Mimicking the Shape and Function of the ClC Chloride Channel Selective Pore by Combining a Molecular Hourglass Shape with Anion-π Interactions.

ClC is the main family of natural chloride channel proteins that transport Cl- across the cell membrane with high selectivity. The chloride transport and selectivity are determined by the hourglass-shaped pore and the filter located in the central and narrow region of the pore. Artificial unimolecular channel that mimics both the shape and function of the ClC selective pore is attractive, because it could provide simple molecular model to probe the intriguing mechanism and structure-function relevance of ClC. Here we elaborated upon the concept of molecular hourglass plus anion-π interactions for this purpose. The concept was validated by experimental results of molecular hourglasses using shape-persistent 1,3-alternate tetraoxacalix[2]arene[2]triazine as the central macrocyclic skeleton to control the conductance and selectivity, and anion-π interactions as the driving force to facilitate the chloride dehydration and movement along the channel.

Reversing the ion transport selectivity through arm modification of an artificial molecular hourglass.

An arm modification strategy, by replacing relatively rigid, electron-deficient side arms with flexible ether chain arms and linking them onto a tetraoxacalix[2]arene[2]triazine skeleton, was utilized to design an artificial molecular hourglass. The planar bilayer experiments confirmed the unimolecular channel mechanism and suggested reversed ion selectivity from the previously reported anion selectivity to weak cation selectivity.

Prenatal DEHP exposure predicts neurological disorders via transgenerational epigenetics.

Recent experimental and observational research has suggested that childhood allergic asthma and other conditions may be the result of prenatal exposure to environmental contaminants, such as di-(2-ethylhexyl) phthalate (DEHP). In a previous epidemiological study, we found that ancestral exposure (F0 generation) to endocrine disruptors or the common plasticizer DEHP promoted allergic airway inflammation via transgenerational transmission in mice from generation F1 to F4. In the current study, we employed a MethylationEPIC Beadchip microarray to examine global DNA methylation in the human placenta as a function of maternal exposure to DEHP during pregnancy. Interestingly, global DNA hypomethylation was observed in placental DNA following exposure to DEHP at high concentrations. Bioinformatic analysis confirmed that DNA methylation affected genes related to neurological disorders, such as autism and dementia. These results suggest that maternal exposure to DEHP may predispose offspring to neurological diseases. Given the small sample size in this study, the potential role of DNA methylation as a biomarker to assess the risk of these diseases deserves further investigation.

An Artificial Single Molecular Channel Showing High Chloride Transport Selectivity and pH-Responsive Conductance.

Inspired by the unique structure and function of the natural chloride channel (ClC) selectivity filter, we present herein the design of a ClC-type single channel molecule. This channel displays high ion transport activity with half-maximal effective concentration, EC50 , of 0.10 µM, or 0.075 mol % (channel molecule to lipid ratio), as determined by fluorescent analysis using lucigenin-encapsulated vesicles. Planar bilayer lipid membrane conductance measurements indicated an excellent Cl- /K+ selectivity with a permeability ratio P Cl - ${{_{{\rm Cl}{^{- }}}}}$ /P K + ${{_{{\rm K}{^{+}}}}}$ up to 12.31, which is comparable with the chloride selectivity of natural ClC proteins. Moreover, high anion/anion selectivity (P Cl - ${{_{{\rm Cl}{^{- }}}}}$ /P Br - ${{_{{\rm Br}{^{- }}}}}$ =66.21) and pH-dependent conductance and ion selectivity of the channel molecule were revealed. The ClC-like transport behavior is contributed by the cooperation of hydrogen bonding and anion-π interactions in the central macrocyclic skeleton, and by the existence of pH-responsive terminal phenylalanine residues.

Quantitative and amplification-free detection of SOCS-1 CpG methylation percentage analyses in gastric cancer by fiber optic nanoplasmonic biosensor.

