A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Mechanistic study of dual-function inhibitors targeting topoisomerase II and Rad51-mediated DNA repair pathway against castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair. METHODS: Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis. RESULTS: A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIß but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51. CONCLUSION: The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.

Engineering Additive Manufacturing and Molding Techniques to Create Lifelike Willis' Circle Simulators with Aneurysms for Training Neurosurgeons.

Neurosurgeons require considerable expertise and practical experience in dealing with the critical situations commonly encountered during difficult surgeries; however, neurosurgical trainees seldom have the opportunity to develop these skills in the operating room. Therefore, physical simulators are used to give trainees the experience they require. In this study, we created a physical simulator to assist in training neurosurgeons in aneurysm clipping and the handling of emergency situations during surgery. Our combination of additive manufacturing with molding technology, elastic material casting, and ultrasonication-assisted dissolution made it possible to create a simulator that realistically mimics the brain stem, soft brain lobes, cerebral arteries, and a hollow transparent Circle of Willis, in which the thickness of vascular walls can be controlled and aneurysms can be fabricated in locations where they are likely to appear. The proposed fabrication process also made it possible to limit the error in overall vascular wall thickness to just 2-5%, while achieving a Young's Modulus closely matching the characteristics of blood vessels (~5%). One neurosurgical trainee reported that the physical simulator helped to elucidate the overall process of aneurysm clipping and provided a realistic impression of the tactile feelings involved in this delicate operation. The trainee also experienced shock and dismay at the appearance of leakage, which could not immediately be arrested using the clip. Overall, these results demonstrate the efficacy of the proposed physical simulator in preparing trainees for the rigors involved in performing highly delicate neurological surgical operations.

Risk prediction for 30-day mortality among patients with Clostridium difficile infections: a retrospective cohort study.

Background: Current guidelines have unsatisfied performance in predicting severe outcomes after Clostridium difficile infection (CDI). Our objectives were to develop a risk prediction model for 30-day mortality and to examine its performance among inpatients with CDI. Methods: This retrospective cohort study was conducted at China Medical University Hospital, a 2111-bed tertiary medical center in central Taiwan. We included adult inpatients who had a first positive C. difficile culture or toxin assay and had diarrhea as the study population. The main exposure of interest was the biochemical profiles of white blood cell count, serum creatinine (SCr), estimated glomerular filtration rate, blood urea nitrogen (BUN), serum albumin, and glucose. The primary outcome was the 30-day all-cause mortality and the secondary outcome was the length of stay in the intensive care units (ICU) following CDI. A multivariable Cox model and a logistic regression model were developed using clinically relevant and statistically significant variables for 30-day mortality and for length of ICU stay, respectively. A risk scoring system was established by standardizing the coefficients. We compared the performance of our models and the guidelines. Results: Of 401 patients, 23.4% died within 30 days. In the multivariable model, malignancy (hazard ratio [HR] = 1.95), ≥ 1.5-fold rise in SCr (HR = 2.27), BUN-to-SCr ratio > 20 (HR = 2.04), and increased glucose (≥ 193 vs < 142 mg/dL, HR = 2.18) were significant predictors of 30-day mortality. For patients who survived the first 30 days of CDI, BUN-to-SCr ratio > 20 (Odds ratio [OR] = 4.01) was the only significant predictor for prolonged (> 9 days) length of ICU stay following CDI. The Harrell's c statistic of our Cox model for 30-day mortality (0.727) was significantly superior to those of SHEA-IDSA 2010 (0.645), SHEA-IDSA 2018 (0.591), and ECSMID (0.650). Similarly, the conventional c statistic of our logistic regression model for prolonged ICU stay (0.737) was significantly superior to that of the guidelines (SHEA-IDSA 2010, c = 0.600; SHEA-IDSA 2018, c = 0.634; ESCMID, c = 0.645). Our risk prediction scoring system for 30-day mortality correctly reclassified 20.7, 32.1, and 47.9% of patients, respectively. Conclusions: Our model that included novel biomarkers of BUN-to-SCr ratio and glucose have a higher predictive performance of 30-day mortality and prolonged ICU stay following CDI than do the guidelines.

