N6-methyladenosine levels in peripheral blood RNA: a potential diagnostic biomarker for colorectal cancer.

BACKGROUND: N6-methyladenosine (m6A) is dysregulated in various cancers, including colorectal cancer (CRC). Herein, we assess the diagnostic potential of peripheral blood (PB) m6A levels in CRC. METHODS: We collected PB from healthy controls (HCs) and patients with CRC, analyzed PB RNA m6A levels and the expression of m6A-related demethylase genes FTO and ALKBH5, cocultured CRC cells with PB mononuclear cells (PBMCs), and constructed an MC38 cancer model. RESULTS: PB RNA m6A levels were higher in the CRC than that in HCs. The area under the curve (AUC) of m6A levels (0.886) in the CRC was significantly larger compared with carbohydrate antigen 199 (CA199; 0.666) and carcinoembryonic antigen (CEA; 0.834). The combination of CEA and CA199 with PB RNA m6A led to an increase in the AUC (0.935). Compared with HCs, the expression of FTO and ALKBH5 was decreased in the CRC. After coculturing with CRC cells, the PBMCs RNA m6A were significantly increased, whereas the expression of FTO and ALKBH5 decreased. Furthermore, m6A RNA levels in the PB of MC38 cancer models were upregulated, whereas the expression of FTO and ALKBH5 decreased. CONCLUSIONS: PB RNA m6A levels are a potential diagnostic biomarker for patients with CRC.

Heat stress combined with lipopolysaccharide induces pulmonary microvascular endothelial cell glycocalyx inflammatory damage in vitro.

Heat stroke is a life-threatening disease with high mortality and complications. Endothelial glycocalyx (EGCX) is essential for maintaining endothelial cell structure and function as well as preventing the adhesion of inflammatory cells. Potential relationship that underlies the imbalance in inflammation and coagulation remains elusive. Moreover, the role of EGCX in heat stroke-induced organ injury remained unclear. Therefore, the current study aimed to illustrate if EGCX aggravates apoptosis, inflammation, and oxidative damage in human pulmonary microvascular endothelial cells (HPMEC). Heat stress and lipopolysaccharide (LPS) were employed to construct in vitro models to study the changes of glycocalyx structure and function, as well as levels of heparansulfate proteoglycan (HSPG), syndecan-1 (SDC-1), heparansulfate (HS), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, Von Willebrand factor (vWF), endothelin-1 (ET-1), occludin, E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and reactive oxygen species (ROS). Here, we showed that heat stress and LPS devastated EGCX structure, activated EGCX degradation, and triggered oxidative damage and apoptosis in HPMEC. Stimulation of heat stress and LPS decreased expression of HSPG, increased levels of SDC-1 and HS in culture supernatant, promoted the production and release of proinflammation cytokines (TNF-α and IL-6,) and coagulative factors (vWF and ET-1) in HPMEC. Furthermore, Expressions of E-selection, VCAM-1, and ROS were upregulated, while that of occludin was downregulated. These changes could be deteriorated by heparanase, whereas they meliorated by unfractionated heparin. This study indicated that EGCX may contribute to apoptosis and heat stroke-induced coagulopathy, and these effects may have been due to the decrease in the shedding of EGCX.

Comprehensive Analysis of KREMEN2 as an Immunotherapeutic and Prognostic Biomarker in Pan-Cancer.

