Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Background: Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Methods: Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. Results: The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Conclusions: Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

Elevated CD4+ T Cell Senescence Associates with Impaired Immune Responsiveness in Severe COVID-19.

Aging is a critical risk factor for unfavorable clinical outcomes among COVID-19 patients and may impact vaccine efficacy. However, whether the senescence of T cells is associated with severe COVID-19 outcome in elderly individuals is unclear. Using flow cytometry, we analyzed the frequency of senescent T cells (Tsens) in peripheral blood from 100 hospitalized elderly COVID-19 patients and compared differences between those with mild/moderate and severe/critical illness. We also assessed correlations between the percentage of Tsens and the quantity and quality of spike-specific antibodies by ELISA, neutralizing antibody test kit, and ELISPOT assay respectively, the cytokine production profile of COVID-19 reactive T cells, and plasma soluble factors by cytometric bead array (CBA). Our study found a significantly elevated level of CD4+ Tsens in patients with severe/critical disease compared to those with mild/moderate illness. Patients with a higher level of CD4+ Tsens (>19.78%) showed a decreased survival rate compared to those with a lower level (≤19.78%). This is more pronounced among patients with breakthrough infections. The percentage of CD4+ Tsens was negatively correlated with spike-specific antibody titers, neutralization ability, and COVID-19 reactive IL-2+CD4+ T cells. In addition, spike-specific antibody levels were positively correlated with IL-2 producing T cells and plasma IL-2 amount. Mechanistically, with defective CD40L, T cells from patients with CD4+ Tsens >19.78% were unable to support B cell proliferation and differentiation. Our data demonstrate that the percentage of CD4+ Tsens in peripheral blood may serve as a reliable biomarker for the prognosis of severe COVID-19 patients, especially in breakthrough infections. Therefore, restoring the immune response of CD4+ Tsens may be key to preventing severe illness and improving vaccine efficacy in older adults.

Integrative analysis of DNA methylomes reveals novel cell-free biomarkers in lung adenocarcinoma.

Lung cancer is a leading cause of cancer-related deaths worldwide, with a low 5-year survival rate due in part to a lack of clinically useful biomarkers. Recent studies have identified DNA methylation changes as potential cancer biomarkers. The present study identified cancer-specific CpG methylation changes by comparing genome-wide methylation data of cfDNA from lung adenocarcinomas (LUAD) patients and healthy donors in the discovery cohort. A total of 725 cell-free CpGs associated with LUAD risk were identified. Then XGBoost algorithm was performed to identify seven CpGs associated with LUAD risk. In the training phase, the 7-CpGs methylation panel was established to classify two different prognostic subgroups and showed a significant association with overall survival (OS) in LUAD patients. We found that the methylation of cg02261780 was negatively correlated with the expression of its representing gene GNA11. The methylation and expression of GNA11 were significantly associated with LAUD prognosis. Based on bisulfite PCR, the methylation levels of five CpGs (cg02261780, cg09595050, cg20193802, cg15309457, and cg05726109) were further validated in tumor tissues and matched non-malignant tissues from 20 LUAD patients. Finally, validation of the seven CpGs with RRBS data of cfDNA methylation was conducted and further proved the reliability of the 7-CpGs methylation panel. In conclusion, our study identified seven novel methylation markers from cfDNA methylation data which may contribute to better prognosis for LUAD patients.

Association between Changes in Plasma Metabolism and Clinical Outcomes of Sepsis.

Current prognostic biomarkers for sepsis have limited sensitivity and specificity. This study aimed to investigate dynamic lipid metabolomics and their association with septic immune response and clinical outcomes of sepsis. This prospective cohort study included patients with sepsis who met the Sepsis 3.0 criteria. On hospitalization days 1 (D1) and 7 (D7), plasma samples were collected, and patients underwent liquid chromatography with tandem mass spectrometry. A total of 40 patients were enrolled in the study, 24 (60%) of whom were men. The median age of the enrolled patients was 81 (68-84) years. Thirty-one (77.5%) patients had a primary infection site of the lung. Participants were allocated to the survivor (25 cases) and nonsurvivor (15 cases) groups based on their 28-day survival status. Ultimately, a total of 113 lipids were detected in plasma samples on D 1 and D 7, of which 42 lipids were most abundant in plasma samples. The nonsurvival group had significantly lower lipid expression levels in lysophosphatidylcholine (LysoPC) (16 : 0, 17 : 0,18 : 0) and 18 : 1 SM than those in the survival group (p < 0.05) on D7-D1. The correlation analysis showed that D7-D1 16 : 0 LysoPC (r = 0.367, p = 0.036),17 : 0 LysoPC (r = 0.389, p = 0.025) and 18 : 0 LysoPC(r = 0.472, p = 0.006) levels were positively correlated with the percentage of CD3+ T cell in the D7-D1. Plasma LysoPC and SM changes may serve as prognostic biomarkers for sepsis, and lipid metabolism may play a role in septic immune disturbances.

