Structural basis of human Nav1.5 gating mechanisms.

Voltage-gated Nav1.5 channels are central to the generation and propagation of cardiac action potentials1. Aberrations in their function are associated with a wide spectrum of cardiac diseases including arrhythmias and heart failure2-5. Despite decades of progress in Nav1.5 biology6-8, the lack of structural insights into intracellular regions has hampered our understanding of its gating mechanisms. Here we present three cryo-EM structures of human Nav1.5 in previously unanticipated open states, revealing sequential conformational changes in gating charges of the voltage-sensing domains (VSDs) and several intracellular regions. Despite the channel being in the open state, these structures show the IFM motif repositioned in the receptor site but not dislodged. In particular, our structural findings highlight a dynamic C-terminal domain (CTD) and III-IV linker interaction, which regulates the conformation of VSDs and pore opening. Electrophysiological studies confirm that disrupting this interaction results in the fast inactivation of Nav1.5. Together, our structure-function studies establish a foundation for understanding the gating mechanisms of Nav1.5 and the mechanisms underlying CTD-related channelopathies.

Structural insights into actin isoforms.

Actin isoforms organize into distinct networks that are essential for the normal function of eukaryotic cells. Despite a high level of sequence and structure conservation, subtle differences in their design principles determine the interaction with myosin motors and actin-binding proteins. Therefore, identifying how the structure of actin isoforms relates to function is important for our understanding of normal cytoskeletal physiology. Here, we report the high-resolution structures of filamentous skeletal muscle α-actin (3.37 Å), cardiac muscle α-actin (3.07 Å), ß-actin (2.99 Å), and γ-actin (3.38 Å) in the Mg2+·ADP state with their native post-translational modifications. The structures revealed isoform-specific conformations of the N-terminus that shift closer to the filament surface upon myosin binding, thereby establishing isoform-specific interfaces. Collectively, the structures of single-isotype, post-translationally modified bare skeletal muscle α-actin, cardiac muscle α-actin, ß-actin, and γ-actin reveal general principles, similarities, and differences between isoforms. They complement the repertoire of known actin structures and allow for a comprehensive understanding of in vitro and in vivo functions of actin isoforms.

The protein actin is important for many fundamental processes in biology, from contracting muscle to dividing a cell in two. As actin is involved in such a variety of roles, human cells have slightly different versions of the protein, known as isoforms. For example, alpha-actin is vital for contracting muscle, while beta- and gamma-actin drive cellular processes in non-muscle cells. In order to carry out its various functions, actin interacts with many other proteins inside the cell, such as myosin motors which power muscle contraction. These interactions rely on the precise chain of building blocks, known as amino acids, that make up the actin isoforms; even subtle alterations in this sequence can influence the behavior of the protein. However, it is not clear how differences in the amino acid sequence of the actin isoforms impact actin's interactions with other proteins. Arora et al. addressed this by studying the structure of four human actin isoforms using a technique called cryo-electron microscopy, where the proteins are flash-frozen and bombarded with electrons. These experiments showed where differences between the amino acid chains of each isoform were located in the protein. Arora et al. then compared their structures with previous work showing the structure of actin bound to myosin. This revealed that the tail-end of the protein (known as the N-terminus) differed in shape between the four isoforms, and this variation may influence how actin binds to others proteins in the cell. These results are an important foundation for further work on actin and how it interacts with other proteins. The structures could help researchers design new tools that can be used to target specific isoforms of actin in different types of laboratory experiments.

Radiographic Evaluation of Regeneration Strategies for the Treatment of Advanced Mandibular Furcation Defects: A Retrospective Study.

Teeth with furcation involvement (FI) present a higher risk of loss and are difficult to maintain. This study evaluated the efficacy of furcation defect regeneration (FDR) as a regeneration strategy. Pre-operative and 6-month postoperative radiographs were collected from patients receiving regeneration therapy for mandibular teeth with degree II and early degree III FI. The linear furcation involvement (LFI), ratio of LFI (RLI), LFI and RLI adjusted bythe alveolar bone crest (ABC), and radiographic intensity were assessed. The effects of demographic characteristics, regeneration treatment strategies, the relationship between furcation and ABC, and adjacent intrabony defect regeneration (AIDR) were evaluated using a generalized linear model and logistic regression. The results demonstrated that 1.5 mm adjusted LFI and 40% adjusted RLI were achieved in both pure furcation defects and combined furcation-angular defects by the combination of bone replacement grafts (BRG) and enamel matrix derivatives (EMD) or collagen membrane (CM); deproteinized bovine bone matrix (DBBM) showed a superior outcome among BRG. In combined furcation-angular defects, EMD appeared more beneficial than CM, and AIDR significantly promoted adjusted LFI and RLI. In conclusion, DBBM with EMD or CM was effective for FDR, and AIDR had a positive effect on FDR in the combined furcation-angular defect.

