Prognostic relevance of ventricular arrhythmias in surgical patients with gastrointestinal tumors.

BACKGROUND: Individuals diagnosed with gastrointestinal tumors are at an increased risk of developing cardiovascular diseases. Among which, ventricular arrhythmia is a prevalent clinical concern. This suggests that ventricular arrhythmias may have predictive value in the prognosis of patients with gastrointestinal tumors. AIM: To explore the prognostic value of ventricular arrhythmias in patients with gastrointestinal tumors receiving surgery. METHODS: We retrospectively analyzed data from 130 patients undergoing gastrointestinal tumor resection. These patients were evaluated by a 24-h ambulatory electrocardiogram (ECG) at the Sixth Affiliated Hospital of Sun Yat-sen University from January 2018 to June 2020. Additionally, 41 general healthy age-matched and sex-matched controls were included. Patients were categorized into survival and non-survival groups. The primary endpoint was all-cause mortality, and secondary endpoints included major adverse cardiovascular events (MACEs). RESULTS: Colorectal tumors comprised 90% of cases. Preoperative ambulatory ECG monitoring revealed that among the 130 patients with gastrointestinal tumors, 100 (76.92%) exhibited varying degrees of premature ventricular contractions (PVCs). Ten patients (7.69%) manifested non-sustained ventricular tachycardia (NSVT). The patients with gastrointestinal tumors exhibited higher PVCs compared to the healthy controls on both conventional ECG [27 (21.3) vs 1 (2.5), P = 0.012] and 24-h ambulatory ECG [14 (1.0, 405) vs 1 (0, 6.5), P < 0.001]. Non-survivors had a higher PVC count than survivors [150.50 (7.25, 1690.50) vs 9 (0, 229.25), P = 0.020]. During the follow-up period, 24 patients died and 11 patients experienced MACEs. Univariate analysis linked PVC > 35/24 h to all-cause mortality, and NSVT was associated with MACE. However, neither PVC burden nor NSVT independently predicted outcomes according to multivariate analysis. CONCLUSION: Patients with gastrointestinal tumors exhibited elevated PVCs. PVCs > 35/24 h and NSVT detected by 24-h ambulatory ECG were prognostically significant but were not found to be independent predictors.

CCDC50 promotes tumor growth through regulation of lysosome homeostasis.

The maintenance of lysosome homeostasis is crucial for cell growth. Lysosome-dependent degradation and metabolism sustain tumor cell survival. Here, we demonstrate that CCDC50 serves as a lysophagy receptor, promoting tumor progression and invasion by controlling lysosomal integrity and renewal. CCDC50 monitors lysosomal damage, recognizes galectin-3 and K63-linked polyubiquitination on damaged lysosomes, and specifically targets them for autophagy-dependent degradation. CCDC50 deficiency causes the accumulation of ruptured lysosomes, impaired autophagic flux, and superfluous reactive oxygen species, consequently leading to cell death and tumor suppression. CCDC50 expression is associated with malignancy, progression to metastasis, and poor overall survival in human melanoma. Targeting CCDC50 suppresses tumor growth and lung metastasis, and enhances the effect of BRAFV600E inhibition. Thus, we demonstrate critical roles of CCDC50-mediated clearance of damaged lysosomes in supporting tumor growth, hereby identifying a potential therapeutic target of melanoma.

Myocardial injury before noncardiac surgery.

Non-cardiac surgical procedures present a significant circulatory stress and can potentially trigger cardiovascular events, such as myocardial infarction and heart failure. Myocardial injury before non-cardiac surgery is associated with an increased risk of mortality and major cardiovascular complications during perioperative period, as well as up to 5 years after non-cardiac surgery. While the definition of preoperative myocardial injury is not yet clear, it is generally understood as myocardial injury resulting from various causes of troponin elevation without acute coronary syndrome prior to surgery. Detecting preoperative myocardial injury through routine troponin monitoring is crucial for reducing perioperative risk, but it is also challenging. The aim of this review is to discuss the definition of preoperative myocardial injury, its pathophysiology, implications on clinical practice and decision-making for patients with elevated troponin levels before non-cardiac surgery.

