Characteristics of Chinese breast cancer patients with double heterozygosity for BRCA1 and BRCA2 germline pathogenic variants.

PURPOSE: Despite of very rare, breast cancer patients with double heterozygosity (DH) variants in BRCA1 and BRCA2 genes have been identified in other ethnic groups and seem to be associated with distinctive phenotypes. However, little is known about the frequency and clinical characteristics of Chinese breast cancer patients with BRCA1/2 DH variants. METHODS: Four hundred and eleven unrelated patients with BRCA1 or BRCA2 pathogenic variants (PVs) were identified in a large series of unselected breast cancer patients. Another two siblings with metachronous bilateral breast cancer were referred for genetic counseling, after which BRCA1/2 DH variants were detected. RESULTS: Four unrelated breast cancer patients with BRCA1/2 DH were identified in the cohort of 411 patients with BRCA1 or BRCA2 PVs, the frequency of BRCA1/2 DH was 0.97%. In total, six BRCA1/2 DH patients from five families were found in this study. In two families, the hereditary pattern of DH was speculated to have originated from both sides of the family. BRCA1/2 DH patients were more likely to have a family history of breast cancer than patients with a BRCA1 (100% vs. 29.2%, P = 0.004) or BRCA2 (100% vs. 29.6%, P = 0.004) single PV. BRCA1/2 DH patients were more likely to be triple-negative breast tumors than patients with single BRCA2 PVs (66.7% vs. 14.1%, P = 0.020), which was comparable to the findings in patients with single BRCA1 PVs (66.7% vs. 56.9%, P = 1.00). CONCLUSION: Chinese patients with BRCA1/2 DH exhibit a high percentage of family history of breast cancer. The tumor pathological features of BRCA1/2 DH carriers are similar to those of BRCA1 PV carriers.

Retinal microcirculation: A window into systemic circulation and metabolic disease.

The retinal microcirculation system constitutes a unique terminal vessel bed of the systemic circulation, and its perfusion status is directly associated with the neural function of the retina. This vascular network, essential for nourishing various layers of the retina, comprises two primary microcirculation systems: the retinal microcirculation and the choroidal microcirculation, with each system supplying blood to distinct retinal layers and maintaining the associated neural function. The blood flow of those capillaries is regulated via different mechanisms. However, a range of internal and external factors can disrupt the normal architecture and blood flow within the retinal microcirculation, leading to several retinal pathologies, including diabetic retinopathy, macular edema, and vascular occlusions. Metabolic disturbances such as hyperglycemia, hypertension, and dyslipidemia are known to modify retinal microcirculation through various pathways. These alterations are observable in chronic metabolic conditions like diabetes, coronary artery disease, and cerebral microvascular disease due to advances in non-invasive or minimally invasive retinal imaging techniques. Thus, examination of the retinal microcirculation can provide insights into the progression of numerous chronic metabolic disorders. This review discusses the anatomy, physiology and pathophysiology of the retinal microvascular system, with a particular emphasis on the connections between retinal microcirculation and systemic circulation in both healthy states and in the context of prevalent chronic metabolic diseases.

The Evolution of the Bone Turnover Marker in Patients Following Recovery from Diabetic Ketoacidosis.

The aim of the study was to investigate whether the biomarkers for bone turnover could rapidly recover during the period of diabetic ketoacidosis (DKA). Bone turnover biomarkers, including 25-hydroxyvitamin D3, N-terminal middle molecular fragment of osteocalcin (NMID), and ß-C terminal cross-linking telopeptide of type 1 collagen were evaluated using in-patient data (n=627) from Shanghai Pudong Hospital from 2018-2022. The comparison was performed between type 2 diabetes (T2D only) (n=602) and DKA (n=25), in which we checked the bone turnover markers at pre-treatment and recovery. After matching by body mass index (BMI), we found that except for 25-OH-VitD3, the age difference, indices of glucose metabolism, and bone turnover were significant between the 2 groups (p<0.05). We found only a significant restoration of NMID (p<0.001). NMID and ß-CTX, when compared with T2D, showed overt distinction between recovery and T2D (p<0.05). In addition, the investigations demonstrated a substantial difference between 25-OH-VitD3 in males and NMID in females, regardless of age (p<0.05). Multilinear regression analysis revealed that 2 hours postprandial plasma C-peptide was an independent predictor of the NMID in both pre-treatment (ß=0.58, p=0.003) and recovery (ß=0.447, p=0.025), although sex was significant in pre-treatment (ß=-0.444, p=0.020). Finally, we found that only age variation affected DKA's fasting plasma glucose level (p<0.05). The study revealed that the bone turnover of DKA is significantly different in pre-treatment and recovery; however, NMID might recover quickly if the patients received appropriate treatment. Importantly, pancreatic function plays a critical role in changing bone turnover biomarkers.