BACKGROUND/AIM: Kremen2 has been shown to play an important role in multiple cancers formation as a negative regulatory factor in the Wnt signaling pathway. Our study aimed to explore the potential value of KREMEN2 in pan-cancer and investigate the molecular mechanisms associated with tumor development, providing a basis for prognostic factors and new therapeutic targets for cancer. MATERIALS AND METHODS: Raw RNA-seq data for 32 types of cancers were obtained from The Cancer Genome Atlas (TCGA), while Xena database provided overall survival (OS) and progression-free survival (PFI) data for TCGA patients. R language was used to identify the association between KREMEN2 and immune response, tumor mutational burden (TMB), and microsatellite instability (MSI). Gene Set Variation Analysis (GSVA) and Gene Set Enrichment Analysis (GSEA) were conducted in pan-cancer. A Nomogram prediction model and weighted gene co-expression network analysis (WGCNA) were constructed in colorectal cancer (CRC). RESULTS: KREMEN2 was found highly expressed in 17 types of tumor tissues compared to normal tissues. KREMEN2 was only correlated with some tumor pathological stages. KREMEN2 with high expression had poor prognosis in pan-cancer. KREMEN2 expression was significantly associated with immune infiltration, immune checkpoints, immune-related genes, commonly regulated tumor-related genes, TMB, and MSI. Moreover, GSVA and GSEA analyses suggested that KREMEN2 played a role in cell cycle in pan-cancer. KREMEN2 expression had a significant impact on the performance of Nomogram prediction model in CRC, and WGCNA analysis indicated that KREMEN2 performed special functions in CRC. CONCLUSION: The comprehensive pan-cancer analysis revealed that KREMEN2 is a promising tumor prognostic biomarker and a potential anti-tumor immunotherapeutic target in human tumors.

Lkb1 loss in regulatory T cells leads to dysregulation of hematopoietic stem cell expansion and differentiation in bone marrow.

The tumor suppressor Lkb1 is known to regulate the expression of forkhead box P3 (Foxp3), thereby maintaining the levels of Foxp3+ regulatory T cells (Treg) that play a crucial role in self-tolerance. However, the effect of Lkb1 in Treg on hematopoietic stem cells (HSCs) in the bone marrow (BM) remains obscure. Here, we demonstrated that conditional deletion of Lkb1 in Treg causes loss of Treg in the BM, which leads to failure of HSC homeostasis and the abnormal expansion. Moreover, the loss of BM Treg results in dysregulation of other developing progenitors/stem cell populations, leading to the defective differentiation of T cells and B cells. In addition, HSC from the BM with Treg loss exhibited poor engraftment efficiency, indicating that loss of Treg leads to irreversible impairment of HSC. Collectively, these results demonstrated the essential role of Lkb1 in Treg for maintaining HSC homeostasis and differentiation in mice. These findings provide insight into the mechanisms of HSC regulation and guidance for a strategy to improve the outcomes and reduce complications of HSC transplantation.

Recent Research Progress of Organic Small-Molecule Semiconductors with High Electron Mobilities.

Organic electronics has made great progress in the past decades, which is inseparable from the innovative development of organic electronic devices and the diversity of organic semiconductor materials. It is worth mentioning that both of these great advances are inextricably linked to the development of organic high-performance semiconductor materials, especially the representative n-type organic small-molecule semiconductor materials with high electron mobilities. The n-type organic small molecules have the advantages of simple synthesis process, strong intermolecular stacking, tunable molecular structure, and easy to functionalize structures. Furthermore, the n-type semiconductor is a remarkable and important component for constructing complementary logic circuits and p-n heterojunction structures. Therefore, n-type organic semiconductors play an extremely important role in the field of organic electronic materials and are the basis for the industrialization of organic electronic functional devices. This review focuses on the modification strategies of organic small molecules with high electron mobility at molecular level, and discusses in detail the applications of n-type small-molecule semiconductor materials with high mobility in organic field-effect transistors, organic light-emitting transistors, organic photodetectors, and gas sensors.

Anchoring red blood cell with tetrahedral DNA nanostructure: Electrochemical biosensor for the sensitive signage of circulating tumor DNA.

Circulating tumor cells (CTCs), as a type of tumor, have attracted wide attention because of their characteristics of shedding from the primary tumor and spreading to other tissues and organs through peripheral blood. The circulating tumor DNA (ctDNA), the DNA released by CTCs and other tumor cells into the peripheral blood, was considered as a promising detection substance for clinical application. By utilizing the biocompatibility of red blood cells to realize the attachment of tetrahedral DNA (TDN), as well as the specific target recognition ability of TDN to enable efficient recognition of targets, a biocompatible electrochemical biosensor for effective and rapid detection of ctDNA was developed using methylene blue (MB) as the signal probe. The current signal and the logarithm of ctDNA concentration were linearly correlated in the range from 1 fM to 100 pM with the detection limit of 0.66 fM. With high specificity, the TDN-based biosensor can detect ctDNA efficiently in the real biological environment such as serum, which provided a potential opportunity for the early clinical diagnosis.

