Computational prediction of protein interactions in single cells by proximity sequencing.

Proximity sequencing (Prox-seq) simultaneously measures gene expression, protein expression and protein complexes on single cells. Using information from dual-antibody binding events, Prox-seq infers surface protein dimers at the single-cell level. Prox-seq provides multi-dimensional phenotyping of single cells in high throughput, and was recently used to track the formation of receptor complexes during cell signaling and discovered a novel interaction between CD9 and CD8 in naïve T cells. The distribution of protein abundance can affect identification of protein complexes in a complicated manner in dual-binding assays like Prox-seq. These effects are difficult to explore with experiments, yet important for accurate quantification of protein complexes. Here, we introduce a physical model of Prox-seq and computationally evaluate several different methods for reducing background noise when quantifying protein complexes. Furthermore, we developed an improved method for analysis of Prox-seq data, which resulted in more accurate and robust quantification of protein complexes. Finally, our Prox-seq model offers a simple way to investigate the behavior of Prox-seq data under various biological conditions and guide users toward selecting the best analysis method for their data.

Musashi-2 potentiates colorectal cancer immune infiltration by regulating the post-translational modifications of HMGB1 to promote DCs maturation and migration.

Post-translational modifications (PTMs) of the non-histone protein high-mobility group protein B1 (HMGB1) are involved in modulating inflammation and immune responses. Recent studies have implicated that the RNA-binding protein (RBP) Musashi-2 (MSI2) regulates multiple critical biological metabolic and immunoregulatory functions. However, the precise role of MSI2 in regulating PTMs and tumor immunity in colorectal cancer (CRC) remains unclear. Here, we present data indicating that MSI2 potentiates CRC immunopathology in colitis-associated colon cancer (CAC) mouse models, cell lines and clinical specimens, specifically via HMGB1-mediated dendritic cell (DC) maturation and migration, further contributes to the infiltration of CD4+ and CD8+ T cells and inflammatory responses. Under stress conditions, MSI2 can exacerbate the production, nucleocytoplasmic transport and extracellular release of damage-associated molecular patterns (DAMPs)-HMGB1 in CRC cells. Mechanistically, MSI2 mainly enhances the disulfide HMGB1 production and protein translation via direct binding to nucleotides 1403-1409 in the HMGB1 3' UTR, and interacts with the cytoplasmic acetyltransferase P300 to upregulate its expression, further promoting the acetylation of K29 residue in HMGB1, thus leading to K29-HMGB1 nucleocytoplasmic translocation and extracellular release. Furthermore, blocking HMGB1 activity with glycyrrhizic acid (Gly) attenuates MSI2-mediated immunopathology and immune infiltration in CRC in vitro and in vivo. Collectively, this study suggests that MSI2 may improve the prognosis of CRC patients by reprogramming the tumor immune microenvironment (TIME) through HMGB1-mediated PTMs, which might be a novel therapeutic option for CRC immunotherapy.

Ultrasensitive electrochemical sensor based on synergistic effect of Ag@MXene and antifouling cyclic multifunctional peptide for PD-L1 detection in serum.

A sensing interface co-constructed from the two-dimensional conductive material (Ag@MXene) and an antifouling cyclic multifunctional peptide (CP) is described. While the large surface area of Ag@MXene loads more CP probes, CP binds to Ag@MXene to form a fouling barrier and ensure the structural rigidity of the targeting sequence. This strategy synergistically enhances the biosensor's sensitivity and resistance to contamination. The SPR results showed that the binding affinity of the CP to the target was 6.23 times higher than that of the antifouling straight-chain multifunctional peptide (SP) to the target. In the 10 mg/mL BSA electrochemical fouling test, the fouling resistance of Ag@MXene + CP (composite sensing interface of CP combined with Ag@MXene) was 30 times higher than that of the bare electrode. The designed electrochemical sensor exhibited good selectivity and wide dynamic response range at PD-L1 concentrations from 0.1 to 50 ng/mL. The lowest detection limit was 24.54 pg/mL (S/N = 3). Antifouling 2D materials with a substantial specific surface area, coupled with non-straight chain antifouling multifunctional peptides, offer a wide scope for investigating the sensitivity and antifouling properties of electrochemical sensors.

