Nucleophosmin 1 promotes mucosal immunity by supporting mitochondrial oxidative phosphorylation and ILC3 activity.

Nucleophosmin 1 (NPM1) is commonly mutated in myelodysplastic syndrome (MDS) and acute myeloid leukemia. Concurrent inflammatory bowel diseases (IBD) and MDS are common, indicating a close relationship between IBD and MDS. Here we examined the function of NPM1 in IBD and colitis-associated colorectal cancer (CAC). NPM1 expression was reduced in patients with IBD. Npm1+/- mice were more susceptible to acute colitis and experimentally induced CAC than littermate controls. Npm1 deficiency impaired the function of interleukin-22 (IL-22)-producing group three innate lymphoid cells (ILC3s). Mice lacking Npm1 in ILC3s exhibited decreased IL-22 production and accelerated development of colitis. NPM1 was important for mitochondrial biogenesis and metabolism by oxidative phosphorylation in ILC3s. Further experiments revealed that NPM1 cooperates with p65 to promote mitochondrial transcription factor A (TFAM) transcription in ILC3s. Overexpression of Npm1 in mice enhanced ILC3 function and reduced the severity of dextran sulfate sodium-induced colitis. Thus, our findings indicate that NPM1 in ILC3s protects against IBD by regulating mitochondrial metabolism through a p65-TFAM axis.

Subgenome-aware analyses reveal the genomic consequences of ancient allopolyploid hybridizations throughout the cotton family.

Malvaceae comprise some 4,225 species in 243 genera and nine subfamilies and include economically important species, such as cacao, cotton, durian, and jute, with cotton an important model system for studying the domestication of polyploids. Here, we use chromosome-level genome assemblies from representatives of five or six subfamilies (depending on the placement of Ochroma) to differentiate coexisting subgenomes and their evolution during the family's deep history. The results reveal that the allohexaploid Helicteroideae partially derive from an allotetraploid Sterculioideae and also form a component of the allodecaploid Bombacoideae and Malvoideae. The ancestral Malvaceae karyotype consists of 11 protochromosomes. Four subfamilies share a unique reciprocal chromosome translocation, and two other subfamilies share a chromosome fusion. DNA alignments of single-copy nuclear genes do not yield the same relationships as inferred from chromosome structural traits, probably because of genes originating from different ancestral subgenomes. These results illustrate how chromosome-structural data can unravel the evolutionary history of groups with ancient hybrid genomes.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Scattered differentiation of unlinked loci across the genome underlines ecological divergence of the selfing grass Brachypodium stacei.

Ecological divergence without geographic isolation, as an early speciation process that may lead finally to reproductive isolation through natural selection, remains a captivating topic in evolutionary biology. However, the pattern of genetic divergence underlying this process across the genome may vary between species and mating systems. Here, we present evidence that Brachypodium stacei, an annual and highly selfing grass model species, has undergone sympatric ecological divergence without geographic isolation. Genomic, transcriptomic, and metabolomic analyses together with lab experiments mimicking the two opposite environmental conditions suggest that diploid B. stacei populations have diverged sympatrically in two slopes characterized by distinct biomes at Evolution Canyon I (ECI), Mount Carmel, Israel. Despite ongoing gene flow, primarily facilitated by seed dispersal, the level of gene flow has progressively decreased over time. This local adaptation involves the scattered divergence of many unlinked loci across the total genome that include both coding genes and noncoding regions. Additionally, we have identified significant differential expressions of genes related to the ABA signaling pathway and contrasting metabolome composition between the arid- vs. forest-adapted B. stacei populations in ECI. These results suggest that multiple small loci involved in environmental responses act additively to account for ecological adaptations by this selfing species in contrasting environments.

Subgenomic Stability of Progenitor Genomes During Repeated Allotetraploid Origins of the Same Grass Brachypodium hybridum.

