Gene-gene interaction network analysis indicates CNTN2 is a candidate gene for idiopathic generalized epilepsy.

Twin and family studies have established the genetic contribution to idiopathic generalized epilepsy (IGE). The genetic architecture of IGE is generally complex and heterogeneous, and the majority of the genetic burden in IGE remains unsolved. We hypothesize that gene-gene interactions contribute to the complex inheritance of IGE. CNTN2 (OMIM* 615,400) variants have been identified in cases with familial adult myoclonic epilepsy and other epilepsies. To explore the gene-gene interaction network in IGE, we took the CNTN2 gene as an example and investigated its co-occurrent genetic variants in IGE cases. We performed whole-exome sequencing in 114 unrelated IGE cases and 296 healthy controls. Variants were qualified with sequencing quality, minor allele frequency, in silico prediction, genetic phenotype, and recurrent case numbers. The STRING_TOP25 gene interaction network analysis was introduced with the bait gene CNTN2 (denoted as A). The gene-gene interaction pair mode was presumed to be A + c, A + d, A + e, with a leading gene A, or A + B + f, A + B + g, A + B + h, with a double-gene A + B, or other combinations. We compared the number of gene interaction pairs between the case and control groups. We identified three pairs in the case group, CNTN2 + PTPN18, CNTN2 + CNTN1 + ANK2 + ANK3 + SNTG2, and CNTN2 + PTPRZ1, while we did not discover any pairs in the control group. The number of gene interaction pairs in the case group was much more than in the control group (p = 0.021). Taking together the genetic bioinformatics, reported epilepsy cases, and statistical evidence in the study, we supposed CNTN2 as a candidate pathogenic gene for IGE. The gene interaction network analysis might help screen candidate genes for IGE or other complex genetic disorders.

Concise Total Synthesis of (-)-Quinocarcin Enabled by Catalytic Enantioselective Reductive 1,3-Dipolar Cycloaddition of Secondary Amides.

A concise asymmetric total synthesis of (-)-quinocarcin has been accomplished with high step economy from commercially available starting materials. A catalytic enantioselective reductive 1,3-dipolar cycloaddition reaction of N-heteroaryl secondary amides with reactive dipolarophiles using iridium/copper relay catalysis was developed to prepare the key chiral pyrrolidine intermediate with three stereocenters. This protocol features excellent regio-, exo- and enantioselectivities, broad substrate scope, and good functional group tolerance. The high efficiency was also ensured by a RhIII -catalyzed C-H activation/cyclization and a tandem diastereoselective hydrogenation/cyclization to construct the tetrahydroisoquinoline-pyrrolidine tetracyclic core unit of quinocarcin.

Chemoselective Reactions of Isocyanates with Secondary Amides: One-Pot Construction of 2,3-Dialkyl-Substituted Quinazolinones.

A facile method for the preparation of 2,3-dialkyl-substituted quinazolinones from readily available N-arylamides and commercial isocyanates was developed. This one-pot procedure involves the chemoselective activation of the secondary amide with Tf2O/2-Br-Pyr, the sequential addition of isocyanate, and cyclization. The mild reaction is general for a wide range of substrates and can be run on a gram scale.

PMPCB Silencing Sensitizes HCC Tumor Cells to Sorafenib Therapy.

Hepatocellular carcinoma (HCC) tumors invariably develop resistance to cytotoxic and targeted agents, resulting in failed treatment and tumor recurrence. Previous in vivo short hairpin RNA (shRNA) screening evidence revealed mitochondrial-processing peptidase (PMPC) as a leading gene contributing to tumor cell resistance against sorafenib, a multikinase inhibitor used to treat advanced HCC. Here, we investigated the contributory role of the ß subunit of PMPC (PMPCB) in sorafenib resistance. Silencing PMPCB increased HCC tumor cell susceptibility to sorafenib therapy, decreased liver tumor burden, and improved survival of tumor-bearing mice receiving sorafenib. Moreover, sorafenib + PMPCB shRNA combination therapy led to attenuated liver tumor burden and improved survival outcome for tumor-bearing mice, and it reduced colony formation in murine and human HCC cell lines in vitro. Additionally, PMPCB silencing enhanced PINK1-Parkin signaling and downregulated the anti-apoptotic protein MCL-1 in sorafenib-treated HCC cells, which is indicative of a healthier pro-apoptotic phenotype. Higher pre-treatment MCL-1 expression was associated with inferior survival outcomes in sorafenib-treated HCC patients. Elevated MCL-1 expression was present in sorafenib-resistant murine HCC cells, while MCL-1 knockdown sensitized these cells to sorafenib. In conclusion, our findings advocate combination regimens employing sorafenib with PMPCB knockdown or MCL-1 knockdown to circumvent sorafenib resistance in HCC patients.