Musashi-2 Deficiency Triggers Colorectal Cancer Ferroptosis by Downregulating the MAPK Signaling Cascade to Inhibit HSPB1 Phosphorylation.

BACKGROUND: Musashi-2 (MSI2) is a critical RNA-binding protein (RBP) whose ectopic expression drives the pathogenesis of various cancers. Accumulating evidence suggests that inducing ferroptosis of tumor cells can inhibit their malignant biological behavior as a promising therapeutic approach. However, it is unclear whether MSI2 regulates cell death in colorectal cancer (CRC), especially the underlying mechanisms and biological effects in CRC ferroptosis remain elusive. METHODS: Experimental methods including qRT‒PCR, immunofluorescence, flow cytometry, western blot, co-immunoprecipitation, CCK-8, colony formation assay, in vitro cell transwell migration and invasion assays, in vivo xenograft tumor experiments, liver and lung CRC metastasis models, CAC mice models, transmission electron microscopy, immunohistochemistry, histopathology, 4D label-free proteomics sequencing, bioinformatic and database analysis were used in this study. RESULTS: Here, we investigated that MSI2 was upregulated in CRC and positively correlated with ferroptosis inhibitor molecules. MSI2 deficiency suppressed CRC malignancy by inhibiting cell proliferation, viability, migration and invasion in vitro and in vivo; and MSI2 deficiency triggered CRC ferroptosis by changing the intracellular redox state (ROS levels and lipid peroxidation), erastin induced cell mortality and viability, iron homeostasis (intracellular total irons and ferrous irons), reduced glutathione (GSH) levels and mitochondrial injury. Mechanistically, through 4D-lable free proteomics analysis on SW620 stable cell lines, we demonstrated that MSI2 directly interacted with p-ERK and MSI2 knockdown downregulated the p-ERK/p38/MAPK axis signaling pathway, which further repressed MAPKAPK2 and HPSB1 phosphorylation, leading to decreased expression of PCNA and Ki67 and increased expression of ACSL4 in cancer cells. Furthermore, HSPB1 could rescue the phenotypes of MSI2 deficiency on CRC ferroptosis in vitro and in vivo. CONCLUSIONS: This study indicates that MSI2 deficiency suppresses the growth and survival of CRC cells and promotes ferroptosis by inactivating the MAPK signaling pathway to inhibit HSPB1 phosphorylation, which leads to downregulation of PCNA and Ki67 and upregulation of ACSL4 in cancer cells and subsequently induces redox imbalance, iron accumulation and mitochondrial shrinkage, ultimately triggering ferroptosis. Therefore, targeted inhibition of MSI2/MAPK/HSPB1 axis to promote ferroptosis might be a potential treatment strategy for CRC.

Lysosome activation in peripheral blood mononuclear cells and prognostic significance of circulating LC3B in COVID-19.

Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide, causing significant mortality. There is a mechanistic relationship between intracellular coronavirus replication and deregulated autophagosome-lysosome system. We performed transcriptome analysis of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients and identified the aberrant upregulation of genes in the lysosome pathway. We further determined the capability of two circulating markers, namely microtubule-associated proteins 1A/1B light chain 3B (LC3B) and (p62/SQSTM1) p62, both of which depend on lysosome for degradation, in predicting the emergence of moderate-to-severe disease in COVID-19 patients requiring hospitalization for supplemental oxygen therapy. Logistic regression analyses showed that LC3B was associated with moderate-to-severe COVID-19, independent of age, sex and clinical risk score. A decrease in LC3B concentration <5.5 ng/ml increased the risk of oxygen and ventilatory requirement (adjusted odds ratio: 4.6; 95% CI: 1.1-22.0; P = 0.04). Serum concentrations of p62 in the moderate-to-severe group were significantly lower in patients aged 50 or below. In conclusion, lysosome function is deregulated in PBMCs isolated from COVID-19 patients, and the related biomarker LC3B may serve as a novel tool for stratifying patients with moderate-to-severe COVID-19 from those with asymptomatic or mild disease. COVID-19 patients with a decrease in LC3B concentration <5.5 ng/ml will require early hospital admission for supplemental oxygen therapy and other respiratory support.