Multi-Parameter Relief Map from High-Resolution DEMs: A Case Study of Mudstone Badland.

Topographic parameters of high-resolution digital elevation models (DEMs) with meter to sub-meter spatial resolution, such as slope, curvature, openness, and wetness index, show the spatial properties and surface characterizations of terrains. The multi-parameter relief map, including two-parameter (2P) or three-parameter (3P) information, can visualize the topographic slope and terrain concavities and convexities in the hue, saturation, and value (HSV) color system. Various combinations of the topographic parameters can be used in the relief map, for instance, using wetness index for upstream representation. In particular, 3P relief maps are integrated from three critical topographic parameters including wetness or aspect, slope, and openness data. This study offers an effective way to explore the combination of topographic parameters in visualizing terrain features using multi-parameter relief maps in badlands and in showing the effects of smoothing and parameter selection. The multi-parameter relief images of high-resolution DEMs clearly show micro-topographic features, e.g., popcorn-like morphology and rill.

A cone-beam computed tomography study of C-shaped root canal systems in mandibular second premolars in a Taiwan Chinese subpopulation.

BACKGROUND/PURPOSE: The purpose of this study is to investigate the prevalence and the morphologic characteristics of the radicular groove and root canal system in human mandibular second premolars with C-shaped root in a Taiwan Chinese subpopulation using cone-beam computed tomographic (CBCT) imaging. METHODS: CBCT images of 580 mandibular second premolars were collected from 317 patients. All of the mandibular second premolars were examined in serial axial sections to identify the presence of any C-shaped root and C-shaped canal systems. The morphologic characteristics of mandibular second premolars with C-shaped roots were studied by performing measurements of serial axial sections. RESULTS: The prevalence rate of mandibular second premolars with a C-shaped root was 3.45% (20/580 teeth) and the rate of those with a C-shaped canal system was 2.24% (13/580 teeth). It was found that 69% of the radicular grooves were located on the lingual half of the root (9/13 teeth) in mandibular second premolars with a C-shaped canal system. In those teeth with a lingual radicular groove, the main canal was toward the buccal side. Frequently, the first C-shaped canal image was found at the mid-root level. The deepest part of the radicular groove was located at about 2.5 mm apical to the first C-shaped canal image. CONCLUSION: There was a 2-3% morphologic variation of the mandibular second premolar with a C-shaped root canal system among the Taiwan Chinese subpopulation investigated in this study. Detailed knowledge of the morphological characteristics of teeth may be valuable when choosing clinical treatments.

Disco spotlight sign: A sign that may be useful for diagnosis of pneumomediastinum.

The use of ultrasonography for the investigation of pneumomediastinum is limited by the presence of air artifacts. Air accumulation in the mediastinum obscures the heart, sometimes leading to misinterpretation as lung tissue. We found that cardiac apical swinging during the heart cycle, however, can create a uniquely characteristic squeezing of mediastinal free air, producing a sonographic B-line that flashes in and out. We named this dynamic finding, the "disco spotlight" sign. This finding may be useful to confirm the diagnosis of pneumomediastinum.

Linear Folliculotropic CD30-Positive Lymphomatoid Drug Reaction.

We report a unique case of probable drug-induced CD30-positive lymphomatoid reaction. A 58-year-old woman presented with bilateral facial eruptions of 3 weeks duration composed of erythematosus papules in a linear distribution. The pathological features demonstrated a dense dermal and follicular infiltrate of many medium- to large-sized atypical CD30-positive lymphoid cells. The rash resolved rapidly after discontinuation of her medication 1 week later and did not recur. This case highlights the importance of clinicopathological correlation.

Validating the operational bias and hypothesis of universal exponent in landslide frequency-area distribution.