The association of preoperative high-sensitivity cardiac troponin i and long-term outcomes in colorectal cancer patients received tumor resection surgery.

BACKGROUND: This study aimed to evaluate the association between preoperative hs-cTnI and long-term mortality and major adverse cardiovascular events (MACE) in colorectal cancer patients. METHODS: This single-center retrospective cohort study included 1105 consecutive colorectal cancer patients who received tumor resection surgery between January 2018 and June 2020. Inclusion criteria were an age ≥ 18 years and had been tested for hs-cTnI on admission within 7 days prior to tumor resection surgery. Exclusion criteria were emergent surgery, failure to received tumor resection surgery, hospital death, there was clinical evidence of unstable coronary artery disease or pulmonary embolism occurred before operation according to medical record. The primary endpoint was all-cause death. Secondary endpoint was major adverse cardiovascular events (MACE). RESULTS: A total of 1105 patients were enrolled: 1032 with normal hs-cTnI and 73 with elevated hs-cTnI. The mean follow-up was 24.4 ± 10.8 months, 176 patients died and 39 patients met MACE. In the elevated troponin group, 50%, 32.1% and 17.9% died from cancer, cardiovascular and other causes, while those in the normal troponin group were 75.7%, 2% and 22.3%, there was statistical difference between 2 groups (P < 0.001). Patients with elevated preoperative hs-cTnI had significantly higher mortality (P < 0.001) and more MACE (P < 0.001) compared with those with normal hs-cTnI. A propensity-matching analysis were performed, resulting in 151 patients with normal hs-cTnI and 60 patients with elevated hs-cTnI. The matched population had the similar results for all-cause death (P = 0.009) and MACE (P = 0.001). The results were consistent after further excluding 147 patients who had received chemoradiotherapy prior to surgery in subgroup analysis. The results of multivariate Cox regression analysis shown that hs-cTnI was one of the best predictors for all-cause death (hazard ratio [HR] 2.278; 95% confidence interval [CI] 1.19-4.361) and MACE (HR, 3.523; 95%CI, 1.477-8.403) in total populations, similar results were found in subgroup analysis. CONCLUSIONS: Colorectal cancer patients without myocardial ischemia manifestation but with elevated hs-cTnI prior to tumor resection surgery were at increased risk for long-term all-cause death and MACE, irrespective of whether they have received chemoradiotherapy prior to surgery.

Risk factors of preoperative myocardial injury in patients with gastrointestinal tumors.

BACKGROUND: Recent studies indicated that the prognosis of patients with gastrointestinal tumors is frequently influenced by its complications, notably myocardial injury. The main object is to investigate the occurrence and risk factors of myocardial injury in patients with gastrointestinal tumor. METHODS: 1126 patients who received gastrointestinal tumor related surgery from May 2018 to June 2020 in the Sixth Affiliated Hospital of Sun Yat-sen University were retrospectively collected and divided into the non-myocardial injury group and the myocardial injury group (high-sensitive cardiac troponin I (hs-cTnI) ≥ 0.028 ng/ml). The occurrence and risk factors of myocardial injury in patients with gastrointestinal tumor are analyzed. The influence of myocardial injury on the ICU detention time in gastrointestinal tumor patients is also studied. RESULTS: In total, 78 (6.93%) patients developed myocardial injuries. Compared with patients in the non-myocardial injury group, patients in the myocardial injury group have a higher prevalence of cardiovascular risk factors (including advanced age and higher smoking ratio), a higher prevalence of comorbidities (such as previous coronary artery disease, hypertension, atrium fibrillation and diabetes), and a higher rate of premedication (such as anticoagulation, ß-blocker, Angiotensin-converting enzyme inhibitor/Angiotensin II receptor blocker, and diuretic) (all with P-value < 0.05). In addition, patients in the myocardial injury group also presented with a higher revised cardiac risk index (Lee index), higher neutrophil granulocyte ratio, lower hemoglobin, and higher likelihood of impaired cardiac structure and function (all with P-value < 0.05). There was a trend of statistical significance in the ICU detention time between the myocardial injury group and the non-myocardial injury group (1[1,3] vs. 2[1,10], P = 0.064). In this study, there were 7 patients presented with clinical symptoms in the myocardial injury group (chest discomfort in 4 cases, non-compressive precordial chest pain in 1 case, dyspnea in 2 cases). In the multivariate analysis, advanced age, increased Lee index score, increased neutrophil granulocyte ratio, decreased left ventricular ejection fraction (LVEF), increased interventricular septum were independent risk factors for myocardial injury. CONCLUSION: In conclusion, advanced age, increased Lee index, increased neutrophil granulocyte ratio, decreased left ventricular ejection fraction, and increased ventricular septum were independent risk factors for preoperative myocardial injury in patients with gastrointestinal tumors. The proportion of clinical symptoms in gastrointestinal tumor patients with myocardial injury was low, indicating the necessity to closely monitor the cardiac status of individuals with gastrointestinal tumors.