Perspectives and trends in advanced optical and electrochemical biosensors based on engineered peptides.

With the advancement of life medicine, in vitro diagnostics (IVD) technology has become an auxiliary tool for early diagnosis of diseases. However, biosensors for IVD now face some disadvantages such as poor targeting, significant antifouling properties, low density of recognized molecules, and poor stability. In recent years, peptides have been demonstrated to have various functions in unnatural biological systems, such as targeting properties, antifouling properties, and self-assembly properties, which indicates that peptides can be engineered. These properties of peptides, combined with their good biocompatibility, can be well applied to the design of biosensors to solve the problems mentioned above. This review provides an overview of the properties of engineered functional peptides and their applications in enhancing biosensor performance, mainly in the field of optics and electrochemistry.

Aptamer-Assisted Protein Orientation on Silver Magnetic Nanoparticles: Application to Sensitive Leukocyte Cell-Derived Chemotaxin 2 Surface Plasmon Resonance Sensors.

Leukocyte cell-derived chemotaxin 2 (LECT2) has been proved to be a potential biomarker for the diagnosis of liver fibrosis. In this work, a sensitive surface plasmon resonance (SPR) assay for LECT2 analysis was developed. Tyrosine kinase with immune globulin-like and epidermal growth factor-like domains 1 (Tie1) is an orphan receptor of LECT2 with a C-terminal Fc tag, which is far away from the LECT2 binding sites. The Fc aptamer was intentionally used to capture the Tie1 through its Fc tag, connecting with Fe3O4-coated silver magnetic nanoparticles (Ag@MNPs) and ensuring the LECT2 binding site to be outward. Attributed to the orientation nature of the captured protein, Ag@MNPs were able to enhance the SPR signal. A sensitive LECT2 sensor was successfully fabricated with a detection limit of 10.93 pg/mL. The results showed that the immobilization method improved the binding efficiency of Tie1 protein. This strategy could be extended to attach antibodies or recombinant Fc label proteins to Fc aptamer-based nanoparticles.

A Highly Accurate Positioning Solution for C-V2X Systems.

Cellular vehicle-to-everything (C-V2X) is essential in enabling safe, reliable, and efficient transportation services. It serves as serve as the foundation for vehicles to communicate with each other and everything around them. One fundamental element in C-V2X is positioning, namely extracting the vehicle's absolute and relative positions concerning other objects such as buildings, pedestrians, traffic signs, and other vehicles. However, its feasibility in enabling vehicular positioning has not been fully explored yet. In this paper, key performance indicators (KPIs) for C-V2X positioning have been described firstly. Then positioning challenges and conventional positioning methods for C-V2X are reviewed. Afterward, two user equipment (UE)-based and UE-assisted C-V2X positioning architectures are proposed, and key technologies are also described. Lastly, testing and typical application cases are provided.

Inhibition of S-Adenosylhomocysteine Hydrolase Induces Endothelial Dysfunction via Epigenetic Regulation of p66shc-Mediated Oxidative Stress Pathway.

