A Simple, Rapid, and Cost-Effective PCR Procedure for Detection of NUDT15 Gene Variants in Vietnamese Patients with Acute Lymphoblastic Leukemia.

Objective The NUDT15 variants impact thiopurine dose selection in acute lymphoblastic leukemia patients. The ability to rapidly detect variants is important in clinical practice. This study aims to develop a simple polymerase chain reaction (PCR) procedure for detecting NUDT15 variants in Vietnamese patients. Materials and Methods Sanger sequencing was used to determine NUDT15 variants from 200 patients. We designed primers and optimized the PCR procedure for detection of wild-type and variant alleles and compared with Sanger sequencing results. Results The inserted variant c.55_56insGAGTCG was detected by differences in size through conventional PCR. The tetra-primer amplification refractory mutation system PCR was successful in detecting two variations, c.52G > A and c.415C > T. The sensitivity and specificity of PCR procedure achieved 100% when compared to 200 Sanger sequencing results. Conclusion Our PCR procedure is suitable for replacing Sanger sequencing to detect the NUDT15 variants in clinical setting.

The influence of NUDT15 variants on 6-mercaptopurine-induced neutropenia in Vietnamese pediatric acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP) serves as the backbone of maintenance therapy in acute lymphoblastic leukemia. The nucleoside diphosphate-linked moiety X-type motif 15 genes (NUDT15) affects the metabolism of 6-MP and thiopurine-related neutropenia in the Asian population. This study reports the influence of these variants on 6MP-induced neutropenia in children with acute lymphoblastic leukemia (ALL). A total of 102 children were enrolled in this retrospective cohort study. NUDT15 variants on exon 1 and exon 3 were identified by Sanger sequencing. We divided the intermediate metabolizer group and the normal metabolizer group base on NUDT15 diplotypes. During the first 3 months of maintenance treatment, medical reports measured treatment-related toxicity (neutropenia) and 6-MP dose decreases. NUDT15 genotyping showed two categories of mutations: wild type (75.5%) and heterozygous variant (24.5%). Neutropenia during the early phase of maintenance therapy in the intermediate metabolizer group (68%) was significantly higher than the normal metabolizer group (18.2%) with 10-fold greater odds. Especially, the c.415C>T heterozygous variant was extremely associated with neutropenia compared with the C>C genotype (odds ratio [OR]: 12; 95% confidence interval [CI]: 3.5-41.7). The tolerated doses of 6-MP after the first 3 months of maintenance therapy related to the intermediate metabolizer group and the normal metabolizer group were 48.7 and 64.3 mg/m2/day, respectively (p < 0.001). One-fourth of individuals had NUDT15 variations. All NUDT15 heterozygous mutations cause neutropenia and need 6-MP dose optimization. Given the frequency of NUDT15 mutations in Vietnamese children and their connection with early neutropenia, testing is indicated.

Amino acid variants of SARS-CoV-2 papain-like protease have impact on drug binding.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused both a health and economic crisis around the world. Its papain-like protease (PLpro) is one of the protein targets utilized in designing new drugs that would aid vaccines in the fight against the virus. Although there are already several potential candidates for a good inhibitor of this protein, the degree of variability of the protein itself is not taken into account. As an RNA virus, SARS-CoV-2 can mutate to a high degree, but PLpro variability has not been studied to date. Based on sequence data available in databases, we analyzed the mutational potential of this protein. We focused on the effect of observed mutations on inhibitors' binding mode and their efficacy as well as protein's activity. Our analysis identifies five mutations that should be monitored and included in the drug design process: P247S, E263D-Y264H and T265A-Y268C.

AlphaKnot: server to analyze entanglement in structures predicted by AlphaFold methods.

AlphaKnot is a server that measures entanglement in AlphaFold-solved protein models while considering pLDDT confidence values. AlphaKnot has two main functions: (i) providing researchers with a webserver for analyzing knotting in their own AlphaFold predictions and (ii) providing a database of knotting in AlphaFold predictions from the 21 proteomes for which models have been published prior to 2022. The knotting is defined in a probabilistic fashion. The knotting complexity of proteins is presented in the form of a matrix diagram which shows users the knot type for the entire polypeptide chain and for each of its subchains. The dominant knot types as well as the computed locations of the knot cores (i.e. minimal portions of protein backbones that form a given knot type) are shown for each protein structure. Based mainly on the pLDDT confidence values, entanglements are classified as Knots, Unsure, and Artifacts. The database portion of the server can be used, for example, to examine protein geometry and entanglement-function correlations, as a reference set for protein modeling, and for facilitating evolutional studies. The AlphaKnot server can be found at https://alphaknot.cent.uw.edu.pl/.

