Asymmetric synthesis of P-stereogenic phosphindane oxides via kinetic resolution and their biological activity.

The importance of P-stereogenic heterocycles has been widely recognized with their extensive use as privileged chiral ligands and bioactive compounds. The catalytic asymmetric synthesis of P-stereogenic phosphindane derivatives, however, remains a challenging task. Herein, we report a catalytic kinetic resolution of phosphindole oxides via rhodium-catalyzed diastereo- and enantioselective conjugate addition to access enantiopure P-stereogenic phosphindane and phosphindole derivatives. This kinetic resolution method features high efficiency (s factor up to >1057), excellent stereoselectivities (all >20:1 dr, up to >99% ee), and a broad substrate scope. The obtained chiral phosphindane oxides exhibit promising therapeutic efficacy in autosomal dominant polycystic kidney disease (ADPKD), and compound 3az is found to significantly inhibit renal cyst growth both in vitro and in vivo, thus ushering in a promising scaffold for ADPKD drug discovery. This study will not only advance efforts towards the asymmetric synthesis of challenging P-stereogenic heterocycles, but also surely inspire further development of P-stereogenic entities for bioactive small-molecule discovery.

Developing Biarylhemiboronic Esters for Biaryl Atropisomer Synthesis via Dynamic Kinetic Atroposelective Suzuki-Miyaura Cross-Coupling.

We herein introduce biarylhemiboronic esters as a new type of bridged biaryl reagent for asymmetric synthesis of axially chiral biaryl structures, and the palladium-catalyzed asymmetric Suzuki-Miyaura cross-coupling of biarylhemiboronic esters is developed. This dynamic kinetic atroposelective coupling reaction exhibits high enantioselectivity, good functional group tolerance, and a broad substrate scope. The synthetic application of the current method was demonstrated by transformations of the product and a programmed synthesis of chiral polyarene. Preliminary mechanistic studies suggested that the reaction proceeded via an enantio-determining dynamic kinetic atroposelective transmetalation step.

Catalytic asymmetric indolization by a desymmetrizing [3 + 2] annulation strategy.

A new catalytic asymmetric indolization reaction by a desymmetrizing [3 + 2] annulation strategy is developed. The reaction proceeds via a rhodium-catalyzed enantioposition-selective addition/5-exo-trig cyclization/dehydration cascade between ortho-amino arylboronic acids and 2,2-disubstituted cyclopentene-1,3-diones to produce N-unprotected cyclopenta[b]indoles bearing an all-carbon quaternary stereocenter in high yields with good enantioselectivities. A quantitative structure-selectivity relationship (QSSR) model was established to identify the optimal chiral ligand, which effectively controlled the formation of the stereocenter away from the reaction site. Density functional theory (DFT) calculations, non-covalent interaction analysis, and Eyring analysis were performed to understand the key reaction step and the function of the ligand.

Culture of cerebrospinal fluid-contacting neurons from neonatal mouse spinal cord.

Cerebrospinal fluid-contacting neurons (CSF-cNs) act crucial role in chemosensory and mechanosensory function in spinal cord. Recently, CSF-cNs were found to be an immature neuron and may be involved in spinal cord injury recovery. But how to culture it and explore its function in vitro are not reported in previous research. Here, we first reported culture and identification of CSF-cNs in vitro. We first established a protocol for in vitro culture of CSF-cNs from the cervical spinal cord of mice within 24 h after birth. Polycystic kidney disease 2-like 1 (PKD2L1)+ cells were isolated by fluorescence-activated cell sorting and expressed the neuron marker ß-tubulin III and CSF-cNs marker GABA. Intriguingly, PKD2L1+ cells formed neurosphere and expressed neural stem cell markers Nestin, Sox2 and GFAP. Thus, our research provided culture and isolation of CSF-cNs and this facilitate the investigation the CSF-cNs function in vitro.

Silencing Notch4 promotes tumorigenesis and inhibits metastasis of triple-negative breast cancer via Nanog and Cdc42.

Elucidation of individual Notch protein biology in specific cancer is crucial to develop safe, effective, and tumor-selective Notch-targeting therapeutic reagents for clinical use [1]. Here, we explored the Notch4 function in triple-negative breast cancer (TNBC). We found that silencing Notch4 enhanced tumorigenic ability in TNBC cells via upregulating Nanog expression, a pluripotency factor of embryonic stem cells. Intriguingly, silencing Notch4 in TNBC cells suppressed metastasis via downregulating Cdc42 expression, a key molecular for cell polarity formation. Notably, downregulation of Cdc42 expression affected Vimentin distribution, but not Vimentin expression to inhibit EMT shift. Collectively, our results show that silencing Notch4 enhances tumorigenesis and inhibits metastasis in TNBC, indicating that targeting Notch4 may not be a potential strategy for drug discovery in TNBC.