The exponent decay in landslide frequency-area distribution is widely used for assessing the consequences of landslides and with some studies arguing that the slope of the exponent decay is universal and independent of mechanisms and environmental settings. However, the documented exponent slopes are diverse and hence data processing is hypothesized for this inconsistency. An elaborated statistical experiment and two actual landslide inventories were used here to demonstrate the influences of the data processing on the determination of the exponent. Seven categories with different landslide numbers were generated from the predefined inverse-gamma distribution and then analyzed by three data processing procedures (logarithmic binning, LB, normalized logarithmic binning, NLB and cumulative distribution function, CDF). Five different bin widths were also considered while applying LB and NLB. Following that, the maximum likelihood estimation was used to estimate the exponent slopes. The results showed that the exponents estimated by CDF were unbiased while LB and NLB performed poorly. Two binning-based methods led to considerable biases that increased with the increase of landslide number and bin width. The standard deviations of the estimated exponents were dependent not just on the landslide number but also on binning method and bin width. Both extremely few and plentiful landslide numbers reduced the confidence of the estimated exponents, which could be attributed to limited landslide numbers and considerable operational bias, respectively. The diverse documented exponents in literature should therefore be adjusted accordingly. Our study strongly suggests that the considerable bias due to data processing and the data quality should be constrained in order to advance the understanding of landslide processes.

Wide-converter lens design for a pico projector.

In this paper, we propose a concept for the design of a 0.702× wide-converter lens for a 0.3 in. digital micromirror device. This 0.702× wide-converter lens is capable of enlarging the size of the original projected image at the same projection distance. This 0.702× wide-converter lens is attached in front of the projection lens of a pico projector (PP). Compared with the projection lens of a PP without the 0.702× wide-converter lens, there is an increase in the size of the original projected image of 42.4% at the same projection distance. In other words, there is a saving of projection distance of 29.78% to obtain a projected image of the same size.

Ligand-functionalization of BPEI-coated YVO4:Bi3+,Eu3+ nanophosphors for tumor-cell-targeted imaging applications.

In this study, surface-functionalized, branched polyethylenimine (BPEI)-modified YVO4:Bi(3+),Eu(3+) nanocrystals (NCs) were successfully synthesized by a simple, rapid, solvent-free hydrothermal method. The BPEI-coated YVO4:Bi(3+),Eu(3+) NCs with high crystallinity show broad-band excitation in the λ=250 to 400 nm near-ultraviolet (NUV) region and exhibit a sharp-line emission band centered at λ=619 nm under excitation at λ=350 nm. The surface amino groups contributed by the capping agent, BPEI, not only improve the dispersibility and water/buffer stability of the BPEI-coated YVO4:Bi(3+),Eu(3+) NCs, but also provide a capability for specifically targeted biomolecule conjugation. Folic acid (FA) and epidermal growth factor (EGF) were further attached to the BPEI-coated YVO4:Bi(3+),Eu(3+) NCs and exhibited effective positioning of fluorescent NCs toward the targeted folate receptor overexpressed in HeLa cells or EGFR overexpressed in A431 cells with low cytotoxicity. These results demonstrate that the ligand-functionalized, BPEI-coated YVO4:Bi(3+),Eu(3+) NCs show great potential as a new-generation biological luminescent bioprobe for bioimaging applications. Moreover, the unique luminescence properties of BPEI-coated YVO4:Bi(3+),Eu(3+) NCs show potential to combine with a UVA photosensitizing drug to produce both detective and therapeutic effects for human skin cancer therapy.

Sample stacking by field-amplified sample injection and sweeping for simultaneous analysis of acidic and basic components in clinic application.

In this study, online sample concentration method, which coupled field-amplified sample injection (FASI) and sweeping technology with micellar electrokinetic chromatography (MEKC), was used to detect and analyze acidic and basic components in a single run. In order to concentrate the acidic and basic components simultaneously in a single run sweeping step, a combination of successive anion- and cation-selective injections were used. Before sample loading, a rinse buffer containing 50 mM Tris buffer (pH 3) with 41% MeOH and 0.1% polyethylene oxide (PEO) was injected in order to suppress the electroosmotic flow (EOF). Sample loading of anionic components was achieved by electrokinetic injection at a negative voltage of -2.5 kV for 80 s, and then the cationic components were injected at a positive voltage of +5 kV for 120 s. Finally, sweeping with SDS micelles from the separation buffer (25 mM Tris buffer with 60 mM SDS, pH 3) was performed at a negative voltage of -20 kV. This capillary electrophoretic methodology was applied to the quantification of acidic and basic drugs in commercial tablets and in plasma samples. The precision and accuracy of the proposed method at different concentrations ranging from high, medium, to low were evaluated on spiked plasma samples. The intra and interday precision and accuracy values at three concentrations were all below 6.1%. The method was also successfully applied to monitor the tested drugs in the plasma of nine elderly cardiovascular and/or Alzheimer's disease patients after oral administration of the commercial products.