Robust combination of liver stereotactic body radiotherapy modulates pharmacokinetics of sorafenib toward preferable parameters.

To evaluate the effect and mechanism of radiotherapy (RT)-sorafenib pharmacokinetics (PK) in different regimens with conventional or high dose irradiation. Between February 2012 and December 2018, 43 patients with portal vein tumor thrombosis treated with sorafenib plus conventional RT (58%) or stereotactic body radiation therapy (SBRT, 42%) were retrospectively reviewed. In vivo and in vitro studies of concurrent and sequential RT with sorafenib were designed. SBRT resulted in a 3-fold increase in complete recanalization compared to conventional RT group (28% vs. 8%, p = 0.014). Compared to the control group, the area under the concentration vs. time curve (AUC) of sorafenib was increased in the concurrent RT2Gy and RT9Gy groups and the sequential RT9Gy group by 132% (p = 0.046), 163% (p = 0.038) and 102% (p = 0.018), respectively; and was decreased by 59% in the sequential RT2Gy group (p = 0.036). Sequential RT2Gy and RT9Gy increased CYP3A4 activity by 82% (p = 0.028) and 203% (p = 0.0004), respectively, compared to that with the corresponding concurrent regimen. SBRT produced better recanalization than conventional RT with sorafenib. The AUC of sorafenib was modulated by RT. P-gp expression was not influenced by RT. The sequential RT regimen increased CYP3A4 activity that may increase the RT-sorafenib synergy effect and overall sorafenib activity. The biodistribution of sorafenib was modulated by local RT with the different regimens.

5-Aroylindoles Act as Selective Histone Deacetylase 6 Inhibitors Ameliorating Alzheimer's Disease Phenotypes.

This paper reports the development of a series of 5-aroylindolyl-substituted hydroxamic acids. N-Hydroxy-4-((5-(4-methoxybenzoyl)-1 H-indol-1-yl)methyl)benzamide (6) has potent inhibitory selectivity against histone deacetylase 6 (HDAC6) with an IC50 value of 3.92 nM. It decreases not only the level of phosphorylation of tau proteins but also the aggregation of tau proteins. Compound 6 also shows neuroprotective activity by triggering ubiquitination. In animal models, compound 6 is able to ameliorate the impaired learning and memory, and it crosses the blood-brain barrier after oral administration. Compound 6 can be developed as a potential treatment for Alzheimer's disease in the future.

Correction: 2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition.

Correction for '2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition' by Kunal Nepali et al., Org. Biomol. Chem., 2016, 14, 716-723.

Antidepressant-like effects of Gan-Mai-Dazao-Tang via monoamine regulatory pathways on forced swimming test in rats.

Depression is a highly prevalent and recurrent mental disorder that impacts all aspects of human life. Undesirable effects of the antidepressant drugs led to the development of complementary and alternative therapies. Gan-Mai-Da-Zao-Tang (, gan mài dà zǎo tang) is a traditional herbal formula commonly used for the treatment of depression, but lack of scientific proof on its mechanism. It consisted of Glycyrrhiza uralensis Fisch. (licorice), Triticum aestivum L. (wheat) and Zizphus jujuba Mill. (jujube). The objective of this study is to investigate the antidepressant effects of Gan-Mai-Dazao-Tang and its ingredients in rats exposed to forced swimming test (FST). The 72 of male Nerl: Wistar rats (8 weeks old) were randomized into control (10 mL/kg bw H2O), licorice (0.4 g/kg bw), wheat (1.6 g/kg bw), jujube (0.5 g/kg bw), Gan-Mai-Da-Zao-Tang (2.5 g/kg bw of licorice: wheat: jujube in ratio of 5:20:6) and Prozac (18 mg/kg bw) groups. Samples were administered by oral gavage for 21 days. FST was performed on 21st day, with 15 min for pretest followed by 5 min for real test. Then, the animals were sacrificed and brain tissues were collected for monoamines analyses. The Gan-Mai-Da-Zao-Tang (LWJ) showed significantly down-regulation of immobility time, 3,4-dihydroxyphenylacetic acid (DOPAC) and DOPAC/dopamine (DA) turnover rates, and also enhanced the concentration of serotonin (5-HT) and DA in brain tissues, as compared with the control. The LWJ stated the potent antidepressant-like effect by modulating these monoamines concentration, while the licorice, wheat and jujube did not reported significant results as compared with control group. The positive control (Prozac) was noted with significantly reduction in body weight and appetite. In conclusion, the antidepressant-like effects of LWJ might be mediated by the regulation of monoamine neurotransmitters. Thus, it could beneficial in depression treatment as a complementary approach.

