Detection of Lymphocytic Choriomeningitis Virus-Specific Memory B Cells Using Antigen Tetramers.

Memory B cells are central to the establishment of immunological memory, providing long-term protection against specific pathogens and playing a vital role in the efficacy of vaccines. Understanding how memory B cell formation is disrupted during persistent infection is essential for new therapeutics. Lymphocytic choriomeningitis virus (LCMV) is an ideal model for investigating memory B cells in acute versus chronic infection. This protocol details techniques to isolate, enrich, and examine LCMV-specific memory B cells in both acute and chronic LCMV infection. Using an antigen tetramer enrichment system and flow cytometry, this method assesses low-frequency, polyclonal antigen-specific memory B cells.

Reaction-Induced Self-Assembly of Polymyxin Mitigates Cytotoxicity and Reverses Drug Resistance.

Polymyxins have been regarded as an efficient therapeutic against many life-threatening, multidrug resistant Gram-negative bacterial infections; however, the cytotoxicity and emergence of drug resistance associated with polymyxins have greatly hindered their clinical potential. Herein, the reaction-induced self-assembly (RISA) of polymyxins and natural aldehydes in aqueous solution is presented. The resulting assemblies effectively mask the positively charged nature of polymyxins, reducing their cytotoxicity. Moreover, the representative PMBA4 (composed of polymyxin B (PMB) and (E)-2-heptenal (A4)) assemblies demonstrate enhanced binding to Gram-negative bacterial outer membranes and exhibit multiple antimicrobial mechanisms, including increased membrane permeability, elevated bacterial metabolism, suppression of quorum sensing, reduced ATP synthesis, and potential reduction of bacterial drug resistance. Remarkably, PMBA4 assemblies reverse drug resistance in clinically isolated drug-resistant strains of Gram-negative bacteria, demonstrating exceptional efficacy in preventing and eradicating bacterial biofilms. PMBA4 assemblies efficiently eradicate Gram-negative bacterial biofilm infections in vivo and alleviate inflammatory response. This RISA strategy offers a practical and clinically applicable approach to minimize side effects, reverse drug resistance, and prevent the emergence of resistance associated with free polymyxins.

Assessing the anti-inflammatory effects of whole-body vibration: a meta-analysis based on pre-clinical and clinical evidences.

BACKGROUND: Whole-body vibration (WBV) is a commonly used physical exercise for disease prevention and rehabilitation. Recent studies indicated the beneficial mechanism of WBV may be associated with its anti-inflammatory potential, however, its regulatory roles on different inflammatory mediators remained controversial. The aim of this study was to perform a meta-analysis to re-confirm the effects of WBV exercise on various inflammatory factors. METHODS: The PubMed, EMBASE and Cochrane Library databases were searched up to September 2023 to collect all articles comparing WBV with control (or post-pre trials). The effect size was expressed as the standardized mean difference (SMD) and 95% confidence intervals (CI). RESULTS: A total of 31 eligible studies were included, including 14 pre-clinical and 17 clinical studies. The meta-analysis of pre-clinical studies showed that compared with the control group, WBV exercise could significantly reduce the level of IL-6 (SMD: -1.03, 95% CI: -1.93, -0.13), TNF-α (SMD: -1.36, 95% CI: -2.54, -0.17) (for disease subgroup), IL-1ß (SMD: -2.20, 95% CI: -3.24, -1.15), IFN-γ (SMD: -1.91, 95% CI: -2.71, -1.12), IL-4 (SMD: -0.71, 95% CI: -1.39, -0.03) and IL-17 (SMD: -1.32, 95% CI: -2.05, -0.59) overall. Pooling of clinical studies revealed WBV exercise significantly reduced the level of TNF-α (WBV vs control: SMD: -1.11, 95% CI: -2.16, -0.06; post vs pre: SMD: -1.29, 95% CI: -1.91, -0.67), CRP (SMD: -3.59, 95% CI: -6.36, -0.82, P = 0.011) and enhanced the level of IL-10 (WBV vs control: SMD: 2.90, 95% CI: 1.10, 4.71; post vs pre: SMD: 1.75, 95% CI: 0.64, 2.87) and IL-6 (SMD: 0.91, 95% CI: 0.31, 1.52) (healthy subgroup). CONCLUSION: WBV may be an effective prevention and rehabilitation tool for inflammatory diseases.