Causal association between obesity and hypothyroidism: a two-sample bidirectional Mendelian randomization study.

Introduction: Previous observational studies have reported a positive correlation between obesity and susceptibility to hypothyroidism; however, there is limited evidence from alternative methodologies to establish a causal link. Methods: We investigated the causal relationship between obesity and hypothyroidism using a two-sample bidirectional Mendelian randomization (MR) analysis. Single-nucleotide polymorphisms (SNPs) associated with obesity-related traits were extracted from a published genome-wide association study (GWAS) of European individuals. Summarized diagnostic data of hypothyroidism were obtained from the UK Biobank. Primary analyses were conducted using the inverse variance-weighted (IVW) method with a random-effects model as well as three complementary approaches. Sensitivity analyses were performed to ascertain the correlation between obesity and hypothyroidism. Results: MR analyses of the IVW method and the analyses of hypothyroidism/myxedema indicated that body mass index (BMI) and waist circumference (WC) were significantly associated with higher odds and risk of hypothyroidism. Reverse MR analysis demonstrated that a genetic predisposition to hypothyroidism was associated with an increased risk of elevated BMI and WC, which was not observed between WC adjusted for BMI (WCadjBMI) and hypothyroidism. Discussion: Our current study indicates that obesity is a risk factor for hypothyroidism, suggesting that individuals with higher BMI/WC have an increased risk of developing hypothyroidism and indicating the importance of weight loss in reducing the risk of hypothyroidism.

Exosomes from bone marrow mesenchymal stem cells decrease chemosensitivity of acute myeloid leukemia cells via delivering miR-10a.

Bone marrow mesenchymal stem cells (BMSCs) are an integral part of the acute myeloid leukemia (AML) bone marrow microenvironment and contribute to AML progression. In this study, we explored the communication between BMSCs and AML cells via exosomes. The AML cells co-cultured with BMSCs-Exos were found to have lower chemosensitivity exposed to cytarabine, suggesting that BMSCs-Exos could protect AML cells from cytarabine. Interestingly, miR-10a was elevated in BMSCs-Exos derived from AML (AML-BMSCs-Exos) compared with that from healthy donor. The expression levels of miR-10a in AML cells was significantly up-regulated after co-culture with BMSCs-Exos. Furthermore, the up-regulated miR-10a was an crucial factor contributing to the chemoresistance of leukemia cells. Down-regulation of miR-10a substantially increase chemosensitivity of AML cells treated with BMSCs-Exos. Chemosensitivity of AML cells was also decreased through down-regulating RPRD1A by miR-10a that ultimately lead to the stimulation of the Wnt/ß-catenin signaling pathway. Collectively, our findings demonstrated that AML-BMSCs could deliver miR-10a to AML cells via exosomes, which could target RPRD1A and activate Wnt/ß-catenin signaling pathway that subsequently decreased chemosensitivity of AML cells.

Critical Role of Lkb1 in the Maintenance of Alveolar Macrophage Self-Renewal and Immune Homeostasis.

Alveolar macrophages (AMs) are pivotal for maintaining lung immune homeostasis. We demonstrated that deletion of liver kinase b1 (Lkb1) in CD11c+ cells led to greatly reduced AM abundance in the lung due to the impaired self-renewal of AMs but not the impeded pre-AM differentiation. Mice with Lkb1-deficient AMs exhibited deteriorated diseases during airway Staphylococcus aureus (S. aureus) infection and allergic inflammation, with excessive accumulation of neutrophils and more severe lung pathology. Drug-mediated AM depletion experiments in wild type mice indicated a cause for AM reduction in aggravated diseases in Lkb1 conditional knockout mice. Transcriptomic sequencing also revealed that Lkb1 inhibited proinflammatory pathways, including IL-17 signaling and neutrophil migration, which might also contribute to the protective function of Lkb1 in AMs. We thus identified Lkb1 as a pivotal regulator that maintains the self-renewal and immune function of AMs.

