Structural Characterization and Cytotoxic Activity Evaluation of Ulvan Polysaccharides Extracted from the Green Algae Ulva papenfussii.

Ulvan, a sulfated heteropolysaccharide with structural and functional properties of interest for various uses, was extracted from the green seaweed Ulva papenfussii. U. papenfussii is an unexplored Ulva species found in the South China Sea along the central coast of Vietnam. Based on dry weight, the ulvan yield was ~15% (w/w) and the ulvan had a sulfate content of 13.4 wt%. The compositional constitution encompassed L-Rhamnose (Rhap), D-Xylose (Xylp), D-Glucuronic acid (GlcAp), L-Iduronic acid (IdoAp), D-Galactose (Galp), and D-Glucose (Glcp) with a molar ratio of 1:0.19:0.35:0.52:0.05:0.11, respectively. The structure of ulvan was determined using High-Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Nuclear Magnetic Resonance spectroscopy (NMR) methods. The results showed that the extracted ulvan comprised a mixture of two different structural forms, namely ("A3s") with the repeating disaccharide [→4)-ß-D-GlcAp-(1→4)-α-L-Rhap 3S-(1→]n, and ("B3s") with the repeating disaccharide [→4)-α-L-IdoAp-(1→4)-α-L-Rhap 3S(1→]n. The relative abundance of A3s, and B3s was 1:1.5, respectively. The potential anticarcinogenic attributes of ulvan were evaluated against a trilogy of human cancer cell lineages. Concomitantly, Quantitative Structure-Activity Relationship (QSAR) modeling was also conducted to predict potential adverse reactions stemming from pharmacological interactions. The ulvan showed significant antitumor growth activity against hepatocellular carcinoma (IC50 ≈ 90 µg/mL), human breast cancer cells (IC50 ≈ 85 µg/mL), and cervical cancer cells (IC50 ≈ 67 µg/mL). The QSAR models demonstrated acceptable predictive power, and seven toxicity indications confirmed the safety of ulvan, warranting its candidacy for further in vivo testing and applications as a biologically active pharmaceutical source for human disease treatment.

Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

The life-history fitness of F1 hybrids of the microcrustacean Daphnia pulex and D. pulicaria (Crustacea, Anomopoda).

Negative interaction between alleles that arise independently in diverging populations (i.e., Dobzhansky-Muller incompatibilities) can cause reduction of fitness in their hybrids. However, heterosis in hybrids can emerge if hybridization breaks down detrimental epistatic interaction within parental lineages. In this study, we examined the life-history fitness of the inter-specific F1s of two recently diverged microcrustacean species Daphnia pulex and D. pulicaria as well as intra-specific crosses of D. pulex. We identified heterosis in two out of five life-history traits in the inter-specific F1s. According to theories that heterosis can transiently emerge in early speciation, the observation of heterosis in these life-history traits suggests that there are no major genetic incompatibilities between these two species affecting these traits and that D. pulex and D. pulicaria are at an early stage of speciation.

The Preparation and Characterization of Expanded Graphite via Microwave Irradiation and Conventional Heating for the Purification of Oil Contaminated Water.

Recently, the graphite based materials have gained interest as excellent platforms to remove aqueous pollutants via adsorption routes. This is given that such materials possess large specific surface area and low density. In the present work, a comparative study of two facile and effective approaches is conventional thermal heating and microwave irradiation methods to fabricate expanded graphite from available flake graphite sources of Vietnam for oil-contaminated water purification. The as-prepared expanded graphite was characterized by using FT-IR, SEM, XRD and BET analysis. The results exhibited that expanded graphite has multilevel pore structures and the surface area of expanded graphite obtained from microwave irradiation and conventional heating was 147.5 (m²/g) and 100.97 (m²/g) under optimal processing conditions. The as-synthesized expanded graphite from the microwave irradiation method was found to have higher adsorption capacities for diesel oil, crude oil, and fuel oil compared to conventional heating method.

Novel Enzyme Actions for Sulphated Galactofucan Depolymerisation and a New Engineering Strategy for Molecular Stabilisation of Fucoidan Degrading Enzymes.