Interleukin-18-primed human umbilical cord-mesenchymal stem cells achieve superior therapeutic efficacy for severe viral pneumonia via enhancing T-cell immunosuppression.

Coronavirus disease 2019 (COVID-19) treatments are still urgently needed for critically and severely ill patients. Human umbilical cord-mesenchymal stem cells (hUC-MSCs) infusion has therapeutic benefits in COVID-19 patients; however, uncertain therapeutic efficacy has been reported in severe patients. In this study, we selected an appropriate cytokine, IL-18, based on the special cytokine expression profile in severe pneumonia of mice induced by H1N1virus to prime hUC-MSCs in vitro and improve the therapeutic effect of hUC-MSCs in vivo. In vitro, we demonstrated that IL-18-primed hUC-MSCs (IL18-hUCMSC) have higher proliferative ability than non-primed hUC-MSCs (hUCMSCcon). In addition, VCAM-1, MMP-1, TGF-ß1, and some chemokines (CCL2 and CXCL12 cytokines) are more highly expressed in IL18-hUCMSCs. We found that IL18-hUCMSC significantly enhanced the immunosuppressive effect on CD3+ T-cells. In vivo, we demonstrated that IL18-hUCMSC infusion could reduce the body weight loss caused by a viral infection and significantly improve the survival rate. Of note, IL18-hUCMSC can also significantly attenuate certain clinical symptoms, including reduced activity, ruffled fur, hunched backs, and lung injuries. Pathologically, IL18-hUCMSC transplantation significantly enhanced the inhibition of inflammation, viral load, fibrosis, and cell apoptosis in acute lung injuries. Notably, IL18-hUCMSC treatment has a superior inhibitory effect on T-cell exudation and proinflammatory cytokine secretion in bronchoalveolar lavage fluid (BALF). Altogether, IL-18 is a promising cytokine that can prime hUC-MSCs to improve the efficacy of precision therapy against viral-induced pneumonia, such as COVID-19.

Establishment of a lysosome-related prognostic signature in breast cancer to predict immune infiltration and therapy response.