(N-Hydroxycarbonylbenylamino)quinolines as Selective Histone Deacetylase 6 Inhibitors Suppress Growth of Multiple Myeloma in Vitro and in Vivo.

A series of bicyclic arylamino/heteroarylamino hydroxamic acids (7-31) have been examined as novel histone deacetylase 6 (HDAC6) inhibitors. One compound (13) exhibits remarkable inhibitory activity of HDAC6 with an IC50 value of 0.29 nM, which is 4,000-43,000 times more selective over other HDAC isoforms. Compound 13 was shown to have antiproliferative activity against human multiple myeloma RPMI 8226, U266, and NCI-H929 cells with no effect on normal bone marrow cells. Compound 13, as a single drug, suppresses the growth of tumors by a %TGI factor of 60.4% in human multiple myeloma RPMI 8226 xenograft models and, in combination with bortezomib, shows significant in vivo antitumor activity (%TGI = 86.2%). Compound 13 also demonstrates good human hepatocytic stability and high permeability, without any effect on mutagenicity and cytotoxicity. Thus, compound 13 is a potent HDAC6 inhibitor that could be developed for the treatment of multiple myeloma in the future.

1,4-Naphthoquinones as inhibitors of Itch, a HECT domain-E3 ligase, and tumor growth suppressors in multiple myeloma.

A series of 1,4-naphthoquinones (10a-10q) were synthesized and evaluated for anticancer activity. Compound 10e was identified as an inhibitor of Itch, a HECT domain-E3 ligase. In an evaluation of in vivo efficacy, 10e exhibited remarkable anticancer activity with TGI values of 98.3% and 100% at 25 mg/kg and 50 mg/kg orally daily, respectively, against human RPMI-8226 multiple myeloma xenograft. Treatment with 10e also showed a decrease of Itch level in human RPMI-8226 multiple myeloma cells. Thus 10e is a lead compound for further development of inhibitors targeting E3 ligase for treatment of multiple myeloma.

3-Aroylindoles display antitumor activity in vitro and in vivo: Effects of N1-substituents on biological activity.

A series of 3-aroylindole hydroxamic acids (10-17) were developed based on the concept of a structural combination of tubulin and histone deacetylase (HDAC) inhibitors. This was accomplished by introducing hydroxamic acid-containing moieties at the N1 position of the tubulin assembly inhibitor, compound 9 (SCB01A, BPR0L075, phase II trial). Most of synthetic compounds produced in this way displayed comparable HDAC inhibitory activity, and four (10, 12-14) of them also inhibit tubulin assembly. Notably, compound 12 possesses not only tubulin and HDAC inhibitory activity but also shows HDAC6 selectivity over other HDAC isoforms. In addition, it exhibits remarkable inhibitory activity against the growth cancer cells in vitro and in vivo (PC3 and RPMI-8226 cells). Notably, it suppresses the growth of multiple myeloma xenografts without leading to the death of teated animals like reference compound. In sum, this study provided potential compounds with safer profiles for cancer treatment.

Combination of OipA, BabA, and SabA as candidate biomarkers for predicting Helicobacter pylori-related gastric cancer.

Helicobacter pylori (H. pylori ) infection is a major cause of chronic gastritis and is highly related to duodenal ulcer (DU) and gastric cancer (GC). To identify H. pylori-related GC biomarkers with high seropositivity in GC patients, differences in levels of protein expression between H. pylori from GC and DU patients were analyzed by isobaric tag for relative and absolute quantitation (iTRAQ). In total, 99 proteins showed increased expression (>1.5-fold) in GC patients compared to DU patients, and 40 of these proteins were categorized by KEGG pathway. The four human disease-related adhesin identified, AlpA, OipA, BabA, and SabA, were potential GC-related antigens, with a higher seropositivity in GC patients (n = 76) than in non-GC patients (n = 100). Discrimination between GC and non-GC patients was improved using multiple antigens, with an odds ratio of 9.16 (95% CI, 2.99-28.07; p < 0.0001) for three antigens recognized. The optimized combination of OipA, BabA, and SabA gave a 77.3% correct prediction rate. A GC-related protein microarray was further developed using these antigens. The combination of OipA, BabA, and SabA showed significant improvement in the diagnostic accuracy and the protein microarray containing above antigens should provide a rapid and convenient diagnosis of H. pylori-associated GC.

2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition.