Catheter ablation of atrial fibrillation in patients with left bundle branch block.

BACKGROUND: Left bundle branch block (LBBB) and atrial fibrillation (AF) are commonly coexisting conditions. The impact of LBBB on catheter ablation of AF has not been well determined. This study aims to explore the long-term outcomes of patients with AF and LBBB after catheter ablation. METHODS: Forty-two patients with LBBB of 11,752 patients who underwent catheter ablation of AF from 2011 to 2020 were enrolled as LBBB group. After propensity score matching in a 1:4 ratio, 168 AF patients without LBBB were enrolled as non-LBBB group. Late recurrence and a composite endpoint of stroke, all-cause mortality, and cardiovascular hospitalization were compared between the two groups. RESULTS: Late recurrence rate was significantly higher in the LBBB group than that in the non-LBBB group (54.8% vs. 31.5%, p = .034). Multivariate analysis showed that LBBB was an independent risk factor for late recurrence after catheter ablation of AF (hazard ratio [HR] 2.19, 95% confidence interval [CI] 1.09-4.40, p = .031). LBBB group was also associated with a significantly higher incidence of the composite endpoint (21.4% vs. 6.5%, HR 3.98, 95% CI 1.64-9.64, p = .002). CONCLUSIONS: LBBB was associated with a higher risk for late recurrence and a higher incidence of composite endpoint in the patients underwent catheter ablation.

Development of a Me2SO-free cryopreservation medium and its long-term cryoprotection on the CAR-NK cells.

Cryopreservation is a crucial step in the supply process of off-the-shelf chimeric antigen receptor engineered natural killer (CAR-NK) cell products. Concerns have been raised over the clinical application of dimethyl sulfoxide (Me2SO) due to the potential for adverse reactions following infusion and limited cell-specific cytotoxic effects if misapplied. In this study, we developed a Me2SO-free cryopreservation medium specifically tailored for CAR-NK cells to address this limitation. The cryopreservation medium was formulated using human serum albumin (HSA) and glycerol as the base components. Following initial screening of seven clinically-compatible solutions, four with cryoprotective properties were identified. These were combined and optimized into a single formulation: IF-M. The viability, phenotype, and function of CAR-NK cells were evaluated after short-term and long-term cryopreservation to assess the effectiveness of IF-M, with Me2SO serving as the control group. The viability and recovery of CAR-NK cells in the IF-M group were significantly higher than those in the Me2SO group within 90 days of cryopreservation. Moreover, after 1 year of cryopreservation the cytotoxic capacity of CAR-NK cells cryopreserved with IF-M was comparable to that of fresh CAR-NK cells and significantly superior to that of CAR-NK cells cryopreserved in Me2SO. The CD107a expression intensity of CAR-NK cells in IF-M group was significantly higher than that of Me2SO group. No statistical differences were observed in other indicators under different cryopreservation times. These results underscore the robustness of IF-M as a suitable replacement for traditional Me2SO-based cryopreservation medium for the long-term cryopreservation and clinical application of off-the-shelf CAR-NK cells.

Gender differences in the association between changes in the atherogenic index of plasma and cardiometabolic diseases: a cohort study.