Evaluation of Technical Feasibility of Reusing Coal Ash in Dense Asphalt Mixes by Assessing Mechanical Performance.

This manuscript evaluates the technical feasibility of reusing coal ash in the formulation design of dense asphalt mixes. Physicochemical and mineralogical properties of fly and bottom ash matrixes prepared with coal ash were analyzed by scanning electron microscopy (SEM), X-Ray diffraction (XRD), semi-quantitative spectrometry by X-Ray fluorescence (XRF) and surface characterization by Nitrogen (N2) adsorption. Filler fractions under 0.075 mm with 6.0% in weight from an entirely gneissic aggregate gradation curve taken as reference were compared to identical mixes prepared with fly and bottom ashes individually and also to a combined sample with 3.0% of each ash type. Tests on compaction ability with gyratory shear press, resistance to action of water and to rutting were carried out to compare mechanical performance. The results indicate that both gneissic and coal ash fillers do not form dipoles of effective electric attractions to bituminous matrixes, resulting in inert and hydrophilic behavior regarding to action of water, respectively. Despite surface and morphologic characteristics underlying the mechanical performance of gneissic fillers, coal ash matrixes have shown, in general, good technical feasibility to be used in asphalt mixes.

Liquid biopsy uncovers distinct patterns of DNA methylation and copy number changes in NSCLC patients with different EGFR-TKI resistant mutations.

Targeted therapy with tyrosine kinase inhibitors (TKI) provides survival benefits to a majority of patients with non-small cell lung cancer (NSCLC). However, resistance to TKI almost always develops after treatment. Although genetic and epigenetic alterations have each been shown to drive resistance to TKI in cell line models, clinical evidence for their contribution in the acquisition of resistance remains limited. Here, we employed liquid biopsy for simultaneous analysis of genetic and epigenetic changes in 122 Vietnamese NSCLC patients undergoing TKI therapy and displaying acquired resistance. We detected multiple profiles of resistance mutations in 51 patients (41.8%). Of those, genetic alterations in EGFR, particularly EGFR amplification (n = 6), showed pronounced genome instability and genome-wide hypomethylation. Interestingly, the level of hypomethylation was associated with the duration of response to TKI treatment. We also detected hypermethylation in regulatory regions of Homeobox genes which are known to be involved in tumor differentiation. In contrast, such changes were not observed in cases with MET (n = 4) and HER2 (n = 4) amplification. Thus, our study showed that liquid biopsy could provide important insights into the heterogeneity of TKI resistance mechanisms in NSCLC patients, providing essential information for prediction of resistance and selection of subsequent treatment.

SARS-CoV-2 Papain-Like Protease Potential Inhibitors-In Silico Quantitative Assessment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC50 values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC50 values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.

Ultra-Deep Massive Parallel Sequencing of Plasma Cell-Free DNA Enables Large-Scale Profiling of Driver Mutations in Vietnamese Patients With Advanced Non-Small Cell Lung Cancer.

Population-specific profiling of mutations in cancer genes is of critical importance for the understanding of cancer biology in general as well as the establishment of optimal diagnostics and treatment guidelines for that particular population. Although genetic analysis of tumor tissue is often used to detect mutations in cancer genes, the invasiveness and limited accessibility hinders its application in large-scale population studies. Here, we used ultra-deep massive parallel sequencing of plasma cell free DNA (cfDNA) to identify the mutation profiles of 265 Vietnamese patients with advanced non-small cell lung cancer (NSCLC). Compared to a cohort of advanced NSCLC patients characterized by sequencing of tissue samples, cfDNA genomic testing, despite lower mutation detection rates, was able to detect major mutations in tested driver genes that reflected similar mutation composition and distribution pattern, as well as major associations between mutation prevalence and clinical features. In conclusion, ultra-deep sequencing of plasma cfDNA represents an alternative approach for population-wide genetic profiling of cancer genes where recruitment of patients is limited to the accessibility of tumor tissue site.

Actionable Mutation Profiles of Non-Small Cell Lung Cancer patients from Vietnamese population.