Substrate-Controlled Chemoselective Reactions of Isocyanoacetates with Amides and Lactams.

Versatile and chemoselective C-C bond forming methods for the one-pot transformation of amides into other classes of compounds are highly demanding. In this report, we demonstrate the reductive addition of isocyanoacetates to common amides and lactams to produce 5-methoxyoxazoles or bicyclic imidazolines. This one-pot procedure involves partial reduction of amides with Schwartz reagent and chemoselective addition of the carbon of isocyanide group or α-carbon in isocyanoacetates. The quite different reactivity of the isocyanoacetate is due to the different steric hindrance of the amides and lactams.

Genistein protects female nonobese diabetic mice from developing type 1 diabetes when fed a soy- and alfalfa-free diet.

The objective of this study was to determine the effects of the phytoestrogen genistein (GEN) on the time of onset and/or the incidence of type 1 diabetes (T1D) in female nonobese diabetic (NOD) mice, when administered GEN by gavage once every day for up to 180 days. Five groups of mice (approximately 24 animals/group; 6-7 weeks of age) were included: naive control, vehicle control (25 mM Na2CO3 in water), and 3 GEN treatment groups (2 mg/kg, 6 mg/kg, and 20 mg/kg). Mice were maintained on a soy- and alfalfa-free diet (5K96) during the study and were monitored for blood glucose changes every week. When compared to the vehicle control, exposure to 2-mg/kg GEN produced significant decreases ranging from 55 to 79% in the total incidences of diabetes (blood glucose ≥ 250 mg/dl) and severe diabetes (blood glucose ≥ 400 mg/dl) starting at week 14 of the study. However, during the later stages of the study (i.e., after week 23), the 2-mg/kg dose had no effect on disease incidence. In animals treated with 6-mg/kg and 20-mg/kg GEN, significant decreases in the total incidence of diabetes were observed starting at week 16, while the incidence of severe diabetes was significantly decreased with the changes being observed initially at weeks 18 and 17 for the 6-mg/kg and 20-mg/kg GEN treatment groups, respectively. Several lines of evidence, including histopathological analysis, suggested that GEN protected the pancreas from autoimmune destruction. However, this protective effect of GEN was absent when female NOD mice were maintained on NTP-2000 rodent diet, which contained 5% soybean meal and 7.5% alfalfa meal (the total concentrations of phytoestrogens ranged between 95 and 134 mg/kg). In summary, oral dosing of GEN reduced the incidence and increased the time to onset of T1D in female NOD mice but only when fed a soy- and alfalfa-free diet.

Comparative Metabolomic Profiling of Hepatocellular Carcinoma Cells Treated with Sorafenib Monotherapy vs. Sorafenib-Everolimus Combination Therapy.

BACKGROUND: Sorafenib-everolimus combination therapy may be more effective than sorafenib monotherapy for hepatocellular carcinoma (HCC). To better understand this effect, we comparatively profiled the metabolite composition of HepG2 cells treated with sorafenib, everolimus, and sorafenib-everolimus combination therapy. MATERIAL AND METHODS: A 2D HRMAS 1H-NMR metabolomic approach was applied to identify the key differential metabolites in 3 experimental groups: sorafenib (5 µM), everolimus (5 µM), and combination therapy (5 µM sorafenib +5 µM everolimus). MetaboAnalyst 3.0 was used to perform pathway analysis. RESULTS: All OPLS-DA models displayed good separation between experimental groups, high-quality goodness of fit (R2), and high-quality goodness of predication (Q2). Sorafenib and everolimus have differential effects with respect to amino acid, methane, pyruvate, pyrimidine, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. The addition of everolimus to sorafenib resulted in differential effects with respect to pyruvate, amino acid, methane, glyoxylate and dicarboxylate, glycolysis or gluconeogenesis, glycerophospholipid, and purine metabolism. CONCLUSIONS: Sorafenib and everolimus have differential effects on HepG2 cells. Sorafenib preferentially affects glycerophospholipid and purine metabolism, while the addition of everolimus preferentially affects pyruvate, amino acid, and glucose metabolism. This phenomenon may explain (in part) the synergistic effects of sorafenib-everolimus combination therapy observed in vivo.