Background: Lysosomes are instrumental in intracellular degradation and recycling, with their functional alterations holding significance in tumor growth. Nevertheless, the precise role of lysosome-related genes (LRGs) in breast cancer (BC) remains elucidated. This study aimed to establish a prognostic model for BC based on LRGs. Methods: Employing The Cancer Genome Atlas (TCGA) BC cohort as a training dataset, this study identified differentially expressed lysosome-related genes (DLRGs) through intersecting LRGs with differential expression genes (DEGs) between tumor and normal samples. A prognostic model of BC was subsequently developed using Cox regression analysis and validated within two Gene Expression Omnibus (GEO) external validation sets. Further analyses explored functional pathways, the immune microenvironment, immunotherapeutic responses, and sensitivity to chemotherapeutic drugs in different risk groups. Additionally, the mRNA and protein expression levels of genes within the risk model were examined by utilizing the Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) databases. Clinical tissue specimens obtained from patients were gathered to validate the expression of the model genes via Real-Time Polymerase Chain Reaction (RT-PCR). Results: We developed a risk model of BC based on five specific genes (ATP6AP1, SLC7A5, EPDR1, SDC1, and PIGR). The model was validated for overall survival (OS) in two GEO validation sets (p=0.00034 for GSE20685 and p=0.0095 for GSE58812). In addition, the nomogram incorporating clinical factors showed better predictive performance. Compared to the low-risk group, the high-risk group had a higher level of certain immune cell infiltration, including regulatory T cells (Tregs) and type 2 T helper cells (Th2). The high-risk patients appeared to respond less well to general immunotherapy and chemotherapeutic drugs, according to the Tumor Immune Dysfunction and Exclusion (TIDE), Immunophenotype Score (IPS), and drug sensitivity scores. The RT-PCR results validated the expression trends of some prognostic-related genes in agreement with the previous differential expression analysis. Conclusion: Our innovative lysosome-associated signature can predict the prognosis for BC patients, offering insights for guiding subsequent immunotherapeutic and chemotherapeutic interventions. Furthermore, it has the potential to provide a scientific foundation for identifying prospective therapeutic targets.

[Effect of Special Structure Bi-Specific Chimeric Antigen Receptor T Cell on Tumor Cells].

OBJECTIVE: To explore and design a novel bi-specific chimeric antigen receptor (CAR) structure. To obtain the corresponding CAR-T cells and verify killing effects on tumor cells in vitro and in vivo. METHODS: Five kinds of bi-specific CAR structures including humanized CD19 scFv and CD79b scFv, CD8 hinge & TM-4-1BB-CD3ζ and/or CD3ε chain intracellular regions were constructed and prepared. CAR-19-79b cells were obtained. Five kinds of CAR-T cells were co-incubated with the 3M-CD19-CD79b-Luc target cells. Luciferase assay and ELISA were used to detecte the killing ability of these five groups of CAR-T cells and the secretion of cytokines and compared. The optimal structure of CAR-T cells was used to treat the leukemia mouse model constructed by Daudi-Luc cells. And the treatment efficacy was evaluated. At the same time, other targets were used in this structure. With the same methods, the stability and effectiveness of the structure were verified. RESULTS: CAR-19-79b-T cells were cultured for 7 days, the expression rates of CAR-19 and CAR-79b were 21.6%-36.3% and 21.7%-37.8%, respectively. The killing rates of 5 kinds of CAR-19-79b-T cells prepared by T cells from 3 healthy donors on 3M-CD19-CD79b-Luc cells were significantly higher than those of the T cell control group at the effect-target ratio of 10∶1. Among them, the killing rates of CAR-19-79b-T cells with No. III and No. IV structures were the strongest. After co-incubation with 3M-CD19-CD79b-Luc target cells, the amount of IFN-γ and TNF-α secreted by CAR-T cells with CAR IV and CARV structures was the lowest. And there was no significance between the two groups (P>0.05). CAR IV cells with remarkable killing effect and low secretion factor had obvious therapeutic effect on Daudi-Luc leukemia mice, extending the survival period of mice to 64 days. And all mice in the T cell control group died at 41.0±2.4 days. The CAR-19-BCMA-T and CAR-19-22-T with the same structure showed significant killing ability and low cytokine expression levels. CONCLUSION: A novel bi-specific CAR structures was successfully designed, which could efficiently kill the corresponding tumor cells and secrete less cytokines (such as TNF-α, IFN-γ). Moreover, it shows obvious therapeutic effect on Daudi lymphoma mouse model. The bi-specific CAR structure shows good killing specificity and safety.