BACKGROUND: Elevated levels of S-adenosylhomocysteine (SAH), the precursor of homocysteine, are positively associated with the risk of cardiovascular disease and with the development and progression of atherosclerosis. However, the role of SAH in endothelial dysfunction is unclear. METHODS: Apolipoprotein E-deficient ( apoE-/-) mice received dietary supplementation with the SAH hydrolase (SAHH) inhibitor adenosine dialdehyde or were intravenously injected with a retrovirus expressing SAHH shRNA. These 2 approaches, along with the heterozygous SAHH gene knockout ( SAHH+/-) mouse model, were used to elevate plasma SAH levels and to examine the role of SAH in aortic endothelial dysfunction. The relationship between plasma SAH levels and endothelial dysfunction was also investigated in human patients with coronary artery disease and healthy control subjects. RESULTS: Plasma SAH levels were increased in SAHH+/- mice and in apoE-/- mice after dietary administration of adenosine dialdehyde or intravenous injection with SAHH shRNA. SAHH+/- mice or apoE-/- mice with SAHH inhibition showed impaired endothelium-dependent vascular relaxation and decreased nitric oxide bioavailability after treatment with acetylcholine; this was completely abolished by the administration of the endothelial nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester. Furthermore, SAHH inhibition induced production of reactive oxygen species and p66shc expression in the mouse aorta and human aortic endothelial cells. Antioxidants and p66shc siRNA prevented SAHH inhibition-induced generation of reactive oxygen species and attenuated the impaired endothelial vasomotor responses in high-SAH mice. Moreover, inhibition of SAHH induced hypomethylation in the p66shc gene promoter and inhibited expression of DNA methyltransferase 1. Overexpression of DNA methyltransferase 1, induced by transduction of an adenovirus, was sufficient to abrogate SAHH inhibition-induced upregulation of p66shc expression. Finally, plasma SAH levels were inversely associated with flow-mediated dilation and hypomethylation of the p66shc gene promoter and positively associated with oxidative stress levels in patients with coronary artery disease and healthy control subjects. CONCLUSIONS: Our findings indicate that inhibition of SAHH results in elevated plasma SAH levels and induces endothelial dysfunction via epigenetic upregulation of the p66shc-mediated oxidative stress pathway. Our study provides novel molecular insight into mechanisms of SAH-associated endothelial injury that may contribute to the development of atherosclerosis. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT03345927.

Live birth after cervical ectopic uterus transplantation in mice.

An ideal animal model is a prerequisite for the basic research of uterus transplantation. This study aimed to develop a new cervical ectopic uterus transplantation mice model, which was established by vascular anastomosis of the right common iliac artery and vein of the donor with the right common carotid artery and external jugular vein of the recipient, respectively, using the cuff method. The survival status of the transplanted uterus was assessed by macroscopic observation and histological examination after surgery, and the function of the graft uterus was tested by verifying whether the pregnancy is possible. A total of 40 transplants were performed, of which only 1 failed due to donor hemorrhage. After 26 transplants, the total operation time reduced to 52.4 ± 3.8 minutes, of which the total ischemia time took 6.6 ± 1.1 minutes. Sixty days after transplantation, all the graft uteri had a good blood supply and spontaneous contraction. The histology showed no significant difference between the transplanted uterus and the native. Embryo transfer experiments have proven that the transplanted uterus has uterine function. In conclusion, this new model is an effective and simple mice model for the studies of the scientific issues related to uterus transplantation.

Combination of N, N'-dicyclohexyl-N-arachidonic acylurea and tacrolimus prolongs cardiac allograft survival in mice.

Current immunosuppressive agents for organ transplantation are not ideal because of their strong toxicity and adverse effects. Hence, there is an urgent need to develop novel immunosuppressive agents. The compound N, N'-dicyclohexyl-N-arachidonic acylurea (DCAAA) is a novel highly unsaturated fatty acid from the traditional Chinese medicinal plant Radix Isatidis. In this study, we systematically investigated the toxicity, immunosuppressive effect and mechanisms underlying the activity of DCAAA. The toxicity tests showed that DCAAA treatment did not lead to red blood cell hemolysis and did not affect the liver and kidney functions in mice. The lymphocyte transformation test showed that DCAAA treatment inhibited lymphocyte proliferation in a dose-dependent manner. An in vivo cardiac allotransplantation experiment showed that DCAAA treatment could suppress the immune rejection and significantly prolong the survival of cardiac allografts in recipient mice by reducing the proportion of CD4+ T cells in the spleen and grafts, concentration of interferon-γ in the supernatant and serum and infiltration of inflammatory cells into the grafts. Moreover, a combination treatment with DCAAA and tacrolimus had a synergistic effect in preventing acute rejection of heart transplants. In vitro molecular biology experiments showed that DCAAA treatment inhibited activation of the T-cell receptor-mediated phosphoinostide 3-kinase-protein kinase B pathway, thereby arresting cell cycle transition from the G1 to the S phase, and inhibiting lymphocyte proliferation. Overall, our study reveals a novel, low-toxicity immunosuppressive agent that has the potential to reduce the toxic side effects of existing immunosuppressive agents when used in combination with them.