Baseline red blood cell distribution width and perforin, dynamic levels of interleukin 6 and lactate are predictors of mortality in patients with sepsis.

BACKGROUND: Sepsis is a critical illness often encountered in the intensive care unit. However, prognostic biomarkers for sepsis have limited sensitivity. This study aimed to identify more sensitive predictors of mortality through repeated monitoring of laboratory parameters. METHODS: Patients with sepsis (Sepsis 3.0 criteria met) were recruited and divided into the survivor and nonsurvivor groups after 28 days. Data on blood biochemistry, lymphocyte subsets, and cytokines were obtained on the first and seventh hospitalization days. Univariate and multivariate Cox regression analyses were performed to explore the correlation between these variables and patient mortality. RESULTS: Forty patients with sepsis were included. The mortality rate was 37.5%. Red blood cell distribution width-standard deviation (RDWSD) (hazard ratio [HR] = 1.107 [95% CI: 1.005-1.219], p = 0.040) and perforin level (HR = 1.001 [95% CI: 1-1.003], p = 0.035) on the first day, as well as lactate (HR = 112.064 [95% CI: 2.192-5729.629], p = 0.019) and interleukin 6 (IL-6) (HR = 1.005 [95% CI: 1.001-1.008], p = 0.014) levels on the seventh day, were independent risk factors of mortality. If the patients were divided into two groups based on RDWSD (normal: n = 31; increased: n = 9), the Kaplan-Meier curves showed that the group with increased RDWSD had a lower survival (p = 0.025). CONCLUSION: Baseline RDWSD and perforin, along with dynamic IL-6 and lactate levels, were independent predictors of mortality in patients with sepsis.

A GRIP-1-EZH2 switch binding to GATA-4 is linked to the genesis of rhabdomyosarcoma through miR-29a.

Terminal differentiation failure is an important cause of rhabdomyosarcoma genesis, however, little is known about the epigenetic regulation of aberrant myogenic differentiation. Here, we show that GATA-4 recruits polycomb group proteins such as EZH2 to negatively regulate miR-29a in undifferentiated C2C12 myoblast cells, whereas recruitment of GRIP-1 to GATA-4 proteins displaces EZH2, resulting in the activation of miR-29a during myogenic differentiation of C2C12 cells. Moreover, in poorly differentiated rhabdomyosarcoma cells, EZH2 still binds to the miR-29a promoter with GATA-4 to mediate transcriptional repression of miR-29a. Interestingly, once re-differentiation of rhabdomyosarcoma cells toward skeletal muscle, EZH2 was dispelled from miR-29a promoter which is similar to that in myogenic differentiation of C2C12 cells. Eventually, this expression of miR-29a results in limited rhabdomyosarcoma cell proliferation and promotes myogenic differentiation. We thus establish that GATA-4 can function as a molecular switch in the up- and downregulation of miR-29a expression. We also demonstrate that GATA-4 acts as a tumor suppressor in rhabdomyosarcoma partly via miR-29a, which thus provides a potential therapeutic target for rhabdomyosarcoma.

Ascitic Senescent T Cells Are Linked to Chemoresistance in Patients With Advanced High-Grade Serous Ovarian Cancer.

