Blockage of bone marrow kinase in chromosome X enhances ABC294640-induced growth inhibition and apoptosis of colorectal cancer cells

Purpose: To investigate the role of bone marrow kinase in chromosome X (BMX) in colorectal cancer (CRC) cell resistance to ABC294640 treatment.Methods: HCT-116R, LS174T and WiDr cells were transfected with either BMX-specific siRNA or scrambled siRNA, and then BMX mRNA and protein expressions were detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. The cells were treated with ABC294640 and cell viability evaluated using cell counting and colony formation assays. Apoptosis wasdetermined by detecting caspase 3/7 activity. To evaluate tumor growth of  HCT-116R cells, a xenograft model was utilized to measure tumor size.Results: Pharmacological inhibition of sphingosine kinase type 2 (SK2) with ABC294640 significantly decreased cell viability (p < 0.001) when compared with control group. SK2 inhibition also remarkably induced apoptosis in HCT-116 CRC cells in a dose-dependent manner (p < 0.01 and p < 0.001). However, no significant effects were observed in HCT-116R, LS174T, or WiDr cells following ABC294640 treatment. BMX mRNA and protein expression increased in ABC294640-resistant cell lines. In addition, silencing BMX expression with siRNA potentiated  ABC294640-induced inhibition of tumor growth in CRC cells in vitro and in vivo.Conclusion: ABC294640-induced BMX upregulation impedes the antitumor effect of ABC294640 in CRC cells. Therefore, these results may provide a novel therapeutic strategy for CRC using a combination of ABC294640 treatment and BMX blockade.Keywords: ABC294640, Apoptosis, Bone marrow kinase in chromosome X, Cell viability, Colorectal cance

Predicting pharmacodynamic effects through early drug discovery with artificial intelligence-physiologically based pharmacokinetic (AI-PBPK) modelling

A mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model links the concentration-time profile of a drug with its therapeutic effects based on the underlying biological or physiological processes. Clinical endpoints play a pivotal role in drug development. Despite the substantial time and effort invested in screening drugs for favourable pharmacokinetic (PK) properties, they may not consistently yield optimal clinical outcomes. Furthermore, in the virtual compound screening phase, researchers cannot observe clinical outcomes in humans directly. These uncertainties prolong the process of drug development. As incorporation of Artificial Intelligence (AI) into the physiologically based pharmacokinetic/pharmacodynamic (PBPK) model can assist in forecasting pharmacodynamic (PD) effects within the human body, we introduce a methodology for utilizing the AI-PBPK platform to predict the PK and PD outcomes of target compounds in the early drug discovery stage. In this integrated platform, machine learning is used to predict the parameters for the model, and the mechanism-based PD model is used to predict the PD outcome through the PK results. This platform enables researchers to align the PK profile of a drug with desired PD effects at the early drug discovery stage. Case studies are presented to assess and compare five potassium-competitive acid blocker (P-CAB) compounds, after calibration and verification using vonoprazan and revaprazan

Three Capsular Polysaccharide Synthesis-Related Glucosyltransferases, GT-1, GT-2 and WcaJ, Are Associated With Virulence and Phage Sensitivity of Klebsiella pneumoniae

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage

Protein und Farbstoffe in begrenzter Geometrie und Protein-Dynamik: Untersuchungen mit Fluoreszenz-Verfahren und NMR-Spektroskopie