A new innovative approach is essential for early and effective diagnosis of gastric cancer, using promoter hypermethylation of the tumor suppressor, SOCS-1, that is frequently inactivated in human cancers. We have developed an amplification-free fiber optic nanoplasmonic biosensor for detecting DNA methylation of the SOCS-1 human genome. The method is based on the fiber optic nanogold-linked sorbent assay of PCR-free DNA from human gastric tumor tissue and cell lines. We designed a specific DNA probe fabricated on the fiber core surface while the other probe is bioconjugated with gold nanoparticles in free form to allow percentage determination and differentiating the methylated and unmethylated cell lines, further demonstrating the SOCS-1 methylation occurs in cancer patients but not in normal cell lines. The observed detection limit is 0.81 fM for methylated DNA, and the detection time is within 15 min. In addition, our data were significantly correlated to the data obtained from PCR-based pyrosequencing, and yet with superior accuracy. Hence our results provide new insight to the quantitative evaluation of methylation status of the human genome and can act as an alternative to PCR with a great potential.

PCDHB15 as a potential tumor suppressor and epigenetic biomarker for breast cancer.

Breast cancer is among the most frequently diagnosed cancer types and the leading cause of cancer-related death in women. The mortality rate of patients with breast cancer is currently increasing, perhaps due to a lack of early screening tools. In the present study, using The Cancer Genome Atlas (TCGA) breast cancer dataset (n=883), it was determined that methylation of the protocadherin ß15 (PCDHB15) promoter was higher in breast cancer samples than that in normal tissues. A negative association between promoter methylation and expression of PCDHB15 was observed in the TCGA dataset and breast cancer cell lines. In TCGA cohort, lower PCDHB15 expression was associated with shorter relapse-free survival times. Treatment with the DNA methyltransferase inhibitor restored PCDHB15 expression in a breast cancer cell line; however, overexpression of PCDHB15 was shown to suppress colony formation. PCDHB15 methylation detected in circulating cell-free DNA (cfDNA) isolated from serum samples was higher in patients with breast cancer (40.8%) compared with that in patients with benign tumors (22.4%). PCDHB15 methylation was not correlated with any clinical parameters. Taken together, PCDHB15 is a potential tumor suppressor in cases of breast cancer, which can be epigenetically silenced via promoter methylation. PCDHB15 methylation using cfDNA is a novel minimally invasive epigenetic biomarker for the diagnosis and prognosis of breast cancer.

The optimal vaccination strategy to control COVID-19: a modeling study in Wuhan City, China.

BACKGROUND: Reaching optimal vaccination rates is an essential public health strategy to control the coronavirus disease 2019 (COVID-19) pandemic. This study aimed to simulate the optimal vaccination strategy to control the disease by developing an age-specific model based on the current transmission patterns of COVID-19 in Wuhan City, China. METHODS: We collected two indicators of COVID-19, including illness onset data and age of confirmed case in Wuhan City, from December 2, 2019, to March 16, 2020. The reported cases were divided into four age groups: group 1, ≤ 14 years old; group 2, 15 to 44 years old; group 3, 44 to 64 years old; and group 4, ≥ 65 years old. An age-specific susceptible-exposed-symptomatic-asymptomatic-recovered/removed model was developed to estimate the transmissibility and simulate the optimal vaccination strategy. The effective reproduction number (Reff) was used to estimate the transmission interaction in different age groups. RESULTS: A total of 47 722 new cases were reported in Wuhan City from December 2, 2019, to March 16, 2020. Before the travel ban of Wuhan City, the highest transmissibility was observed among age group 2 (Reff = 4.28), followed by group 2 to 3 (Reff = 2.61), and group 2 to 4 (Reff = 1.69). China should vaccinate at least 85% of the total population to interrupt transmission. The priority for controlling transmission should be to vaccinate 5% to 8% of individuals in age group 2 per day (ultimately vaccinated 90% of age group 2), followed by 10% of age group 3 per day (ultimately vaccinated 90% age group 3). However, the optimal vaccination strategy for reducing the disease severity identified individuals ≥ 65 years old as a priority group, followed by those 45-64 years old. CONCLUSIONS: Approximately 85% of the total population (nearly 1.2 billion people) should be vaccinated to build an immune barrier in China to safely consider removing border restrictions. Based on these results, we concluded that 90% of adults aged 15-64 years should first be vaccinated to prevent transmission in China.