Crystal structure and mutational analysis of aminoacylhistidine dipeptidase from Vibrio alginolyticus reveal a new architecture of M20 metallopeptidases.

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.

Expression and characterization of the biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase from Vibrio alginolyticus.

The biofilm-related and carnosine-hydrolyzing aminoacylhistidine dipeptidase (pepD) gene from Vibrio alginolyticus was cloned and sequenced. The recombinant PepD protein was produced and biochemically characterized and the putative active-site residues responsible for metal binding and catalysis were identified. The recombinant enzyme, which was identified as a homodimeric dipeptidase in solution, exhibited broad substrate specificity for Xaa-His and His-Xaa dipeptides, with the highest activity for the His-His dipeptide. Sequence and structural homologies suggest that the enzyme is a member of the metal-dependent metallopeptidase family. Indeed, the purified enzyme contains two zinc ions per monomer. Reconstitution of His.Tag-cleaved native apo-PepD with various metal ions indicated that enzymatic activity could be optimally restored when Zn2+ was replaced with other divalent metal ions, including Mn2+, Co2+, Ni2+, Cu2+ and Cd2+, and partially restored when Zn2+ was replaced with Mg2+. Structural homology modeling of PepD also revealed a 'catalytic domain' and a 'lid domain' similar to those of the Lactobacillus delbrueckii PepV protein. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173 and His461 in metal binding and Asp82 and Glu149 in catalysis. In addition, individual substitution of Glu149 and Glu150 with aspartic acid resulted in the partial retention of enzymatic activity, indicating a functional role for these residues on the catalysis and zinc ions, respectively. These effects may be necessary either for the activation of the catalytic water molecule or for the stabilization of the substrate-enzyme tetrahedral intermediate. Taken together, these results may facilitate the design of PepD inhibitors for application in antimicrobial treatment and antibody-directed enzyme prodrug therapy.

Identification of a Vibrio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity.

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.

Identification of galectin I and thioredoxin peroxidase II as two arsenic-binding proteins in Chinese hamster ovary cells.

In this study, we report the identification of two arsenic-binding proteins from Chinese hamster ovary (CHO) cells. The crude extract derived from CHO and SA7 (arsenic-resistant CHO cells) was applied to a phenylarsine oxide-agarose affinity column, and after extensive washing, the absorbed proteins were eluted with buffers containing 20 mM 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). Three differentially expressed proteins, galectin 1 (Gal-1; in the 2-ME-eluted fraction from CHO cells), glutathione S-transferase P-form (GST-P) and thioredoxin peroxidase II (TPX-II), respectively in the 2-ME- and DTT-eluted fractions from SA7 cells, were identified by partial amino acid sequence analysis after separation by SDS/PAGE. The GST-P protein has been previously shown to facilitate the excretion of sodium arsenite [As(III)] from SA7 cells. TPX II was detected predominately in SA7 cells [routinely cultured in As(III)-containing medium], but not in CHO or SA7N (a revertant of SA7 cells cultured in regular medium) cells. In contrast, Gal-1 was specifically identified in CHO and SA7N cells, but not in SA7 cells. The preferential expression of Gal-1 in CHO cells and TPX-II in SA7 cells was further illustrated by quantitative PCR analysis. The binding of Gal-1 and TPX-II with As(III) was further verified by both co-immunoprecipitation and co-elution of Gal-1 and TPX-II with As(III). It is suggested that Gal-1 and TPX-II are two proteins that serve as high-affinity binding sites for As(III) and thus both may be involved in the biological action of As(III).