Phase diagrams guide synthesis of highly ordered intermetallic electrocatalysts: separating alloying and ordering stages.

Supported platinum intermetallic compound catalysts have attracted considerable attention owing to their remarkable activities and durability for the oxygen reduction reaction in proton-exchange membrane fuel cells. However, the synthesis of highly ordered intermetallic compound catalysts remains a challenge owing to the limited understanding of their formation mechanism under high-temperature conditions. In this study, we perform in-situ high-temperature X-ray diffraction studies to investigate the structural evolution in the impregnation synthesis of carbon-supported intermetallic catalysts. We identify the phase-transition-temperature (TPT)-dependent evolution process that involve concurrent (for alloys with high TPT) or separate (for alloys with low TPT) alloying/ordering stages. Accordingly, we realize the synthesis of highly ordered intermetallic catalysts by adopting a separate annealing protocol with a high-temperature alloying stage and a low-temperature ordering stage, which display a high mass activity of 0.96 A mgPt-1 at 0.9 V in H2-O2 fuel cells and a remarkable durability.

Modified Grisotti flap technique in centrally located breast cancer: case report.

Traditionally, breast cancer patients with centrally located mass always receive mastectomy or the combination of central excision and primary closure. With the development of modern oncoplastic breast-conserving techniques, these patients can conserve their breast, and achieve satisfactory cosmetic outcome as well as clear margin. A variety of techniques are available to deal with centrally located breast cancers (CLBCs). Among these techniques, Grisotti flap technique is special, because it is easy to handle, and only causes minor injury by using a local rotational dermoglandular flap to fill the defection of central part. However, in our clinical practice, we find a lot of women in south China have special properties. Such as short distance from inframammary liner to the nipple, long distance from midclavicular to the nipple, and large breast diameter. Simply apply the Grisotti flap technique to those patients is not very suitable that drive us to modify this technique to suit our patients. We adopt the idea that use pedicled skin flap with skin island to replace the central defection to modify Grisotti flap technique. And applied this technique to two patients. We find modified Grisotti flap technique for Paget's disease or CLBC had good cosmetic results as well as safety in suitable patients. In the future, we can use superior pedicle with skin island for ptotic breasts, and lateral pedicle is suitable for patients without large and ptotic breasts.

The predictive value of high-sensitive troponin I for perioperative risk in patients undergoing gastrointestinal tumor surgery.

BACKGROUND: The incidence of cardiovascular events in perioperative period of gastrointestinal tumor surgery cannot be ignored, and studies have shown that level of postoperative troponin is related to the postoperative risk of non-cardiac surgery. However, the relationship between pre-operative troponin levels and perioperative risk of gastrointestinal tumor surgery is unclear. Thus, we aimed to evaluate the value of high-sensitive cardiac troponin I (hs-cTnI) prior to gastrointestinal tumor surgery for perioperative risk assessment. METHODS: In this retrospective cohort study, 1259 patients who underwent gastrointestinal tumor surgery and had been tested for hs-cTnI on admission within 7 days prior to surgery were retrospectively recruited from January 2018 to June 2020. The primary combined endpoint including in-hospital all-cause mortality, acute myocardial infarction, cardiac arrest or ventricular fibrillation and acute decompensated heart failure. The secondary endpoint included total hospital stay and requirement of intensive care treatment. FINDINGS: Compared with patients with normal hs-cTnI, those with elevated hs-cTnI (> 0·028 ng/ml) were more likely to experience the combined endpoint (28·2% versus 2·7%, P < 0·001) and there was also an increasing rate of in mortality in elevated hs-cTnI group (2·4% versus 0·3%, P = 0·057). The length of total hospital stay was significantly longer in patients with elevated hs-cTnI (24·8 ± 16·3 versus 19·5 ± 7·9, P = 0·003) and the number of patients requiring intensive care treatment was also higher (22·6% versus 4·2%, P < 0·001). The area under the ROC curve assessing hs-cTnI in predicting in-hospital mortality was 0·787 [95% confidence interval (CI) 0·612-0·963, P = 0·015] and for combined endpoint was 0·822 [95% CI 0·766-0·879, P < 0·001]. Hs-cTnI > 0·028 ng/ml was associated with significantly higher cardiovascular event rate in patients with the revised cardiac index ≤ 1. The positive likelihood ratio of hs-cTnI (> 0·028 ng/ml) for predicting combined endpoint reaches 10.5 in patients with Lee index = 0. In multivariate logistic analyses, hs-cTnI was one of the best predictors for the combined endpoint [odds ratio (OR) 5·924 (95%CI: 2·869-12·233), P < 0·001]. INTERPRETATION: Hs-cTnI provides powerful prognostic information for patients undergoing gastrointestinal tumor surgery, and therefore provides reliable prognostic information incremental to revised cardiac index.