OBJECTIVE: The relationship between changes in Atherogenic Index of Plasma (AIP) and cardiometabolic diseases (CMD) in middle-aged and elderly individuals remains unclear. This study aims to explore the association between changes in AIP and CMD. METHODS: This study included 3,791 individuals aged over 45 years from CHARLS. Participants were divided into four groups using the K-Means clustering method. Cumulative AIP was used as a quantitative indicator reflecting changes in AIP. Differences in baseline data and CMD incidence rates among these four groups were compared. Multifactorial logistic regression models were used to assess the relationship between changes in AIP and CMD, and subgroup analysis and interaction tests were conducted to evaluate potential relationships between changes in AIP and CMD across different subgroups. Restricted cubic splines (RCS) were used to assess the dose-response relationship between cumulative AIP and CMD. RESULTS: Changes in AIP were independently and positively associated with CMD. In males, the risk significantly increased in class4 compared to class1 (OR 1.75, 95%CI 1.12-2.73). In females, changes in AIP were not significantly associated with CMD. Cumulative AIP was positively correlated with CMD (OR 1.15, 95%CI 1.01-1.30), with significant gender differences in males (OR 1.29, 95%CI 1.07-1.55) and females (OR 1.03, 95%CI 0.87-1.23) (p for interaction = 0.042). In addition, a linear relationship was observed between cumulative AIP and CMD in male. CONCLUSION: Substantial changes in AIP may increase the risk of CMD in middle-aged and elderly Chinese males. Dynamic monitoring of AIP is of significant importance for the prevention and treatment of CMD.

Utilize multi-metabolic parameters as determinants for prediction of skeletal muscle mass quality in elderly type2 diabetic Chinese patients.

BACKGROUND: Sarcopenia, an age-related disorder characterized by loss of skeletal muscle mass and function, is recently recognized as a complication in elderly patients with type 2 diabetes mellitus (T2DM). Skeletal muscles play a crucial role in glycemic metabolism, utilizing around 80% of blood glucose. Accordingly, we aimed to explore the relationship between glucose metabolism and muscle mass in T2DM. METHODS: We employed the AWGS 2019 criteria for diagnosing low muscle mass and 1999 World Health Organization (WHO) diabetes diagnostic standards. This study included data of 191 individuals aged 60 and above with T2DM of Shanghai Pudong Hospital from November 2021 to November 2022. Fasting C-peptide (FPCP), fasting plasma glucose (FPG), 2-hour postprandial plasma glucose (PPG) and postprandial 2-hour C-peptide (PPCP), glycated hemoglobin A1c (HbA1c), glycated albumin (GA), serum lipids spectrum, renal and hepatic function, hemoglobin, and hormone were measured. Based on the findings of univariate analysis, logistic regression and receiver operating characteristic (ROC) curves were established. RESULTS: Participants with low muscle mass had significantly lower alanine and aspartate aminotransferase, and both FPCP and PPCP levels (P < 0.05). Compared with those without low muscle mass, low muscle mass group had significantly higher FPG, HbA1c, GA levels (P < 0.05). Body fat (BF, OR = 1.181) was an independent risk factor for low muscle mass. PPCP (OR = 0.497), BMI (OR = 0.548), and female (OR = 0.050) were identified as protective factors for low skeletal muscle. The AUC of BMI was the highest, followed by the PPCP, gender and BF (0.810, 0.675, 0.647, and 0.639, respectively), and the AUC of the combination of the above four parameters reached 0.895. CONCLUSIONS: In this cross-sectional study, BMI, Female, and PPCP associated with T2DM were protective factors for low muscle mass. BF was associated with T2DM and risk factor for low muscle mass.

Quantitative imaging for predicting hematoma expansion in intracerebral hemorrhage: A multimodel comparison.