Transcriptomic and proteomic profiling of bi-partite and tri-partite murine iPSC-derived neurospheroids under steady-state and inflammatory condition.

induced-pluripotent stem cell (iPSC)-derived neurospheroid (NSPH) models are an emerging in vitro toolkit to study the influence of inflammatory triggers on neurodegeneration and repair in a 3D neural environment. In contrast to their human counterpart, the absence of murine iPSC-derived NSPHs for profound characterisation and validation studies is a major experimental research gap, even though they offer the only possibility to truly compare or validate in vitro NSPH responses with in vivo brain responses. To contribute to these developments, we here describe the generation and characterisation of 5-week-old CX3CR1eGFP+/- CCR2RFP+/- murine (m)iPSC-derived bi-partite (neurons + astrocytes) and tri-partite (neurons + astrocytes + microglia) NSPH models that can be subjected to cellular activation following pro-inflammatory stimulation. First, cytokine analysis demonstrates that both bi-partite and tri-partite NSPHs can be triggered to release IL6 and CXCL10 following three days of stimulation with, respectively, TNFα + IL1ß + IFNγ and LPS + IFNγ. Additionally, immunocytochemical analysis for G3BP1 and PABPC1 revealed the development of stress granules in both bi-partite and tri-partite NSPHs after 3 days of stimulation. To further investigate the observed signs of inflammatory response and cellular stress, we performed an untargeted transcriptomic and proteomic analysis of bi- and tri-partite NSPHs under steady-state and inflammatory conditions. Here, using the combined differential gene and protein expression profiles between unstimulated and stimulated NSPHs, Ingenuity Pathway Analysis (IPA) confirms the activation of canonical pathways associated with inflammation and cellular stress in both bi-partite and tri-partite NSPHs. Moreover, our multi-omics analysis suggests a higher level of downstream inflammatory responses, impairment of homeostatic and developmental processes, as well as activation of cell death processes in stimulated tri-partite NSPHs compared to bi-partite NSPHs. Concluding, these results emphasise the advantages of including microglia in NSPH research to study inflammation-induced neurodegeneration in a 3D neural environment.

Unveiling the dance of evolution: Pla-mediated cleavage of Ymt modulates the virulence dynamics of Yersinia pestis.

Yersinia pestis has recently evolved into a highly lethal flea-borne pathogen through the pseudogenization of extensive genes and the acquisition of exogenous plasmids. Particularly noteworthy are the newly acquired pPCP1 and pMT1 plasmids, which encode the virulence determinants Pla and Yersinia murine toxin (Ymt), crucial for subcutaneous infection and survival within flea vector of Y. pestis, respectively. This study reveals that Pla can cleave Ymt at K299 both in vivo and in vitro. Y. pestis expressing YmtK299A displays enhanced in vitro biofilm formation and increased blood survival, indicating significant roles of Pla-mediated Ymt cleavage in these phenotypes. Intriguingly, although both the ancestral form of Pla and the prevalent Pla-I259T variant in modern Y. pestis strains are capable of cleaving Ymt at K299, the cleavage efficiency of Pla-I259T is only half that of the ancestral variant. In subcutaneous infection, mice infected with Δymt::ymt-K299A show significantly prolonged survival compared to those infected with Δymt::ymt. Similarly, infection with Δpla::pla-I259T also results in extended survival compared to Δpla::pla infection. These data demonstrate that the I259T substitution of Pla mitigates the enhanced virulence of Y. pestis in mice caused by Pla-mediated Ymt cleavage, thereby prolonging the survival period of infected animals and potentially conferring advantages on the transmission of Y. pestis to the next host. These findings deepen our understanding of the intricate interplay between two newly acquired plasmids and shed light on the positive selection of the Pla-I259T mutation, providing new insights into the virulence dynamics and transmission mechanisms of Y. pestis. IMPORTANCE: The emergence of Y. pestis as a highly lethal pathogen is driven by extensive gene pseudogenization and acquisition of exogenous plasmids pPCP1 and pMT1. However, the interplay between these two plasmids during evolution remains largely unexplored. Our study reveals intricate interactions between Ymt and Pla, two crucial virulence determinants encoded on these plasmids. Pla-mediated cleavage of Ymt significantly decreases Y. pestis survival in mouse blood and enhances its virulence in mice. The prevalent Pla-I259T variant in modern strains displays reduced Ymt cleavage, thereby extending the survival of infected animals and potentially increasing strain transmissibility. Our findings shed light on the nuanced evolution of Y. pestis, wherein reduced cleavage efficiency is a positive selection force, shaping the pathogen's natural trajectory.

