Recent advances in Zn2+ imaging: From organelles to in vivo applications.

Zn2+ is involved in various physiological and pathological processes in living systems. Monitoring the dynamic spatiotemporal changes of Zn2+ levels in organelles, cells, and in vivo is of great importance for the investigation of the physiological and pathological functions of Zn2+. However, this task is quite challenging since Zn2+ in living systems is present at low concentrations and undergoes rapid dynamic changes. In this review, we summarize the design and application of fluorescent probes for Zn2+ imaging in organelles, cells, and live organisms reported over the past two years. We aim to provide inspiration for the design of novel Zn2+ probes for multi-level monitoring and deepen the understanding of Zn2+ biology.

Naringenin prevents pregnancy-induced hypertension via suppression of JAK/STAT3 signalling pathway in mice.

BACKGROUND: Pregnancy-induced hypertension (PIH) is characterized by high blood pressure during pregnancy, which causes perinatal and maternal mortality. Inflammation, oxidative stress and the JAK2/STAT3 signalling pathway have been reported to play critical roles in the pathogenies of PIH. Due to the safety and side effects of current treatments for PIH, searching for new therapeutic agents is urgently needed. Naringenin is a flavonoid with anti-inflammation and anti-oxidation activities. In the current study, the effects of naringenin on PIH were investigated. METHODS: We established the PIH mouse model and administrated naringenin to these mice. The blood pressure, total urine protein, plasma levels of vasodilation converting enzyme (VCE), α-1A adrenergic receptor (α-ADR) and angiotensin, inflammatory cytokines, oxidative stress markers were measured. The protein levels of reactive oxygen species proto-oncogene 1 (ROS1), superoxide dismutase 2 (SOD2), signal transducer and activator of transcription 3 (STAT3), phospho-STAT3, Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1), Janus kinase 2 (JAK2) and phospho-JAK2, in vascular endothelium cells were detected by western blot. RESULTS: Administration of naringenin significantly decreased blood pressure, total urine protein level, plasma levels of VCE, α-ADR and angiotensin in PIH mice. Naringenin decreased serum levels of pro-inflammatory cytokines interleukin (IL)-2, IL-6 and tumour necrosis factor alpha (TNF-α), while increased IL-10. Naringenin decreased serum levels of ROS, endothelin while increased SOD and nitric oxide levels. Western blot analysis showed that naringenin inhibited ROS expression, while increased SOD expression in vascular endothelial cells of mice. In addition, western blot also showed that naringenin inhibited JAK2/STAT3 signalling by suppressing SHP-1 expression in vascular endothelial cells of mice. CONCLUSION: Naringenin suppressed the activation of JAK2/STAT3 signalling pathway and promoted SHP-1 expression, leading to ameliorated hypertension in pregnancy.

Identification of Diagnostic CpG Signatures in Patients with Gestational Diabetes Mellitus via Epigenome-Wide Association Study Integrated with Machine Learning.

BACKGROUND: Gestational diabetes mellitus (GDM) is the most prevalent metabolic disease during pregnancy, but the diagnosis is controversial and lagging partly due to the lack of useful biomarkers. CpG methylation is involved in the development of GDM. However, the specific CpG methylation sites serving as diagnostic biomarkers of GDM remain unclear. Here, we aimed to explore CpG signatures and establish the predicting model for the GDM diagnosis. METHODS: DNA methylation data of GSE88929 and GSE102177 were obtained from the GEO database, followed by the epigenome-wide association study (EWAS). GO and KEGG pathway analyses were performed by using the clusterProfiler package of R. The PPI network was constructed in the STRING database and Cytoscape software. The SVM model was established, in which the ß-values of selected CpG sites were the predictor variable and the occurrence of GDM was the outcome variable. RESULTS: We identified 62 significant CpG methylation sites in the GDM samples compared with the control samples. GO and KEGG analyses based on the 62 CpG sites demonstrated that several essential cellular processes and signaling pathways were enriched in the system. A total of 12 hub genes related to the identified CpG sites were found in the PPI network. The SVM model based on the selected CpGs within the promoter region, including cg00922748, cg05216211, cg05376185, cg06617468, cg17097119, and cg22385669, was established, and the AUC values of the training set and testing set in the model were 0.8138 and 0.7576. The AUC value of the independent validation set of GSE102177 was 0.6667. CONCLUSION: We identified potential diagnostic CpG signatures by EWAS integrated with the SVM model. The SVM model based on the identified 6 CpG sites reliably predicted the GDM occurrence, contributing to the diagnosis of GDM. Our finding provides new insights into the cross-application of EWAS and machine learning in GDM investigation.