Artificial Chloride-Selective Channel: Shape and Function Mimic of the ClC Channel Selective Pore.

An artificial channel molecule 1 that mimics the shape and function of the ClC channel selective pore was described. To facilitate the transport of chloride along a unimolecular pathway, anion-π interactions were introduced as the noncovalent driving force. The hourglass-like shape of 1 was constructed with 1,3-alternate tetraoxacalix[2]arene[2]triazine as the narrowest (central) unit. Two diglycolamine-linked imide arms were tethered as the extending part, and phenylalanine moieties were fixed as the terminal anchoring groups. The ion transport activity was examined by a combination of vesicle and planar bilayer conductance techniques (BLM). The fluorescence analysis on the vesicle indicated that 1 is an efficient chloride transporter with high activity (EC50 = 1.50 µM; 1/lipid = 1:89). The ion channel characteristics were confirmed by BLM measurements, showing an average conductance of 20.8 ± 1.0 pS in symmetric KCl solutions (cis/trans = 1.0 M/1.0 M). Anion/cation selectivity with a permeability ratio PCl-/PK+ = 1.90 in an asymmetric KCl solution (cis/trans = 1.0 M/0.25 M) and anion/anion selectivity with PCl-/PBr- = 22.83 in a KCl/KBr solution (cis/trans = 1.0 M KCl/1.0 M KBr) were demonstrated.

Improving the accuracy and efficacy of diagnosing polycystic ovary syndrome by integrating metabolomics with clinical characteristics: study protocol for a randomized controlled trial.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine syndrome with poorly understood mechanisms. To provide patients with PCOS with individualized therapy, it is critical to precisely diagnose the phenotypes of the disease. However, the criteria for diagnosing the different phenotypes are mostly based on symptoms, physical examination and laboratory results. This study aims to compare the accuracy and efficacy of diagnosing PCOS by integrating metabolomic markers with common clinical characteristics. METHODS: This is a prospective, multicenter, analyst-blinded, randomized controlled trial. Participants will be grouped into (1) people without PCOS (healthy control group), (2) patients diagnosed with PCOS based on clinical indices (experimental group 1), and (3) patients diagnosed with PCOS based on metabolomic indices (experimental group 2). A total of 276 participants, including 60 healthy people and 216 patients with PCOS, will be recruited. The 216 patients with PCOS will be randomly assigned to the two experimental groups in a 1:1 ratio, and each group will receive a different 6-month treatment. The primary outcome for the experimental groups will be the effect of PCOS treatment. DISCUSSION: The results of this trial should help to determine whether using metabolomic indices is more accurate and effective than using clinical characteristics in diagnosing the phenotypes of PCOS. The results could provide a solid foundation for the accurate diagnosis of different PCOS subgroups and for future research on individualized PCOS therapy. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ID: ChiCTR-INR-1800016346. Registered 26 May 2018.

Anion Transporters Based on Noncovalent Balance including Anion-π, Hydrogen, and Halogen Bonding.

Anion transmembrane transport mediated by novel noncovalent interactions is of central interest in supramolecular chemistry. In this work, a series of oxacalix[2]arene[2]triazine-derived transporters 1 and 2 bearing anion-π-, hydrogen-, and halogen-bonding sites in rational proximity were designed and synthesized by a one-pot strategy starting from gallic acid ester derivatives and mono- or di-halogen-substituted triazines. 1H NMR titrations demonstrated efficient binding of 1 and 2 toward Cl- and Br- in solution, giving association constants in the range of 102-104 M-1. Cooperation of anion-π, hydrogen, and halogen bonding was revealed as a driving force for anion binding by single-crystal structures of two complexes and density functional theory calculations. Fluorescence assays indicated that compounds 1 are efficient chloride transporters with effective concentrations (EC50) falling in the range of 3.1-7.4 µM and following an order of 1a > 1b > 1c > 1d. The contribution of halogen bonding and cooperative noncovalent bonds to ion transport was then discussed. Significantly, transporters 1 exhibit high anticancer activity. In the presence of 1 and KCl (60 mM), the cell survival of HCT116 reduces to 11.9-24.9% with IC50 values in the range of 52.3-66.4 µM.

