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 ± 20 billion cells per day), is crucial for maintaining individual health. Any disruption in this pathway can have significant consequences, leading to health issues. According to the World Health Organization, an estimated 25% of the global population presents symptoms of anemia. This protocol describes how to generate human erythroid cells both in vitro using hematopoietic stem and progenitor cells (HSPCs) from sources such as umbilical cord blood (UCB) or blood taken from healthy donors and ex vivo with HSPCs isolated from patients' bone marrow. Using genetic approach, genes of interest can be modulated in HSPCs, and their impact on erythropoiesis can be monitored at various stages of the differentiation process. This method allows for the screening of compounds perturbing, enhancing, or rescuing the capacity of HSPCs to differentiate into mature erythroid cells and to investigate the role of genes of interest during the erythroid differentiation process.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%-40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

Transgender (TG) people are individuals whose gender identity and sex assigned at birth do not match. They often undergo gender-affirming hormone therapy (GAHT), a medical intervention that allows the acquisition of secondary sex characteristics more aligned with their individual gender identity, providing consistent results in the improvement of numerous socio-psychological variables. However, GAHT targets different body systems, and some side effects are recorded, although not yet fully identified and characterized. Therefore, TG people undergoing GAHT may be considered as a susceptible sub-group of population and specific attention should be paid in the frame of risk assessment, e.g., through the use of targeted animal models. The present work describes the procedures set to implement two rat models mimicking GAHT: the demasculinizing-feminizing model (dMF) mimicking the GAHT for TG women and the defeminizing-masculinizing model (dFM) mimicking the GAHT for TG men. The models have been implemented through the administration of the same hormones used for human GAHT, namely, β-estradiol plus cyproterone acetate for dMF and testosterone for dFM, by the same routes of exposure for a 2 week period. Rats are checked daily during the treatment to evaluate health status and potentially aggressive behaviors. At sacrifice, blood and target tissues have been sampled and stored for biochemical, molecular, and histopathological analysis. Sex-specific parameters, namely, sperm count and clitoral dimensions, have also been evaluated. In addition, CYP450 isoforms, exclusively and/or preferentially expressed in male and female rat liver, are identified and characterized as novel biomarkers to verify the success of GAHT and to set the model. Thyroid involvement has also been explored as a key target in the endocrine system.

Non-invasive assessment of pulmonary nodule malignancy remains a critical challenge in lung cancer diagnosis. Traditional methods often lack precision in differentiating benign from malignant nodules, particularly in the early stages. This study introduces an approach using multifractal spectrum analysis to quantitatively evaluate pulmonary nodule characteristics. A fractal-based protocol was developed to process computed tomography (CT)-digital imaging and communications in medicine (DICOM) data, enabling three-dimensional (3D) visualization and analysis of pulmonary nodule's multifractal spectrum. The method involves 3D volume reconstruction, precise ROI delineation, and calculation of fractal dimensions across multiple scales. Multifractal spectra were computed for both early-stage and late-stage lung adenocarcinoma nodules, with comparative analysis performed using data tip tool quantification. Analysis revealed that the fractal dimension of a pulmonary nodule's 3D digital matrix varies continuously with different voxel scales, forming a distinctive multifractal spectrum. Significant differences were observed between early-stage and late-stage nodules. Late-stage nodules demonstrated a wider scale range (longer X-axis) and higher extreme points in their multifractal spectra. These distinctions were quantitatively confirmed, indicating the method's potential for precise staging. The multifractal spectrum analysis provides a highly significant and precise quantitative method for staging pulmonary nodules, effectively differentiating between benign and malignant cases. This non-invasive technique shows promise for improving early diagnosis and accurate staging of lung cancer, potentially enhancing clinical decision-making in pulmonary oncology.

The iChip isolation technique uses an in-situ isolation device that increases the cultivability of previously unculturable microorganisms. Microorganisms are an important source of novel chemistries and potentially bioactive molecules. However, only 1% of environmental microorganisms can be cultured using conventional laboratory methods. With the rise in antimicrobial resistance, finding new drugs to combat infections and diseases is of foremost importance, and a critical method to finding new molecules is the discovery of new microorganisms. By incubating colonies of soil microorganisms in the wells of a 96-well plate, sealed with a semipermeable membrane and incubated on top of soil, the microbes are in contact with water and growth factors from the soil, allowing for the isolation of novel microbes in a laboratory setting. After a period of domestication in an iChip, microorganisms can potentially be subcultured onto conventional media and used for further study. This device is valuable to bioactive molecule discovery and soil microbiome research and has been used previously in both applications.

The abnormal alternation of pulmonary angiogenesis is related to lung microvascular dysfunction and is deeply linked to vascular wall integrity, blood flow regulation, and gas exchange. In murine models, lung lobes exhibit significant differences in size, shape, location, and vascularization, yet existing methods lack consideration for these variations when quantifying microvascular density. This limitation hinders the comprehensive study of lung microvascular dysfunction and the potential remodeling of microvasculature circulation across different lobules. Our protocol addresses this gap by employing two sectioning methods to quantify pulmonary microvascular density changes, leveraging the size, shape, and distribution of airway branches across distinct lobes in mice. We then utilize Isolectin B4 (IB4) staining to label lung microvascular endothelial cells on different slices, followed by unbiased microvascular density analysis using the freely available software ImageJ. The results presented here highlight varying degrees of microvascular density changes across lung lobules with aging, comparing young and old mice. This protocol offers a straightforward and cost-effective approach for unbiased quantification of pulmonary microvascular density, facilitating research on both physiological and pathological aspects of lung microvasculature.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.