XPD Functions as a Tumor Suppressor and Dysregulates Autophagy in Cultured HepG2 Cells.

BACKGROUND: Recent clinical studies have linked polymorphisms in the xeroderma pigmentosum group D (XPD) gene, a key repair gene involved in nucleotide excision repair, to increased risk of hepatocellular carcinoma (HCC). However, the cellular effects of XPD expression in cultured HCC cells remain largely uncharacterized. Therefore, the aim of this study was to characterize the in vitro cellular effects of XPD expression on the HCC cell line HepG2. MATERIAL AND METHODS: HepG2 cells were transfected as follows to create four experimental groups: pEGFP-N2/XPD plasmid (XPD) group, EGFP-N2 plasmid (N2) control group, lipofectamine™ 2000 (lipid) control group, and non-transfected (CON) control group. An MTT cell proliferation assay, Annexin V-APC apoptosis assay, colony formation assay, scratch wound migration assay, Transwell migration assay, and Western blotting of the autophagic proteins LC3 and p62 were conducted. RESULTS: XPD expression significantly inhibited HepG2 cell proliferation (p<0.05), significantly promoted HepG2 cell apoptosis (p<0.05), significantly inhibited HepG2 colony formation (p<0.05), significantly decreased HepG2 cells' migratory ability (p<0.05), and significantly lowered HepG2 cells' invasive capacity (p<0.05). Western blotting showed that XPD expression significantly increased LC3 expression (p<0.05) and significantly reduced p62 expression (p<0.05). CONCLUSIONS: XPD expression serves as a tumor suppressor and dysregulates autophagic protein degradation in HepG2 cells in vitro. Further in vivo pre-clinical studies and clinical trials are needed to validate XPD's potential as a tumor-suppressive gene therapy.

The CXCLs-CXCR2 axis modulates the cross-communication between tumor-associated neutrophils and tumor cells in cervical cancer.

OBJECTIVE: This study aimed to check the expression profile of the C-X-C motif chemokine ligands (CXCLs)-C-X-C motif chemokine receptor 2 (CXCR2) axis in cervical cancer and to explore the cross-talk between cervical cancer cells and neutrophils via CXCLs-CXCR2 axis. METHODS: Available RNA-sequencing data based on bulk tissues and single-cell/nucleus RNA-sequencing data were used for bioinformatic analysis. Cervical cancer cell lines Hela and SiHa cells were utilized for in vitro and in vivo studies. RESULTS: Except for neutrophils, CXCR2 mRNA expression is limited in other types of cells in the cervical tumor microenvironment. CXCLs bind to CXCR2 and are mainly expressed by tumor cells. CXCL1, 2, 3, 5, 6, and 8, which are consistently associated with neutrophil infiltration, are also linked to poor prognosis. SB225002 (a CXCR2 inhibitor) treatment significantly impairs SiHa cell-induced neutrophil migration. CXCL1, CXCL2, CXCL5, or CXCL8 neutralized conditioned medium from SiHa cells have weaker recruiting effects. The conditioned medium of neutrophils from healthy donors can slow cancer cell proliferation. Conditioned medium of tumor-associated neutrophils (TANs) can drastically enhance cervical cancer cell growth in vitro and in vivo. CONCLUSIONS: The CXCLs-CXCR2 axis is critical in neutrophil recruitment and tumor cell proliferation in the cervical cancer microenvironment.

Long-term efficacy, safety and biocompatibility of a novel sirolimus eluting iron bioresorbable scaffold in a porcine model.