Mesoporöse Materialien mit geordneter Porenstruktur sind von Interesse im Hinblick auf ihr breites Anwendungspotenzial in verschiedenen Feldern, unter anderem als Träger von Wirkstoffen oder künstlichen Licht-Antennen, sowie in der Biokatalyse. MCM-41 Silica-Nanoteilchen mit definierter Porenweite im Bereich von 3 bis 4 nm weisen vielversprechende Eigenschaften als Trägerpartikel für pharmazeutische Wirkstoffe auf, doch bleibt die Entwicklung eines einfachen und bio-kompatiblen Porenverschluss-Systems eine Herausforderung. Wir haben gefunden, dass Lysozym-Moleküle als pH-responsive Nanoschalter zum Verschließen und Öffnen der Poreneingänge von MCM-41 Teilchen wirken können. Experimente mit dem Farbstoff Rhodamin B als Modell-Wirkstoff zeigen, dass die Wirkung von Lysozym als molekularer Schalter auf einer pH-induzierten Konformationsänderung beruht, durch welche die effektive Größe der Proteinmoleküle in reversibler Weise beeinflusst wird. Dieser Effekt könnte die Basis für Systeme zur kontrollierten Wirkstoff-Abgabe ohne Notwendigkeit einer chemischen Modifizierung der Porenwände oder des Porenverschlusses bilden. Mesoporöse Silica-Materialien sind auch von Interesse als Wirtsmaterialien zum Studium photochemischer Prozesse in räumlich begrenzter Geometrie. Es wurde der Fluoreszenz-Resonanzenergietransfer (FRET) von Fluorescein (Donor) auf Rhodamin B (Akzeptor) in MCM-41 Silica mittels der Fluoreszenz-Lebenszeit-Imaging-Mikroskopie (FLIM) studiert. Messungen für eine konstante Donorkonzentration und verschiedene Akzeptor-Konzentrationen wurden mittels multi-exponentiellen Abfall-Funktionen analysiert. Aus den FRET-Daten wurde der Donor-Akzeptor Abstand rDA ermittelt und mit dem aus der (Gesamt-) Farbstoff-Konzentrationen bestimmten mittleren Teilchenabstand d verglichen. Bei hohen Farbstoff-Konzentrationen ergeben sich für rDA Werte ähnlich zu d, bei geringen Farbstoff-Konzentrationen dagegen deutlich kleiner als d. Hieraus lassen sich Rückschlüsse auf die Verteilung der Moleküle in der porösen Matrix ziehen. Diese Untersuchungen bilden eine Basis für weiterführende Untersuchungen zum Studium der Verteilung von Proteinmolekülen in den Poren von geordnet mesoporösen Silica-Materialien. Obwohl das aktuelle Verständnis von Enzym-Funktion überwiegend auf der aus der Röntgen-Strukturbestimmung erhaltenen statischen Proteinstruktur basiert, wird allgemein vermutet, dass auch die Protein-Dynamik von essentieller Bedeutung für ihre biologische Funktion ist. Dehaloperoxidase-Hämoglobin (DHP A) kann halogenierte Aromaten oxidativ dehalogenieren unter Beibehaltung ihrer Sauerstoff-Speicherfunktion. In dieser Arbeit wurden heteronukleare NMR Methoden benutzt, um die Dynamik von DHP A in Lösung in Gegenwart des Substrats 2, 4, 6-trichlorophenol (TCP) zu studieren. Für die NMR Messungen wurde mit 15N angereichertes DHP A hergestellt. Die NMR Relaxationsparameter T1 und T2 und {1H}-15N NOE wurden mittels reduced spectral density mapping und der Lipari-Szabo modellfreien Methode analysiert. Die in dieser Arbeit gewonnenen 15N-Relaxationsdaten geben Hinweise auf langsam ablaufende Konformationsänderungen in DHP A in Gegenwart von TCP. Weitere Unter-suchungen sind nötig, um diese Ergebnisse abzusichern und ihren Bezug zur katalytischen Oxidation abzuklären.There has been ever increasing interest in ordered mesoporous materials because of their high applications potential in many fields, such as drug delivery, artificial light-harvesting and biocatalysis. MCM-41 mesoporous silica nanoparticles with pore widths in the 3 to 4 nm range hold great promise as a drug carrier vehicle because of the chemical stability, well-defined pore geometry, and tunable pore size of the particles, but the development of simple and biocompatible capping system for the pores has remained a challenge. We have found that lysozyme molecules can act as a pH-responsive nano-valve to block and unlock the pore entrances of MCM-41 nanoparticles for guest molecules. Experiments with the dye rhodamine B as a model drug indicate that pore blocking by lysozyme is due to a pH induced conformational change by which the effective size of the protein is changed in a reversible manner. This effect may form the basis of a controlled-release system without the need to functionalize the pore mouth and caps. Mesoporous silica materials have also attracted interest as a host for nano-confined dye molecules, which undergo enhanced energy transfer and exhibit optical functionality. Measurements of the fluorescence resonance energy transfer (FRET) from fluorescein (donor) to rhodamine b (acceptor) molecules embedded in the pore space of MCM-41 silica were performed by fluorescence lifetime imaging microscopy (FLIM) and are analyzed in terms of a two- and three-exponential decay function. Results are presented for a given donor concentration and a wide range of acceptor concentrations. The mean donor-to-acceptor distance rDA as calculated from the FRET efficiency is compared with the mean separation of dye molecules d as obtained from the measured uptake. It is found that rDA is similar to d at high overall dye concentrations but significantly smaller than d at low concentrations. These findings are discussed in terms of the distribution of molecular distances of the guest molecules in the silica matrix. These FRET studies with dye molecules form a basis for an investigation into the mean distance between protein molecules in the pore space of ordered mesoporous silica materials. Although current understanding of enzyme function is based mostly on static protein structures as obtained from X-ray crystallography, the dynamics of protein molecules is believed to be essential to their biological function. Dehaloperoxidase-hemoglobin (DHP A) can oxidatively dehalogenate haloaromatic compounds while maintaining its oxygen storage function. We have used hetero-nuclear NMR methods to probe the dynamics of DHP A in solution in the presence of the substrate 2, 4, 6-trichlorophenol (TCP). To facilitate the NMR measurements, DHP A was labeled with 15N, and NMR relaxation parameters T1 and T2 and {1H}-15N NOE have been analyzed using reduced spectral density mapping and the Lipari-Szabo model free method. The preliminary 15N spin-relaxation data reveal evidence for the presence of slow conformational exchange in DHP A in the presence of TCP. Further work is needed to corroborate these findings and their significance for the catalytic oxidization reaction