Fucoidans from brown macroalgae have beneficial biomedical properties but their use as pharma products requires homogenous oligomeric products. In this study, the action of five recombinant microbial fucoidan degrading enzymes were evaluated on fucoidans from brown macroalgae: Sargassum mcclurei, Fucus evanescens, Fucus vesiculosus, Turbinaria ornata, Saccharina cichorioides, and Undaria pinnatifida. The enzymes included three endo-fucoidanases (EC 3.2.1.-GH 107), FcnA2, Fda1, and Fda2, and two unclassified endo-fucoglucuronomannan lyases, FdlA and FdlB. The oligosaccharide product profiles were assessed by carbohydrate-polyacrylamide gel electrophoresis and size exclusion chromatography. The recombinant enzymes FcnA2, Fda1, and Fda2 were unstable but were stabilised by truncation of the C-terminal end (removing up to 40% of the enzyme sequence). All five enzymes catalysed degradation of fucoidans containing α(1→4)-linked l-fucosyls. Fda2 also degraded S. cichorioides and U. pinnatifida fucoidans that have α(1→3)-linked l-fucosyls in their backbone. In the stabilised form, Fda1 also cleaved α(1→3) bonds. For the first time, we also show that several enzymes catalyse degradation of S. mcclurei galactofucan-fucoidan, known to contain α(1→4) and α(1→3) linked l-fucosyls and galactosyl-ß(1→3) bonds in the backbone. These data enhance our understanding of fucoidan degrading enzymes and their substrate preferences and may assist development of enzyme-assisted production of defined fuco-oligosaccharides from fucoidan substrates.

Density-functional description of polymer crystals: A comparative study of recent van der Waals functionals.

Due to the lack of treatment of long-range dispersion energies, density functional theory with local and semilocal approximations of exchange-correlation energy is known to fail in describing van der Waals complexes, including polymer crystals. This limitation can be overcome by using a different class of functionals, called van der Waals density functional (vdW-DF), originally developed by Dion et al. [Phys. Rev. Lett. 92, 246401 (2004)]. In this work, we performed a systematic study of structural properties of polymeric crystals using the original vdW-DF functional by Dion et al. and its variants and refinements. Our study shows that this class of functional outperforms the conventional LDA or PBE functionals and gives results with similar accuracy to that of empirical dispersion-corrected schemes such as DFT-D. This study suggests the use of vdW-DF2 functional - a revised version of vdW-DF functional - to obtain a high-fidelity prediction of structural and other properties of polymeric materials.

An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer.

B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.

Efficient CRISPR genome editing and integrative genomic analyses reveal the mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.

Despite the revolutionary impacts of CRISPR-Cas gene editing systems, the effective and widespread use of CRISPR technologies in emerging model organisms still faces significant challenges. These include the inefficiency in generating heritable mutations at the organismal level, limited knowledge about the genomic consequences of gene editing, and an inadequate understanding of the inheritance patterns of CRISPR-Cas-induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for CRISPR editing in the microcrustacean Daphnia pulex; 2) assessing the editing efficiency of Cas9 and Cas12a nucleases, examining mutation inheritance patterns, and analyzing the local and global mutation spectrum in the scarlet mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet underlying their swimming behavior changes. Our reengineered CRISPR microinjection method results in efficient biallelic editing with both nucleases. While indels are dominant in Cas-induced mutations, a few on-site large deletions (>1kb) are observed, most likely caused by microhomology-mediated end joining repair. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progenies. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes (e.g., NMDA1, ABAT, CNTNAP2) involved in human neurodegenerative diseases. This study expands our understanding of the dynamics of gene editing in the tractable model organism Daphnia and highlights its promising potential as a neurological disease model.

Deep learning-based optimization of a microfluidic membraneless fuel cell for maximum power density via data-driven three-dimensional multiphysics simulation.

A deep learning-based method for optimizing a membraneless microfluidic fuel cell (MMFC)performance by combining the artificial neural network (ANN) and genetic algorithm (GA) was for the first time introduced. A three-dimensional multiphysics model that had an accuracy equivalent to experimental results (R2 = 0.976) was employed to generate the ANN's training data. The constructed ANN is equivalent to the simulation (R2 = 0.999) but with far better computation resource efficiency as the ANN's execution time is only 0.041 s. The ANN model is then used by the GA to determine the inputs (microchannel length = 10.040 mm, width = 0.501 mm, height = 0.635 mm; temperature = 288.210 K, cell voltage = 0.309 V) that lead to the maximum power density of 0.263 mWcm-2 (current density of 0.852 mAcm-2) of the MMFC. The ANN-GA and numerically calculated maximum power densities differed only by 0.766%.

