Alginate Hydrogel-Embedded Capillary Sensor for Quantitative Immunoassay with Naked Eye.

We have developed an alginate hydrogel-embedded capillary sensor (AHCS) for naked eye-based quantification of immunoassay. Alkaline phosphatase (ALP) can modulate gel-sol transformation to increase the permeability of Cu2+-cross-linked alginate hydrogel film in the AHCS, followed by solution exchange into the capillary. Through measuring the length of the liquid phase of the microfluidics in the capillary at a given time, the concentration of the ALP could be quantified with the naked eye. Since ALP is widely applied as a signal reporter for immunoassays, the AHCS could easily accommodate conventional immune sensing platforms. We justify the practicality of AHCS with hepatitis B virus surface antigen (HBsAg) in serum samples and got comparable results with commercialized immunoassay. This AHCS is easy to make and use, effective in cost, and robust in quantification with the naked eye, showing great promise for next generation point-of-care testing.

Toward Atomistic Modeling of Irreversible Covalent Inhibitor Binding Kinetics.

Covalent inhibitors have emerged as an important drug class in recent years, largely due to their many unique advantages as compared to noncovalent inhibitors, including longer duration of action, lower prolonged systemic exposure, higher potency, and selectivity. However, the potential off-target toxicity of covalent inhibitors, particularly of irreversible covalent inhibitors, represents a great challenge in covalent drug development. Therefore, accurate calculation of protein covalent inhibitor reaction kinetics to guide the design of selective inhibitors would greatly benefit covalent drug discovery efforts. In the present paper, we present a computational method to calculate the relative reaction kinetics between congeneric irreversible covalent inhibitors and their protein receptors. The method combines density functional theory calculations of the transition state barrier height of the rate-limiting step for reaction between the warhead of the inhibitor and a single protein residue, and molecular-mechanics-based free energy calculations to account for the interactions between the ligand in the transition state and the protein environment. The method was tested on four pharmaceutically interesting irreversible covalent binding systems involving 28 ligands; the mean unsigned error (MUE) of the relative reaction rate for all pairs of ligands between the predictions and experimental results for these tested systems is 0.79 log unit. This is to our knowledge the first time where the reaction kinetics of protein irreversible covalent inhibition have been directly calculated with physics-based free energy calculation methods and transition state theory. We anticipate the outstanding accuracy demonstrated here across a broad range of target classes will have a strong impact on the design of selective covalent inhibitors.

Are induced fit protein conformational changes caused by ligand-binding predictable? A molecular dynamics investigation.

In this work, the ability of molecular dynamics simulations (MD) to prospectively predict regions of ligand binding sites that could undergo induced fit effects was investigated. Conventional MD was run on 39 apo structures (no ligand), and the resulting trajectories were compared to a set of 147 holo X-ray structures (ligand-bound). It was observed from the simulations, in the absence of the ligands, that structures exhibiting large residue conformational changes indicated higher likelihood of induced fit effects. Nevertheless, the simulation results did not perform better than using the normalized crystallographic structural factors as predictors of active-site rigid residues (87% predictive power) and mobile residues (47% predictive power). While the simulations could not produce full active sites conformations similar to holo-like states, it was found that the simulations could reproduce bound state conformations of individual residues. These results suggest potential issues in the use of unligated simulation frames directly for drug design applications such as ligand docking, and an overall caution in the use of protein flexibility in docking protocols should be emphasized. © 2017 Wiley Periodicals, Inc.

Biological Characteristics of Cluster of Differentiation 147 and Its Relationship with Tumour.

Cluster of differentiation 147(CD147)/extracellular matrix metalloproteinase inducer (EMMPRIN) is a widely distributed transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is highly enriched on the surface of malignant tumour cells. A major function of CD147 is to stimulate matrix metalloproteinase production in stromal fibroblasts and endothelial cells. CD147 promotes growth,invasion,metastasis,and glycolysis of malignant cells,induces angiogenesis,multidrug resistance,and anoikis resistance,and inhibits starvation-induced autophagy et al. This review focuses on the structural and biological characteristics of CD147 as well as recent advances in its multiple functions in malignant tumours and underlining mechanisms.