Rh-catalyzed ring-opening coupling of cyclic vinyl ethers with organometallic reagents.

The rhodium-catalyzed ring-opening coupling of cyclic vinyl ethers, including 2,3-dihydrofuran and benzofuran, with organometallic reagents to give homoallylic alcohols and stilbenoids was reported. The suitable organometallic reagent for 2,3-dihydrofuran and benzofuran was found to be substrate-dependent, and a plausible mechanism involving different active organorhodium intermediates was proposed for these coupling reactions.

INTERLEUKIN-35 DOWNREGULATES THE IMMUNE RESPONSE OF EFFECTOR CD4 + T CELLS VIA RESTRICTING HIGH MOBILITY GROUP BOX-1 PROTEIN-DEPENDENT AUTOPHAGY IN SEPSIS.

ABSTRACT: Background: Immunosuppression is critically involved in the development of sepsis and is closely associated with poor outcomes. The novel role of the anti-inflammatory cytokine IL-35 in sepsis was examined. Methods: Sepsis was induced by in C57BL/6 mice cecal ligation and puncture (CLP). The impacts of IL-35 on effector CD4 + T cells were investigated by examining cell proliferation and the Th1/Th2 ratio in the presence of recombinant IL-35 (rIL-35) or anti-IL-35 (EBI3). The regulatory effect of IL-35 on autophagy was evaluated by measuring autophagy markers and autophagic flux in CLP mice in vivo and in activated effector CD4 + T cells in vitro . Results: IL-35 levels were significantly increased in the serum and spleens of septic mice. rIL-35 administration after CLP further decreased proliferation and the Th1/Th2 ratio in effector CD4 + T cells and significantly shortened the survival time. Sepsis-induced autophagy activation was protective in effector CD4 + T cells and was blocked by rIL-35. The inhibitory effect of IL-35 on autophagy was observed in activated effector CD4 + T cells in vitro , and this effect was mediated by restricting high mobility group box-1 protein (HMGB1) translocation. Conclusion: IL-35 is an immunosuppressive cytokine that impairs CD4 + T-cell proliferation and differentiation in sepsis, and the effect might be mediated by reducing HMGB1-dependent autophagy.

Macrophage-derived extracellular vesicles regulate follicular activation and improve ovarian function in old mice by modulating local environment.

In mammals, ovarian function is dependent on the primordial follicle pool and the rate of primordial follicle activation determines a female's reproductive lifespan. Ovarian ageing is characterised by chronic low-grade inflammation with accelerated depletion of primordial follicles and deterioration of oocyte quality. Macrophages (Mφs) play critical roles in multiple aspects of ovarian functions; however, it remains unclear whether Mφs modulate the primordial follicle pool and what is their role in ovarian ageing. Here, by using super- or naturally ovulated mouse models, we demonstrated for the first time that ovulation-induced local inflammation acted as the driver for selective activation of surrounding primordial follicles in each estrous cycle. This finding was related to infiltrating Mφs in ovulatory follicles and the dynamic changes of the two polarised Mφs, M1 and M2 Mφs, during the process. Further studies on newborn ovaries cocultured with different subtypes of Mφs demonstrated the stimulatory effect of M1 Mφs on primordial follicles, whereas M2 Mφs maintained follicles in a dormant state. The underlying mechanism was associated with the differential regulation of the Phosphatidylinositol 3-kinase/Mechanistic target of rapamycin (PI3K/mTOR) signaling pathway through secreted extracellular vesicles (EVs) and the containing specific miRNAs miR-107 (M1 Mφs) and miR-99a-5p (M2 Mφs). In aged mice, the intravenous injection of M2-EVs improved ovarian function and ameliorated the inflammatory microenvironment within the ovary. Thus, based on the anti-ageing effects of M2 Mφs in old mice, M2-EVs may represent a new approach to improve inflammation-related infertility in women.

Patient-derived primary breast cancer cells and their potential for predicting sensitivity to chemotherapy.