Arsenic trioxide ameliorates experimental autoimmune encephalomyelitis in C57BL/6 mice by inducing CD4+ T cell apoptosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system characterized by severe white matter demyelination. Because of its complex pathogenesis, there is no definite cure for MS. Experimental autoimmune encephalomyelitis (EAE) is an ideal animal model for the study of MS. Arsenic trioxide (ATO) is an ancient Chinese medicine used for its therapeutic properties with several autoimmune diseases. It is also used to inhibit acute immune rejection due to its anti-inflammatory and immunosuppressive properties. However, it is unclear whether ATO has a therapeutic effect on EAE, and the underlying mechanisms have not yet been clearly elucidated. In this study, we attempted to assess whether ATO could be used to ameliorate EAE in mice. METHODS: ATO (0.5 mg/kg/day) was administered intraperitoneally to EAE mice 10 days post-immunization for 8 days. On day 22 post-immunization, the spinal cord, spleen, and blood were collected to analyze demyelination, inflammation, microglia activation, and the proportion of CD4+ T cells. In vitro, for mechanistic studies, CD4+ T cells were sorted from the spleen of naïve C57BL/6 mice and treated with ATO and then used for an apoptosis assay, JC-1 staining, imaging under a transmission electron microscope, and western blotting. RESULTS: ATO delayed the onset of EAE and alleviated the severity of EAE in mice. Treatment with ATO also attenuated demyelination, alleviated inflammation, reduced microglia activation, and decreased the expression levels of IL-2, IFN-γ, IL-1ß, IL-6, and TNF-α in EAE mice. Moreover, the number and proportion of CD4+ T cells in the spinal cord, spleen, and peripheral blood were reduced in ATO-treated EAE mice. Finally, ATO induced CD4+ T cell apoptosis via the mitochondrial pathway both in vitro and in vivo. Additionally, the administration of ATO had no adverse effect on the heart, liver, or kidney function, nor did it induce apoptosis in the spinal cord. CONCLUSIONS: Overall, our findings indicated that ATO plays a protective role in the initiation and progression of EAE and has the potential to be a novel drug in the treatment of MS.

Associations between dietary patterns and the risk of breast cancer: a systematic review and meta-analysis of observational studies.

BACKGROUND: Epidemiologic evidence suggests that certain dietary patterns were associated with breast cancer risk, but the results have been inconclusive. We assessed the associations between different dietary patterns and the risk of breast cancer by conducting a meta-analysis of observational studies. METHODS: Relevant articles were searched in PubMed, Embase, and Cochrane library databases through September 2017. Multivariable-adjusted relative risks (RRs) and 95% confidence intervals (CIs) comparing the highest and lowest categories of Western and prudent dietary patterns were combined by using the random-effects meta-analyses. RESULTS: We identified 32 eligible articles including 14 cohort and 18 case-control studies (34 Western and 35 prudent studies). The pooled analyses found that a Western dietary pattern was associated with a 14% increased risk (RR 1.14, 95% CI 1.02, 1.28), whereas a prudent dietary pattern was associated with an 18% reduced risk of breast cancer (RR 0.82, 95% CI 0.75, 0.89). In addition, sub-group analyses showed that the positive association between a Western dietary pattern and breast cancer risk was significant among postmenopausal (RR 1.20, 95% CI 1.06, 1.35), but not premenopausal women (RR 1.18, 95% CI 0.99, 1.40), and significant for hormone receptor-positive tumors (RR 1.18, 95% CI 1.04, 1.33), but not receptor-negative tumors (RR 0.97, 95% CI 0.83, 1.12). In contrast, the inverse association between a prudent dietary pattern and breast cancer was significant in premenopausal (RR 0.77, 95% CI 0.61, 0.98), but not postmenopausal women (RR 0.88, 95% CI 0.74, 1.03), and significant for both hormone receptor-positive and receptor-negative tumors. CONCLUSIONS: The results of the current meta-analysis suggest a possible increased risk of breast cancer associated with a Western dietary pattern and a reduced risk with a prudent dietary pattern. Large-scale cohort studies with a high quality need to be conducted to further confirm the findings of the current meta-analysis. As dietary patterns are modifiable, these findings may provide viable strategies for breast cancer prevention through changes in dietary intake.