Comprehensive profiling of actionable mutations in non-small cell lung cancer (NSCLC) is vital to guide targeted therapy, thereby improving the survival rate of patients. Despite the high incidence and mortality rate of NSCLC in Vietnam, the actionable mutation profiles of Vietnamese patients have not been thoroughly examined. Here, we employed massively parallel sequencing to identify alterations in major driver genes (EGFR, KRAS, NRAS, BRAF, ALK and ROS1) in 350 Vietnamese NSCLC patients. We showed that the Vietnamese NSCLC patients exhibited mutations most frequently in EGFR (35.4%) and KRAS (22.6%), followed by ALK (6.6%), ROS1 (3.1%), BRAF (2.3%) and NRAS (0.6%). Interestingly, the cohort of Vietnamese patients with advanced adenocarcinoma had higher prevalence of EGFR mutations than the Caucasian MSK-IMPACT cohort. Compared to the East Asian cohort, it had lower EGFR but higher KRAS mutation prevalence. We found that KRAS mutations were more commonly detected in male patients while EGFR mutations was more frequently found in female. Moreover, younger patients (<61 years) had higher genetic rearrangements in ALK or ROS1. In conclusions, our study revealed mutation profiles of 6 driver genes in the largest cohort of NSCLC patients in Vietnam to date, highlighting significant differences in mutation prevalence to other cohorts.

Evaluation of physicochemical and hydromechanical properties of MSWI bottom ash for road construction.

Municipal Solid Waste Incinerator (MSWI) Bottom Ash has been used as a substitute for traditional aggregates in road construction; however, this material is little understood. The work presented in this paper pursues the study on the mechanical performance of bottom ash, proven by Le et al. (2017). Using a coupling technique for the first time, the physicochemical aspects and hydromechanical resistance of bottom ash were evaluated and analyzed. Physicochemical tests were first carried out, followed by oedometer tests under a wetting path. This coupled evaluation underlined the role of principal mineralogical components of the studied bottom ash as well as the link with its hydromechanical properties. Tests results showed that the principal constituent of bottom ash is SiO2, which thus affects the characteristics of bottom ash. Given the physical stability of SiO2 which generated a compacted material being less sensitive to water and chemical reactions, and bottom ash's other characteristics, this demonstrates why bottom ash could be a viable material in roadworks.

Kinetics of the reactions of soot surface-bound polycyclic aromatic hydrocarbons with O3.

The kinetics of the heterogeneous reactions of O(3) with 17 polycyclic aromatic hydrocarbons (PAHs) present on laboratory generated kerosene soot surface was studied at T=255-330 K in a low pressure flow reactor combined with an electron-impact mass spectrometer. The kinetics of soot-bound PAHs consumption in reaction with O(3) was monitored using off-line HPLC measurements of their concentrations in soot samples as a function of time of exposure to O(3). Concentration of ozone in the gas phase was analyzed by mass spectrometry. The first-order rate constants measured for individual PAHs ranged from 0.004 to 0.008 s(-1) and were found to be independent of the ozone concentration ([O(3)]=(0.5-92) x 10(12) mol cm (-3)) and temperature (255-330 K). Results show that reaction with ozone can be an important degradation pathway of the particulate PAHs in the atmosphere.

Desorption of polycyclic aromatic hydrocarbons from soot surface: five- and six-ring (C22, C24) PAHs.

The kinetics of thermal desorption of five soot-bound nonvolatile (C(22), C(24)) polycyclic aromatic hydrocarbons (benzo(ghi)perylene (BghiP), indeno(1,2,3-cd)pyrene (IdP), anthanthrene (Antha), dibenz(ah)anthracene (DBahA), dibenzo(ae)pyrene (DBaeP)) identified in laboratory-generated kerosene soot were studied over the temperature range 335-370 K in a low pressure flow reactor. The PAH desorption rate constants were measured using off-line HPLC monitoring of their concentrations in soot samples as a function of desorption time. The following Arrhenius expressions were determined for the desorption rate constants of the soot-bound PAHs studied: k(des) = 8.4 x 10(15) exp[-(129.7 +/- 4.4)/RT], 1.0 x 10(16) exp[-(130.6 +/- 6.1)/RT], 1.1 x 10(16) exp[-(131.6 +/- 3.2)/RT], 1.0 x 10(16) exp[-(128.0 +/- 3.9)/RT], and 1.4 x 10(16) exp[-(134.2 +/- 10.7)/RT] (k(des) are in s(-1) and activation energies in kJ mol(-1)) for BghiP, IdP, Antha, DBahA, and DBaeP, respectively. Analysis of the present and previous experimental data showed that PAH-soot binding energies linearly correlate with the number of PAH carbon atoms. The present data and those from previous studies are discussed in the frame of the existing theoretical gas-to-particle partitioning model.