Effects of anesthesia with sevoflurane on outcome parameters in murine experimental studies.

PURPOSE: Multiple murine studies modelling the immuno-pathophysiological consequences of trauma, shock, burn or sepsis were performed during the last decades. Almost every animal model requires anesthesia for practical and ethical reasons. Furthermore, often, corresponding control groups involve untreated animals without or with a limited exposure to anesthetics. However, the influences of anesthetic drugs on immuno-pathophysiological reactions remain insufficiently investigated. Therefore, we aimed to closer characterize the anesthetic impact exemplified by sevoflurane on the organ performance in mice and thereby investigate the influence of anesthesia itself on major outcome parameters in animal studies. METHODS: C57/BL6 mice were subjected either to 270 min of sevoflurane narcosis or directly euthanized. Plasma, BAL-fluids, lungs, kidneys, liver and intestine were collected and examined for immunological, functional and morphological changes. RESULTS: Systemic levels of the cytokine keratinocyte chemoattractant (KC) were raised in the narcosis group, while concentrations of high mobility group box protein 1 (HMGB-1) as a major inflammatory marker were reduced. In the lungs, levels of HMGB-1 and interleukin 6 (IL-6) were reduced. In contrast, systemic concentrations of intestinal fatty acid binding-protein (i-FABP) as an intestinal damage marker were elevated. Furthermore, liver-type fatty acid binding-protein (L-FABP) levels were lower in the narcosis animals, and inflammatory markers were reduced in liver tissues. Anesthesia also ameliorated the inflammatory reaction in renal tissues, while plasma levels of urea and creatinine were elevated, reflecting either dehydration and/or impaired renal function. CONCLUSION: As anesthesia with sevoflurane exhibited distinct effects in different organs, it is difficult to predict its specific impact on targets of interest in in vivo studies. Therefore, further studies are required to clarify the effects of different anesthetic drugs. Overall, the inclusion of a control group subjected to the same anesthesia protocol as the experimental groups of interest seems helpful to precisely define the inherent impact of the anesthetic when investigating immuno-pathophysiologic conditions in vivo.

Exploring the effects of taurolidine on tumor weight and microvessel density in a murine model of osteosarcoma.

Background: Osteosarcoma is the most common malignant primary bone tumor. The prognosis for patients with disseminated disease remains very poor despite recent advancements in chemotherapy. Moreover, current treatment regimens bear a significant risk of serious side effects. Thus, there is an unmet clinical need for effective therapies with improved safety profiles. Taurolidine is an antibacterial agent that has been shown to induce cell death in different types of cancer cell lines. Methods: In this study, we examined both the antineoplastic and antiangiogenic effects of taurolidine in animal models of osteosarcoma. K7M2 murine osteosarcoma cells were injected, both intramuscular and intraperitoneal, into 60 BALB/c mice on day zero. Animals were then randomized to receive treatment with taurolidine 2% (800 mg/kg), taurolidine 1% (400 mg/kg), or NaCl 0.9% control for seven days by intravenous or intraperitoneal administration. Results: After 35 days, mice were euthanized, and the tumors were harvested for analysis. Eighteen mice were excluded from the analysis due to complications. Body weight was significantly lower in the 2% taurolidine intraperitoneal treatment group from day 9 to 21, consistent with elevated mortality in this group. Intraperitoneal tumor weight was significantly lower in the 1% (p = 0.003) and 2% (p = 0.006) intraperitoneal taurolidine treatment groups compared to the control. No antineoplastic effects were observed on intramuscular tumors or for intravenous administration of taurolidine. There were no significant differences in microvessel density or mitotic rate between treatment groups. Reduced body weight and elevated mortality in the 2% taurolidine intraperitoneal group suggest that the lower 1% dose is preferable. Conclusions: In conclusion, there is no evidence of antiangiogenic activity, and the antitumor effects of taurolidine on osteosarcoma observed in this study are limited. Moreover, its toxic profile grants further evaluation. Given these observations, further research is necessary to refine the use of taurolidine in osteosarcoma treatment.