Glycyrrhizin potentially suppresses the inflammatory response in preeclampsia rat model.

OBJECTIVE: The expression of high-mobility group box 1 (HMGB1) in trophoblasts is elevated, which contributes to the development of preeclampsia. Thus, this study aimed to investigate the effect of glycyrrhizin, a natural HMGB1 inhibitor, on the development of preeclampsia. METHODS: Preeclampsia was induced in pregnant Lewis rats through oral administration of L-NAME (50 mg/kg/day) on gestational day (GD) 13-19. Glycyrrhizin (10, 30, or 60 mg/kg/day) was given by oral gavage on GD 10-19. Systolic blood pressure (SBP), diastolic blood pressure (DBP), 24-hour proteinuria, live pup birth ratio, pup weight, pup body length, and placental weight were measured. Also, the expression levels of inflammatory factors (TNF-α, iNOS, IL-1, and IL-6), HMGB1, and TLR4 in the placenta or in the serum were analyzed by enzyme-linked immunosorbent assay, RT-PCR, and Western blot analysis. RESULTS: Glycyrrhizin significantly reduced the SBP, DBP, and 24-hour proteinuria on GD 16 and 20 in a dose-dependent manner and ameliorated the pregnancy outcomes in preeclampsia rats. The elevated inflammatory molecule levels were markedly decreased by glycyrrhizin not only in the serum but also in the placenta. Moreover, the upregulated HMGB1 and TLR4 expression levels were diminished by glycyrrhizin administration. CONCLUSION: This study shows that glycyrrhizin could alleviate preeclampsia and the preeclampsia-associated inflammatory reaction, and this effect could be attributed to HMGB1 inhibition.

The bioflavonoid troxerutin prevents gestational hypertension in mice by inhibiting STAT3 signaling.

Gestational hypertension is a high-risk disease for women, and the current treatments have limited efficacies. Here, we aimed to evaluate troxerutin, which is a natural monomer of flavone, in the treatment of gestational hypertension. Pregnant mice with or without pregnancy-induced hypertension (PIH) were treated with troxerutin (20 and 40 mg/kg) or vehicle. Blood pressure and proteinuria were monitored during treatment. The expression of vasodilation converting enzyme (VCE), angiotensin, TNFα, IL-6, IL-1ß and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was assessed by measuring the reactive oxygen species (ROS) levels and antioxidant enzyme concentrations. Western blot analysis was used to assess the expression of p-STAT3, STAT3, SHP-1, and RNF6. Troxerutin reduced blood pressure and the expression of VCE, angiotensin, urinary protein and pro-inflammatory cytokines in a dose-dependent manner while increasing the expression of anti-inflammatory cytokines. The levels of ROS were decreased, and the levels of antioxidant enzymes were increased. Troxerutin treatment significantly suppressed STAT3/RNF6 signaling. Overexpression of RNF6 attenuated the effects of troxerutin in ameliorating inflammation and oxidative stress. Our data support the use of troxerutin for reducing gestational hypertension due to the role of troxerutin in reducing inflammation and oxidative stress.

Regulative effect of maternal serum fatty acid-binding protein 4 on insulin resistance and the development of gestational diabetes mellitus.