EMP18 functions in mitochondrial atp6 and cox2 transcript editing and is essential to seed development in maize.

RNA editing plays an important role in organellar gene expression in plants, and pentatricopeptide repeat (PPR) proteins are involved in this function. Because of its large family size, many PPR proteins are not known for their function and roles in plant growth and development. Through genetic and molecular analyses of the empty pericarp18 (emp18) mutant in maize (Zea mays), we cloned the Emp18 gene, revealed its molecular function, and defined its role in the mitochondrial complex assembly and seed development. Emp18 encodes a mitochondrial-localized DYW-PPR protein. Null mutation of Emp18 arrests embryo and endosperm development at an early stage in maize, resulting in embryo lethality. Mutants are deficient in the cytidine (C)-to-uridine (U) editing at atp6-635 and cox2-449, which converts a Leu to Pro in ATP6 and a Met to Thr in Cox2. The atp6 gene encodes the subunit a of F1 Fo -ATPase. The Leu to Pro alteration disrupts an α-helix of subunit a, resulting in a dramatic reduction in assembly and activity of F1 Fo -ATPase holoenzyme and an accumulation of free F1 -subcomplex. These results demonstrate that EMP18 functions in the C-to-U editing of atp6 and cox2, and is essential to mitochondrial biogenesis and seed development in maize.

Design, synthesis, and biological evaluation of novel nitric oxide releasing dehydroandrographolide derivatives.

A series of new hybrids of dehydroandrographolide (TAD), a biologically active natural product, bearing nitric oxide (NO)-releasing moieties were synthesized and designated as NO-donor dehydroandrographolide. The biological activities of target compounds were studied in human erythroleukemia K562 cells and breast cancer MCF-7 cells. Biological evaluation indicated that the most active compound I-5 produced high levels of NO and inhibited the proliferation of K562 (IC50 1.55 µmol·L-1) and MCF-7 (IC50 2.91 µmol·L-1) cells, which were more potent than the lead compound TAD and attenuated by an NO scavenger. In conclusion, I-5 is a novel hybrid with potent antitumor activity and may become a promising candidate for future intensive study.

The pentatricopeptide repeat protein EMP9 is required for mitochondrial ccmB and rps4 transcript editing, mitochondrial complex biogenesis and seed development in maize.

Pentatricopeptide repeat (PPR) proteins comprise a large family of sequence-specific RNA binding proteins in land plants. Because of its large family size and frequent embryo lethality in the mutants, molecular functions and physiological roles of many PPR proteins are unknown. Through characterization of an empty pericarp9 (emp9) mutant in maize (Zea mays), we defined the functions of EMP9 in mitochondrial RNA editing, respiratory complex formation and seed development. Mu insertions in different regions of Emp9 facilitated dissection of the domain functions of the EMP9. Through genetic and functional analyses of multiple alleles, we showed that deletions of two N-terminal PPR motifs and partial E+ domain do not eliminate the editing function of EMP9. Emp9 encodes an E+ subclass PPR protein that is localized in mitochondria. Loss of EMP9 function abolishes the C-to-U editing of ccmB-43 and rps4-335 sites in mitochondria. The loss of editing at ccmB-43 and rps4-335 affects the maturation of cytochrome c and impairs the biogenesis of mitochondrial respiratory complexes, particularly complex III. This work extends our understanding of PPR-E+ protein in editing function and seed development, and provides insights into the molecular function of mitochondrial CcmB protein in higher plants.