Non-Structural Protein 5 of Zika Virus Interacts with p53 in Human Neural Progenitor Cells and Induces p53-Mediated Apoptosis.

Zika virus (ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells (NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins, the nonstructural protein 5 (NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of hNPCs. Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.

An unconventional role of an ASB family protein in NF-κB activation and inflammatory response during microbial infection and colitis.

Nuclear factor κB (NF-κB)-mediated signaling pathway plays a crucial role in the regulation of inflammatory process, innate and adaptive immune responses. The hyperactivation of inflammatory response causes host cell death, tissue damage, and autoinflammatory disorders, such as sepsis and inflammatory bowel disease. However, how these processes are precisely controlled is still poorly understood. In this study, we demonstrated that ankyrin repeat and suppressor of cytokine signaling box containing 1 (ASB1) is involved in the positive regulation of inflammatory responses by enhancing the stability of TAB2 and its downstream signaling pathways, including NF-κB and mitogen-activated protein kinase pathways. Mechanistically, unlike other members of the ASB family that induce ubiquitination-mediated degradation of their target proteins, ASB1 associates with TAB2 to inhibit K48-linked polyubiquitination and thereby promote the stability of TAB2 upon stimulation of cytokines and lipopolysaccharide (LPS), which indicates that ASB1 plays a noncanonical role to further stabilize the target protein rather than induce its degradation. The deficiency of Asb1 protects mice from Salmonella typhimurium- or LPS-induced septic shock and increases the survival of mice. Moreover, Asb1-deficient mice exhibited less severe colitis and intestinal inflammation induced by dextran sodium sulfate. Given the crucial role of ASB proteins in inflammatory signaling pathways, our study offers insights into the immune regulation in pathogen infection and inflammatory disorders with therapeutic implications.

A novel selective autophagy receptor, CCDC50, delivers K63 polyubiquitination-activated RIG-I/MDA5 for degradation during viral infection.

Autophagy is a conserved process that delivers cytosolic substances to the lysosome for degradation, but its direct role in the regulation of antiviral innate immunity remains poorly understood. Here, through high-throughput screening, we discovered that CCDC50 functions as a previously unknown autophagy receptor that negatively regulates the type I interferon (IFN) signaling pathway initiated by RIG-I-like receptors (RLRs), the sensors for RNA viruses. The expression of CCDC50 is enhanced by viral infection, and CCDC50 specifically recognizes K63-polyubiquitinated RLRs, thus delivering the activated RIG-I/MDA5 for autophagic degradation. The association of CCDC50 with phagophore membrane protein LC3 is confirmed by crystal structure analysis. In contrast to other known autophagic cargo receptors that associate with either the LIR-docking site (LDS) or the UIM-docking site (UDS) of LC3, CCDC50 can bind to both LDS and UDS, representing a new type of cargo receptor. In mouse models with RNA virus infection, CCDC50 deficiency reduces the autophagic degradation of RIG-I/MDA5 and promotes type I IFN responses, resulting in enhanced viral resistance and improved survival rates. These results reveal a new link between autophagy and antiviral innate immune responses and provide additional insights into the regulatory mechanisms of RLR-mediated antiviral signaling.