Fatty acid binding protein 4 (FABP4) was found to be closely correlated with gestational diabetes mellitus (GDM), a severe pregnancy syndrome. However, safe and efficient treatment for GDM is limited. We aimed to investigate whether inhibition of FABP4 could ameliorate GDM and the underlying mechanism. An evaluation of blood samples from a total of 109 patients showed significantly positive correlations between serum FABP4 and biochemical parameters known to associate with GDM. This correlation was subsequently explored in vitro. FABP4 inhibition was achieved using BMS309403 in GDM mice. GDM related symptoms, including insulin resistance and macrophage infiltration in the adipose tissues, were measured. Lipid metabolism in 3T3-L1 adipocytes was tested. We firstly confirmed the positive correlations between serum FABP4, insulin resistance and inflammation cytokines, including tumor necrosis factor-α (TNF) and interleukin-6 (IL-6), in GDM patients. Surprisingly, inhibition of FABP4 by BMS309403 resulted in significant alleviation of GDM symptoms in GDM mouse model. BMS309403 improved glucose and insulin tolerance and transcriptionally repressed the levels of TNF-α and IL-6, suggesting a role of FABP4 in inflammation. Furthermore, macrophage infiltration into the adipose tissues was dramatically decreased in the BMS309403-treated GDM mice compared to untreated GDM mice. Interestingly, incubation of 3T3-L1 adipocytes with FABP4 protein decreased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), which was absent when BMS309403 was used. However, lipid accumulation was promoted in FABP4-treated 3T3-L1 adipocytes which showed no change in the presence of BMS309403. In conclusion, inhibition of FABP4 by BMS309403 could be an effective treatment to alleviate GDM.

Chemoprophylaxis, diagnosis, treatments, and discharge management of COVID-19: An evidence-based clinical practice guideline (updated version).

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a rapidly spreading illness, coronavirus disease 2019 (COVID-19), affecting more than seventeen million people around the world. Diagnosis and treatment guidelines for clinicians caring for patients are needed. In the early stage, we have issued "A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia (standard version)"; now there are many direct evidences emerged and may change some of previous recommendations and it is ripe for develop an evidence-based guideline. We formed a working group of clinical experts and methodologists. The steering group members proposed 29 questions that are relevant to the management of COVID-19 covering the following areas: chemoprophylaxis, diagnosis, treatments, and discharge management. We searched the literature for direct evidence on the management of COVID-19, and assessed its certainty generated recommendations using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Recommendations were either strong or weak, or in the form of ungraded consensus-based statement. Finally, we issued 34 statements. Among them, 6 were strong recommendations for, 14 were weak recommendations for, 3 were weak recommendations against and 11 were ungraded consensus-based statement. They covered topics of chemoprophylaxis (including agents and Traditional Chinese Medicine (TCM) agents), diagnosis (including clinical manifestations, reverse transcription-polymerase chain reaction (RT-PCR), respiratory tract specimens, IgM and IgG antibody tests, chest computed tomography, chest x-ray, and CT features of asymptomatic infections), treatments (including lopinavir-ritonavir, umifenovir, favipiravir, interferon, remdesivir, combination of antiviral drugs, hydroxychloroquine/chloroquine, interleukin-6 inhibitors, interleukin-1 inhibitors, glucocorticoid, qingfei paidu decoction, lianhua qingwen granules/capsules, convalescent plasma, lung transplantation, invasive or noninvasive ventilation, and extracorporeal membrane oxygenation (ECMO)), and discharge management (including discharge criteria and management plan in patients whose RT-PCR retesting shows SARS-CoV-2 positive after discharge). We also created two figures of these recommendations for the implementation purpose. We hope these recommendations can help support healthcare workers caring for COVID-19 patients.

Chemoprophylaxis, diagnosis, treatments, and discharge management of COVID-19: an evidence-based clinical practice guideline (updated version)

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a rapidly spreading illness, coronavirus disease 2019 (COVID-19), affecting more than seventeen million people around the world. Diagnosis and treatment guidelines for clinicians caring for patients are needed. In the early stage, we have issued "A rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-nCoV) infected pneumonia (standard version)"; now there are many direct evidences emerged and may change some of previous recommendations and it is ripe for develop an evidence-based guideline. We formed a working group of clinical experts and methodologists. The steering group members proposed 29 questions that are relevant to the management of COVID-19 covering the following areas: chemoprophylaxis, diagnosis, treatments, and discharge management. We searched the literature for direct evidence on the management of COVID-19, and assessed its certainty generated recommendations using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Recommendations were either strong or weak, or in the form of ungraded consensus-based statement. Finally, we issued 34 statements. Among them, 6 were strong recommendations for, 14 were weak recommendations for, 3 were weak recommendations against and 11 were ungraded consensus-based statement. They covered topics of chemoprophylaxis (including agents and Traditional Chinese Medicine (TCM) agents), diagnosis (including clinical manifestations, reverse transcription-polymerase chain reaction (RT-PCR), respiratory tract specimens, IgM and IgG antibody tests, chest computed tomography, chest x-ray, and CT features of asymptomatic infections), treatments (including lopinavir-ritonavir, umifenovir, favipiravir, interferon, remdesivir, combination of antiviral drugs, hydroxychloroquine/chloroquine, interleukin-6 inhibitors, interleukin-1 inhibitors, glucocorticoid, qingfei paidu decoction, lianhua qingwen granules/capsules, convalescent plasma, lung transplantation, invasive or noninvasive ventilation, and extracorporeal membrane oxygenation (ECMO)), and discharge management (including discharge criteria and management plan in patients whose RT-PCR retesting shows SARS-CoV-2 positive after discharge). We also created two figures of these recommendations for the implementation purpose. We hope these recommendations can help support healthcare workers caring for COVID19 patients