BACKGROUND: Several studies report that radiomics provides additional information for predicting hematoma expansion in intracerebral hemorrhage (ICH). However, the comparison of diagnostic performance of radiomics for predicting revised hematoma expansion (RHE) remains unclear. METHODS: The cohort comprised 312 consecutive patients with ICH. A total of 1106 radiomics features from seven categories were extracted using Python software. Support vector machines achieved the best performance in both the training and validation datasets. Clinical factors models were constructed to predict RHE. Receiver operating characteristic curve analysis was used to assess the abilities of non-contrast computed tomography (NCCT) signs, radiomics features, and combined models to predict RHE. RESULTS: We finally selected the top 21 features for predicting RHE. After univariate analysis, 4 clinical factors and 5 NCCT signs were selected for inclusion in the prediction models. In the training and validation dataset, radiomics features had a higher predictive value for RHE (AUC = 0.83) than a single NCCT sign and expansion-prone hematoma. The combined prediction model including radiomics features, clinical factors, and NCCT signs achieved higher predictive performances for RHE (AUC = 0.88) than other combined models. CONCLUSIONS: NCCT radiomics features have a good degree of discrimination for predicting RHE in ICH patients. Combined prediction models that include quantitative imaging significantly improve the prediction of RHE, which may assist in the risk stratification of ICH patients for anti-expansion treatments.

Root caries in older adults: A co-citation network analysis (1980-2023).

BACKGROUND: Root caries affect the oral health and quality of life of older adults. This study examines the breadth of global research on this topic, aiming to clarify its expansive scope and to shed light on pertinent trends for new researchers in the field. OBJECTIVE: To identify key advances in root caries research as highlighted in high-quality articles from the Social Science Citation Index (SSCI) as well as to explore emerging trends and perspectives. MATERIALS AND METHODS: Using the Web of Science (WoS) database, we conducted a comprehensive review of articles related to root caries in older adults. Our focus was on finding high-quality SSCI articles, identifying major contributors, journals and research trends and exploring areas such as dentistry, oral surgery and medicine for potential future research. RESULTS: Our analysis included 192 articles, each of which was subjected to bibliometric and VOS viewer evaluations. The results revealed a concentration of studies in dentistry, oral surgery and medicine, with gaps identified in areas like anthropology, biochemistry, molecular biology and chemistry. A notable deficiency was found in root caries management. CONCLUSION: We discuss research gaps and propose future directions based on our findings, emphasising interdisciplinary research approaches.

FDC-SP as a diagnostic and prognostic biomarker and modulates immune infiltrates in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC), one of the top 10 causes of cancer death, is responsible for more than 90% of all cases of primary renal cancer worldwide. Follicular dendritic cell-secreted protein (FDC-SP) specifically binds to activated B cells and regulates the generation of antibodies. It is also thought to promote cancer cell invasion and migration, which could help with tumor metastases. This study aimed to assess the efficacy of FDC-SP in the diagnosis and prognosis of RCC and to investigate the relationship between immune infiltration in RCC and these outcomes. RESULTS: RCC tissues had significantly higher levels of FDC-SP protein and mRNA than normal tissues. The high level of FDC-SP expression was linked to the T stage, histological grade, pathological stage, N stage, M stage, and OS event. Functional enrichment analysis identified the major pathways that were enriched as immune response regulation, complement, and coagulation. Immunological checkpoints and immune cell infiltration were observed to substantially correlate with the levels of FDC-SP expression. FDC-SP expression levels showed the ability to precisely distinguish high-grade or high-stage renal cancer (area under the curve (AUC) = 0.830, 0.722), and RCC patients with higher FDC-SP expression levels had worse prognoses. The AUC values for one-, two-, and five-year survival rates were all greater than 0.600. Moreover, the FDC-SP expression is an independent predictive biomarker of OS in RCC patients. CONCLUSION: FDC-SP may be a prospective therapeutic target in RCC as well as a possible diagnostic and prognostic biomarker associated with immune infiltration.

Risk Factors for Left Ventricular Thrombus Formation in Patients with Dilated Cardiomyopathy.