Cryopreservation of peripheral blood mononuclear cells using uncontrolled rate freezing.

Peripheral blood mononuclear cells are widely used as source material for anticancer immunotherapies. The conventional cryopreservation method for peripheral blood mononuclear cells is time-consuming and expansive, which involves controlled rate freezing followed by storage in liquid nitrogen. Instead, the convenient uncontrolled rate freezing cryopreservation method had been reported successfully in peripheral blood hematopoietic stem cells and peripheral blood progenitor cells. Therefore, we hypothesized that uncontrolled rate freezing cooling method maybe also applied to peripheral blood mononuclear cells cryopreservation. In this study, we evaluated the performance of uncontrolled rate freezing and controlled rate freezing cooling methods through cell recovery rate, viability, differentiation potential into cytokine-induced killer cells and the cellular properties of the cultured cytokine-induced killer cells. The results showed similar post-thaw viability and recovery rate in both controlled rate freezing and uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells. Importantly, the uncontrolled rate freezing cryopreserved peripheral blood mononuclear cells exhibited higher growth ratio and earlier cell clustering during ex-vivo cytokine-induced killer cell culture than the controlled rate freezing ones. These two groups of expanded cytokine-induced killer cells also exhibited similar effector cell subset ratio and tumoricidal activity. In general, the performance of cryopreserved peripheral blood mononuclear cells using uncontrolled rate freezing cooling method, with the commercial cryoprotective agent CellBanker 2, was equal or better than the controlled rate freezing method. Our study implied that the combined use of cryoprotective agent CellBanker 2 and uncontrolled rate freezing could be a convenient cryopreservation method for peripheral blood mononuclear cells.

Initiation of stress granule assembly by rapid clustering of IGF2BP proteins upon osmotic shock.

Stress granules (SGs) are membraneless organelles formed in the cytoplasm by liquid-liquid phase separation (LLPS) of translationally-stalled mRNA and RNA-binding proteins during stress response. Understanding the mechanisms governing SG assembly requires imaging SG formation in real time. Although numerous SG proteins have been identified, the kinetics of their recruitment during SG assembly has not been well established. Here we used live cell imaging and super-resolution imaging to visualize SG assembly in human cells. We found that IGF2BP proteins formed microscopically visible clusters in living cells almost instantaneously after osmotic stress, followed by fusion of clusters and the recruitment of G3BP1 and TIA1. Rapid clustering of IGF2BP1 was reduced in cells pretreated with emetine that stabilizes polysomes on mRNA. The KH3/4 di-domain and an intrinsically disordered region (IDR) of IGF2BP1 were found to mediate its clustering. Super-resolution imaging confirmed the formation of IGF2BP clusters associated with mRNA at 40 s after osmotic stress. In mature SGs, multiple clusters of poly(A) mRNA were found to associate with the periphery and the interior of a dense granule formed by IGF2BP1. Taken together, our findings revealed a novel, multi-stage LLPS process during osmotic stress, in which rapid clustering of IGF2BP proteins initiates SG assembly.

Recovery and functionality of cryopreserved peripheral blood mononuclear cells using five different xeno-free cryoprotective solutions.

In this study, we compared three commercially available and two widely used CPAs for their ability of cryopreserving PBMCs. Similar survival (81.0%) and recovery rate (73.7%) were observed among cells using these five CPAs. However, all the cryopreserved PBMCs exhibited a significantly lower survival rate when compared with the fresh samples (94.3%). We further evaluated effector cell subpopulation and tumoricidal activity of PBMC-derived cytokine-induced killing (CIK) cells and natural killing (NK) cells. Similar and high survival (CIK: 88.6%; NK: 87.5%) and recovery (CIK: 99.5%; NK: 99.7%) rates were detected in CIK and NK cells prepared from cryopreserved PBMCs using the five CPAs. The CD3+CD56+ effector percentage (27.3%) of cryopreserved PBMC-derived CIK cells using the five different CPAs and their tumoricidal activities on melanoma CHL-1 cells (45.7%) and bladder cancer cell line T-24 (44.7%) were similar but significantly lower than those of the fresh PBMC-derived controls (effector: 30.7%; CHL-1: 84.2%; T-24: 82.2%). Cryopreserved PBMC-derived NK cells also exhibited similar tumoricidal activities (CHL-1: 73.8%; T-24: 71.9%) but was significantly lower than that of the fresh control group. We were not able to identify a specific CPA that performed superior than others in PBMC cryopreservation.