Series of Mitochondria/Lysosomes Self-Targetable Near-Infrared Hemicyanine Dyes for Viscosity Detection.

Six mitochondria/lysosomes self-targetable and viscosity-sensitive dyes (1a-1f) were developed via simple structure modification on cyanine-derived dyes. They all showed remarkable OFF-ON fluorescent response to viscosity in the near-infrared region (652-690 nm) and exhibited good linear relationship with solution viscosity. The transient absorption spectra were used to evaluate the excited-state lifetime of dye 1a in different viscosity environments. Furthermore, cellular imaging assays indicated that different derivatives (1a-1f) with the same chromophore core exhibited different organelle-targeting abilities. Among them, dyes 1a-1c could sense lysosomal viscosity fluctuations while dyes 1d-1f could be applied in mitochondrial viscosity detections.

A novel xanthylene-based effective mitochondria-targeting ratiometric cysteine probe and its bioimaging in living cells.

In this study, a mitochondria-specific fluorescent probe for efficient ratiometric detection of Cys was designed and investigated. Probe 1 is composed of a xanthylene skeleton and a benzyl group containing an acryloyl moiety. The probe showed excellent water solubility, good selectivity and sensitivity toward Cys over other analytes, and afforded an extremely low detection limit of 33.7 nM. The possible detection mechanism was ascertained by HRMS analysis. Moreover, probe 1 had excellent mitochondrial-targeting ability (the Pearson's correlation coefficient was 0.96), and was capable of monitoring endogenous Cys in living HeLa cells by dual channel ratiometric bioimaging, demonstrating its significant potential in biological applications.

Benzoxazine-based fluorescent probes with different auxochrome groups for cysteine detection.

Three 5H-benzo[a]phenoxazin-5-one-based (benzoresorufin and nile-red) Cysteine (Cys) detection probes have been comparatively designed and synthesized in this paper. The optical experiments exhibit probe 1b with a crotonoyl group has no response toward Cys; while probes 1a and 1c have the same reaction site (acryloyl group), their optical responses to Cys are quite different. The benzoresorufin-based-probe 1a shows a turn-on fluorescence response (118-fold) to Cys at 631 nm and affords a very low detection limit (DL = 19.8 nM). Compared with probe 1a, the nile-red-based probe 1c displays gradually diminishing fluorescence intensity with increased Cys concentration at 665 nm. And the notable different fluorescence response mechanisms of probes 1a and 1c toward Cys can be interpreted by HRMS and time-dependent density functional theorety (TDDFT) calculations. Furthermore, both of the two probes indicate high sensitivity and selectivity toward Cys over other similar structured amino acids including homocysteine (Hcy) and glutathione (GSH). Further cellular applications of the two probes have been successfully performed in HeLa cells.

A Pilot Study of Parameter-Optimized Low-Intensity Pulsed Ultrasound Stimulation for the Bone Marrow Mesenchymal Stem Cells Viability Improvement.

To investigate how a back propagation neural network based on genetic algorithm (GA-BPNN) optimizes the low-intensity pulsed ultrasound (LIPUS) stimulation parameters to improve the bone marrow mesenchymal stem cells (BMSCs) viability further. The LIPUS parameters were set at various frequencies (0.6, 0.8, 1.0, and 1.2 MHz), voltages (5, 6, 7, and 8 V), and stimulation durations (3, 6, and 9 minutes). As only some discrete points can be set up in the experiments, the optimal LIPUS stimulation parameter may not be in the value of these settings. The GA-BPNN algorithm is used to optimize parameters of LIPUS to increase the BMSCs viability further. The BMSCs viability of the LIPUS-treated group was improved up to 19.57% (P < 0.01). With the optimization via the GA-BPNN algorithm, the viability of BMSCs was further improved by about 5.36% (P < 0.01) under the optimized condition of 6.92 V, 1.02 MHz, and 7.3 min. LIPUS is able to improve the BMSCs viability, which can be improved further by LIPUS with parameter optimization via GA-BPNN algorithm.