Iron is considered as an attractive alternative material for bioresorbable scaffolds (BRS). The sirolimus eluting iron bioresorbable scaffold (IBS), developed by Biotyx Medical (Shenzhen, China), is the only iron-based BRS with an ultrathin-wall design. The study aims to investigate the long-term efficacy, safety, biocompatibility, and lumen changes during the biodegradation process of the IBS in a porcine model. A total of 90 IBSs and 70 cobalt-chromium everolimus eluting stents (EES) were randomly implanted into nonatherosclerotic coronary artery of healthy mini swine. The multimodality assessments including coronary angiography, optical coherence tomography, micro-computed tomography, magnetic resonance imaging, real-time polymerase chain reaction (PCR), and histopathological evaluations, were performed at different time points. There was no statistical difference in area stenosis between IBS group and EES group at 6 months, 1year, 2 years and 5 years. Although the scaffolded vessels narrowed at 9 months, expansive remodeling with increased mean lumen area was found at 3 and 5 years. The IBS struts remained intact at 6 months, and the corrosion was detectable at 9 months. At 5 years, the iron struts were completely degraded and absorbed in situ, without in-scaffold restenosis or thrombosis, lumen collapse, aneurysm formation, and chronic inflammation. No local or systemic toxicity and abnormal histopathologic manifestation were found in all experiments. Results from real-time PCR indicated that no sign of iron overload was reported in scaffolded segments. Therefore, the IBS shows comparable efficacy, safety, and biocompatibility with EES, and late lumen enlargement is considered as a unique feature in the IBS-implanted vessels.

Long-term safety and absorption assessment of a novel bioresorbable nitrided iron scaffold in porcine coronary artery.

This study aimed to investigate the long-term biocompatibility, safety, and degradation of the ultrathin nitrided iron bioresorbable scaffold (BRS) in vivo, encompassing the whole process of bioresorption in porcine coronary arteries. Fifty-two nitrided iron scaffolds (strut thickness of 70 µm) and 28 Vision Co-Cr stents were randomly implanted into coronary arteries of healthy mini-swine. The efficacy and safety of the nitrided iron scaffold were comparable with those of the Vision stentwithin 52 weeks after implantation. In addition, the long-term biocompatibility, safety, and bioresorption of the nitrided iron scaffold were evaluated by coronary angiography, optical coherence tomography, micro-computed tomography, scanning electron microscopy, energy dispersive spectrometry and histopathological evaluations at 4, 12, 26, 52 weeks and even at 7 years after implantation. In particular, a large number of struts were almost completely absorbed in situ at 7 years follow-up, which were first illustrated in this study. The lymphatic drainage pathway might serve as the potential clearance way of iron and its corrosion products.

A concise, protection-free and divergent approach for the enantioselective syntheses of two pheromonal epoxide components of the fall webworm moth and other species.

On the basis of Zhou's modified Sharpless asymmetric epoxidation, sequential coupling reactions, and a divergent strategy, the protection-free syntheses of two main pheromonal components 1 and 5, found in the fall webworm moth, Hyphantria cunea, and other species have been accomplished in 10 steps (for two compounds). The overall yields are 31% for 1, 28% for 5, and 25% for both 1 and 5, respectively. The ee values of the final products 1 and 5 are at least 99%.

Pentraxin 3 contributes to neurogenesis after traumatic brain injury in mice.

Emerging evidence indicates that pentraxin 3 is an acute-phase protein that is linked with the immune response to inflammation. It is also a newly discovered marker of anti-inflammatory A2 reactive astrocytes, and potentially has multiple protective effects in stroke; however, its role in the adult brain after traumatic brain injury is unknown. In the present study, a moderate model of traumatic brain injury in mice was established using controlled cortical impact. The models were intraventricularly injected with recombinant pentraxin 3 (the recombinant pentraxin 3 group) or an equal volume of vehicle (the control group). The sham-operated mice underwent craniotomy, but did not undergo the controlled cortical impact. The potential neuroprotective and neuroregenerative roles of pentraxin 3 were investigated on days 14 and 21 after traumatic brain injury. Western blot assay showed that the expression of endogenous pentraxin 3 was increased after traumatic brain injury in mice. Furthermore, the neurological severity test and wire grip test revealed that recombinant pentraxin 3 treatment reduced the neurological severity score and increased the wire grip score, suggesting an improved recovery of sensory-motor functions. The Morris water maze results demonstrated that recombinant pentraxin 3 treatment reduced the latency to the platform, increased the time spent in the correct quadrant, and increased the number of times traveled across the platform, thus suggesting an improved recovery of cognitive function. In addition, to investigate the effects of pentraxin 3 on astrocytes, specific markers of A2 astrocytes were detected in primary astrocyte cultures in vitro using western blot assay. The results demonstrated that pentraxin 3 administration activates A2 astrocytes. To explore the protective mechanisms of pentraxin 3, immunofluorescence staining was used. Intraventricular injection of recombinant pentraxin 3 increased neuronal maintenance in the peri-injured cortex and ipsilateral hippocampus, increased the number of doublecortin-positive neural progenitor cells in the subventricular and subgranular zones, and increased the number of bromodeoxyuridine (proliferation) and neuronal nuclear antigen (mature neuron) double-labeled cells in the hippocampus and peri-injured cortex. Pentraxin 3 administration also increased the number of neurospheres and the number of bromodeoxyuridine and doublecortin double-labeled cells in neurospheres, and enhanced the proliferation of neural progenitor cells in primary neural progenitor cell cultures in vitro. In conclusion, recombinant pentraxin 3 administration activated A2 astrocytes, and consequently improved the recovery of neural function by increasing neuronal survival and enhancing neurogenesis. All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China on March 1, 2016.