Canine Influenza Virus is Mildly Restricted by Canine Tetherin Protein.

Tetherin (BST2/CD317/HM1.24) has emerged as a key host-cell ·defence molecule that acts by inhibiting the release and spread of diverse enveloped virions from infected cells. We analysed the biological features of canine tetherin and found it to be an unstable hydrophilic type I transmembrane protein with one transmembrane domain, no signal peptide, and multiple glycosylation and phosphorylation sites. Furthermore, the tissue expression profile of canine tetherin revealed that it was particularly abundant in immune organs. The canine tetherin gene contains an interferon response element sequence that can be regulated and expressed by canine IFN-α. A CCK-8 assay showed that canine tetherin was effective in helping mitigate cellular damage caused by canine influenza virus (CIV) infection. Additionally, we found that the overexpression of canine tetherin inhibited replication of the CIV and that interference with the canine tetherin gene enhanced CIV replication in cells. The impact of canine tetherin on CIV replication was mild. However, these results elucidate the role of the innate immune factor, canine tetherin, during CIV infection for the first time.

Topical delivery of l-theanine ameliorates TPA-induced acute skin inflammation via downregulating endothelial PECAM-1 and neutrophil infiltration and activation.

l-theanine, the most abundant free amino acid in tea, has been documented to possess many different bioactive properties through oral or intragastrical delivery. However, little is known about the effect of topical delivery of l-theanine on acute inflammation. In the present study, by using 12-O-tetradecanoylphorbol-13-acetate (TPA, 2.5 µg/ear)-induced ear edema model in mice, we first found that single-dose local pretreatment of l-theanine 30 min before TPA time- and dose-dependently suppressed the increases in both skin thickness and weight. Subsequently l-theanine ameliorated TPA-induced erythema, vascular permeability increase, epidermal and dermal hyperplasia, neutrophil infiltration and activation via downregulating the expression of PECAM-1 (a platelet endothelial adhesion molecule-1) in blood vessels and the production of pro-inflammatory cytokines IL-1ß, TNF-α, and mediator cyclooxygenase-2 (COX-2), which is mainly expressed in neutrophils. It highlighted the potential of l-theanine as a locally administrable therapeutic agent for acute cutaneous inflammation.

cfa-miR-143 Promotes Apoptosis via the p53 Pathway in Canine Influenza Virus H3N2-Infected Cells.

MicroRNAs regulate multiple aspects of the host response to viral infection. This study verified that the expression of cfa-miR-143 was upregulated in vivo and in vitro by canine influenza virus (CIV) H3N2 infection. To understand the role of cfa-miR-143 in CIV-infected cells, the target gene of cfa-miR-143 was identified and assessed for correlations with proteins involved in the apoptosis pathway. A dual luciferase reporter assay showed that cfa-miR-143 targets insulin-like growth factor binding protein 5 (Igfbp5). Furthermore, a miRNA agomir and antagomir of cfa-miR-143 caused the downregulation and upregulation of Igfbp5, respectively, in CIV-infected madin-darby canine kidney (MDCK) cells. This study demonstrated that cfa-miR-143 stimulated p53 and caspase3 activation and induced apoptosis via the p53 pathway in CIV H3N2-infected cells. In conclusion, CIV H3N2 induced the upregulation of cfa-miR-143, which contributes to apoptosis via indirectly activating the p53-caspase3 pathway.