Single nucleotide polymorphism rs3746444 in miR­499a affects susceptibility to non­small cell lung carcinoma by regulating the expression of CD200.

The present study evaluated the association between single nucleotide polymorphism (SNP) rs3746444 and the risk of non­small cell lung carcinoma (NSCLC) in a Chinese population. Computational analyses and luciferase assays were performed to investigate the regulatory relationship between miR­499a and CD200. In addition, reverse transcription­quantitative polymerase chain reaction and western blot assays were performed to examine the effect of rs3746444 on the expression of miR­499a and CD200. The results demonstrated a significant difference in the smoking history of patients carrying malignant pulmonary nodules and those carrying benign pulmonary nodules. Furthermore, CD200 was demonstrated to be a direct target of miR­499a, and a miR­499a binding site was located in the 3'UTR of CD200. Notably, the levels of miR­499a in malignant pulmonary nodules were higher compared with benign pulmonary nodules, while the levels of CD200 were higher in benign pulmonary nodules compared with malignant pulmonary nodules. In addition, the subjects carrying the AA genotype of SNP rs3746444 exhibited upregulated miR­499a expression and reduced CD200 expression, compared with the subjects carrying AG and GG genotypes. These findings indicate that the SNP rs3746444 in miR­499a could affect the prognosis of NSCLC patients by regulating the expression of CD200.

Totally robotic-assisted non-circumferential tracheal resection and anastomosis for leiomyoma in an elderly female.

We describe a novel technique of totally robotic-assisted non-circumferential tracheal resection and running anastomosis with coverage of anastomosis with anterior mediastinal fat flap. A 71-year-old female presented with cough and CT scan revealed a mass at the intra-thoracic trachea. A complete robotic-assisted tracheal resection and anastomosis was performed. The postoperative course was uneventful. The final pathologic examination confirmed the diagnosis of primary tracheal leiomyoma.

Molecular target characterization and antimyeloma activity of the novel, insulin-like growth factor 1 receptor inhibitor, GTx-134.

PURPOSE: Therapeutic strategies that target insulin-like growth factor 1 receptor (IGF-1R) hold promise in a wide variety of cancers including multiple myeloma (MM). In this study, we describe GTx-134, a novel small-molecule inhibitor of IGF-1R and insulin receptor (IR) and characterized its antitumor activity in preclinical models of MM. EXPERIMENTAL DESIGN: The activity of GTx-134 as a single agent and in combination was tested in MM cell lines and primary patient samples. Downstream effector proteins and correlation with apoptosis was evaluated. Cytotoxcity in bone marrow stroma coculture experiments was assessed. Finally, the in vivo efficacy was evaluated in a human myeloma xenograft model. RESULTS: GTx-134 inhibited the growth of 10 of 14 myeloma cell lines (<5 µmol/L) and induced apoptosis. Sensitivity to GTx-134 correlated with IGF-1R signal inhibition. Expression of MDR-1 and CD45 were associated with resistance to GTx-134. Coculture with insulin-growth factor-1 (IGF-1) or adherence to bone marrow stroma conferred modest resistance, but did not overcome GTx-134-induced cytotoxicity. GTx-134 showed in vitro synergies when combined with dexamethasone or lenalidomide. Further, GTx-134 enhanced the activity of PD173074, a fibroblast growth factor receptor 3 (FGFR3) inhibitor, against t(4;14) myeloma cells. Therapeutic efficacy of GTx-134 was shown against primary cells and xenograft tumors. Although dysregulation of glucose homeostasis was observed in GTx-134-treated mice, impairment of glucose tolerance was modest. CONCLUSIONS: These studies support the potential therapeutic efficacy of GTx-134 in MM. Further, they provide a rationale for clinical application in combination with established antimyeloma treatments and novel targeted therapies.