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain.

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

Knowledge-Based Strategy to Improve Ligand Pose Prediction Accuracy for Lead Optimization.

Accurately predicting how a small molecule binds to its target protein is an essential requirement for structure-based drug design (SBDD) efforts. In structurally enabled medicinal chemistry programs, binding pose prediction is often applied to ligands after a related compound's crystal structure bound to the target protein has been solved. In this article, we present an automated pose prediction protocol that makes extensive use of existing X-ray ligand information. It uses spatial restraints during docking based on maximum common substructure (MCS) overlap between candidate molecule and existing X-ray coordinates of the related compound. For a validation data set of 8784 docking runs, our protocol's pose prediction accuracy (80-82%) is almost two times higher than that of one unbiased docking method software (43%). To demonstrate the utility of this protocol in a project setting, we show its application in a chronological manner for a number of internal drug discovery efforts. The accuracy and applicability of this algorithm (>70% of cases) to medicinal chemistry efforts make this the approach of choice for pose prediction in lead optimization programs.

What general conclusions can we draw from kinase profiling data sets?

Understanding general selectivity trends across the kinome has implications ranging from target selection, compound prioritization, toxicity and patient tailoring. Several recent publications have described the characterization of kinase inhibitors via large assay panels, offering a range of generalizations that influenced kinase inhibitor research trends. Since a subset of profiled inhibitors overlap across reports, we evaluated the concordance of activity results for the same compound-kinase pairs across four data sources generated from different kinase biochemical assay technologies. Overall, 77% of all results are within 3 fold or qualitatively in agreement across sources. However, the agreement for active compounds is only 37%, indicating that different profiling panels are in better agreement to determine a compound's lack of activity rather than degree of activity. Low concordance is also found when comparing the promiscuity of kinase targets evaluated from different sources, and the pharmacological similarity of kinases. In contrast, the overall promiscuity of kinase inhibitors was consistent across sources. We highlight the difficulty of drawing general conclusions from such data by showing that no significant selectivity difference distinguishes type I vs. type II inhibitors, and limited kinase space similarity that is consistent across different sources. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Photosynthetic and Self-Draining Biohybrid Dressing for Accelerating Healing of Diabetic Wound.

Wound healing is a well-orchestrated progress associated with angiogenesis, epithelialization, inflammatory status, and infection control, whereas these processes are seriously disturbed in diabetic wounds. In this study, a biohybrid dressing integrating the inherent ability of Bromeliad leaf (photosynthesis and self-draining) with the therapeutic effect of artificial materials (glucose-degrading and ROS-scavenging) is presented. The dressing consists of double-layered structures as follows: 1) Outer layer, a Bromeliad leaf substrate full of alginate hydrogel-immobilized glucose oxidase (GOx@Alg@Bromeliad substrate, abbreviated as BGA), can generate oxygen to guarantee the GOx-catalyzed glucose oxidation by photosynthesis, reducing local hyperglycemia to stabilize hypoxia inducible factor-1 alpha (HIF-1α) for angiogenesis and producing hydrogen peroxide for killing bacteria on the surface of wound tissue. The sophisticated structure of the leaf drains excessive exudate away via transpiration-mimicking, preventing skin maceration and impeding bacterial growth. 2) Inner layer, microneedles containing catalase (CAT-HA MNs, abbreviated as CHM), reduces excessive oxidative stress in the tissue to promote the proliferation of fibroblasts and inhibits proinflammatory polarization of macrophages, improving re-epithelialization of diabetic wounds. Together, the biohybrid dressing (BGA-CHM, abbreviated as BCHM) can enhance angiogenesis, strengthen re-epithelialization, alleviate chronic inflammation, and suppress bacterial infection, providing a promising strategy for diabetic wound therapy.