Linearized and nonlinearized modellings for comparative uptake assessment of metal-organic framework-derived nanocomposite towards sulfonamide antibiotics.

The emergent occurrence of sulfonamide species involving sulfadiazine (SDZ) and sulfamethazine (SMZ) in aquatic systems can cause a wide range of potential risks; hence, remediation strategies need to be necessary. Here, we develop the novel metal-organic framework-derived nanocomposite, and apply for the adsorption of SDZ and SMZ antibiotics. To assess the best-fitting kinetic (pseudo first-order, pseudo second-order) and isotherm (Langmuir, Freundlich, Temkin, Dubinin-Radushkevich, Redlich-Peterson, Sips, Toth, and Khan) models, a series of numerous statistical analysis was performed. Numerous error functions including squares of the errors (SSE), hybrid fractional error function (HYBRID), Marquardt's percent standard deviation (MPSD), and mean relative error (MRE) were also analyzed to assess the linear and nonlinear models. The results indicated that both linear and nonlinear kinetic models were mostly fitted well with pseudo second-order models (Radj)2 > 0.97. Although linear kinetics gave better (Radj)2, error functions (MRE, SSE, HYBRID, and MPSD) were mostly higher than those of nonlinear kinetics. For adsorption isotherm, nonlinear Redlich-Peterson was the most compatible model with extremely high adjusted coefficients of determination (Radj)2 ~ 1.0000. While nonlinear Langmuir model gave relatively high (Radj)2 (0.9898-0.9960) and acceptable error functions, we found the considerable difference of error functions and parameters among four types of linear Langmuir (Types I, II, III, IV). The findings indicate potential errors as selecting one of linearized Langmuir types in equilibrium study. It is suggested that nonlinear models should be applied for better fitness.

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure.

Fucoidans are sulfated, fucose-rich marine polysaccharides primarily found in cell walls of brown seaweeds (macroalgae). Fucoidans are known to possess beneficial bioactivities depending on their structure and sulfation degree. Here, we report the first functional characterization and the first crystal structure of a prokaryotic sulfatase, PsFucS1, belonging to sulfatase subfamily S1_13, able to release sulfate from fucoidan oligosaccharides. PsFucS1 was identified in the genome of a Pseudoalteromonas sp. isolated from sea cucumber gut. PsFucS1 (57 kDa) is Ca2+ dependent and has an unusually high optimal temperature (68 °C) and thermostability. Further, the PsFucS1 displays a unique quaternary hexameric structure comprising a tight trimeric dimer complex. The structural data imply that this hexamer formation results from an uncommon interaction of each PsFucS1 monomer that is oriented perpendicular to the common dimer interface (~ 1500 Å2) that can be found in analogous sulfatases. The uncommon interaction involves interfacing (1246 Å2) through a bundle of α-helices in the N-terminal domain to form a trimeric ring structure. The high thermostability may be related to this unusual quaternary hexameric structure formation that is suggested to represent a novel protein thermostabilization mechanism.

Anti-LYPD1/CD3 T-Cell-Dependent Bispecific Antibody for the Treatment of Ovarian Cancer.

Ovarian cancer is a diverse class of tumors with very few effective treatment options and suboptimal response rates in early clinical studies using immunotherapies. Here we describe LY6/PLAUR domain containing 1 (LYPD1) as a novel target for therapeutic antibodies for the treatment of ovarian cancer. LYPD1 is broadly expressed in both primary and metastatic ovarian cancer with ∼70% prevalence in the serous cancer subset. Bispecific antibodies targeting CD3 on T cells and a tumor antigen on cancer cells have demonstrated significant clinical activity in hematologic cancers. We have developed an anti-LYPD1/CD3 T-cell-dependent bispecific antibody (TDB) to redirect T-cell responses to LYPD1 expressing ovarian cancer. Here we characterize the nonclinical pharmacology of anti-LYPD1/CD3 TDB and show induction of a robust polyclonal T-cell activation and target dependent killing of LYPD1 expressing ovarian cancer cells resulting in efficient in vivo antitumor responses in PBMC reconstituted immune-deficient mice and human CD3 transgenic mouse models. Anti-LYPD1/CD3 TDB is generally well tolerated at high-dose levels in mice, a pharmacologically relevant species, and showed no evidence of toxicity or damage to LYPD1 expressing tissues.