ADH2 Is Downregulated by Methylation and Acts as a Novel Biomarker for Breast Carcinoma Prognosis.

Breast carcinoma (BC) ranks the second leading cause of cancer death in females. Alcohol is consistent risk factor for BC. The alcohol dehydrogenases (ADHs) family is associated with alcohol metabolism in vivo. However, whether ADHs can act as biomarkers for BC and the underlying mechanism of them affecting BC are unclear. In the present study, the expression levels, prognostic values, epigenetic and genetic alterations, and regulatory networks of ADHs were explored in BC using public online database. Among ADHs family, the expression level of ADH2 is remarkably decreased in the BC and high expression level of ADH2 is significantly associated with better overall survival in BC. Decreasing mRNA expression level of ADH2 is due to DNA hypermethylation in the promoter rather than genetic alterations. ADH2 strongly correlates with pathways in glycolysis/gluconeogenesis, fatty acid metabolism, and cytochrome P450 pathways in BC. Our finding provided novel insights into ADH2 in BC and implied that ADH2 could act as a novel biomarker for BC prognosis.

Modified Culture System and Combination of FICTION Could Increase the Detection Rate of Abnormalities in Multiple Myeloma.

OBJECTIVE: Conventional karyotyping of multiple myeloma (MM) is hampered by the low mitotic index of plasma cells (PCs), and low proportion of PCs in some specimens may lead to false negative results in fluorescence in situ hybridisation (FISH) detection. METHODS: Bone marrow cells were cultured in an ordinary medium for 24 h or in a medium containing 10 ng/mL IL-6 and 40 ng/mL GM-CSF for 6 d. Fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasms (FICTION) was also conducted, combining CD138 fluorescent immunophenotype and FISH. RESULTS: Under modified culture conditions, the successful rate of culture and abnormality detection rate during karyotype analysis increased to 86.4% and 40.9%, respectively. The abnormality detection rate of FICTION (89.5%) was significantly higher than that of FISH (60.0%). The genetic abnormality detection rate increased to 92.3% when FICTION and karyotyping were conducted under modified culture conditions. CONCLUSION: The established modified culture system could improve karyotyping quality in MM. Due to its obvious advantages compared with FISH, FICTION is recommended for detecting genetic abnormalities in MM.

[Analysis of genetic and clinical characteristics of nine cases of myelodysplastic syndrome with near tetraploid/tetraploidy karyotype].

OBJECTIVE: To explore the genetic and clinical characteristics of near-tetraploidy/tetraploidy karyotype (NT/T) in patients with myelodysplastic syndrome (MDS). METHODS: Cytogenetic findings of 1576 inpatients with primary MDS were retrospective analyzed, among which 9 were diagnosed with NT/T. Clinical data including gender, age, morphology, genetic feature and prognosis were analyzed. RESULTS: The prevalence of MDS patients with NT/T (NT/T-MDS) among all cases was 0.57%. Karyotyping analysis suggested that eight MDS patients had sole NT/T, while the remainder one had a complex karyotype. In addition to the typical morphology of MDS, NT/T-MDS had unique morphology including huge blast, double-nuclear cell and irregular nuclear membrane. One NT/T-MDS patient gave up therapy, and the remaining eight underwent the first course of treatment, albeit with poor prognosis. Only one patient had complete remission, one had partial remission, three had no remission; and three had converted to acute myeloid leukemia. CONCLUSION: NT/T-MDS is rare and has unique morphology. Generally, NT/T-MDS patients have poor prognosis. However, NT/T cannot be simply classified as high-risk group, but with consideration whether they have affected particular chromosomal structures as well as other clinical data.