Genome-wide identification and analysis of MAPK and MAPKK gene family in Chinese jujube (Ziziphus jujuba Mill.)

Abstract Background Chinese jujube (Ziziphus jujuba Mill.) is one of the most important members in the Rhamnaceae family. The whole genome sequence and more than 30,000 proteins of Chinese jujube have been obtained in 2014. Mitogen-activated protein kinase cascades are universal signal transduction modules in plants, which is rapidly activated under various biotic and abiotic stresses. To date, there has been no comprehensive analysis of the MAPK and MAPKK gene family in Chinese jujube at the whole genome level. Results By performing a series of bioinformatics analysis, ten MAPK and five MAPKK genes were identified from the genome database of Chinese jujube, and then compared with the homologous genes from Arabidopsis. Phylogenetic analysis showed that ZjMAPKs was classified into four known groups, including A, B, C and D. ZjMAPKs contains five members of the TEY phosphorylation site and five members with the TDY motif. The ZjMAPKK family was subsequently divided into three groups, A, B and D. The gene structure, conserved motifs, functional annotation and chromosome distribution of ZjMAPKs and ZjMAPKKs were also predicted. ZjMAPKs and ZjMAPKKs were distributed on nine pseudo-chromosomes of Chinese jujube. Subsequently, expression analysis of ZjMAPK and ZjMAPKK genes using reverse transcription PCR and quantitative real-time PCR was carried out. The majority of ZjMAPK and ZjMAPKK genes were expressed in all tested organs/tissues with considerable differences in transcript levels indicating that they might be constitutively expressed. Moreover, ZjMKK5 was specific expressed in early development stage of jujube flower bud, indicating it plays some roles in reproductive organs development. The transcript expression of most ZjMAPK and ZjMAPKK genes was down-regulated in response to plant growth regulators, darkness treatment and phytoplasma infection. Conclusions We identified ten ZjMAPK and five ZjMAPKK genes from the genome database of Chinese jujube, the research results shown that ZjMPKs and ZjMKKs have the different expression patterns, indicating that they might play different roles in response to various treatments. The results provide valuable information for the further elucidation of physiological functions and biological roles of jujube MAPKs and MAPKKs

Association between dyslipidemia and risk of type 2 diabetes mellitus in middle-aged and older Chinese adults: a secondary analysis of a nationwide cohort

Aims To evaluate the type 2 diabetes mellitus (T2DM) risk of individuals with different types of dyslipidaemia and compare the predictive value of distinct lipid parameters in predicting T2DM.Methods We conducted a secondary analysis of data from the China Health and Retirement Longitudinal Study (CHARLS). 17 708 individuals over 45 years old were interviewed, and 11 847 blood samples were collected at the baseline survey (2011–2012). Outcome of T2DM was confirmed during two follow-up surveys (2013–2014 and 2015–2016). The HRs and 95% CI of T2DM associated with dyslipidaemia were estimated by Cox proportional hazards regressions model. The discriminatory value of eight lipid parameters were compared by the area under the receiver operating characteristic (ROC) curve (AUC).Results A total of 7329 participants were enrolled in our analysis; during the mean follow-up time of 3.4 years, 387 (5.28%) participants developed new-onset diabetes. Compared with participants in normal lipid levels, the T2DM risk of those with hypercholesterolaemia, hypertriglyceridaemia and low high-density lipoprotein cholesterol (HDL-C) were significantly increased (HRs (95% CI) were 1.48 (1.11 to 1.96), 1.92 (1.49 to 2.46) and 1.67 (1.35 to 2.07), respectively). The AUCs of non-HDL-C (0.685, 95% CI 0.659 to 0.711), triglyceride (TG) (0.684, 95% CI 0.658 to 0.710), total cholesterol (TC)/HDL-C (0.685, 95% CI 0.659 to 0.712) and TG/HDL-C (0.680, 95% CI 0.654 to 0.706) were significantly (p<0.005) larger than that of other lipid parameters.Conclusion Middle-aged and elderly adults with hypertriglyceridaemia, hypercholesterolaemia and low HDL-C were at higher risk for developing diabetes. Non-HDL-C, TG, TC/HDL and TG/HDL have greater performance than other lipid parameters in predicting T2DM incidence