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation.

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.

Delivery methods for CRISPR/Cas9 gene editing in crustaceans.

In this mini-review we provide an up-to-date overview of the delivery methods that have been used for CRISPR/Cas9 genomic editing in crustacean species. With embryonic microinjection as the main workforce for delivering CRISPR/Cas9 reagents, biologists working with crustacean species have to tackle the technical challenges involved in microinjection. We use examples of three crustacean species (the branchiopod Daphnia, amphipod Parhyale hawaiensis, and decapod Exopalaemon carinicauda) to provide a technical guide for embryonic microinjection. Moreover, we outline two potentially useful new techniques for delivering CRISPR/Cas9 components into crustaceans, i.e., Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and electroporation.

Transcriptional profile of isoproterenol-induced cardiomyopathy and comparison to exercise-induced cardiac hypertrophy and human cardiac failure.

BACKGROUND: Isoproterenol-induced cardiac hypertrophy in mice has been used in a number of studies to model human cardiac disease. In this study, we compared the transcriptional response of the heart in this model to other animal models of heart failure, as well as to the transcriptional response of human hearts suffering heart failure. RESULTS: We performed microarray analyses on RNA from mice with isoproterenol-induced cardiac hypertrophy and mice with exercise-induced physiological hypertrophy and identified 865 and 2,534 genes that were significantly altered in pathological and physiological cardiac hypertrophy models, respectively. We compared our results to 18 different microarray data sets (318 individual arrays) representing various other animal models and four human cardiac diseases and identified a canonical set of 64 genes that are generally altered in failing hearts. We also produced a pairwise similarity matrix to illustrate relatedness of animal models with human heart disease and identified ischemia as the human condition that most resembles isoproterenol treatment. CONCLUSION: The overall patterns of gene expression are consistent with observed structural and molecular differences between normal and maladaptive cardiac hypertrophy and support a role for the immune system (or immune cell infiltration) in the pathology of stress-induced hypertrophy. Cross-study comparisons such as the results presented here provide targets for further research of cardiac disease that might generally apply to maladaptive cardiac stresses and are also a means of identifying which animal models best recapitulate human disease at the transcriptional level.

Cotargeting of MEK and PDGFR/STAT3 Pathways to Treat Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human diseases and remains largely refractory to available drug treatments. Insufficient targeting of the known oncogenic drivers and activation of compensatory feedback loops and inability to prevent metastatic spread contribute to poor prognosis for this disease. The KRAS-driven MEK pathway is mutationally activated in most pancreatic cancers and is an important target for therapeutics. Using a two-dimensional monolayer culture system as well as three-dimensional spheroid culture system, we conducted a screen of a large panel of anticancer agents and found that MAP2K (MEK) inhibitors were most effective in targeting PDAC spheroids in comparison with monolayer cultures. Combination treatment with an MEK inhibitor and the multikinase inhibitor ponatinib was effective in targeting pancreatic cancer cells both in monolayer and spheroids by effectively blocking signaling via the PDGFRα and MEK kinases, while also preventing the activation of STAT3- and S6-mediated compensatory feedback loops in cancer cells. Furthermore, using xenograft models, we demonstrate that cotreatment with a MEK inhibitor and ponatinib causes significant tumor regression. PDAC patient samples also provided evidence of increased STAT3 activation in PDAC tumors and MAPK1 (ERK) activation in liver metastases, implicating STAT3 and ERK as key drivers in primary tumors and metastases, respectively. These results reveal a combination drug treatment strategy that may be effective in pancreatic cancer. Mol Cancer Ther; 16(9); 1729-38. ©2017 AACR.

Genome Sequences of Mycobacteriophages Findley, Hurricane, and TBond007.