Clinical characteristics of early and late drug-eluting stent in-stent restenosis and mid-term prognosis after repeated percutaneous coronary intervention.

BACKGROUND: The mechanism and characteristics of early and late drug-eluting stent in-stent restenosis (DES-ISR) have not been fully clarified. Whether there are different outcomes among those patients being irrespective of their repeated treatments remain a knowledge gap. METHODS: A total of 250 patients who underwent initial stent implantation in our hospital, and then were readmitted to receive treatment for the reason of recurrent significant DES-ISR in 2016 were involved. The patients were categorized as early ISR (<12 months; E-ISR; n = 32) and late ISR (≥12 months; L-ISR; n = 218). Associations between patient characteristics and clinical performance, as well as clinical outcomes after a repeated percutaneous coronary intervention (PCI) were evaluated. Primary composite endpoint of major adverse cardiac events (MACEs) included cardiac death, non-fatal myocardial infarction (MI), or target lesion revascularization (TLR). RESULTS: Most baseline characteristics are similar in both groups, except for the period of ISR, initial pre-procedure thrombolysis in myocardial infarction, and some serum biochemical indicators. The incidence of MACE (37.5% vs. 5.5%; P < 0.001) and TLR (37.5% vs. 5.0%; P < 0.001) is higher in the E-ISR group. After multivariate analysis, E-ISR (odds ratio [OR], 13.267; [95% CI 4.984-35.311]; P < 0.001) and left ventricular systolic dysfunction (odds ratio [OR], 6.317; [95% CI 1.145-34.843]; P = 0.034) are the independent predictors for MACE among DES-ISR patients in the mid-term follow-up of 12 months. CONCLUSIONS: Early ISR and left ventricular systolic dysfunction are associated with MACE during the mid-term follow-up period for DES-ISR patients. The results may benefit the risk stratification and secondary prevention for DES-ISR patients in clinical practice.

(S)-5-Hexyl-1-[(S)-2-hydr-oxy-1-phenyl-ethyl]-4-meth-oxy-1H-pyrrol-2(5H)-one.

The title compound, C(19)H(27)NO(3), was obtained by the reaction of (3S,7aR)-7a-hexyl-7-meth-oxy-3-phenyl-2,3-dihydro-pyrrolo[2,1-b]oxazol-5(7aH)-one and triethyl-silane using titanium(IV) chloride as catalyst. In the mol-ecule, the phenyl and dihydro-pyrrolone rings form a dihedral angle of 83.8 (1)°. O-H⋯O hydrogen-bonding inter-actions lead to the formation of a chain parallel to the a axis.

Organocatalytic, Enantioselective Reductive Bis-functionalization of Secondary Amides: One-Pot Construction of Chiral 2,2-Disubstituted 3-Iminoindoline.

We report the first catalytic, enantioselective reductive bis-functionalization of common amides, which provides a facile access to a variety of 2,2-disubstituted 3-iminoindolines in good yields and with excellent enantioselectivities. The reaction conditions are quite mild and can be run on a gram scale. In this one-pot reaction, three C-C bonds, one ring, and one nitrogen-containing tetrasubstituted carbon stereocenter are created in a catalytic enantioselective manner.