Trends and Prospects of Stem Cell Research in China.

Great progresses have been made in fundamental and clinical stem cell research in China in recent years. The official policy on stem cells, which was announced in 2015, seems as the spring of stem cell therapy in China. However, the regulation, governance, and management of clinical expectations are still challenging. This review summarized the current stem cell research and development in the field, as well as its rapidly evolving commercial, regulatory and ethical environment in China. As expected, the prospects of stem cells in China look prospective.

[Synthesis and anti-proliferative activity of fluoroquinolone (rhodanine unsaturated ketone) amide derivatives].

To discover novel antitumor rhodanine unsaturated ketones, a series of fluoroquinolone (rhodanine α, ß-unsaturated ketone) amine derivatives (5a-5r) were designed and synthesized with fluoroquinolone amide scaffold as a carrier. The structures of eighteen title compounds were characterized by elemental analysis, 1H NMR and MS. The in vitro anti-proliferative activity against Hep-3B, Capan-1 and HL60 cells was evaluated by MTT assay. The results showed that the title compounds not only had more significant anti-proliferative activity against three tested cancer cell lines than that of the parent ciprofloxacin 1, but also exhibited the highest activity against Capan-1 cells. The SAR revealed that some compounds carrying aromatic heterocyclic rings or phenyl attached to an electron-withdrawing carboxyl or sulfonamide substituent were comparable to or better than comparison doxorubicin against Capan-1 cells. As such, it suggests that fluoroquinolone (rhodanine α, ß-unsaturated ketone) amines are promising leads for the development of novel antitumor fluoroquinolones or rhodanine analogues.

[Synthesis and anti-proliferative activity of fluoroquinolone C-3 fused heterocyclic α,ß-unsaturated ketones derived from ciprofloxacin].

To discover novel antitumor fluoroquinolone lead compounds from a rational modification for antibacterial fluoroquinolones, a fused heterocyclic ketone corresponding to thiazolo[2,3- b][1,2,4]triazolone used as a bioisosteric replacement of the C-3 carboxylic acid group of ciprofloxacin 1, and further modification by a Claisen condensation reaction with substituted benzaldehydes formed novel fluoroquinolone C-3 fuse heterocyclic α, ß-unsaturated ketones as the title compounds (6a-6r), separately. The structures of eighteen title compounds were characterized by elemental analysis, 1H NMR and MS, and the in vitro anti-proliferative activity against human hepatoma Hep-3B cells, pancreatic Capan-1 cells and leukemia HL60 cells was evaluated by a MTT assay. The preliminary results showed that the title compounds not only had more significant anti-proliferative activity against three tested cancer cell lines than that of the parent ciprofloxacin 1, but also exhibited the highest activity against Capan-1 cells. In particular, compounds carrying an electron-withdrawing carboxyl (6k, 6m) or sulfonamide substituent (6q, 6r) attached to benzene ring were comparable to or better than constractive drug doxorubicin against Capan-1 cells. As such, it suggests that it is favorable for a fused heterocyclic α, ß-unsaturated ketone scaffold instead of the C-3 carboxylic acid group to improve the antitumor activity of fluoroquinolones.

[Design, synthesis, antibacterial and anti-cell proliferation activities of [1,2,4]triazino[3,4-h] [1,8]naphthyridine-8-one-7-carboxylic acid derivatives].