Primary and Orthotopic Murine Models of Nasopharyngeal Carcinoma Reveal Molecular Mechanisms Underlying its Malignant Progression.

Nasopharyngeal carcinoma (NPC), a squamous cell carcinoma originating in the nasopharynx, is a leading malignancy in south China and other south and east Asia areas. It is frequently associated with Epstein-Barr virus (EBV) infection, while there are also some NPC patients without EBV infection. Here, it is shown that the EBV+ (EBV positive) and EBV- (EBV negative) NPCs contain both shared and distinct genetic abnormalities, among the latter are increased mutations in TP53. To investigate the functional roles of NPC-associated genetic alterations, primary, orthotopic, and genetically defined NPC models were developed in mice, a key tool missed in the field. These models, initiated with gene-edited organoids of normal nasopharyngeal epithelium, faithfully recapitulated the pathological features of human disease. With these models, it is found that Trp53 and Cdkn2a deficiency are crucial for NPC initiation and progression. And latent membrane protein1 (LMP1), an EBV-coding oncoprotein, significantly promoted the distal metastasis. Further, loss of TGFBR2, which is frequently disrupted both in EBV- and EBV+ NPCs, dramatically accelerated the progression and lung metastasis of NPC probably by altering tumor microenvironment. Taken together, this work establishes a platform to dissect the genetic mechanisms underlying NPC pathogenesis and might be of value for future translational studies.

Plasmacytoid Dendritic Cells Mediate CpG-ODN Induced Increase in Survival in a Mouse Model of Lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a devastating disease primarily found in women of reproductive age that leads to cystic destruction of the lungs. Recent work has shown that LAM causes immunosuppression and that checkpoint inhibitors can be used as LAM treatment. Toll-like receptor (TLR) agonists can also re-activate immunity and the TLR9 agonist, CpG-ODN, has been effective in treating lung cancer in animal models. Here we investigate the use of TLR9 agonist CpG-ODN as LAM immunotherapy in combination with checkpoint inhibitor, anti-PD1, standard of care rapamycin and determine the immune mechanisms underlying therapeutic efficacy. We used survival studies, flow cytometry, ELISA, and histology to assess immune response and survival after intranasal treatment with CpG-ODN in combination with rapamycin or anti-PD1 therapy in a mouse model of metastatic LAM. We found that local administration of CpG-ODN enhances survival in a mouse model of LAM. We found that a lower dose led to longer survival likely due to fewer local side effects but increased LAM nodule count and size compared to the higher dose. CpG-ODN treatment also reduced regulatory T cells and increased the number of Th17 helper T cells as well as cytotoxic T cells. These effects appear to be mediated in part by plasmacytoid dendritic cells (pDCs), as depletion of pDCs reduces survival and abrogates Th17 T cell response. Finally, we found that CpG-ODN treatment is effective in early stage and progressive disease and is additive with anti-PD1 therapy and rapamycin. In summary, we have found that TLR9 agonist CpG-ODN can be used as LAM immunotherapy and effectively synergizes with rapamycin and anti-PD1 therapy in LAM.

Development of two novel on-site detection visualization methods for murine hepatitis virus based on the multienzyme isothermal rapid amplification.

Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/µL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/µL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.

Modeling the CD8＋ T cell immune response to influenza infection in adult and aged mice.

The CD8＋ T cell response is the main determinant of viral clearance during influenza infection. However, influenza viral dynamics and the respective immune responses are affected by the host's age. To investigate age-related differences in the CD8＋ T cell immune response dynamics, we propose 16 ordinary differential equation models of existing experimental data. These data consist of viral titer and CD8＋ T cell counts collected periodically over a period of 19 days from adult and aged mice infected with influenza A/Puerto Rico/8/34 (H1N1). We use the corrected Akaike Information Criterion to identify the models which best represent the considered data. Our model selection process indicates differences in mechanisms which reduce the CD8＋ T cell response: linear downregulation is favored for adult mice, while baseline exponential decay is favored for aged mice. Parameter fitting of the top ranked models suggests that the aged population has reduced CD8＋ T cell proliferation compared to the adult population. More experimental work is needed to determine the specific immunological features through which age might cause these differences. A better understanding of the immunological mechanisms by which aging leads to discrepant CD8＋ T cell dynamics may inform future treatment strategies.