Both homeologous exchanges and homeologous expression bias are generally found in most allopolyploid species. Whether homeologous exchanges and homeologous expression bias differ between repeated allopolyploid speciation events from the same progenitor species remains unknown. Here, we detected a third independent and recent allotetraploid origin for the model grass Brachypodium hybridum. Our homeologous exchange with replacement analyses indicated the absence of significant homeologous exchanges in any of the three types of wild allotetraploids, supporting the integrity of their progenitor subgenomes and the immediate creation of the amphidiploids. Further homeologous expression bias tests did not uncover significant subgenomic dominance in different tissues and conditions of the allotetraploids. This suggests a balanced expression of homeologs under similar or dissimilar ecological conditions in their natural habitats. We observed that the density of transposons around genes was not associated with the initial establishment of subgenome dominance; rather, this feature is inherited from the progenitor genome. We found that drought response genes were highly induced in the two subgenomes, likely contributing to the local adaptation of this species to arid habitats in the third allotetraploid event. These findings provide evidence for the consistency of subgenomic stability of parental genomes across multiple allopolyploidization events that led to the same species at different periods. Our study emphasizes the importance of selecting closely related progenitor species genomes to accurately assess homeologous exchange with replacement in allopolyploids, thereby avoiding the detection of false homeologous exchanges when using less related progenitor species genomes.

Ultrastable Luminol-OH--(Ni-WOx-CNT) ECL System with High Strength and Its Applications in Sensing.

In traditional luminol electrochemiluminescence (ECL) systems, hydrogen peroxide (H2O2) and dissolved oxygen (DO) are the commonly used coreactants to generate reactive oxygen species (ROS) for ECL emission. However, the self-decomposition of hydrogen peroxide and the limited solubility and content of oxygen in solution undoubtedly restrict the luminescence efficiency and stability of the luminol ECL system. Inspired by the ROS-mediated ECL mechanism, we pioneered hydroxide ion as an advanced luminol ECL coreactant using nickel-doped and carbon nanotube-modified tungsten oxide (Ni-WOx-CNT) as the coreactant accelerator. Owing to the excellent catalytic activity of Ni-WOx-CNT, amounts of ROS were generated from OH- at a low excitation voltage, which subsequently reacted with luminol anion radicals and triggered intense ECL signals. Experiments confirmed an impressive ECL behavior in terms of high luminescent intensity (85,563 a.u.) and super stability over 1300 consecutive tests; both are superior to those recently reported luminol-H2O2 and luminol-DO systems with smaller ECL intensities and consecutive tests less than 25 times. To validate the feasibility and versatility of the developed system in sensor, traditional three-electrodes system and closed bipolar electrodes system with various sensing strategies of direct oxidation, "gate-effect" of molecularly imprinted polymer, immune reaction, and enzyme-catalyzed reaction were proposed to monitor uric acid (UA), C-reactive protein (CRP), immunoglobulin G (IgG), and glucose (Glu). The superior sensing performances confirmed the great application potential of the developed ROS-mediated ECL system.

Multifunctional Self-Signaling nanoMIP and Its Application for a Washing-Free Assay of Human Angiotensin-Converting Enzyme 2.

Molecular imprinting techniques have attracted a lot of attention as a potential biomimetic technology, but there are still challenges in protein imprinting. Herein, multifunctional nanosized molecularly imprinted polymers (nanoMIPs) for human angiotensin-converting enzyme 2 (ACE2) were prepared by epitope imprinting of magnetic nanoparticles-anchored peptide (magNP-P) templates, which were further applied to construct a competitive displacement fluorescence assay toward ACE2. A cysteine-flanked dodecapeptide sequence was elaborately selected as an epitope for ACE2, which was immobilized onto the surface of magnetic nanoparticles and served as a magNP-P template for imprinting. During polymerization, fluorescent monomers were introduced to endow fluorescence responsiveness to the prepared self-signaling nanoMIPs. A competitive displacement fluorescence assay based on the nanoMIPs was established and operated in a washing-free manner, yielding a wide range for ACE2 (0.1-6.0 pg/mL) and a low detection limit (0.081 pg/mL). This approach offers a promising avenue in the preparation of nanoMIPs for macromolecule recognition and expands potential application of an MIP in the detection of proteins as well as peptides.

High-Valence Metals Accelerate the Reaction Kinetics for Boosting Water Oxidation.

The transition metal with high valence state in oxyhydroxides can accelerate the reaction kinetics, enabling highly intrinsic OER activity. However, the formation of high-valence transition-metal ions is thermodynamically unfavorable in most cases. Here, a novel strategy is proposed to realize the purpose and reveal the mechanism by constructing amorphous phase and incorporating of elements with the characteristic of Lewis acid or variable charge state. A model catalyst, CeO2-NiFeOxHy, is presented to achieve the modulation of valence state of active site (Ni2+→Ni3+→Ni4+) for improved OER, leading to dominant active sites with high valence state. The CeO2-NiFeOxHy electrode exhibits superior OER performance with overpotential of 214 and 659 mV at 10 and 500 mA cm-2, respectively (without IR correction), and high stability, which are much better than those of NiOxHy, NiFeOxHy and CeO2-NiOxHy. These findings provide an effective strategy to achieve the active metals with high-valence state for highly efficient OER.