Preclinical Evaluation of a Novel Sirolimus-Eluting Iron Bioresorbable Coronary Scaffold in Porcine Coronary Artery at 6 Months.

OBJECTIVES: The aim of this study was to investigate the operability, 6-month efficacy, and safety of the novel sirolimus-eluting iron bioresorbable coronary scaffold (IBS) system compared with a cobalt-chromium everolimus-eluting stent (EES) (XIENCE Prime stent) in porcine coronary arteries. BACKGROUND: Bioresorbable scaffolds have been considered the fourth revolution in percutaneous coronary intervention. However, the first-generation bioresorbable scaffold showed suboptimal results. METHODS: Forty-eight IBS and 48 EES were randomly implanted into nonatherosclerotic swine. The operability, efficacy, and safety of the IBS and EES were evaluated using coronary angiography, optical coherence tomography, micro-computed tomography, scanning electron microscopy, and histopathologic evaluation at 7, 14, 28, 90, and 180 days after implantation. RESULTS: The operability of the ultrathin IBS (∼70 µm) was comparable with that of the EES, except for its visibility. There was no statistically significant difference in area stenosis between the IBS and EES from 28 to 180 days. The IBS maintained its integrity up to 90 days without corrosion, while corrosion was observed in a few struts in 2 of 10 IBS at 180 days. The percentage of endothelialization of IBS was higher than that of XIENCE Prime stents within 14 days after implantation. The fibrin score was higher in the IBS group at 28 days but comparable with the EES group at 90 and 180 days. No scaffold or stent thrombosis was seen in either group. No abnormal histopathologic changes in scaffolded or stented vessel segments and 5 main remote organs were observed in either group. CONCLUSIONS: Preclinical results suggest that the novel IBS has comparable operability, mid-term efficacy, and safety with the EES, and its corrosion profile in porcine coronary arteries is reasonable, which could support initial clinical study of the IBS.

(3S,7aR)-7-Meth-oxy-7a-methyl-3-phenyl-2,3-dihydro-pyrrolo[2,1-b]oxazol-5(7aH)-one.

In the title chiral butterfly-like bicyclic lactam, C(14)H(15)NO(3), the phenyl and methyl groups are syn with respect to each other. The dihydro-pyrrrole ring adopts a boat conformation, whereas the oxazole ring has a slightly distorted boat conformation. The packing of mol-ecules in the crystal structure is stabilized by inter-molecular C-H⋯O hydrogen bonds.

(S)-1,5-Dibenzyl-3-tert-butyl-imidazol-idin-4-one.

The title compound, C(21)H(26)N(2)O, was obtained as an unexpected by-product when attempting to prepare (S)-2-benzyl-N-tert-butyl-1,2,3,4-tetra-hydro-isoquinoline-3-carboxamide from (S)-2-benzyl-amino-N-tert-butyl-3-phenyl-propanamide and dimethoxy-methane. The mol-ecules are linked by weak C-H⋯O hydrogen bonds, generating linear chains parallel to the b axis. C-H⋯π inter-actions provide further stability for the crystal structure. The planes of the two phenyl rings make a dihedral angle of 84.1 (1)°. The absolute configuration was known from the starting material.

(1S,3S,4S)-tert-Butyl N-[1-benzyl-3-hydr-oxy-5-phenyl-4-(picolinamido)pent-yl]carbamate.

The title compound, C(29)H(35)N(3)O(4), was obtained by the reaction of (2S,4S,5S)-tert-butyl N-(4-amino-1-benzyl-3-hydr-oxy-5-phenyl-pent-yl)carbamate and picolinic acid using oxalyl chloride as a chlorinating reagent to activate the carboxyl group. In the crystal structure there are two mol-ecules in the asymmetric unit, which are aligned edge-to-face. In one mol-ecule, the pyridyl ring forms a dihedral angle of 22.0 (1)° with the phenyl ring of the terminal benzyl group and 14.3 (1)° with the other phenyl ring; in the other mol-ecule, the corresponding angles are 12.1 (1) and 10.6 (1)°, respectively. The packing is stabilized by inter-molecular hydrogen bonds and C-H⋯π inter-actions.