To discover novel fluoroquinolone lead compounds as possible anti-infective or/and antitumor chemotherapies, combination principle of pharmacophore-based drug design, a series of novel tricyclic fluoroquinolone title compounds, [1,2,4]triazino[3,4-h][1,8]naphthyridine-8-one-7-carboxylic acid derivatives ( 5a-5p), were designed and synthesized with a fused [1,2,4]-triazine ring unit. Their structures were characterized by spectral data and elemental analysis and the in vitro antibacterial and anti-cell proliferation activities were also evaluated. The results showed that the titled compounds exhibited more significant inhibitory activities against drug-resistant bacteria (Methicillin-resistant Staphylococcus aureus and multi drug-resistant Escherichia coli strains) and three tested cancer cell lines (human hepatoma SMMC-7721, murine leukemia L1210 and human murine leukemia HL60 cells). Interestingly, SAR showed that compounds with electron-donating groups attached to benzene ring had stronger antibacterial activity than antitumor activity, but electron-withdrawing compounds displayed more potential antitumor activity than antibacterial activity, especially antitumor activity of nitro compounds was comparable to comparison doxorubicin. Thus, novel triazine-fused tricyclic fluoroquinolones as potent anti-infective or/and antitumor lead compounds are valuable to pay attention and for further development.

[Synthesis and antitumor activity of fluoroquinolon-3-yl-s-triazole sulfide ketones and their derivatives from ciprofloxacin].

To discover an efficient strategy for the conversion of the antibacterial activity of fluoroquinolones into the antitumor activity, the three series of C-3 s-triazole-based derivatives including sulfide ketones (6a-6g), thiosemicarbazones (7a-7g) and fused heterocyclic thiazolotriazoles (8a-8g) were synthesized from ciprofloxacin (1), respectively. The structures were characterized by elemental analysis and spectral data. The antitumor activity was tested against three tumor cell lines (Hep-3B, Capan-1 and HL60) using the MTT assay. The three types of compounds all exhibited stronger anti-proliferative activities than ciprofloxacin in the test. The order of their activities was in compounds 7>8>6, and the order of selectivity against cancer cell lines was Capan-1, Hep-3B and HL60. Meanwhile, the SAR revealed that some compounds with electron-drawing group substituted such as fluoro- and nitro-phenyl compounds (6f, 7f, 8f) and (6g, 7g, 8g) displayed more significant activity than the control compounds, especially the IC50 values of thiosemicarbazone compounds 7f and 7g against Capan-1 was comparable to doxorubicin. Thus, a five-membered triazole as the C-3 bioisostere modified with the functionalized side-chain of sulfide-ketone thiosemicarbazone warrants special attention and further investigation.

Biological activity studies of the novel glucagon-like peptide-1 derivative HJ07.

AIM: To identify the glucose lowering ability and chronic treatment effects of a novel coumarin-glucagon-like peptide-1 (GLP-1) conjugate HJ07. METHOD: A receptor activation experiment was performed in HEK 293 cells and the glucose lowering ability was evaluated with hypoglycemic duration and glucose stabilizing tests. Chronic treatment was performed by daily injection of exendin-4, saline, and HJ07. Body weight and HbA1c were measured every week, and an intraperitoneal glucose tolerance test was performed before treatment and after treatment. RESULTS: HJ07 showed well-preserved receptor activation efficacy. The hypoglycemic duration test showed that HJ07 possessed a long-acting, glucose-lowering effect and the glucose stabilizing test showed that the antihyperglycemic activity of HJ07 was still evident at a predetermined time (12 h) prior to the glucose challenge (0 h). The long time glucose-lowering effect of HJ07 was better than native GLP-1 and exendin-4. Furthermore, once daily injection of HJ07 to db/db mice achieved long-term beneficial effects on HbA1c lowering and glucose tolerance. CONCLUSION: The biological activity results of HJ07 suggest that HJ07 is a potential long-acting agent for the treatment of type 2 diabetes.

Synthesis and in vitro evaluation of 1,3,4-thiadiazol-2-yl urea derivatives as novel AChE inhibitors.

1,3,4-Thiadiazole and urea group were hybridized to form new molecular skeleton and 11 compounds were synthesized and evaluated as acetylcholinesterase (AChE) inhibitors. Most of them showed comparable effects in inhibition of AChE, especially compound 6b which exhibited activity with IC50 value 1.17 µM, as strong as galanthamine. This information offered by our research would be valuable for further investigation of structure-activity relationship (SAR) and useful in future research of AChE inhibitors.