Roles of LncRNAs in the Pathogenesis of Pulmonary Hypertension.

Pulmonary hypertension (PH) is a persistently progressive, incurable, multifactorial associated fatal pulmonary vascular disease characterized by pulmonary vascular remodeling. Long noncoding RNAs (lncRNAs) are involved in regulating pathological processes such as pulmonary vasoconstriction, thickening, remodeling, and inflammatory cell infiltration in PH by acting on different cell types. Because of their differential expression in PH patients, as demonstrated by the observation that some lncRNAs are significantly upregulated while others are significantly downregulated in PH patients, lncRNAs are potentially useful biomarkers for assessing disease progression and diagnosis or prognosis in PH patients. This article provides an overview of the different mechanisms by which lncRNAs are involved in the pathogenesis of PH.

Immunogenicity evaluation of viral peptides via nonspecific interactions between anti-peptide IgYs and non-cognate peptides.

Immunogenicity can be evaluated by detecting antibodies (Abs) induced by an antigen. Presently deployed assays, however, do not consider the negative impacts of Ab poly-specificity, which is well established at the monoclonal antibody level. Here, we studied antibody poly-specificity at the serum level (i.e. nonspecific Ab-probe interactions, NSIs), and ended up establishing a new platform for viral peptide immunogenicity evaluation. We first selected three peptides of high, medium and low immunogenicity, using a 'vaccine serum response rate'-based approach (i.e. the gold standard). These three peptides (Pi) in the bovine serum albumin-Pi form were used to immunize chickens, resulting in longitudinal serum samples for screening with a non-cognate peptide library. The signal intensity of Ab-peptide specific binding and 'NSI count' was used to evaluate the viral peptides' immunogenicity. Only the NSI count agreed with the gold standard. The NSI count also provides more informative data on antibody production than the aggregated signal intensity by whole-protein-based indirect enzyme-linked immunosorbent assay.

Modified submucosal tunneling endoscopic septum division for symptomatic esophageal diverticula: a prospective case series (with video).

BACKGROUND AND AIMS: Submucosal tunneling endoscopic septum division (STESD) is a valid endoscopic modality for treating esophageal diverticula. However, it requires highly skilled endoscopists. Here, we propose a modified STESD method. METHODS: This is a single-center, prospective case series. We consecutively enrolled 8 patients diagnosed with esophageal diverticula. All patients underwent the modified STESD by 1 experienced endoscopist. RESULTS: Only 1 patient had intraoperative bleeding, which was successfully stopped endoscopically. The mean ± standard deviation size of the diverticula was 3.16 ± 1.14 cm. The operation time ranged from 27 to 68 minutes, with a mean value of 40.88 minutes. The number of clips ranged from 4 to 8. The success rate was 100%. None of the patients had symptom recurrences during 2 to 25 months of follow-up. CONCLUSIONS: The modified STESD method for esophageal diverticula is effective and safe; further large prospective controlled studies are needed.

Identification and characterization of two Bacillus anthracis bacteriophages.

Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.

Factors affecting the incidence of pulmonary tuberculosis based on the GTWR model in China, 2004-2021.

Contra-posing panel data on the incidence of pulmonary tuberculosis (PTB) at the provincial level in China through the years of 2004-2021 and introducing a geographically and temporally weighted regression (GTWR) model were used to explore the effect of various factors on the incidence of PTB from the perspective of spatial heterogeneity. The principal component analysis (PCA) was used to extract the main information from twenty-two indexes under six macro-factors. The main influencing factors were determined by the Spearman correlation and multi-collinearity tests. After fitting different models, the GTWR model was used to analyse and obtain the distribution changes of regression coefficients. Six macro-factors and incidence of PTB were both correlated, and there was no collinearity between the variables. The fitting effect of the GTWR model was better than ordinary least-squares (OLS) and geographically weighted regression (GWR) models. The incidence of PTB in China was mainly affected by six macro-factors, namely medicine and health, transportation, environment, economy, disease, and educational quality. The influence degree showed an unbalanced trend in the spatial and temporal distribution.

Oxidative stress in acute pulmonary embolism: emerging roles and therapeutic implications.