Desorption of polycyclic aromatic hydrocarbons from a soot surface: three- to five-ring PAHs.

The kinetics of the thermal desorption of a set of three- to five-ring polycyclic aromatic hydrocarbons (PAHs) from a laboratory-generated kerosene soot surface was studied over the temperature range 250-355 K in a low-pressure flow reactor combined with an electron-impact mass spectrometer. Two methods were used to measure the desorption rate constants: monitoring of the surface-bound PAH decays due to desorption using off-line HPLC measurements of their concentrations in soot samples and monitoring of the desorbed molecules (anthracene and phenanthtrene) in the gas phase using in situ mass spectrometric detection. The Arrhenius parameters (A factors and activation energies) for the desorption rate constants of 10 soot-bound PAHs were determined. The PAH-soot binding energies were found to be similar for PAHs with the same number of carbon atoms and to increase with increasing number of PAH carbon atoms. The experimental data are discussed in the frame of the existing theoretical gas to particle partitioning model.

Characterization of a subpopulation of colon cancer cells with stem cell-like properties.

The biology of the normal colonic mucosa suggests that colon cancer originates from normal colon stem cells. CD44 cancer stem cells have been identified in breast and prostate cancer, and we therefore examined whether CD44 similarly identified colon cancer stem cells. Initial assays found CD44(hi) colon tumor cells to have enhanced soft agar colony-forming ability. Subsequently, CD44(hi) cells isolated from 4 primary colon adenocarcinoma xenografts were found to be highly tumorigenic in immune deficient mice. CD44(hi) cells consistently formed tumors with 1,000 cells, and in multiple experiments, as few as 10 and 100 CD44(hi) cells formed tumors in 7/10 and 21/28 mice, respectively. In contrast, CD44(-) colon tumor cells were either nontumorigenic or 10-50-fold less tumorigenic. CD44(hi) cells could be serially passaged up to 4 times in vivo, suggesting self-renewal capacity, and formed tumors that recapitulated the heterogeneity of the original patient tumor. CD44(hi) cells were significantly enriched for nuclear activated beta-catenin, a key element in normal stem/progenitor cells and in early colon tumor progression. Bromodeoxyuridine (BrdU) labeling studies indicated that CD44(hi) cells divide slowly relative to the CD44(-) cells, suggesting their tumorigenicity is not simply due to faster proliferation. Aldehyde dehydrogenase (ALDH) sort further increased the tumorigenicity of CD44(hi) cells from 2/2 patient tumors, but CD133 tumor cells in our hands did not have increased tumorigenicity. Our observations indicate that CD44 is a marker of stem-like cells in colon cancer, and support the use of additional markers to further purify colon cancer stem cells.

Desorption of polycyclic aromatic hydrocarbons from soot surface: pyrene and fluoranthene.

The kinetics of thermal desorption of two four-ring polycyclic aromatic hydrocarbons, fluoranthene, and pyrene from well-characterized laboratory-generated kerosene soot surface was studied over the temperature range 260-320 K in a low-pressure flow reactor combined with an electron-impact mass spectrometer. Two methods were used to measure the desorption rate constants: monitoring of the surface-bound fluoranthene and pyrene decays due to desorption using off-line HPLC measurements of their concentrations in soot samples, and monitoring of the desorbed molecules in the gas phase using in situ mass spectrometric detection. Results obtained with the two methods were in good agreement and yielded the following Arrhenius expressions for the desorption rate constants: k(des) (fluoranthene) = 4 x 10(14) exp[-(93900 +/- 1700)/RT] and k(des) (pyrene) = 6 x 10(14) exp[-(95200 +/- 1800)/RT] (k(des) are in units of s(-1), and activation energies are in J mol(-1)). In addition, the combined uptake coefficient of fluoranthene and pyrene on soot (calculated using specific surface area) was estimated to be near 5 x 10(-3) at T = 310 K.