BACKGROUND: Left ventricular thrombus (LVT) is a common complication of dilated cardiomyopathy (DCM), causing morbidity and mortality. METHODS: This study retrospectively analyzed patients with DCM from January 2002 to August 2020 in Beijing Anzhen Hospital. Clinical characteristics were compared between the LVT group and the age and sex 1:4 matched with the LVT absent group. The receiver operator characteristic (ROC) curve was plotted to evaluate the diagnostic value of D-dimer predicting LVT occurrence in DCM. RESULTS: A total of 3,134 patients were screened, and LVT was detected in 72 (2.3%) patients on echocardiography. The patients with LVT had higher D-dimer, fibrinogen, and lower systolic blood pressure than those without LVT. The ejection fraction (EF) was lower and left ventricular end-systolic diameter was larger in the LVT group. Severe mitral regurgitation (MR) was more common in the LVT absent groups. The prevalence of atrial fibrillation was lower in the LVT group. The ROC curve analysis yielded an optimal cut-off value of 444 ng/mL DDU (D-dimer units) for D-dimer to predict the presence of LVT. Multivariable binary logistic regression analysis revealed that EF (OR = 0.90, 95% CI = 0.86-0.95), severe MR (OR = 0.19, 95% CI = 0.08-0.48), and D-dimer level (OR = 15.4, 95% CI = 7.58-31.4) were independently associated with LVT formation. CONCLUSION: This study suggested that elevated D-dimer levels (>444 ng/mL DDU) and reduced EF were independently associated with increased risk of LVT formation. Severe MR could decrease the incidence of LVT.

CircRNA High Mobility Group At-hook 2 regulates cell proliferation, metastasis and glycolytic metabolism of nonsmall cell lung cancer by targeting miR-331-3p to upregulate High Mobility Group At-hook 2.

Increasing circular RNAs (circRNAs) have been identified as pivotal players in nonsmall cell lung cancer (NSCLC). The study will explore the function and mechanism of circRNA High Mobility Group AT-hook 2 (circHMGA2) in NSCLC. The circHMGA2, microRNA-331-3p (miR-331-3p) and HMGA2 expression analyses were performed via quantitative real-time PCR. Cell proliferation was assessed via Cell Counting Kit-8 and colony formation assays. Transwell migration/invasion assays were used for measuring cell metastasis. Glucose consumption and lactate production were determined for glycolytic evaluation. Western blot was used to detect the protein expression of HMGA2 and glycolytic markers. Target analysis was performed by dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Xenograft tumor assay in mice was conducted for the investigation of circHMGA2 in vivo . CircHMGA2 was overexpressed in NSCLC, and high circHMGA2 level might be related to NSCLC metastasis and poor prognosis. In-vitro assays suggested that NSCLC cell growth, metastasis and glycolysis were retarded by downregulation of circHMGA2. Upregulation of HMGA2 was shown to return the anticancer response of circHMGA2 knockdown in NSCLC cells. Through interacting with miR-331-3p, circHMGA2 could regulate the expression of HMGA2. In addition, circHMGA2/miR-331-3p and miR-331-3p/HMGA2 axes were affirmed in NSCLC regulation. In-vivo analysis indicated that circHMGA2 inhibition also reduced tumorigenesis and glycolysis of NSCLC via the miR-331-3p/HMGA2 axis. This study disclosed the oncogenic role of circHMGA2 and the regulatory circHMGA2/miR-331-3p/HMGA2 axis in NSCLC.

Prehospital Ultra-Early Neurological Deterioration in Intracerebral Hemorrhage: Definition, Prevalence, and Association with Outcomes.

INTRODUCTION: The objective of this study was to define prehospital ultra-early neurological deterioration (UND) and to investigate the association with functional outcomes in patients with intracerebral hemorrhage (ICH). METHODS: We conducted a prospective cohort study of consecutive acute ICH patients. The stroke severity at onset and hospital admission was assessed using the Chongqing Stroke Scale (CQSS), and prehospital UND was defined as a CQSS increase of ≥2 points between symptoms onset and admission. Early neurological deterioration (END) was defined as the increase of ≥4 points in NIHSS score within the first 24 h after admission. Poor outcome was defined as a modified Rankin Scale (mRS) of 4-6 at 3 months. RESULTS: Prehospital UND occurred in 29 of 169 patients (17.2%). Patients with prehospital UND had a median admission NIHSS score of 17.0 as opposed to those without prehospital UND with a median NIHSS score of 8.5. There were three patterns of neurological deterioration: prehospital UND only in 21 of 169 patients (12.4%), END but without prehospital UND in 20 of 169 patients (11.8%), and continuous neurological deterioration in both phases in 8 patients (4.7%). Prehospital UND was associated with worse 3-month outcomes (median mRS score, 4.0 vs. 2.0, p = 0.002). After adjusting for age, time from onset to admission, END, and systolic blood pressure, prehospital UND was an independent predictor of poor outcome (odds ratio [OR] 3.27, 95% confidence interval [CI] 1.26-8.48, p = 0.015). CONCLUSION: Prehospital UND occurs in approximately 1 in 7 patients between symptom onset and admission and is associated with poor functional outcome in patients with ICH. Further research is needed to investigate the prehospital UND in the prehospital phase in the triage of patients with ICH.