Mechanisms of interleukin-10 induction in murine spleen and RAW264 cells by Latilactobacillus curvatus K4G4 isolated from fermented Brassica rapa L.

Lactic acid bacteria (LAB) are commonly used in fermented foods, and some LAB modulate the immune response. We aimed to investigate the mechanism by which LAB isolates from fermented Brassica rapa L. induce the production of anti-inflammatory interleukin (IL)-10 by the murine spleen and RAW264 cells. Spleen cells from BALB/c mice or the mouse macrophage cell line RAW264 were cultured with heat-killed LAB isolated from fermented B. rapa L., and the IL-10 level in the supernatant was measured. Latilactobacillus curvatus K4G4 provided the most potent IL-10 induction among 13 isolates. Cell wall components of K4G4 failed to induce IL-10, while treatment of the bacteria with RNase A under a high salt concentration altered K4G4 induction of IL-10 by spleen cells. In general, a low salt concentration diminished the IL-10 induction by all strains, including K4G4. In addition, chloroquine pretreatment and knock down of toll-like receptor 7 through small interfering RNA suppressed K4G4 induction of IL-10 production by RAW264 cells. Our results suggest that single-stranded RNA from K4G4 is involved, via endosomal toll-like receptor 7, in the induction of IL-10 production by macrophages. K4G4 is a promising candidate probiotic strain that modulates the immune response by inducing IL-10 from macrophages.

Biomechanical and Biochemical Changes in Murine Skin During Development and Aging.

Aging leads to biochemical and biomechanical changes in skin, with biological and functional consequences. Despite extensive literature on skin aging, there is a lack of studies which investigate the maturation of the tissue and connect the microscopic changes in the skin to its macroscopic biomechanical behavior as it evolves over time. The present work addresses this knowledge gap using multiscale characterization of skin in a murine model considering newborn, adult and aged mice. Monotonic uniaxial loading, tension relaxation with change of bath, and loading to failure tests were performed on murine skin samples from different age groups, complemented by inflation experiments and atomic force microscopy indentation measurements. In parallel, skin samples were characterized using histological and biochemical techniques to assess tissue morphology, collagen organization, as well as collagen content and cross-linking. We show that 1-week-old skin differs across nearly all measured parameters from adult skin, showing reduced strain stiffening and tensile strength, a thinner dermis, lower collagen content and altered crosslinking patterns. Surprisingly, adult and aged skin were similar across most biomechanical parameters in the physiologic loading range, while aged skin had lower stiffening behavior at large force values and lower tensile strength. This correlates with altered collagen content and cross-links. Based on a computational model, differences in mechanocoupled stimuli in the skin of the different age groups were calculated, pointing to a potential biological significance of the age-induced biomechanical changes in regulating the local biophysical environment of dermal cells. STATEMENT OF SIGNIFICANCE: Skin microstructure and the emerging mechanical properties change with age, leading to biological, functional and health-related consequences. Despite extensive literature on skin aging, only very limited quantitative data are available on microstructural changes and the corresponding macroscopic biomechanical behavior as they evolve over time. This work provides a wide-range multiscale mechanical characterization of skin of newborn, adult and aged mice, and quantifies microstructural correlations in tissue morphology, collagen content, organization and cross-linking. Remarkably, aged skin retained normal hydration and biomechanical function in the physiological loading range but showed significantly reduced properties at super-physiological loading. Our data show that age-related microstructural differences have a profound effect not only on tissue-level properties but also on the cell-level biophysical environment.

Site-Specific Glycosylation Analysis of Human and Murine Fcγ Receptor II Family Members Reveals Variant-Specific N-Glycosylation.