Senescent T cells are reported to be increased in patients with cancer and are poor prognostic indicators. However, the distribution of senescent T cells and their correlation with clinical features in high-grade serous ovarian cancer (HGSOC) is unknown. We detected the percentage of senescent T cells in the peripheral blood and ascites of patients with advanced HGSOC (n = 86) at diagnosis by flow cytometry. Compared with healthy donors, patients with HGSOC exhibited an accumulation of CD28-CD57+ (Tsen) CD8+ T cells in the peripheral blood and ascites. The frequency of Tsen CD8+ T cells in the peripheral blood was positively correlated with age and pretreatment serum CA125 and increased in patients with large volume ascites, whereas the frequency of Tsen CD8+ T cells in ascites was elevated in patients with lymph node metastasis. Patients with Tsen-high in ascites (>19.92%), but not in the peripheral blood, were more likely to be resistant to chemotherapy and had shorter progression-free survival. Tsen CD8+ T cells exhibited common senescence features including increased SA-ß-gal activity, declines in proliferation, loss of CD27 and gain of KLRG-1, and the production of cytokines. In ascites, the percentage of Tsen CD8+ T cells was positively correlated with levels of interleukin-10 and granzyme B. This study suggests the potential of ascitic Tsen CD8+ T cells at diagnosis as a prognostic biomarker in HGSOC.

Transcriptome analysis of arterial and venous circulating miRNAs during hypertension.

Most current circulating miRNA biomarkers are derived from peripheral venous blood, whereas miRNA deregulation in arterial blood in disease conditions has been largely ignored. To explore whether peripheral venous blood miRNAs could represent a bona fide specific miRNA deregulation pattern, we selected hypertension, a disease that is particularly associated with vessels, as the model. Circulating miRNA profiles of arterial and venous blood from spontaneously hypertensive (SHR) rats and their corresponding controls (i.e., WKY rats) were investigated by next-generation miRNA sequencing. Little miRNAs were observed between arterial and venous circulating miRNAs in WKY rats. Interestingly, this number was enhanced in SHR hypertensive rats. Bioinformatical analysis of disease association, enriched target genes and the regulatory transcription factors of these differentially expressed miRNAs implied a potential functional link with cardiovascular disease-related functions. Comparisons between arterial and venous miRNAs in hypertension-versus-control conditions also revealed prominent disease association of circulating miRNAs and their target genes in arteries but not in veins. Moreover, a young non-hypertensive animal model in SHR background (i.e. JSHR) was used as a second control for SHR. Additional transcriptomic analysis and droplet digital PCR validation of arterial and venous deregulated miRNAs among SHR and its two controls (WKY, JSHR) revealed a noticeable consensus of artery-deregulated miRNAs in hypertension and two novel arterial circulating signatures (miR-455-3p and miR-140-3p) of hypertension. These results suggest the necessity of re-evaluating the efficacy of certain venous miRNAs identified in previous studies as potential biomarkers in cardiovascular diseases or a wider disease spectrum.

Transcriptome analysis of common and diverged circulating miRNAs between arterial and venous during aging.

Circulating miRNAs have received extensive attention as non-invasive biomarkers for prediction and diagnosis of disease. However, most samples have been obtained from peripheral venous blood. To evaluate whether peripheral venous miRNAs represent circulating miRNAs from all blood vessels under a given condition, such as aging, we compared the miRNA profiles of venous and arterial plasma between young and aged rats by Illumina next-generation sequencing. The DEseq2 tool was used to obtain differentially-expressed miRNAs. We observed 105 aging-related deregulated miRNAs in vein and 62 in artery, which were highly associated with cell survival and inflammation, respectively. On the other hand, the young and aged groups exhibited a unique arterial-venous bias. There were 54 differentially-expressed miRNAs in the young group and 42 in the aged group; only 8 miRNAs were shared. Further transcriptional factors enrichment analysis found that the shared miRNAs could be partially upregulated by NF-κB and SIRT1. These transcriptional factors could be organ-specific and/or regulated in physiological and aging states as possible causal factors. This study suggested the potential application of circulating miRNAs, which reflect the systematic response to certain conditions, such as aging, and the importance of origin selection for candidate circulating miRNAs.

Compromise or not? A case report of successful treatment of pembrolizumab-induced hepatitis in a patient with non-small cell lung cancer with low-dose methylprednisolone and bicyclol.