Chemotherapy resistance exposes patients to side effects and delays the effect of therapy in patients. So far, there are no predictive tools to predict resistance to chemotherapy and select sensitive chemotherapeutic drugs for the patient. Here, we aim to develop an in-vitro primary cell culture model from breast cancer patients to predict sensitivity to chemotherapy. We created the primary breast cancer cell medium BCMI and culture system with higher efficiency of the model establishment. Immunofluorescence staining of ERa, PR and HER2 were done to identify the primary breast cancer cell from the counterpart breast cancer patient. The killing assay showed that these primary breast cancer cells responded differently to doxorubicin and pirarubicin treatment. These results indicate that our established primary breast cancer cell model holds great promise for predicting breast cancer sensitivity to chemotherapy drugs.

The neural stem cell properties of Pkd2l1+ cerebrospinal fluid-contacting neurons in vivo.

The neural stem cells (NSCs) in the ventricular-subventricular zone of the adult mammalian spinal cord may be of great benefit for repairing spinal cord injuries. However, the sources of NSCs remain unclear. Previously, we have confirmed that cerebrospinal fluid-contacting neurons (CSF-cNs) have NSC potential in vitro. In this study, we verified the NSC properties of CSF-cNs in vivo. In mouse spinal cords, Pkd2l1+ CSF-cNs localized around the central canal express NSC markers. In vitro, Pkd2l1+ CSF-cNs form a neurosphere and express NSC markers. Activation and proliferation of CSF-cNs can be induced by injection of the neurotrophic factors basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) into the lateral ventricle. Spinal cord injury (SCI) also induces NSC activation and proliferation of CSF-cNs. Collectively, our results demonstrate that Pkd2l1+ CSF-cNs have NSC properties in vivo and may be involved in SCI recovery.

Fecal microbiota transplantation from young donor mice improves ovarian function in aged mice.

Advanced maternal age is characterized by declines in the quantity and quality of oocytes in the ovaries, and the aging process is accompanied by changes in gut microbiota composition. However, little is known about the relationship between gut microbiota and ovarian aging. By using fecal microbiota transplantation (FMT) to transplant material from young (5-week-old) into aged (42-week-old) mice, we find that the composition of gut microbiota in FMT-treated mice presents a "younger-like phenotype" and an increase of commensal bacteria, such as Bifidobacterium and Ruminococcaceae. Moreover, the FMT-treated mice show increased anti-inflammatory cytokine IL-4 and decreased pro-inflammatory cytokine IFN-γ. Fertility tests for assessing ovarian function reveal that the first litter size of female FMT-treated mice is significantly higher than that of the non-FMT group. Morphology analysis demonstrates a dramatic decrease in follicle atresia and apoptosis as well as an increase in cellular proliferation in the ovaries of the FMT-treated mice. Our results also show that FMT improves the immune microenvironment in aged ovaries, with decreased macrophages and macrophage-derived multinucleated giant cells (MNGCs). These results suggest that FMT from young donors could be a good choice for delaying ovarian aging.

Rhodium-Catalyzed Chemodivergent Pyridylation of Alkynes with Pyridylboronic Acids.

The pyridylation of alkynes with pyridylboronic acids is realized under rhodium catalysis. Chemodivergent pyridylation products, including alkenylpyridines produced via the hydropyridylation pathway and cyclopenta[c]pyridines produced via the pyridylation/cyclization pathway, were selectively produced by fine-tuning the reaction conditions. A mechanistic study revealed that 1,4-rhodium migration to the pyridine ring was involved as the key step in the chemodivergent synthesis.

Mutational analysis of compound heterozygous mutation p.Q6X/p.H232R in SRD5A2 causing 46,XY disorder of sex development.

BACKGROUND: Over 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration. METHODS: The two 46,XY DSD cases were identified and sequenced. In order to identify the source of the compound heterozygous p.Q6X/p.H232R mutation, the parents, maternal grandparents, and maternal uncle were sequenced. Since p.Q6X mutation is a nonsense mutation, p.H232R mutation was transfected into HEK293 cells and dihydrotestosterone (DHT) production were analyzed by liquid chromatography-mass spectrometry (LC-MS) for 5α-reductase 2 enzyme activities test. Apparent michaelis constant (Km) were measured of p.H232R mutation to analyze the binding ability change of 5α-reductase 2 enzyme with testosterone (T) or NADPH. RESULTS: The sequence results showed that the two 46,XY DSD cases were the compound heterozygous p.Q6X/p.H232R mutation, of which the heterozygous p.Q6X mutation originating from maternal family and heterozygous p.H232R mutation originating from the paternal family. The function analysis confirmed that p.H232R variant decreased the DHT production by LC-MS test. The Km analysis demonstrated that p.H232R mutation affected the binding of SRD5A2 with T or NADPH. CONCLUSIONS: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