Fcγ-receptors (FcγRs) including FcγRII (CD32) gene family members are expressed on leukocytes, bind the crystallizable fragment (Fc) region of immunoglobulin G (IgG), and bridge humoral and cellular immunity. FcγRIIA and FcγRIIB have opposing roles, with the former responsible for activation and the latter for inhibition of immune cell signaling and effector functions. The extracellular domains of human and murine FcγRIIs share multiple conserved N-glycosylation sites. Understanding the role(s) of FcγRIIA and FcγRIIB glycosylation in autoimmune diseases is precluded by a lack of effective methods to study disease-associated changes in glycosylation. To address this barrier, we developed a method to assess site-specific glycosylation of human FcγRIIA and FcγRIIB, and the mouse ortholog of human FcγRIIB. Among the receptors, conserved glycosylation sites are compared, with the N144/145 site displaying predominantly complex glycans in recombinant FcγRIIs. Differences in sialylation between recombinant human FcγRIIA H/R134 (H/R131) variants at a nearby N145 N-glycosylation site are reported. Further, a potential human FcγRIIA O-glycosylation site, S179 (S212), is reported in recombinant FcγRIIA. The robust method to assess site-specific glycosylation of FcγRIIs reported here, can be utilized to study the potential role of FcγRII family glycosylation in disease. Data are available via ProteomeXchange with identifier PXD049429.

Orthotopic Models Using New, Murine Lung Adenocarcinoma Cell Lines Simulate Human Non-Small Cell Lung Cancer Treated with Immunotherapy.

Understanding tumor-host immune interactions and the mechanisms of lung cancer response to immunotherapy is crucial. Current preclinical models used to study this often fall short of capturing the complexities of human lung cancer and lead to inconclusive results. To bridge the gap, we introduce two new murine monoclonal lung cancer cell lines for use in immunocompetent orthotopic models. We demonstrate how our cell lines exhibit immunohistochemical protein expression (TTF-1, NapA, PD-L1) and common driver mutations (KRAS, p53, and p110α) seen in human lung adenocarcinoma patients, and how our orthotopic models respond to combination immunotherapy in vivo in a way that closely mirrors current clinical outcomes. These new lung adenocarcinoma cell lines provide an invaluable, clinically relevant platform for investigating the intricate dynamics between tumor and the immune system, and thus potentially contributes to a deeper understanding of immunotherapeutic approaches to lung cancer treatment.

Preclinical human and murine models of hepatocellular carcinoma (HCC).

Hepatocellular carcinoma (HCC) is the most frequent liver cancer, which account for more than 90 % of all liver cancer cases. It is the fifth leading cause of cancer globally and the second leading cause of cancer-related mortality in men. The availability of competent HCC preclinical models is fundamental to the success of mechanistic studies, molecular target identification, and drug testing. However, there are challenges associated with the use of these models. In this review, we provided updates on various cell lines, animals, and human HCC models, their specific preclinic use and associated potential challenges. Overall, the understanding of the merits and demerits of a particular HCC model will improve model selection for various preclinical studies.

Whole Blood Aggregometry in Mice.

Aggregometry plays a crucial role in both clinical diagnostics and research within hematology, serving as a fundamental tool for understanding platelet function and its implications in physiological and pathological processes. In research, aggregometry provides insights into platelet aggregation dynamics and aids in understanding the underlying mechanisms of hemostasis, thrombosis, and related disorders. Light transmission aggregometry (LTA) and lumi-aggregometry, as well as whole blood aggregometry, are commonly employed methods. While LTA and lumi-aggregometry allow for specific platelet function assessment under controlled conditions, whole blood aggregometry provides a more physiologically relevant approach by evaluating platelet aggregation within the context of whole blood. Although both methodologies offer unique advantages, whole blood aggregometry allows for preservation of the native cellular environment, simplicity, and potential for better clinical correlation. In a clinical setting, with human blood samples, protocols are established for both LTA and whole blood aggregometry as they are frequently used diagnostic tools. A protocol for LTA and lumi-aggregometry in murine models has been described; however, to date, there is no standardized protocol for whole blood aggregometry in murine models accessible to hematology researchers. This article aims to outline a simple, basic protocol for murine whole blood aggregometry, offering an alternative method to the commonly used LTA aggregometry in research settings. Standardizing whole blood aggregometry protocols in murine models could enhance experimental reliability and facilitate translational research efforts in hematology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Whole blood aggregometry in mice Support Protocol: Phenylhydrazine-induced anemia in wild-type mice Basic Protocol 2: Hematocrit percentage in mice.

Jackfruit Leaf Protein Hydrolysates Obtained by Enzymatic Hydrolysis of Leaf Protein Concentrate with Pepsin and Pancreatin: Molecular Weight, Cytotoxicity, Antiproliferative Activity, and Oxidative Stress.