Studies of dynamic binding of amino acids to TiO2 nanoparticle surfaces by Solution NMR and Molecular Dynamics Simulations

Adsorption of biomolecules onto material surfaces involves a potentially complex mechanism where molecular species interact to varying degrees with a heterogeneous material surface. Surface adsorption studies by atomic force microscopy (AFM), Sum Frequency Generation (SFG) spectroscopy, and solid state NMR (ssNMR), detect the structures and interactions of biomolecular species that are bound to material surfaces and which, in the absence of a solid liquid interface, do not exchange rapidly between surface-bound forms and free molecular species in bulk solution. Solution NMR has the potential to complement these techniques by detecting and studying transiently bound biomolecules at the liquid-solid interface. Herein we show that dark-state exchange saturation transfer (DEST) NMR experiments on gel-stabilized TiO2 nanoparticle (NP) samples detect several forms of biomolecular adsorption onto titanium (IV) oxide surfaces. Specifically, we use the DEST approach to study the interaction of amino acids arginine (Arg), lysine (Lys), leucine (Leu), alanine (Ala), and aspartic acid (Asp) with TiO2 rutile nanoparticle surfaces. Whereas Leu, Ala, and Asp display only a single weakly interacting form in the presence of TiO2 nanoparticles , Arg and Lys displayed at least two distinct bound forms: a species that is surface bound and retains a degree of reorientational motion, and a second more tightly bound form characterized by broadened DEST profiles upon addition of TiO2 nanoparticles. Molecular Dynamics simulations indicate different surface bound states for both Lys and Arg depending on the degree of TiO2 surface hydroxylation, but only a single bound state for Asp regardless of the degree of surface hydroxylation, in agreement with results obtained from analysis of DEST profiles

Genomic C-Value Variation Analysis in Jujube (<i>Ziziphus jujuba</i> Mill.) in the Middle Yellow River Basin

Chinese jujube (Ziziphus jujuba Mill.) originated in the Yellow River basin (YRB) of the Shanxi–Shaanxi region. The genomic C-value is a crucial indicator for plant breeding and germplasm evaluation. In this study, we used flow cytometry to determine the genomic C-values of jujube germplasms in the YRB of the Shanxi–Shaanxi region and evaluated their differences in different sub-regions. Of the 29 sub-regions, the highest and lowest variations were in Linxian and Xiaxian, respectively. The difference between jujube germplasms was highly significant (F = 14.89, p < 0.0001) in Linxian. Cluster analysis showed that both cluster 2 and 4 belonged to Linxian, which were clearly separated from other taxa but were cross-distributed in them. Linxian County is an important gene exchange center in the YRB of the Shanxi–Shaanxi region. Principal component analysis showed that cluster 1 had low genomic C-values and single-fruit weights and cluster 2 had high genomic C-values and vitamin C contents. The genomic C-value was correlated with single-fruit weight and vitamin C content. In addition, the genomic C-value was used to predict fruit agronomic traits, providing a reference for shortening the breeding cycle and genetic diversity-related studies of jujube germplasm

Effects of microgels fabricated by microfluidic on the stability, antioxidant, and immunoenhancing activities of aquatic protein

Aquatic products are considered a potential source of novel bioactive proteins, which are used as therapeutic drugs for the treatment of different diseases (such as oxidative stress, immunocompromised, and inflammation), as well as nutraceuticals and cosmetics. However, the physical and chemical properties of proteins are unstable, and they are easily denatured by the influence of external high temperature and polar pH during processing, resulting in the loss of their functional activity. Herein, Fenneropenaeus chinensis water-soluble protein (FCWP) and Lateolabrax japonicus water-soluble protein (LJWP) were encapsulated within spherical biopolymer microgels composed of pectin and chitosan produced by the microfluidic device. The encapsulated samples remained inside the microgels when they were exposed to upper gastrointestinal but were released when they were exposed to simulated colonic fluid due to the hydrolysis effect by enzymes secreted by the colonic microflora. The results showed that microgels improve the thermal stability of FCWP and LJWP due to the interaction between polysaccharides and proteins in the microgels. In addition, microgels encapsulation did not affect the antioxidant and immunoenhancing activities of FCWP and LJWP. In summary, these microgels are suitable for oral colon-specific delivery in functional foods and supplements