Trash into Treasure: Nano-coating of Catheter Utilizes Urine to Deprive H2S Against Persister and Rip Biofilm Matrix.

Bacteria-derived hydrogen sulfide (H2S) often contributes to the emergence of antibiotic-recalcitrant bacteria, especially persister (a sub-population of dormant bacteria), thus causing the treatment failure of Catheter-associated urinary tract infection (CAUTI). Here, an H2S harvester nanosystem to prevent the generation of persister bacteria and disrupt the dense biofilm matrix by the self-adaptive ability of shape-morphing is prepared. The nanosystem possesses a core-shell structure that is composed of liquid metal nanoparticle (LM NP), AgNPs, and immobilized urease. The nanosystem decomposes urea contained in urine to generate ammonia for eliminating bacteria-derived H2S. Depending on the oxidative layer of liquid metal, the nanosystem also constitutes a long-lasting reservoir for temporarily storing bacteria-derived H2S, when urease transiently overloads or in the absence of urine in a catheter. Depriving H2S can prevent the emergence of persistent bacteria, enhancing the bacteria-killing efficiency of Ga3+ and Ag+ ions. Even when the biofilm has formed, the urine flow provides heat to trigger shape morphing of the LM NP, tearing the biofilm matrix. Collectively, this strategy can turn trash (urea) into treasure (H2S scavengers and biofilm rippers), and provides a new direction for the antibacterial materials application in the medical field.

Mannan-conjugated adenovirus enhanced gene therapy effects on murine hepatocellular carcinoma cells in vitro and in vivo.

The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. For some patients for whom surgical treatments are not appropriate, one can only rely on chemotherapy. In the conventional chemotherapy, side effects usually occurred in most cases due to high toxicity levels. Moreover, the development of drug resistance toward chemotherapeutic agents often prevents the successful long-term use of chemotherapy for HCC. Gene therapy represents the exciting biotechnological advance that may revolutionize the conventional fashion of cancer treatment. Overexpression of phosphatase and tensin homologue (PTEN) in cancer cells carrying deletion/mutant type of it can induce the apoptosis of cancer cells and inhibit cell proliferation. In this work, in order to make full use of the high transfectivity of adenovirus, we managed to conjugate the polysaccharide mannan (polymannose) to the surface of the adenovirus chemically under appropriate oxidizing conditions to prepare the mannan-modified adenovirus (Man-Ad5-PTEN). The cytotoxicity and anticancer activity of Man-Ad5-PTEN were assessed in vitro. Reporter gene expression of LacZ transferred by Man-Ad5-LacZ was verified on mannose receptor-deficient NIH/3T3 cells versus mannose receptor-efficient macrophages. Hepatocellular carcinoma cell lines transduced by mannan-modified adenovirus were assayed for cell cycle, apoptosis, invasion, and migration. Further, we detected the antitumor effect on intraperitoneal H22 tumor-bearing mice treated by Man-Ad5-PTEN alone or combined with chemotherapeutic agent of doxorubicin. The results demonstrated that cell growth suppression was not observed in Chang normal hepatocyte cells and the cell killing by Man-Ad5-PTEN is tumor selective. Further, the results showed that the strategy of mannan conjugation could enhance adenovirus-mediated PTEN gene therapy effects on murine hepatocellular carcinoma cells in vitro and in vivo.

Machine learning-based models for predicting gas breakthrough pressure of porous media with low/ultra-low permeability.