Pembrolizumab, an anti-programmed cell death protein 1 (PD-1) antibody, has been shown to improve survival in patients with non-small cell lung cancer (NSCLC) with high expression of programmed death-ligand 1 (PD-L1). Corticosteroids are the mainstay for most high-grade immune-related adverse events (irAEs) such as pembrolizumab-induced hepatitis. However, the dose and duration of corticosteroid therapy are not well defined. The objective of this case report was to describe a new treatment pattern for severe immune checkpoint inhibitor-associated hepatitis. Here, we report the case of a patient with metastatic lung adenocarcinoma who developed grade 3 immunotherapy-induced hepatitis after the first cycle of pembrolizumab. Alanine aminotransferase (ALT) levels peaked at 233 U/L. Hepatitis was alleviated after the administration of methylprednisolone. Therefore, we retreated the patient with pembrolizumab. However, aminotransferase levels increased again after the initiation of low-dose methylprednisolone or the reuse of pembrolizumab. Finally, hepatitis was controlled with low-dose methylprednisolone plus bicyclol, a Chinese hepatoprotective agent. Although the patient had been on low-dose methylprednisolone therapy for about six months, he showed a prompt response. During this period, we also found a dramatic decrease in the neutrophil-lymphocyte ratio (NLR), senescent T cells (CD8+ CD28- CD57+ ), and myeloid-derived suppressor cells (MDSCs) in the peripheral blood of the patient. To our knowledge, this is the first case report of successful management of grade 3 pembrolizumab-induced hepatitis with a combination of low-dose corticosteroids and bicyclol. The durable clinical response and changes in blood biomarkers indicate that low doses of corticosteroids do not compromise the efficacy of immune checkpoint inhibitors (ICIs). Therefore, this case may provide a new treatment pattern for severe immunotherapy-induced hepatitis.

Circular RNA expression in isoproterenol hydrochloride-induced cardiac hypertrophy.

Circular RNA (circRNA) is a novel class of noncoding RNAs, and the roles of circRNAs in the development of cardiac hypertrophy remain to be explored. Here, we investigate the potential roles of circRNAs in cardiac hypertrophy. By circRNA sequencing in left ventricular specimens collected from 8-week-old mice with isoproterenol hydrochloride-induced cardiac hypertrophy, we found 401 out of 3323 total circRNAs were dysregulated in the hypertrophic hearts compared with the controls. Of these, 303 circRNAs were upregulated and 98 were downregulated. Moreover, the GO and KEGG analyses revealed that the majority of parental gene of differentially expressed circRNAs were not only related to biological process such as metabolic process and response to stimulus, but also related to pathway such as circulatory system and cardiovascular diseases. On the other hand, total 1974 miRNAs were predicted to binding to these differentially expressed circRNAs, and the possible target mRNAs of those miRNAs were also predicted and analyzed in terms of functional annotation. Finally, we identified that ANF and miR-23a are downstream targets of circRNA wwp1, suggesting that circRNA wwp1 exerts inhibitory roles of cardiac hypertrophy via down-regulation of ANF and miR-23a, which underlying the potential mechanisms whereby circRNA regulates cardiac hypertrophy.

Myeloid-derived suppressor cells: Roles in the tumor microenvironment and tumor radiotherapy.

Cancer progression is closely related to the tumor microenvironment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, signaling molecules and the extracellular matrix. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can impact the growth and evolution of cancerous cells. One of major cell components in the tumor microenvironment is myeloid-derived suppressor cells (MDSCs), which promote tumor growth and metastasis directly or indirectly by recognizing other immune cells, producing cytokines and exerting their immunosuppression functions. MDSCs have emerged as major regulators of immune responses in cancer and key targets for treating cancer. There are many limitations and side-effect in approaches of conventional cancer therapy, including radiotherapy. It has grown up to be a burgeoning field that a combination of radiotherapy and immunotherapy applied to cancer therapy. Therefore, it is fundamental to explore the immune mechanism in the process of cancer treatment. Here, we reviewed the recent progress of MDSCs in roles of the tumor microenvironment and tumor radiotherapy.

miR-29a attenuates cardiac hypertrophy through inhibition of PPARδ expression.