Twist1 promotes dendritic cell-mediated antitumor immunity.

Dendritic cells (DCs) play a central role in autoimmunity, immune homeostasis, and presentation of tumor antigens to T cells in order to prime antitumor responses. The number of tumor-infiltrating DCs is associated with survival and prognosis in cancer. Twist1 is a well-known regulator of tumor initiation and promotion, but whether and how DC-derived Twist1 regulates antitumor responses remains poorly understood. Here, we generated a mouse line with Twist1 conditionally depleted in DCs and found that Twist1-deficiency in DCs did not affect the DCs and T cell homeostasis under steady-state conditions; however, in melanoma models, the proportion of conventional DCs (cDCs) in draining lymph nodes (DLNs) was significantly decreased. Accordingly, a decreased ratio and number of tumor-infiltrating cDCs were observed, which reduced the recruitment of tumor-infiltrating T cells. Furthermore, production of IFN-γ, a crucial antitumor factor, by T cells, was dramatically decreased, which can further dampen the T cell antitumor functions. Collectively, our data indicate that Twist1 in DCs regulates antitumor functions by maintain the number of tumor-infiltrating DCs and T cells, and their antitumor activity.

Lkb1 is an important regulator of Treg differentiation and proliferation of amniotic mesenchymal stem cells.

In this study, we aimed to explore the role of liver kinase b1 (Lkb1) in the biological characteristics and immune regulation of amniotic mesenchymal stem cells (AMSCs). AMSCs were identified via the cell surface markers using flow cytometry. We knocked down the expression of Lkb1 in AMSCs using lentivirus-mediated Lkb1-specific shRNA. The efficiency of the knockdown was detected by flow cytometry, RT-qPCR, and western blot. The AMSC-related phenotype was determined by flow cytometric analysis via staining surface markers. Fibroblast colony-forming cells (CFU-F) assay and Ki-67 intracellular staining assay were used to determine the proliferative capacity. The differentiated and immunosuppressive capabilities were determined by conditional induction of differentiation and co-culture experiments. We observed that AMSCs along with Lkb1 knockdown (AMSCs-Lkb1) displayed similar cellular morphology and surface antigen expression patterns as those observed in AMSCs. However, AMSCs-Lkb1 exhibited an enhanced differentiation capacity towards osteogenesis and chondrogenesis while it showed defective proliferation and increased apoptosis. Furthermore, AMSCs-Lkb1 showed an enhanced immunosuppressive capacity by directly inhibiting conventional T cells and indirectly inducing production of regulatory T cells (Treg). Interestingly, Treg produced by AMSCs-Lkb1 displayed stronger proliferative capacity as compared to those produced by AMSCs. Our results indicate that Lkb1 plays a vital role in maintaining self-renewal of AMSCs and regulating immune equivalence, and may hold potential for the clinical management of diseases such as GVHD.

Lkb1 in dendritic cells restricts CD8+Foxp3+regulatory T cells expansion in vivo.

Liver kinase B1 (Lkb1) in dendritic cells (DCs) plays a key role in maintaining immunity homeostasis and adaptive immunity by controlling the CD4+Foxp3+T regulatory cell (CD4+Tregs) pool and T cells activation. However, the function of Lkb1 in DCs for the regulation of CD8+Foxp3+T regulatory cells (CD8+Tregs) has not been addressed. Herein, we found that Lkb1-deficient DCs could lead to excessive CD8+Tregs expansion in multiple organs. We found that OX40 expression was significantly higher in Lkb1-deficient DCs compared with that in wild-type (WT) mice, suggesting a potential pathway of CD8+Treg expansion. Moreover, we found that CD8+Tregs from mice with conditional deletion Lkb1 in DCs (KO) displayed an activated phenotype and expressed higher levels of specific markers, including ICOS and CD103. Interestingly, compared with the WT mice without lipopolysaccharide(LPS) treatment, we found that CD8+Tregs population increased in the WT mice with LPS treatment which can selectively delete Lkb1 protein in DCs. However, there was no significant difference in CD8+Tregs population in the KO mice between LPS treatment group and non-LPS treatment. Collectively, our findings identified Lkb1 in DCs as a crucial regulator of CD8+Treg expansion.

