A negative feedback loop centered on SMAD3 expression in transforming growth factor ß1-induced corneal myofibroblast differentiation.

SMAD3 downregulation is documented in transforming growth factor ß1 (TGF-ß1)-induced corneal fibroblasts differentiation to myofibroblasts ("fibroTOmyoDiff") or corneal wound healing. However, the exact regulatory mechanism of TGF-ß1/SMAD3 pathway in this context remains unclear. Here, we investigated the role and related mechanism of SMAD3 down-regulation in TGF-ß1-induced human corneal fibroTOmyoDiff. By detecting expression changes of SMAD family during this process, we demonstrated that SMAD3 protein expression was dramatically decreased in the process and the decrease occurred mainly in SMAD3 gene transcription. Furthermore, SMAD3 overexpression using lentivirus infection and knockdown using sgRNA lentivirus infection or siRNAs revealed that SMAD3 overexpression enhanced TGF-ß1-induced corneal fibroTOmyoDiff and vice versa. In addition, specific siRNAs and inhibitors targeting particular signaling pathway were used to figure out the intracellular signaling pathway regulating SMAD3, and the result showed that the decease of SMAD3 induced by TGF-ß1 stimulation in human corneal fibroblasts (HCFs) was strikingly prevented by SMAD4 knockdown or p38 signaling inhibitor SB203580 treatment. Collectively, these results demonstrate that, in TGF-ß1 induced corneal fibroTOmyoDiff, down-regulation of SMAD3 expression regulated by SMAD4 and p38 signaling pathways forms a negative feedback loop of TGFß signaling to avoid excessive activation of the signaling, which suggest that SMAD3 may be a key target for corneal fibrosis treatment.

MSCs helped reduce scarring in the cornea after fungal infection when combined with anti-fungal treatment.

BACKGROUND: Fungal Keratitis (FK) is an infective keratopathy with extremely high blindness rate. The damaging effect of this disease is not only the destruction of corneal tissue during fungal infection, but also the cornea scar formed during the healing period after infection control, which can also seriously affect a patient's vision. The purpose of the study was to observe the effect of umbilical cord mesenchymal stem cells (uMSCs) on corneal scar formation in FK. METHODS: The FK mouse model was made according to a previously reported method. Natamycin eye drops were used for antifungal treatment 24 h after modeling. There are four groups involved in the study, including control group, FK group, vehicleinj FK group and uMSCsinj FK group. Mice in uMSCsinj FK group received repeated subconjunctival injections of uMSCs for 3 times at the 1d, 4d and 7d after FK modeling. At 14d, 21d and 28d after trauma, clinical observation, histological examination, second harmonic generation and molecular assays were performed. RESULTS: The uMSCs topical administration reduced corneal scar formation area and corneal opacity, accompanying with decreased corneal thickness and inflammatory cell infiltration, following down-regulated fibrotic-related factors α-SMA, TGFß1, CTGF, and COLI and finally inhibited phosphorylation of TGFß1/Smad2 signaling pathway during FK corneal fibrosis. CONCLUSION: The results confirmed that uMSCs can improve corneal opacity during the scar formation stage of FK, and exert anti-inflammatory and anti-fibrotic effects.

Multi­omics approach to improve patient­tailored therapy using immune checkpoint blockade and cytokine­induced killer cell infusion in an elderly patient with lung cancer: A case report and literature review.

The 5-year survival rate of patients with advanced non-small cell lung cancer (NSCLC) remains low, despite recent advances in targeted therapy and immunotherapy. Therefore, there is a need to identify alternative strategies to improve treatment outcomes. Modern diagnostics can significantly facilitate the selection of treatment plans to improve patient outcomes. In the present study, multi-form diagnostic methodologies were adopted, including next-generation sequencing-based actionable gene sequencing, programmed death ligand 1 (PD-L1) immunohistochemistry, a circulating tumor cell (CTC) assay, flow cytometric analysis of lymphocyte subsets and computed tomography, to improve disease management in an 86-year-old female patient with relapsed metastatic NSCLC. High expression of PD-L1, elevated CTC tmutations, were observed. Based on these results, the patient was initially treated with the programmed death protein 1 blocking antibody sintilimab for two cycles, resulting in the stabilization of their condition, although the patient still exhibited severe pain and other symptoms, including fatigue, malaise, a loss of appetite and poor mental state. Informed by dynamic monitoring of the patient's response to treatment, the treatment plan was subsequently adjusted to a combination therapy with sintilimab and autologous cytokine-induced killer cell infusion, which eventually led to improved outcomes in both the management of the cancer and quality of life. In conclusion, multi-omics analysis may be used to establish patient-tailored therapies to improve clinical outcomes in hard-to-treat elderly patients with metastatic NSCLC.