Low CO2 partial pressure steers CHO cells into a defective metabolic state.

PURPOSE: The accumulation of carbon dioxide during large-scale culture of animal cells brings adverse effects, appropriate aeration strategies alleviate CO2 accumulation while improper reactor operation may lead to the presence of low CO2 partial pressure (pCO2) condition as occurs in many industrial cases. Thus, this study aims to reveal the in-depth influence of low pCO2 on Chinese Hamster Ovary (CHO) cells for providing a reference for design space determination of CO2 control with regard to the Quality by Design (QbD) guidelines. METHODS AND RESULTS: The headspace air over purging caused the ultra-low pCO2 (ULC) where the monoclonal antibody production as well as the aerobic metabolic activity were reduced. Intracellular metabolomics analysis indicated a less efficient aerobic glucose metabolic state under ULC conditions. Based on the increase of intracellular pH and lactate dehydrogenase activity, the shortage of intracellular pyruvate could be the cause of the deficient aerobic metabolism, which could be partially mitigated by pyruvate addition under ULC conditions. Finally, a semi-empirical mathematical model was used to better understand, predict and control the occurrence of extreme pCO2 conditions during the cultures of CHO cells. CONCLUSION: Low pCO2 steers CHO cells into a defective metabolic state. A predictive relation among pCO2, lactate, and pH control was applied to get new insights into CHO cell culture for better and more robust metabolic behavior and process performance and the determination of QbD design space for CO2 control.

Enhanced vitamin K2 production by engineered Bacillus subtilis during leakage fermentation.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation.

Factors associated with higher falling risk in elderly diabetic patients with lacunar stroke.

PURPOSE: The aim of this study is to explore the factors associated with the fall risk in type 2 diabetes (T2D) patients with a lacunar stroke. MATERIALS AND METHODS: We compiled data of 146 T2D patients (mean age 68 years), including the Morse fall scale data (MFS), nutrition score, self-care scale, laboratory data, and data from continuous glucose monitoring system (CGMS) from 2019 to 2021 in Shanghai Pudong Hospital. Thereby, we evaluated the associations between MFS and other clinical parameters. RESULTS: The analyses showed that there were significantly increased size and numbers of lacunar infarction (p < 0.05). Furthermore, the greater risk group had an older mean age (p < 0.05), and significant decreased estimated glomerular filtration rate (eGFR), total triglyceride (TG), while increased microalbuminuria, magnesium, lipoprotein A (LP(a)), anti-thyroid peroxidase antibody (TPOAb) (p < 0.05). However, the time in range (TIR) was very comparable (p > 0.05). The correlational study revealed the higher score of MFS was associated with the age (r = 0.41), number of lacunar infarction (r = 0.18), nutrition score (r = 0.20), self-care score (r = - 0.43), serum creatine level (r = 0.19), eGFR (r = - 0.26) (p < 0.05). The total numbers of lacunar infarction were associated with age (r = 0.36), eGFR (r = - 0.40), homocysteine level (r = 0.33) (p < 0.05). CONCLUSIONS: Age, nutrition, self-care ability, and renal function are all critical factors associated with the risk of fall in T2D with lacunar infarction. The age, eGFR, and homocysteine are closely associated with lacunar infarction, suggesting that in T2D, evaluation of kidney dysfunction, homocysteine level in the elderly can predict lacunar infarcts and falls.