Jackfruit leaf protein hydrolysates obtained from the enzymatic hydrolysis of leaf protein concentrate with gastrointestinal enzymes have shown good techno-functional properties and high antioxidant capacity. However, molecular weight, antiproliferative activity, cytotoxicity and the ability to reduce reactive oxygen species (ROS) are still unknown. Therefore, this study aimed to evaluate the effect of jackfruit leaf protein hydrolysates obtained by enzymatic hydrolysis with pepsin and pancreatin at different hydrolysis times (30-240 min) on molecular weights, cytotoxicity, antiproliferation of cancer cells, and the reduction of reactive oxygen species in H2O2-induced HaCaT cells. The electrophoretic profile indicated that H-Pep contains peptides with molecular weights between 25 - 20 kDa. Meanwhile, H-Pan is composed of molecular weight products between 25 - 20 kDa and < 20 kDa. H-Pan and H-Pep (125-500 µg/mL) did not show significant cytotoxicity on HaCaT (human keratinocytes) and J774A.1 (murine macrophage cells). Antiproliferative activity was achieved in human cervical, ovarian, and liver cancer cells. H-Pan-240 min (1000 µg/mL) reduced the cell viability of cervical cancer cells by 23% while H-Pan-60 min significantly reduced cell viability of ovarian and liver cancer cells by 14.5 (500 µg/mL) and 17% (1000 µg/mL), respectively (P < 0.05). The protective effect against oxidative stress on H2O2-stressed HaCaT cells was obtained with H-Pep-60 min, which reduced 25% of ROS at 250 µg/mL (P < 0.05). The findings demonstrate the safe use of green biomass as a source of plant protein hydrolysates.

A modified method for isolating sinoatrial node myocytes from adult mice.

Sinoatrial node (SAN) is the pacemaker of the heart in charge of initiating spontaneous electronical activity and controlling heart rate. Myocytes from SAN can generate spontaneous rhythmic action potentials, which propagate through the myocardium, thereby triggering cardiac myocyte contraction. Acutely, the method for isolating sinoatrial node myocytes (SAMs) is critical in studying the protein expression and function of myocytes in SAN. Currently, the SAMs were isolated by transferring SAN tissue directly into the digestion solution, but it is difficult to judge the degree of digestion, and the system was unstable. Here, we present a modified protocol for the isolation of SAMs in mice, based on the collagenase II and protease perfusion of the heart using a Langendorff apparatus and subsequent dissociation of SAMs. The appearance and droplet flow rate of the heart could be significantly changed during enzymatic digestion via perfusion, which allowed us to easily judge the degree of digestion and avoid incomplete or excessive digestion. The SAMs with stable yield and viability achieved from our optimized approach would facilitate the follow-up experiments.

Animal Models and Molecular Pathogenesis of Arrhythmogenic Cardiomyopathy Associated with Pathogenic Variants in Intercalated Disc Genes.

Arrhythmogenic cardiomyopathy (ACM) is a rare genetic cardiac disease characterized by the progressive substitution of myocardium with fibro-fatty tissue. Clinically, ACM shows wide variability among patients; symptoms can include syncope and ventricular tachycardia but also sudden death, with the latter often being its sole manifestation. Approximately half of ACM patients have been found with variations in one or more genes encoding cardiac intercalated discs proteins; the most involved genes are plakophilin 2 (PKP2), desmoglein 2 (DSG2), and desmoplakin (DSP). Cardiac intercalated discs provide mechanical and electro-metabolic coupling among cardiomyocytes. Mechanical communication is guaranteed by the interaction of proteins of desmosomes and adheren junctions in the so-called area composita, whereas electro-metabolic coupling between adjacent cardiac cells depends on gap junctions. Although ACM has been first described almost thirty years ago, the pathogenic mechanism(s) leading to its development are still only partially known. Several studies with different animal models point to the involvement of the Wnt/ß-catenin signaling in combination with the Hippo pathway. Here, we present an overview about the existing murine models of ACM harboring variants in intercalated disc components with a particular focus on the underlying pathogenic mechanisms. Prospectively, mechanistic insights into the disease pathogenesis will lead to the development of effective targeted therapies for ACM.