Emerging erm(B)-Mediated Macrolide Resistance Associated with Novel Multidrug Resistance Genomic Islands in Campylobacter.

The rapid dissemination of the macrolide resistance gene erm(B) will likely compromise the efficacy of macrolides as the treatment of choice for campylobacteriosis. More importantly, erm(B) is always associated with several multidrug resistance genomic islands (MDRGIs), which confer resistance to multiple other antimicrobials. Continuous monitoring of the emergence of erm(B) and analysis of its associated genetic environments are crucial for our understanding of macrolide resistance in Campylobacter In this study, 290 Campylobacter isolates (216 Campylobacter coli isolates and 74 Campylobacter jejuni isolates) were obtained from 1,039 fecal samples collected in 2016 from pigs and chickens from three regions of China (344 samples from Guangdong, 335 samples from Shanghai, and 360 samples from Shandong). Overall, 74 isolates (72 C. coli isolates and 2 C. jejuni isolates) were PCR positive for erm(B). Combined with data from previous years, we observed a trend of increasing prevalence of erm(B) in C. coli Pulsed-field gel electrophoresis analyses suggested that both clonal expansion and horizontal transmission were involved in the dissemination of erm(B) in C. coli, and three novel types of erm(B)-associated MDRGIs were identified among the isolates. Furthermore, 2 erm(B)-harboring C. jejuni isolates also contained an aminoglycoside resistance genomic island and a multidrug-resistance-enhancing efflux pump, encoded by RE-cmeABC Antimicrobial susceptibility testing showed that most of the isolates were resistant to all clinically important antimicrobial agents used for the treatment of campylobacteriosis. These findings suggest that the increasing prevalence of erm(B)-associated MDRGIs might further limit treatment options for campylobacteriosis.

Generation of bioartificial hearts using decellularized scaffolds and mixed cells.

BACKGROUND: Patients with end-stage heart failure must receive treatment to recover cardiac function, and the current primary therapy, heart transplantation, is plagued by the limited supply of donor hearts. Bioengineered artificial hearts generated by seeding of cells on decellularized scaffolds have been suggested as an alternative source for transplantation. This study aimed to develop a tissue-engineered heart with lower immunogenicity and functional similarity to a physiological heart that can be used for heart transplantation. MATERIALS AND METHODS: We used sodium dodecyl sulfate (SDS) to decellularize cardiac tissue to obtain a decellularized scaffold. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and identified by flow cytometric labeling of their surface markers. At the same time, the multi-directional differentiation of MSCs was analyzed. The MSCs, endothelial cells, and cardiomyocytes were allowed to adhere to the decellularized scaffold during perfusion, and the function of tissue-engineered heart was analyzed by immunohistochemistry and electrocardiogram. RESULTS: MSCs, isolated from rats differentiated into cardiomyocytes, were seeded along with primary rat cardiomyocytes and endothelial cells onto decellularized rat heart scaffolds. We first confirmed the pluripotency of the MSCs, performed immunostaining against cardiac markers expressed by MSC-derived cardiomyocytes, and completed surface antigen profiling of MSC-derived endothelial cells. After cell seeding and culture, we analyzed the performance of the bioartificial heart by electrocardiography but found that the bioartificial heart exhibited abnormal electrical activity. The results indicated that the tissue-engineered heart lacked some cells necessary for the conduction of electrical current, causing deficient conduction function compared to the normal heart. CONCLUSION: Our study suggests that MSCs derived from rats may be useful in the generation of a bioartificial heart, although technical challenges remain with regard to generating a fully functional bioartificial heart.

Outline of the biosynthesis and regulation of ergosterol in yeast.

Sterols are crucial functional components for eukaryotic cell membrane. Due to versatile activities, sterols show wide applications in food and pharmaceutical industries. Ergosterol not only reflects cell growth but also serves as the precursor for manufacturing steroid drugs. To date, the ergosterol biosynthetic pathway in yeast has been reported, and the industrial production of ergosterol is achieved by yeast fermentation or extraction from fungal mycelia. Here, we summarize its biosynthesis, regulation, transportation, and subcellular location of enzymes in yeast. In particular, we review the regulation of ergosterol biosynthesis at transcriptional, translational and post-translational levels. Furthermore, we advocate metabolic engineering and fermentation strategies for high-level production of ergosterol. This study may provide evaluable insights into metabolic engineering of yeast for scaled-up fermentation production of ergosterol or beyond.