Efficient Synthesis and Evaluation of Novel Sulfonated Dihydropyrimidinthione Derivatives for the Treatment of UV-B-Induced Corneal Damage.

UV-B-induced corneal damage remains a challenge in clinics, and it is needed to develop novel and effective medicines against UV-B induced photodamage. 3,4-Dihydropyrimidine-2(1H)-thione derivatives have shown many interesting biological activities, including antibacterial, anti-inflammatory, antioxidant, etc. In order to find a promising anticorneal photodamage agent, we designed and synthesized two novel sulfonated dihydropyrimidinthione derivatives to evaluate cytoprotective effect against UV-B-mediated photodamage. With simple structure, compound 6 possessed good water solubility, photostability and biocompatibility. We demonstrated that 6 exhibited significant cytoprotective effects against UV-B-mediated photodamage and the cell viability was up to 93% at 0.2 mg mL-1 . The corneal cells were highly sensitive to UV-B radiation, resulting in the release of inflammatory mediators and DNA damage, which were significantly reversed by 6. Moreover, compound 6 reduced Bax and cleaved Caspase-3 expressions to suppress UV-B mediated the intrinsic apoptosis pathway. Our findings suggest that 6 is a promising UV-B resistant agent with potential to be a promising drug candidate for the treatment of corneal photodamage.

The clinical significance of circulating tumor cells and T lymphocyte subtypes in pancreatic cancer patients.

Circulating tumor cells (CTCs) are sensitive and reliable biomarkers for tracing relapsed and metastatic cancer. Here, we explore the clinical significance of CTCs and T lymphocyte subtypes in patients with pancreatic cancer. A total of 106 patients with the pancreatic cancer were enrolled in this study. The enrichment and identification of CTCs were achieved before treatment by a PatrolCTC detection technique. Flow cytometry (FACS) was used to characterize CD4, CD8, natural killer (NK) cells, and Tregulatory (Treg) lymphocyte subtypes. Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-17A (IL-17A), Interleukin-10 (IL-10), and Interferon γ (IFN-γ) were measured by meso-scale discovery (MSD) assay. Among these patients, 44 (41.5%) patients with pancreatic ductal adenocarcinoma (PDAC) were female and 62 (58.5%) cases were male. Case numbers with II-IV tumor-node-metastasis (TNM) stages were 32 (30.2%), 50 (47.2%), and 24 (22.6%), respectively. The positive rate of CTCs before surgery was 37.5% (12/32), 88.0% (44/50) and 100% (24/24) in stage II, III, and IV patients, respectively. Total CTCs, mixed CTCs, and mesenchymal CTCs (MCTCs) were strongly relevant to shorter progression-free survival (PFS) of the patients. In addition, total CTCs (≥6) and positive MCTCs were also significantly correlated with recurrence and metastasis. The patients with high CTCs also had low levels of CD4, CD4/CD8 ratio, NK cells, IL-2, and IFNγ. In contrast, Treg cells had significant elevation in PDAC patients. These results indicated that CTCs number in PDAC patients was an independent indicator for worse PFS. High CTCs also had strong correlation with weak cellular immunity functions.

A bifunctional fluorescent probe based on "AND logic" for the simultaneous recognition of H2S/HNO and its bioimaging applications.

The reaction of NO and H2S to form HNO is a classical pathway in physiological conditions. The reported single recognition-type fluorescent probes are difficult to track precisely the relationships of H2S and HNO. It is necessary to develop a bifunctional fluorescence probe (NJA) for monitoring simultaneously the production of endogenous HNO and H2S. Using 7-Nitrobenzofurazan (NBD) and 2-(diphenylphosphine) benzoate as recognition sites, the obatined NJA can detect specifically HS- and HNO. The detection limit of HS- and HNO are 0.46 µM and 1.42 µM, respectively. Based on the dual recognition sites and input signals of the probe, a molecular "AND" logic gate was established to detect successfully H2S and HNO in MCF-7 cells. NJA based on "AND logic" provided a simple and robust tool for monitoring the production of endogenous HNO correlative with H2S and NO in living cells.

A bifunctional fluorescent probe based on PET & ICT for simultaneously recognizing Cys and H2S in living cells.