Wnt7a promotes the osteogenic differentiation of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the ability of differentiating into osteoblasts. Elucidating the molecular mechanisms of MSC differentiation into osteoblasts may provide novel therapeutic strategies for bone­related diseases. Increasing evidence has confirmed that Wnt signaling plays the key role in osteoblast differentiation; however, the role of individual Wnt proteins in osteogenesis needs to be investigated. The present study thus aimed to explore the role of Wnt7a in bone formation. For this purpose, human bone­derived MSCs were identified by flow cytometry and the cell differentiation potential, including osteogenic and adipogenic differentiation was examined. In order to explore the role of Wnt7a in MSC osteogenic differentiation, Wnt7a expression was measured at the mRNA and protein level following treatment with the osteogenic inducer, bone morphogenetic protein (BMP)4/7, and following the induction of osteogenic or adipogenic differentiation. The ectopic expression of Wnt7a in MSCs was confirmed and its influence on MSC osteogenic differentiation was detected using osteocyte markers and by Alizarin Red S staining. Mechanistically, the influence of Wnt7a on Runt­related transcription factor 2 (RUNX2) expression was examined at the mRNA and protein level. The regulatory effects of Wnt7a on RUNX2 promoter activities were examined by promoter reporter assay, and by examining the binding of TCF1, a downstream target of Wnt, to the RUNX2 promoter by ChIP assay. The results revealed that the knockdown of Wnt7a in MSCs decreased the expression of osteocyte markers and inhibited osteogenic differentiation. In accordance, the overexpression of Wnt7a in MSCs increased the expression of osteocyte markers and promoted osteogenic differentiation. Mechanistically, the knockdown of Wnt7a in MSCs reduced RUNX2 expression and the overexpression of Wnt7a in MSCs promoted RUNX2 expression. Furthermore, it was confirmed that Wnt7a regulated RUNX2 promoter activities by promoter report assay, and by examining the binding of TCF1 to the RUNX2 promoter by ChIP assay. On the whole, the present study demonstrates that Wnt7a plays a key role in MSC differentiation into osteoblasts and the findings presented herein may provide a promising therapy target for bone­related diseases.

The Neural Stem Cell Properties of PKD2L1+ Cerebrospinal Fluid-Contacting Neurons in vitro.

Cerebrospinal fluid-touching neurons (CSF-cNs) exist in the region surrounding the central canal of the spinal cord, which locate in the adult neurogenic niche. Previous research showed that CSF-cNs expressed the molecular markers of immature neural cells in vivo. Here, we explored the potential of CSF-cNs as neural stem cell in intro. We first found that PKD2L1+ CSF-cNs, isolating by FACS using the molecular marker PKD2L1 of CSF-cNs, expressed neural stem cells markers like Nestin, Sox2, and GFAP by immunofluorescence staining. PKD2L1+ CSF-cNs were able to form neurospheres and passaged in vitro. Immunofluorescence staining showed that the neurospheres forming by PKD2L1+ CSF-cNs also expressed neural stem cell markers Nestin, Sox2 and GFAP. The neurospheres expressed proliferation markers Ki67 and PCNA by immunofluorescence staining, indicating that the neurospheres forming by PKD2L1+ CSF-cNs were proliferative. The neurospheres, forming by CSF-cNs, had the ability of differentiation into neurons, astrocytes, and oligodendrocytes. Collectively, our data suggested that PKD2L1+ CSF-cNs have the properties of neural stem cells in vitro and may provide a promising approach for the repair of spinal cord injury.

Wnt7a Promotes the Occurrence and Development of Colorectal Adenocarcinoma.