Gas breakthrough pressure is a significant parameter for the gas exploration and safety evaluation of engineering barrier systems in the carbon dioxide storage, remediation of contaminated sites, and deep geological repository for disposal of high-level nuclear waste, etc. Test for determining gas breakthrough pressure is very difficult and time-consuming, due to the low/ultra-low conductivity of the specimen. It is also difficult to get a comprehensive and high-precision model based on limited results obtained through individual experiments, as the measurements of gas breakthrough pressure were influenced by many factors. In this study, a collected database was built that covered a lot of former test data, and then, two models were developed by the random forest (RF) algorithm and multiexpression programming (MEP) method. The MEP model constructed with explicit expressions for the gas breakthrough pressure overcame the drawbacks of common "black box" models. Meanwhile, five significant indicators were selected from ten common features using the permutation importance algorithm. The RF model was interpreted by the Shapley value and the PDP/ICE plots, while the MEP model was analyzed through the proposed explicit expression, showing strong consistence with that in former studies. Finally, robustness analysis was conducted, and stability of the proposed two models was verified.

Glucose-driven transformable complex eliminates biofilm and alleviates inflamm-aging for diabetic periodontitis therapy.

Diabetic periodontitis is a major complication of diabetes, which has a deep involvement in teeth loss and more serious systematic diseases, including Alzheimer's disease, atherosclerosis and cancers. Diabetic periodontitis is difficult to treat because of recalcitrant infection and hyperglycemia-induced tissue dysfunction. Current treatments fail to completely eliminate infection due to the diffusion-reaction inhibition of biofilm, and ignore the tissue dysfunction. Here, we design a glucose-driven transformable complex, composed of calcium alginate (CaAlg) hydrogel shell and Zeolitic imidazolate framework-8 (ZIF-8) core encapsulating Glucose oxidase (GOx)/Catalase (CAT) and Minocycline (MINO), named as CaAlg@MINO/GOx/CAT/ZIF-8 (CMGCZ). The reaction product of glucose-scavenging, gluconic acid, could dissolve ZIF-8 core and transform CMGCZ from inflexible to flexible, facilitating the complex to overcome the diffusion-reaction inhibition of biofilm. Meanwhile, reduced glucose concentration could ameliorate the pyroptosis of macrophages to decrease the secretion of pro-inflammatory factors, thereby reducing inflamm-aging to alleviate periodontal dysfunction.

Assessment of free energy predictors for ligand binding to a methyllysine histone code reader.

Methyllysine histone code readers constitute a new promising group of potential drug targets. For instance, L3MBTL1, a malignant brain tumor (MBT) protein, selectively binds mono- and di-methyllysine epigenetic marks (KMe, KMe(2) ) that eventually results in the negative regulation of multiple genes through the E2F/Rb oncogenic pathway. There is a pressing need in potent and selective small-molecule probes that would enable further target validation and might become therapeutic leads. Such an endeavor would require efficient tools to assess the free energy of protein-ligand binding. However, due to an unparalleled function of the MBT binding pocket (i.e., selective binding to KMe/KMe(2) ) and because of its distinctive structure representing a small aromatic "cage," an accurate assessment of its binding affinity to a ligand appears to be a challenging task. Here, we report a comparative analysis of computationally affordable affinity predictors applied to a set of seven small-molecule ligands interacting with L3MBTL1. The analysis deals with novel ligands and targets, but applies widespread computational approaches and intuitive comparison metrics that makes this study compatible with and incremental to earlier large scale accounts on the efficiency of affinity predictors. Ultimately, this study has revealed three top performers, far ahead of the other techniques, including two scoring functions, PMF04 and PLP, along with a simulation-based method MM-PB/SA. We discuss why some methods may perform better than others on this target class, the limits of their application, as well as how the efficiency of the most CPU-demanding techniques could be optimized.

Estimating binding affinities by docking/scoring methods using variable protonation states.

To investigate the effects of multiple protonation states on protein-ligand recognition, we generated alternative protonation states for selected titratable groups of ligands and receptors. The selection of states was based on the predicted pK(a) of the unbound receptor and ligand and the proximity of titratable groups of the receptor to the binding site. Various ligand tautomer states were also considered. An independent docking calculation was run for each state. Several protocols were examined: using an ensemble of all generated states of ligand and receptor, using only the most probable state of the unbound ligand/receptor, and using only the state giving the most favorable docking score. The accuracies of these approaches were compared, using a set of 176 protein-ligand complexes (15 receptors) for which crystal structures and measured binding affinities are available. The best agreement with experiment was obtained when ligand poses from experimental crystal structures were used. For 9 of 15 receptors, using an ensemble of all generated protonation states of the ligand and receptor gave the best correlation between calculated and measured affinities.