Most reported probes that respond to Cysteine (Cys) and Hydrogen sulfide (H2S) can only identify one analyte, or they were interfered with homocysteine (Hcy) and glutathione (GSH) when recognizing Cys and H2S. In addition, nitrobenzoxadiazole (NBD) ether, as one of thiols recognition sites, inevitably encounters the situation that Cys, GSH and H2S cannot be distinguished on the same channel at the cellular level. In this work, by introducing NBD ether and NBD amine, we constructed a bifunctional fluorescent probe NJB for dual-site response to Cys and H2S via PET & ICT processes. NJB has wonderful selectivity for identifying Cys and HS-, with limits of detection as low as 58.4 nM and 81.1 nM, respectively. Interestingly, NJB has been successfully applied to detect Cys and HS- in MCF-7 cells. Therefore, the probe that serves as a great tool for inquiring the physiological and pathological functions of Cys and H2S in living cells is promising.

Multi-mtDNA Variants May Be a Factor Contributing to Mitochondrial Function Variety in the Skin-Derived Fibroblasts of Leber's Hereditary Optic Neuropathy Patients.

Heterogeneity is a major feature of Leber's hereditary optic neuropathy (LHON) and has a significant impact on the manifestation and diagnosis of the disease. This study explored whether multiple variations in mitochondrial genes were associated with the heterogeneity, mainly phenotypic heterogeneity. Ophthalmic examinations were conducted in two probands with LHON with G11778A and multiple mitochondrial DNA gene (mtDNA) variants. Skin fibroblast cell lines were generated from patients and age- and sex-matched controls. ROS levels, mitochondrial membrane potential, cell energy respiration, and metabolic functions were measured. Flow cytometry and cell viability tests were performed to evaluate the cell apoptosis levels and fate. We found that cells with more mtDNA variants had higher ROS levels, lower mitochondrial membrane potential, and weaker respiratory function. Flow cytometry and cell viability testing showed that multiple mtDNA variants are associated with different levels of cell viability and apoptosis. In conclusion, we found that skin-derived fibroblast cells from G11778A LHON patients could be used as models for LHON research. Multi-mtDNA variants contribute to mitochondrial function variety, which may be associated with heterogeneity in patients with LHON.

Association of Clinical Features of Colorectal Cancer with Circulating Tumor Cells and Systemic Inflammatory Markers.

Background: Circulating tumor cells (CTCs) in peripheral blood have been shown to reflect the prognosis of patients with colorectal cancer, and epithelial and mesenchymal markers further predict the likelihood of cancer dissemination. This study was conducted to identify possible association of clinical features of colorectal cancer with CTC counts, their subtypes, and systemic inflammatory markers. Methods: Blood samples of 316 colorectal cancer patients were used for CTC detection and subtyping with EpCAM, CK8/18/19, vimentin, and twist as biomarkers. The neutrophil/lymphocyte ratio, platelet/lymphocyte ratio, C-reactive protein/albumin ratio, lymphocyte/monocyte ratio, and systemic immune-inflammation index (SII) were also measured. The relationship between clinical data and these markers or parameters was analyzed. Results: Total CTC counts were correlated with whether there was lymph node involvement but was not correlated with TNM staging. There was a difference in mesenchymal CTCs between patients with and without lymph node involvement (P < 0.05). Also, more patients with metastasis tested positive for mesenchymal CTCs (P < 0.05). Of the systemic inflammatory markers, platelet/lymphocyte ratio was positively correlated with CTC counts (P < 0.01), and lymphocyte/monocyte ratio was negatively correlated with CTC counts (P < 0.05). Conclusions: Colorectal cancer patients with the mesenchymal markers on their CTCs are more likely to have lymph node involvement or distant metastasis than those without these markers.

Identification of Critical Pathways and Potential Key Genes in Poorly Differentiated Pancreatic Adenocarcinoma.