OBJECTIVE: The expression of Wnt7a in colorectal cancer tissues and cell lines was analyzed, and the effect of Wnt7a on the proliferation of colorectal cancer cells was studied, so as to confirm the relationship between Wnt7a and the occurrence and development of colorectal cancer. METHODS: (1) Immunohistochemical method was used to compare the expression of Wnt7a in different tissues and its relationship with the clinicopathology of colorectal adenocarcinoma. (2) The expression levels of Wnt7a in colorectal cancer cell lines HT-29 and HCT 116 were detected by qRT-PCR. (3) The down-regulated Wnt7A expression vector was constructed, and the down-regulated Wnt7A expression cell line was established. The regeneration ability of cancer cells was detected by stem cell ball formation assay, and the influence of plate cloning assay on the proliferation ability of colorectal cancer cells was detected. RESULTS: (1) The positive rates of Wnt7a in normal colorectal mucosa, colorectal adenoma and colorectal adenocarcinoma tissues gradually increased,Wnt7a are closely related to the degree of colorectal adenocarcinoma differentiation, lymph node metastasis and Duke stage. (2) The expression level of Wnt7a in colorectal cancer cells was higher than that in normal colorectal epithelial cells. (3) The down-regulation of Wnt7A reduced the proliferation ability of colorectal cancer cells. CONCLUSIONS: Wnt7a promotes the occurrence and development of colorectal adenocarcinoma.

Mild and Selective Rhodium-Catalyzed Transfer Hydrogenation of Functionalized Arenes.

Diboron-mediated rhodium-catalyzed transfer hydrogenation of functionalized arenes is reported. In addition to good functional group tolerance, the reaction features operational simplicity and controllable chemoselectivity. The general applicability of this procedure is demonstrated by the selective hydrogenation of a range of arenes, including functionalized benzenes, biphenyls, and polyaromatics.

A rhodium-catalysed conjugate addition/cyclization cascade for the asymmetric synthesis of 2-amino-4H-chromenes.

The enantioselective synthesis of 2-amino-4H-chromenes via the cascade rhodium-catalysed conjugate addition/hetero Thorpe-Ziegler reaction is reported. Moderate to good yields (up to 98%) and high enantioselectivities (up to 92% ee) were obtained with a chiral diene-coordinated rhodium complex as the catalyst. This protocol remedies the methodological deficiency in the asymmetric synthesis of 4-aryl 2-amino-4H-chromenes.

Inactivation of tumor suppressor gene Clusterin leads to hyperactivation of TAK1-NF-κB signaling axis in lung cancer cells and denotes a therapeutic opportunity.

Purpose: Clinical success of precision medicine is severely limited by de novo or acquired drug resistance. It remains a clinically unmet need to treat these patients. Tumor suppressor genes (TSGs) play a critical role in tumorigenesis and impact the therapeutic effect of various treatments. Experimental Design: Using clinical data, in vitro cell line data and in vivo mouse model data, we revealed the tumor suppressive role of Clusterin in lung cancer. We also delineated the signaling cascade elicited by loss of function of CLU in NSCLC cells and tested precision medicine for CLU deficient lung cancers. Results:CLU is a potent and clinically relevant TSG in lung cancer. Mechanistically, CLU inhibits TGFBR1 to recruit TRAF6/TAB2/TAK1 complex and thus inhibits activation of TAK1- NF-κB signaling axis. Lung cancer cells with loss of function of CLU show exquisite sensitivity to TAK1 inhibitors. Importantly, we show that a significant portion of Kras mutation positive NSCLC patients are concurrently deficient of CLU and that TAK1 kinase inhibitor synergizes with existing drugs to treat this portion of lung cancers patients. Conclusions: Combinational treatment with TAK1 inhibitor and MEK1/2 inhibitor effectively shrank Kras mutation positive and CLU deficient NSCLC tumors. Moreover, we put forward a concept that loss of function of a TSG rewires signaling network and thereby creates an Achilles' heel in tumor cells which could be exploited in precision medicine.

A Renewed Focus on GDF11 Level Fluctuation in Human Serum in Relation to Physical Examination Indicators.

Growth and differentiation factor 11 (GDF11) is a member of the transforming growth factor ß superfamily. Previous studies have shown that GDF11 decreases with age and has antiaging effects; however, such reports are controversial. We choose 152 subjects covering a large age range (2 hours to 75 years) to measure serum GDF11. Twenty-two hematological variables and 13 biochemical values were measured. Pearson's analysis found a significant correlation between GDF11 and age (p = .0000, r = .4898), as well as serum creatinine, uric acid, triglycerides, red blood cell count, hemoglobin, hematocrit, and platelet volume distribution width. GDF11 negatively correlated with aspartate transaminase, white blood cell count, platelet count, lymphocyte count, monocyte count, mean platelet volume, and plateletcrit. Interestingly, we found GDF11 increases in people aged 20-30 years, holds steady in people aged 30-50 years, and increases in people older than 50 years. The results suggest that GDF11 serves different roles along the life span. The current actual evidence supports that GDF11 is helpful to promote aging.