Oxidative stress is an imbalance between the body's reactive oxygen species and antioxidant defense mechanisms. Oxidative stress is involved in the development of several cardiovascular diseases, such as pulmonary hypertension, atherosclerosis, and diabetes mellitus. A growing number of studies have suggested the potential role of oxidative stress in the pathogenesis of pulmonary embolism. Biomarkers of oxidative stress in pulmonary embolism have also been explored, such as matrix metalloproteinases, asymmetric dimethylarginine, and neutrophil/lymphocyte ratio. Here, we comprehensively summarize some oxidative stress mechanisms and biomarkers in the development of acute pulmonary embolism and summarize related treatments based on antioxidant stress to explore effective treatment strategies for acute pulmonary embolism.

Association between sensory processing sensitivity and quality of life among cancer patients: a mediation and moderation of resilience and social determinants.

BACKGROUND: Individuals with sensory processing sensitivity (SPS) tend to be overreactive in response to negative environmental stimuli. More is known about the positive relationship between SPS and quality of life (QoL); nevertheless, less is known regarding the roles of resilience and social determinants in this association. This research aimed to investigate the potential mediation effect of resilience and the moderation effect of social determinants on the relationship between SPS and QoL in a large sample of Chinese cancer patients. METHODS: We used the most recent datasets from an ongoing project conducted in southwest China. A two-stage random sampling strategy with a probability proportionate to sample size (PPS) design was adopted. The associations between resilience, SPS, and QoL were evaluated using a linear regression model. Path analysis was adopted to examine the mediation of resilience. RESULTS: Resilience was positively associated with quality of life, while increased sensory processing sensitivity was negatively associated with quality of life. The restricted cubic spline analysis revealed that as resilience increased, the coefficients of quality of life rapidly increased across all domains. Conversely, the coefficients for quality of life gradually decreased with the escalation of sensory processing sensitivity. Resilience was a significant mediator, accounting for 21.88% of the total SPS-QoL association. The mediation effect of resilience varied across ethnicity and sex. CONCLUSION: Sensory processing sensitivity was significantly associated with quality of life in cancer patients, and promoting resilience could mitigate this negative impact. However, the effect of resilience varies across sex and ethnicity. Therefore, targeted resilience promotion interventions, especially those integrating social characteristics, should be considered for implementation.

The association between smoking and family health with the mediation role of personality among Chinese people: nationwide cross-sectional study.

BACKGROUND: There may be unexplored interactions between family health, personality, and smoking that could help provide new perspectives on tobacco control. OBJECTIVE: To examine the relationship between the health of one's family and their smoking habits, as well as investigate the potential influence of personality on this relationship. METHODS: For this cross-sectional investigation, a national survey conducted in China in 2022 recruited a total of 21,916 individuals. The Family Health Scale was utilized to assess the health of the family. The 10-item Big Five Inventory scale was utilized to assess the Big five personality traits. The relationship between big five personality, family health, and smoking were investigated using binary and linear logistic regression. The indirect effects mediated by Big five personality were analyzed using mediation analysis with Sobel tests, and the indirect effects were composited using the Karlson-Holm-Breen method. RESULTS: The overall prevalence of smoking in the study population was 14.87%, 26.19% for males and 3.54% for females. Urban and rural smoking prevalence was 13.81% and 16.10% respectively. Binary logistic regression analysis revealed a significant negative relationship between smoking and family health (odds ratio 0.964, 95% CI 0.959, 0.970, P < 0.001) with covariates controlled. The Karlson-Holm-Breen composition facilitated the connection between extraversion (47.81%) and nervousness (52.19%). CONCLUSIONS: Preventive interventions for smoking behavior should prioritize family health and the Big five personality as significant areas to focus on. According to this study, in addition to implementing various interventions for different personalities, family health should be strengthened to reduce smoking behavior.

Risk profiling of tobacco epidemic and estimated number of smokers living in China: a cross-sectional study based on PBICR.