Biophysical probes reveal a "compromise" nature of the methyl-lysine binding pocket in L3MBTL1.

Histone lysine methylation (Kme) encodes essential information modulating many biological processes including gene expression and transcriptional regulation. However, the atomic-level recognition mechanisms of methylated histones by their respective adaptor proteins are still elusive. For instance, it is unclear how L3MBTL1, a methyl-lysine histone code reader, recognizes equally well both mono- and dimethyl marks but ignores unmodified and trimethylated lysine residues. We made use of molecular dynamics (MD) and free energy perturbation (FEP) techniques in order to investigate the energetics and dynamics of the methyl-lysine recognition. Isothermal titration calorimetry (ITC) was employed to experimentally validate the computational findings. Both computational and experimental methods were applied to a set of designed "biophysical" probes that mimic the shape of a single lysine residue and reproduce the binding affinities of cognate histone peptides. Our results suggest that, besides forming favorable interactions, the L3MBTL1 binding pocket energetically penalizes both methylation states and has most probably evolved as a "compromise" that nonoptimally fits to both mono- and dimethyl-lysine marks.

Accounting for ligand conformational restriction in calculations of protein-ligand binding affinities.

The conformation adopted by a ligand on binding to a receptor may differ from its lowest-energy conformation in solution. In addition, the bound ligand is more conformationally restricted, which is associated with a configurational entropy loss. The free energy change due to these effects is often neglected or treated crudely in current models for predicting binding affinity. We present a method for estimating this contribution, based on perturbation theory using the quasi-harmonic model of Karplus and Kushick as a reference system. The consistency of the method is checked for small model systems. Subsequently we use the method, along with an estimate for the enthalpic contribution due to ligand-receptor interactions, to calculate relative binding affinities. The AMBER force field and generalized Born implicit solvent model is used. Binding affinities were estimated for a test set of 233 protein-ligand complexes for which crystal structures and measured binding affinities are available. In most cases, the ligand conformation in the bound state was significantly different from the most favorable conformation in solution. In general, the correlation between measured and calculated ligand binding affinities including the free energy change due to ligand conformational change is comparable to or slightly better than that obtained by using an empirically-trained docking score. Both entropic and enthalpic contributions to this free energy change are significant.

[Clinical observation of dry eye syndrome treated with acupuncture].

OBJECTIVE: To compare the therapeutic effect between acupuncture and hyaluronic acid sodium eye drops for dry eye syndrome. METHODS: A total of 60 patients with dry eye were randomized into an acupuncture group and a western medication group, 30 cases in each one. In the acupuncture group, acupoints around the eyes, abdomen and limbs were selected, such as Jingming (BL 1), Cuanzhu (BL 2), Fengchi (GB 20), Taiyang (EX-HN 5), Guanyuan (CV 4), Qihai (CV 6) and Hegu (LI 4), once 2 days for 1 month, 15 times in total. In the western medication group, hyaluronic acid sodium eye drops was applied, 3 times a day, one drop every time, for 1 month. The tear meniscus height (TMH), tear break-up time (BUT) and conjunctival congestion scores were observed before and after treatment in the two groups, and the clinical effects of the two groups were compared. RESULTS: The total effective rate in the acupuncture group was 68.3% (41/60), which was superior to 30.0% (18/60) in the western medication group (P<0.05). Compared before treatment, the TMH was increased, BUT was prolonged and conjunctival congestion score was reduced after treatment in the acupuncture group (P<0.05, P<0.01). After treatment, the TMH and BUT in the acupuncture group were higher (P<0.05, P<0.01), and the conjunctival congestion score was lower than those in the western medication group (P<0.05). CONCLUSION: The therapeutic effect of acupuncture is superior to hyaluronic acid sodium eye drops. Acupuncture can promote lacrimal gland secretion and increase the tear film stability.