Human iPSC-MSC-Derived Xenografts Modulate Immune Responses by Inhibiting the Cleavage of Caspases.

Mesenchymal stem cells (MSCs) negatively modulate immune properties. Induced pluripotent stem cells (iPSCs)-derived MSCs are alternative source of MSCs. However, the effects of iPSC-MSCs on T cells phenotypes in vivo remain unclear. We established an iPSC-MSC-transplanted host versus graft reaction mouse model using subcapsular kidney injection. Th1, Th2, regulatory T cells (Treg), and Th17 phenotypes and their cytokines were investigated in vivo and in vitro. The role of caspases and the soluble factors involved in the effects of MSCs were examined. We found that iPSC-MSC grafts led to more cell survival and less infiltration of inflammatory cells in mice. iPSC-MSC transplantation inhibited T cell proliferation, decreased Th1 and Th2 phenotypes and cytokines, upregulated Th17 and Treg subsets. Moreover, iPSC-MSCs inhibited the cleavage of caspases 3 and 8 and inhibition of caspases downregulated Th1, Th2 responses and upregulated Th17, Treg responses. Soluble factors were determined using protein array and TGF-ß1/2/3, IL-10, and MCP-1 were found to be highly expressed in iPSC-MSCs. The administration of the soluble factors decreased Th1/2 response, upregulated Treg response and inhibited the cleavage of caspases. Our results demonstrate that iPSC-MSCs regulate T cell responses as a result of a combined action of the above soluble factors secreted by iPSC-MSCs. These factors suppress T cell responses by inhibiting the cleavage of caspases. These data provide a novel immunomodulatory mechanism for the underlying iPSC-MSC-based immunomodulatory effects on T cell responses. Stem Cells 2017;35:1719-1732.

A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation.

Xenotransplantation is an effective way to solve the problem of donor shortage in clinical transplantation. However, clinical use of xenotransplantation is currently limited due to immunological challenges such as acute vascular rejection and cell-mediated rejection. To finally surpass this immunological barrier, more preclinical research is needed into the molecular mechanisms of rejection and the possible effects of new immunosuppressants. Our aim was to create a refined, highly reproducible protocol to establish the most suitable rat-to-mouse heterotopic heart transplantation model using the cuff technique.

Preparation and characterization of small-diameter decellularized scaffolds for vascular tissue engineering in an animal model.

BACKGROUND: The development of a suitable extracellular matrix (ECM) scaffold is the first step in vascular tissue engineering (VTE). Synthetic vascular grafts are available as an alternative to autologous vessels in large-diameter arteries (>8 mm) and medium-diameter arteries (6-8 mm). In small-diameter vessels (<6 mm), synthetic vascular grafts are of limited use due to poor patency rates. Compared with a vascular prosthesis, natural tissue ECM has valuable advantages. Despite considerable progress in recent years, identifying an optimal protocol to create a scaffold for use in small-diameter (<6 mm) fully natural tissue-engineered vascular grafts (TEVG), remains elusive. Although reports on different decellularization techniques have been numerous, combination of and comparison between these methods are scarce; therefore, we have compared five different decellularization protocols for making small-diameter (<6 mm) ECM scaffolds and evaluated their characteristics relative to those of fresh vascular controls. RESULTS: The protocols differed in the choice of enzymatic digestion solvent, the use of non-ionic detergent, the durations of the individual steps, and UV crosslinking. Due to their small diameter and ready availability, rabbit arteria carotis were used as the source of the ECM scaffolds. The scaffolds were subcutaneously implanted in rats and the results were evaluated using various microscopy and immunostaining techniques. CONCLUSIONS: Our findings showed that a 2 h digestion time with 1× EDTA, replacing non-ionic detergent with double-distilled water for rinsing and the application of UV crosslinking gave rise to an ECM scaffold with the highest biocompatibility, lowest cytotoxicity and best mechanical properties for use in vivo or in situ pre-clinical research in VTE in comparison.