We report here the genome sequences of three newly isolated phages that infect Mycobacterium smegmatis mc2155. Phages Findley, Hurricane, and TBond007 were discovered in geographically distinct locations and are related to cluster K mycobacteriophages, with Findley being similar to subcluster K2 phages and Hurricane and TBond007 being similar to subcluster K3 phages.

Quantitative in situ hybridization of tissue microarrays.

Tissue microarrays enable the rapid histological localization of gene expression in hundreds of archival samples by in situ hybridization. However, the scoring of tissue microarray data may be influenced by intra- and inter-observer variations, and categorizing continuous variables risks discarding potentially meaningful information. Quantitation imposes a greater degree of objectivity, is more reproducible than subjective discriminations, and facilitates the communication and clarity of definitions. Phosphorimaging has been successfully used to quantitate the hybridization signal intensity from arrayed tissues. The process is rapid and has a wide dynamic range, surpassing the densitometric analysis of autoradiograms. This paper presents a detailed method for quantitative isotopic in situ hybridization on formalin-fixed paraffin-embedded tissue microarrays. In addition, the method includes a protocol for the development of synthetic agarose cores to control for the specificity and sensitivity of hybridization.

Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients.

Significant improvements in the outcome of non-small cell lung carcinoma (NSCLC) have been reported in patients treated with the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. To discover biomarkers for the enrichment of patients who might benefit from treatment, a pharmacogenomic approach was used to identify gene signatures that may predict erlotinib activity using in vitro model systems. Erlotinib sensitivity in a panel of 42 NSCLC cell lines was determined by EGFR-mediated proliferative potential, EGFR mutations, and/or EGFR gene amplification, thus supporting an underlying biological mechanism of receptor activation. A strong multigene signature indicative of an epithelial to mesenchymal transition (EMT) was identified as a determinant of insensitivity to erlotinib through both supervised and unsupervised gene expression approaches. This observation was further supported by expression analysis of classic EMT marker proteins, including E-cadherin and vimentin. To investigate the clinical relevance of these findings, we examined expression of the epithelial marker E-cadherin by immunohistochemistry on primary tumor samples from subjects enrolled in a randomized NSCLC clinical trial in which erlotinib in combination with chemotherapy previously failed to show clinical activity. The majority (75%) of the 87 subjects tested showed strong E-cadherin staining and exhibited a significantly longer time to progression (hazard ratio, 0.37; log rank P=0.0028) and a nonsignificant trend toward longer survival with erlotinib plus chemotherapy treatment versus chemotherapy alone. These data support a potential role for EMT as a determinant of EGFR activity in NSCLC tumor cells and E-cadherin expression as a novel biomarker predicting clinical activity of the EGFR inhibitor erlotinib in NSCLC patients.

Thyrotropin-releasing hormone is induced in the left ventricle of rats with heart failure and can provide inotropic support to the failing heart.

BACKGROUND: We reported previously that left ventricular gene expression for thyrotropin-releasing hormone (TRH) precursor was increased in rats with heart failure 8 weeks after myocardial infarction (MI) and that early ACE inhibition tended to cause further myocardial induction of this gene. METHODS AND RESULTS: Here, we show that after MI, the expression of pro-TRH is induced in the heart coordinately with the protease PC1, an important enzyme in TRH biosynthesis. Pro-TRH gene expression was induced in cardiac interstitial cells after MI, and this effect was restricted to the heart, because no increase in TRH mRNA abundance was observed in the hypothalamus, kidney, or lung. Transcript abundance of pro-TRH can be increased in cultured cardiac fibroblasts by several adrenergic agonists, indicating that the adrenergic axis may play a regulatory role in cardiac TRH production. Acute intravenous administration of TRH to rats with ischemic cardiomyopathy caused a significant increase in heart rate, mean arterial pressure, cardiac output, stroke volume, and cardiac contractility. CONCLUSIONS: Taken together, these results indicate that TRH is specifically induced in the heart after MI and that it can increase cardiac performance in rats with ischemic cardiomyopathy. Thus, in addition to catecholamine and angiotensin II, pro-TRH/TRH may be another important axis that affects hemodynamics and cardiac function in heart failure.