Although cardiac hypertrophy is widely recognized as a risk factor that leads to cardiac dysfunction and, ultimately, heart failure, the complex mechanisms underlying cardiac hypertrophy remain incompletely characterized. The nuclear receptor peroxisome proliferator-activated receptor δ (PPARδ) is involved in the regulation of cardiac lipid metabolism. Here, we describe a novel PPARδ-dependent molecular cascade involving microRNA-29a (miR-29a) and atrial natriuretic factor (ANF), which is reactivated in cardiac hypertrophy. In addition, we identify a novel role of miR-29a, in which it has a cardioprotective function in isoproterenol hydrochloride-induced cardiac hypertrophy by targeting PPARδ and downregulating ANF. Finally, we provide evidence that miR-29a reduces the isoproterenol hydrochloride-induced cardiac hypertrophy response, thereby underlining the potential clinical relevance of miR-29a in which it may serve as a potent therapeutic target for heart hypertrophy treatment.

Histone methylation in DNA repair and clinical practice: new findings during the past 5-years.

DNA double-strand breaks (DSBs) are highly toxic lesions that can impair cellular homeostasis and genome stability to result in tumorigenesis for inappropriate repair. Although DSBs are repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ), the related mechanisms are still incompletely unclear. Indeed, more and more evidences indicate that the methylation of histone lysine has an important role in choosing the pathways of DNA repair. For example, tri-methylated H3K36 is required for HR repair, while di-methylated H4K20 can recruit 53BP1 for NHEJ repair. Here, we reviewed the recent progress in the molecular mechanisms by which histone methylation functions in DNA double-strand breaks repair (DSBR). The insight into the mechanisms of histone methylation repairing DNA damage will supply important cues for clinical cancer treatment.

MTOPVIB interacts with AtPRD1 and plays important roles in formation of meiotic DNA double-strand breaks in Arabidopsis.

Meiotic recombination is initiated from the formation of DNA double-strand breaks (DSBs). In Arabidopsis, several proteins, such as AtPRD1, AtPRD2, AtPRD3, AtDFO and topoisomerase (Topo) VI-like complex, have been identified as playing important roles in DSB formation. Topo VI-like complex in Arabidopsis may consist of subunit A (Topo VIA: AtSPO11-1 and AtSPO11-2) and subunit B (Topo VIB: MTOPVIB). Little is known about their roles in Arabidopsis DSB formation. Here, we report on the characterization of the MTOPVIB gene using the Arabidopsis mutant alleles mtopVIB-2 and mtopVIB-3, which were defective in DSB formation. mtopVIB-3 exhibited abortion in embryo sac and pollen development, leading to a significant reduction in fertility. The mtopVIB mutations affected the homologous chromosome synapsis and recombination. MTOPVIB could interact with Topo VIA proteins AtSPO11-1 and AtSPO11-2. AtPRD1 interacted directly with Topo VI-like proteins. AtPRD1 also could interact with AtPRD3 and AtDFO. The results indicated that AtPRD1 may act as a bridge protein to interact with AtPRD3 and AtDFO, and interact directly with the Topo VI-like proteins MTOPVIB, AtSPO11-1 and AtSPO11-2 to take part in DSB formation in Arabidopsis.

A Strong Donor-Acceptor System Based on a Metal Chalcogenide Cluster and Porphyrin.

Although great progress has been made for charge transfer (CT) compounds of various organic donor-acceptor systems, no CT compounds containing both inorganic chalcogenide cluster anions and organic porphyrin cations have been reported. Herein, a germanium chalcogenide cluster (Ge4S104-) is chosen as an electron donor and a methylated tetrakis(4-pyridyl)porphyrin (5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin, TMPyP) is selected as an electron acceptor to create chalcogenide cluster-porphyrin CT compounds (TMPyP-Ge4S10)·5H2O (1) and (MnTMPyP-Ge4S10)·13H2O (2). Their crystal structures have been characterized by single-crystal X-ray diffraction. Compound 1 is an ionic CT salt assembled through interion interactions, and compound 2 is a neutral CT dyad formed by metal-ligand axial coordination of the chalcogenide cluster with manganese porphyrin. The strong charge transfer properties are revealed by electronic spectra, theoretical calculations, 1H NMR, and ESR. The CT intensity of the chalcogenide cluster-porphyrin system can be modulated by metalation. The fluorescence and photocurrent response properties of 1 and 2 are related to the CT intensity.

3D Copper Tetrathiafulvalene Redox-Active Network with 8-Fold Interpenetrating Diamond-like Topology.

A tetrathiafulvalene derivative has been incorporated into a diamond-like structure for the first time. The coordination network shows highly unusual 8-fold interpenetration with redox-active and photoelectric properties.