High minichromosome maintenance protein 7 proliferation indices: a powerful predictor of progression in pancreatic neuroendocrine neoplasms without distant metastasis at the time of surgery.

Pancreatic neuroendocrine neoplasms (PanNENs) have an unpredictable clinical course that varies from indolent to highly malignant. No immunohistochemical markers are available for reliable prediction of the biological behavior of early stage PanNENs. Minichromosome maintenance protein 7 (MCM7) is a putative powerful marker of cell proliferation. Whether the expression of MCM7 is related to the risk of PanNENs progression remains unclear. We assessed the clinical behavior of 156 PanNENs with respect to stage, grade, Ki-67 index, MCM7 index, and other pathologic features. A high MCM7 index was significantly associated with larger tumor size (P < .001), nonfunctioning tumor (P < .001), increased grade (P < .0001), and later TNM stage (P < .001). In multivariate analysis, G2/G3 (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.35-3.62; P < .001), stage III/IV (HR, 2.11; 95% CI, 1.31-3.41; P < .001), and MCM7 labeling index >5% (HR, 3.81; 95% CI, 1.30-11.17; P = .02) were independent negative prognostic factors related to the risk of tumor progression in stage I-IV disease. MCM7 labeling index >5% was associated with an increased risk of progression in stages I-V, I-III, and I-II. Our study confirms that MCM7 is a valuable marker for assessing the progression of PanNENs, especially in patients with early stage disease and without distant metastasis.

Predicting the Conformational Variability of Abl Tyrosine Kinase using Molecular Dynamics Simulations and Markov State Models.

Understanding protein conformational variability remains a challenge in drug discovery. The issue arises in protein kinases, whose multiple conformational states can affect the binding of small-molecule inhibitors. To overcome this challenge, we propose a comprehensive computational framework based on Markov state models (MSMs). Our framework integrates the information from explicit-solvent molecular dynamics simulations to accurately rank-order the accessible conformational variants of a target protein. We tested the methodology using Abl kinase with a reference and blind-test set. Only half of the Abl conformational variants discovered by our approach are present in the disclosed X-ray structures. The approach successfully identified a protein conformational state not previously observed in public structures but evident in a retrospective analysis of Lilly in-house structures: the X-ray structure of Abl with WHI-P154. Using a MSM-derived model, the free energy landscape and kinetic profile of Abl was analyzed in detail highlighting opportunities for targeting the unique metastable states.

Silencing of uPAR via RNA interference inhibits invasion and migration of oral tongue squamous cell carcinoma.

Overexpression of urokinase-type plasminogen activator receptor (uPAR) has been implicated in promoting tumor invasion in various cancer types, including oral tongue squamous cell carcinoma (OTSCC); therefore, the effect of suppressing uPAR expression on the invasive and metastatic potential of OTSCC was investigated. A total of 65 paraffin-embedded tissues were obtained from patients with OTSCC. Immunohistochemistry was used to determine the expression level of uPAR. The Ts cells transfected with short hairpin RNA targeting uPAR were constructed and validated. The cells were used in a number of in vitro analyses, including migration, invasion and western blot analysis assays. Furthermore, a mouse lung metastatic model was used to detect the metastatic ability of OTSCC cells in the lungs. OTSCC cell metastasis and relapse were determined to be associated with uPAR immunopositivity. Inhibition of uPAR expression in Ts cells demonstrated a 40% decrease in migration and a 60% decrease in invasion in vitro, with an associated downregulation of matrix metalloprotease (MMP)-2, MMP-9 and phosphorylated extracellular signal-regulated kinase. In vivo analysis indicated a 90% decrease in the number of mice bearing macroscopic lung metastases. In conclusion, the present study demonstrated that the targeting of uPAR-inhibited cellular proliferation and invasion would provide a potential treatment for OTSCC in the future.