Bone mesenchymal stromal cells exhibit functional inhibition but no chromosomal aberrations in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasia characterized by the presence of the Philadelphia (Ph) chromosome in hematopoietic cells (HCs). As one of the most important components of the bone marrow microenvironment (BMM), bone mesenchymal stromal cells (BMSCs) are critical in the development of leukemia and essential in the regulation of hematopoiesis. However, little is known regarding the alterations of BMSCs in CML. The current study performed Cell Counting Kit-8 and colony-forming unit fibroblast assays to evaluate the proliferative ability of BMSCs. The percentage of senescent BMSCs was evaluated by a senescence-associated ß-galactosidase staining assay. Subsequently, a long-term culture-initiating cell assay was designed to explore the HC-supporting capacity of the BMSCs. Furthermore, cytogenetics were detected by conventional cytogenetic analysis and fluorescence in situ hybridization analysis. The current results revealed that CML-BMSCs exhibited decreased cell proliferation and impaired HC-support capacity, as well as increased susceptibility to senescence. No chromosomal aberrations, including the absence of the Ph chromosome, were noted in all CML-BMSCs. In conclusion, the current study demonstrated functional inhibition of CML-BMSCs; however, no signs of chromosomal aberrations were observed, thereby providing insight into the changes occurring in the CML-BMM.

[Preliminary analysis of genetics and clinical features of patients with acute myeloid leukemia and near-tetraploid/tetraploidy karyotype].

OBJECTIVE: To explore the genetic and clinical features of patients with acute myeloid leukemia (AML) and near-tetraploidy/tetraploidy (NT/T) karyotype. METHODS: Cytogenetic findings of 1836 cases of primary AML were retrospectively analyzed. Karyotypes of the identified cases were confirmed by fluorescence in situ hybridization (FISH). Clinical data including gender, age, morphology, immunophenotype, genetics, and prognosis were reviewed. RESULTS: Nine male and two female patients with NT/T were identified with a median age of 63 years. Microscopically, the patients were characterized by large blasts and irregular nuclear contours. All patients expressed CD34, and nine of them expressed HLA-DR. Ten patients had complete remission during the first course of treatment. One patient showed primary drug resistance. CONCLUSION: NT/T AML primarily occurs in elder males and has a characteristic morphology and genetics. The prognosis is better than AML patients with complex karyotypes.

Chromosome Bandings.

Chromosome banding is an essential technique used in chromosome karyotyping to identify normal and abnormal chromosomes for clinical and research purposes. Giemsa (G)-, reverse (R)-, and centromere (C)-banding are the most commonly dye-based chromosome-banding techniques. G-banding involves the staining of trypsin-treated chromosomes and R-banding involves denaturing in hot acidic saline followed by Giemsa staining. C-banding is specifically used for identifying heterochromatin by denaturing chromosomes in a saturated alkaline solution followed by Giemsa staining. Different banding techniques may be selected for the identification of chromosomes.

[Pathogenetic mechanism for a female patient with hemophilia A].

OBJECTIVE: To explore the pathogenetic mechanism for a female patient affected with hemophilia A (HA). METHODS: Potential genetic defect was detected with inverse shifting-polymerase chain reaction (IS-PCR). The pattern of X chromosome inactivation was determined with a human androgen receptor assay (HUMARA assay). G-banded karyotyping was carried out to exclude potential chromosome aberrations. RESULTS: IS-PCR showed that the defect of FVIII gene was the distal type of intron 22 inversion. The HUMARA assay showed that the X chromosome inactivation was non-random, and that the mother's X chromosome activity was lower than that of the father's X chromosome which has carried the inverted FVIII gene. No abnormalities were found with G-banded chromosomes. CONCLUSION: The prevalence of female HA patient may be caused by non-random inactivation of X chromosomes.