INTRODUCTION: The poorly differentiated pancreatic adenocarcinoma (PDAC) is an extremely lethal neoplasm without effective biomarkers for early detection and prognosis prediction, which is characteristically unresponsive to chemotherapeutic regimens. This study aims at searching for key genes which could be applied as novel prognostic biomarkers and therapeutic targets in PDAC. METHODS: Clinical samples were collected and a comprehensive differential analysis of seven PDAC samples by integrating RNA-seq data of tumor tissues and matched normal tissues from both our cohort and gene expression profiling interactive analysis (GEPIA) were performed to discover potential prognostic genes in PDAC. Pathway enrichment analysis was carried out to determine the biological function of PDAC differentially expressed genes (DEGs), and protein-protein interaction (PPI) network was constructed for functional modules analysis. Real-time PCR was performed to validate expression of hub genes. RESULTS: A total of 126 PDAC-specific expressed genes identified from seven PDAC samples were predominantly enriched in cell adhesion, integral component of membrane, signal transduction and chemical carcinogenesis, IL-17 signaling pathway, indicating that obtained genes might play a unique role in PDAC tumorigenesis. Furthermore, survival analysis revealed that five genes (CEACAM5, KRT6A, KRT6B, KRT7, KRT17) which exhibited high expression levels in tumor tissues were obviously correlated with the prognosis of PDAC patients and KRT7 was positively correlated with KRT6A, KRT6B, KRT17 expression. In addition, real-time PCR demonstrated that the expression level of the hub genes was consistent with RNA-seq analysis. DISCUSSION: The current study suggested that CEACAM5, KRT6A, KRT6B, KRT7, and KRT17 may represent novel prognostic biomarkers as well as novel therapeutic targets for poorly differentiated PDAC.

Cytoprotective Effects of Water Soluble Dihydropyrimidinthione Derivative Against UV-B Induced Human Corneal Epithelial Cell Photodamage.

Excessive UV-B exposure is well known to be a risk factor for corneal phototoxicity including direct DNA damage and disturbances in the antioxidant balance. Here, we showed a successful synthesis of a water-soluble and biocompatible small molecule DHPM 1 with dihydropyrimidinthione skeleton, which could effectively protect human corneal epithelial (HCE-2) cells from UV-B damage. In separate experiments, DHPM 1 absorbed UV-B rays and exhibited scavenging activity against intracellular ROS induced by UV-B radiation, thereby reducing the levels of DNA fragmentation. Additionally, UV-B exposure increased the expression of cleaved caspase-3, as well as the ratio of Bax/Bcl-2 at protein levels, while pretreatment with DHPM 1 significantly reversed these changes. To the best of our knowledge, this is the first report of a study based on dihydropyrimidinthione derivatives to develop a promising eye drops, which may well find extensive applications in UV-B caused corneal damage.

Identification of HDAC6 selective inhibitors: pharmacophore based virtual screening, molecular docking and molecular dynamics simulation.

HDAC6 regulates the expression and activity of various tumor-related proteins, but currently there is no selective inhibitor targeting HDAC6 for clinical application. In order to discover novel HDAC6 inhibitors, virtual screening methods comprised of pharmacophore based virtual screening, molecular docking and molecular dynamics (MD) simulations were employed. 15 molecules were obtained after virtual screening. After in vitro bioassays, two of the hits showed inhibition activity against HDAC6, among which the inhibition activity of G1 to HDAC6 reached 81% at concentration of 20 µM. In addition, the inhibitory activity against HDAC1 and HDAC10 demonstrated that G1 and G10 were highly selective to HDAC6. The analysis of the binding modes of G1 and G10 provides a reference for further development of highly active HDAC6 inhibitors. Communicated by Ramaswamy H. Sarma.

Dehydrins from wheat x Thinopyrum ponticum amphiploid increase salinity and drought tolerance under their own inducible promoters without growth retardation.

Dehydrins confer abiotic stress tolerance in seedlings, but few dehydrins have been studied by transgenic analysis under their own promoters in relation to abiotic stress tolerance. Also the inducible promoters for transgenic engineering are limited. In this study, we isolated from wheat three salt-induced YSK2 dehydrin genes and their promoters. The cDNA sequences were 711, 785, and 932 bp in length, encoding proteins containing 133, 166 and 231 amino acids, respectively, and were named TaDHN1, TaDHN2, and TaDHN3. TaDHN2 doesn't contain introns, while the other two genes each contain one. Semi-quantitative reverse transcription PCR analysis revealed all three dehydrin genes are substantially induced by ABA and NaCl, but only TaDHN2 is induced in seedlings by PEG and by cold (4 °C). Regulatory sequences upstream of the first translation codon (775, 1615 and 889 bp) of the three dehydrin genes were also cloned. Cis-element prediction indicated the presence of ABRE and other abiotic-stress-related elements. Histochemical analysis using GUS expression demonstrated that all three promoters were induced by ABA, cold or NaCl. Ectopic over-expression of TaDHN1 or TaDHN3 in Arabidopsis under their own inducible promoters enhanced NaCl- and drought-stress tolerance without growth retardation.