This study reports the synthesis of a series of 2-aroylquinoline-5,8-diones (11-23) on the basis of scaffold hopping. The presence of a methoxy group at C6 assists the highly regioselective incorporation with various amines, and simplifies the structural identification process. Among the synthetic compounds, 6-dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (12) and 7-pyrrolidin-1-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (23) exhibit remarkable anti-proliferative activity against the cancer cell lines tested with mean IC50 values of 0.14 and 0.27 µM, respectively. Compound 23 showed moderate inhibitory activity against tubulin polymerization with an IC50 value of 5.9 µM. In a western blot analysis, 23 caused induction of HSP70 and degradation of Akt, revealing that it possesses HSP90 inhibitory activity.

Pyrimidinedione-mediated selective histone deacetylase 6 inhibitors with antitumor activity in colorectal cancer HCT116 cells.

We synthesized a series of pyrimidinedione derivatives and evaluated their activities. The results indicate that compound 6, 4-[5-fluoro-2,6-dioxo-3-(tetrahydro-furan-2-yl)-3,6-dihydro-2H-pyrimidin-1-ylmethyl]-N-hydroxy-benzamide, exhibits potent antiproliferative activity, apoptosis induction with cleavage of caspase and PARP, and enhanced tendency to inhibit HDAC6 (IC50 = 12.4 nM) activity over HDAC1 (IC50 = 1710 nM) and HDAC2 (IC50 = 5500 nM). Compound 6 also inhibits tumor growth and is less toxic than parent 4 in vivo. These data provide compelling evidence that compound 6 is a potential antitumor compound with HDAC6 targeted inhibitory activity and may be tested for preclinical investigation for cancer treatment.

Trypsin-induced proteome alteration during cell subculture in mammalian cells.

BACKGROUND: It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. METHODS: In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. RESULTS: 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. CONCLUSIONS: In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

Functional phenotype of macrophages depends on assay procedures.

Macrophages display different phenotypes that can switch in response to their micro-environment. In our earlier study (Chiang, C. S., Liu, W. C. and Jung, S. M., 2005. Compartmental responses after thoracic irradiation of mice: strain differences. Int. J. Radiat. Oncol. Biol. Phys. 62:862) on radiation-induced cytokine expression in lung lavage samples, there was a suggestion that the procedures used to harvest lung macrophages affected the profiles they expressed. To further explore this issue, we examined gene expression by cell populations, mainly macrophages, isolated by lavage from lung and peritoneal cavity following either in vivo or in vitro stimulation with LPS, IFN-gamma or irradiation. We found that expression of mRNA for tumor necrosis factor-alpha, IL-1 alpha/beta and IL-6 varied several fold depending on whether the assay was performed on cells immediately after isolation or after in vitro manipulation. The relative level of inducible nitric oxide synthase (iNOS) to arginase I (Arg I), which is frequently used as index of the M1 versus M2 functional macrophage phenotype, also varied. LPS stimulation in vivo was able to change the profile from Arg I expression to one where the iNOS pathway became dominant, but was unable to do this in vitro. This contrasts with the ability of IFN-gamma to generate an iNOS-dominant pathway in vitro, but not in vivo. This study cautions that the expression of inflammatory cytokines and the iNOS to Arg I ratio, which is often used as an index of their functional capacity, varies with the experimental conditions.

Macrophages from irradiated tumors express higher levels of iNOS, arginase-I and COX-2, and promote tumor growth.

PURPOSE: To investigate the effects of single and fractionated doses of radiation on tumors and tumor-associated macrophages (TAMs), and to elucidate the potential of TAMs to influence tumor growth. METHODS AND MATERIALS: A murine prostate cell line, TRAMP-C1, was grown in C57Bl/6J mice to 4-mm tumor diameter and irradiated with either 25 Gy in a single dose, or 60 Gy in 15 fractions. The tumors were removed at the indicated times and assessed for a variety of markers related to TAM content, activation status, and function. RESULTS: In tumors receiving a single radiation dose, arginase (Arg-I), and cycloxygenase-2 (COX-2) mRNA expression increased as a small transient wave within 24 h and a larger persistent wave starting after 3 days. Inducible nitric oxide synthase (iNOS) mRNA was elevated only after 3 days and continued to increase up to 3 weeks. After fractionated irradiation, Arg-1 and COX-2 mRNA levels increased within 5 days, whereas iNOS was increased only after 10 fractions of irradiation had been given. Increased levels of Arg-I, COX-2, and, to a lesser extent, iNOS protein were found to associate with TAMs 1-2 weeks after tumor irradiation. Function of TAMs were compared by mixing them with TRAMP-C1 cells and injecting them into mice; TRAMP-C1 cells mixed with TAMs from irradiated tumors appeared earlier and grew significantly faster than those mixed with TAMs from unirradiated tumors or TRAMP-C1 alone. CONCLUSIONS: Tumor-associated macrophages in the postirradiated tumor microenvironment express higher levels of Arg-1, COX-2, and iNOS, and promote early tumor growth in vivo.