Continuous NF-κB pathway inhibition promotes expansion of human phenotypical hematopoietic stem/progenitor cells through metabolism regulation.

Hematopoietic stem/progenitor cells (HSPCs) ex vivo expansion is critical in facilitating their widespread clinical application. NF-κB pathway is implicated in the energy homeostasis and metabolic adaptation. To explore the effect of NF-κB pathway on the ex vivo HSPC expansion and metabolism, the 50 nM-1 µM inhibitor of NF-κB pathway TPCA-1 was used to expand cord blood derived CD34+ cells in serum-free culture. The expansion folds, function, mitochondrial profile and metabolism of HSPCs were determined. After 10 days of culture with 100 nM TPCA-1, the expansion of total cells， CD34+CD38- cells， and CD34+CD38-CD45RA-CD90+CD49f+ cells were significantly increased compared to the cytokine priming alone. Notably, TPCA-1 treatment generated ~ 2-fold greater percentage of CD34+EPCR+ and CD34+CD38-CD45RA-CD90+CD49f+ cells compared to cytokine only conditions. Moreover, TPCA-1 expanded CD34+ cells displayed enhanced serial colonies forming potential and secondary expansion capability. NF-κB inhibition increased the expression of self-renewal related genes, while downregulated the expression of mitochondrial biogenesis regulator (Pgc1α) and mitochondrial chaperones and proteases (ClpP, Hsp10, Hsp60). Mitochondrial mass and membrane potential were markedly decreased with TPCA-1 treatment, leading to the reduced mitochondrial reactive oxygen species (ROS) level in HSPCs. NF-κB inhibition displayed augmented glycolysis rate with compromising mitochondrial metabolism. This study demonstrated that NF-κB pathway inhibition improved glycolysis and limited ROS production that promoted the ex vivo expansion and maintenance of functional HSPCs.

Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches.

Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.

MEK1 activation enhances the ex vivo proliferation of haematopoietic stem/progenitor cell.

Haematopoietic stem/progenitor cell (HSPC) integrates intracellular signal network from growth factors (GFs) and utilizes its proliferation feature to generate high yields of transplantable cells upon ex vivo culture. However, the molecular basis for HSPC activation and proliferation is not completely understood. The goal of this study was to investigate proliferation regulator in the downstream of GFs and develop HSPC expansion strategy. Microarray and Ingenuity Pathway Analysis were performed to evaluate differentially expressed genes in cytokine-induced CD34+ cells after ex vivo culture. We identified that MEK1 was a potential HSPC proliferation regulator, which represented indispensable roles and MEK1 silence attenuated the proliferation of HSPC. Notably, 500 nM MEK1 agonist, PAF C-16, increased the numbers of phenotypic HSPC and induced cell cycling of HSPC. The PAF C-16 expanded HSPC demonstrated comparative clonal formation ability and secondary expansion capacity compared to the vehicle control. Our results provide insights into regulating the balance between proliferation and commitment of HSPC by targeting the HSPC proliferation-controlling network. This study demonstrates that MEK1 critically regulates HSPC proliferation and cell production in the ex vivo condition for transplantation.

Trehalose glycopolymers for cryopreservation of tissue-engineered constructs.

The development of an effective cryopreservation method to achieve off-the-shelf and bioactive tissue-engineered constructs (TECs) is important to meet the requirements for clinical applications. The trehalose, a non-permeable cryoprotectant (CPA), has difficulty in penetrating the plasma membranes of mammalian cells and has only been used in combination with other cell penetrating CPA (such as DMSO) to cryopreserve mammalian cells. However, the inherent cytotoxicity of DMSO results in increasing risks with respect to cryopreserved cells. Therefore, in this study, permeable trehalose glycopolymers were synthesised for cryopreservation of TECs. The trehalose glycopolymers exhibited good ice inhibiting activities and biocompatibilities. Furthermore, the viability and function of TECs after cryopreservation with 5.0 wt% S2 were similar to those of the non-cryopreserved TECs. We developed an effective preservation strategy for the off-the-shelf availability of TECs.