BACKGROUND: Evidence on the prevalence of smoking in China remains insufficient, with most previous studies focusing on a single region. However, smoking prevalence exhibits significant inequalities across the entire country. This study aimed to evaluate the risk of tobacco prevalence across the country, taking into account spatial inequalities. METHODS: The data used in this study were collected in 23 provinces, 5 autonomous regions, and 4 municipalities directly under the central government in 2022. Large population survey data were used, and a Bayesian geostatistical model was employed to investigate smoking prevalence rates across multiple spatial domains. FINDINGS: Significant spatial variations were observed in smokers and exposure to secondhand smoke across China. Higher levels of smokers and secondhand smoke exposure were observed in western and northeastern regions. Additionally, the autonomous region of Tibet, Shanghai municipality, and Yunnan province had the highest prevalence of smokers, while Tibet, Qinghai province, and Yunnan province had the highest prevalence of exposure to secondhand smoke. CONCLUSION: We have developed a model-based, high-resolution nationwide assessment of smoking risks and employed rigorous Bayesian geostatistical models to help visualize smoking prevalence predictions. These prediction maps provide estimates of the geographical distribution of smoking, which will serve as strong evidence for the formulation and implementation of smoking cessation policies. HIGHLIGHTS: Our study investigated the prevalence of smokers and exposure to secondhand smoke in different spatial areas of China and explored various factors influencing the smoking prevalence. For the first time, our study applied Bayesian geostatistical modeling to generate a risk prediction map of smoking prevalence, which provides a more intuitive and clear understanding of the spatial disparities in smoking prevalence across different geographical regions, economic levels, and development status. We found significant spatial variations in smokers and secondhand smoke exposure in China, with higher rates in the western and northeastern regions.

ZIPK collaborates with STAT5A in p53-mediated ROS accumulation in hyperglycemia-induced vascular injury.

In this study we investigate the role of Zipper-interacting protein kinase (ZIPK) in high glucose-induced vascular injury, focusing on its interaction with STAT5A and its effects on p53 and inducible nitric oxide synthase (NOS2) expression. Human umbilical vein endothelial cells (HUVECs) are cultured under normal (5 mM) and high (25 mM) glucose conditions. Protein and gene expression levels are assessed by western blot analysis and qPCR respectively, while ROS levels are measured via flow cytometry. ZIPK expression is manipulated using overexpression plasmids, siRNAs, and shRNAs. The effects of the ZIPK inhibitor TC-DAPK6 are evaluated in a diabetic rat model. Our results show that high glucose significantly upregulates ZIPK, STAT5A, p53, and NOS2 expressions in HUVECs, thus increasing oxidative stress. Silencing of STAT5A reduces p53 and NOS2 expressions and reactive oxygen species (ROS) accumulation. ZIPK is essential for high glucose-induced p53 expression and ROS accumulation, while silencing of ZIPK reverses these effects. Overexpression of ZIPK combined with STAT5A silencing attenuates glucose-induced alterations in p53 and NOS2 expression, thereby preventing cell damage. Coimmunoprecipitation reveals a direct interaction between ZIPK and STAT5A in the nucleus under high-glucose condition. In diabetic rats, TC-DAPK6 treatment significantly decreases ZIPK, p53, and NOS2 expressions. Our findings suggest that ZIPK plays a critical role in high glucose-induced vascular injury via STAT5A-mediated pathways, proposing that ZIPK is a potential therapeutic target for diabetic vascular complications.

P-glycoprotein-mediated herb-drug interaction evaluation between Tenacissoside G and paclitaxel.

P-glycoprotein (P-gp)-mediated herb-drug interactions (HDIs) may impact drug efficacy and safety. Tenacissoside G (Tsd-G), a major active component of Marsdenia tenacissima, exhibits anticancer activity. To analyze the effect of Tsd-G on the pharmacokinetics of paclitaxel (PTX), researchers selected 30 Sprague-Dawley (SD) rats, randomized into a solvent control group, a verapamil positive control group, and 20, 40, and 60 mg/kg Tsd-G groups. After seven consecutive days of intraperitoneal injection of verapamil or Tsd-G, a single dose of 6 mg/kg PTX was injected intravenously. Plasma samples were collected at different time points, and proteins were precipitated using a methanol-acetonitrile solution. An ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed, with docetaxel as an internal standard, and quantified using positive ion multiple reaction monitoring (MRM) mode. This analytical method's specificity, accuracy, precision, recovery, matrix effect, and sample stability meet the requirements for biological sample determination. After Tsd-G administration in rats, the mean residence time of PTX was significantly prolonged. And Tsd-G can stably bind to P-gp by forming hydrogen bonds and inhibiting the expression of P-gp in rat liver. Although the metabolites of PTX were not detected in this study, the above results still indicate the existence of HDIs between Tsd-G and PTX, and P-gp may be the main target to mediate HDIs.

Hydrogel-based fluorescence assay kit for simultaneous determination of ceftazidime and avibactam.

Monitoring the concentration of antibiotics rapidly and cost-effectively is crucial for accurate clinical medication and timely identification of drug-induced illnesses. Here, we constructed a novel fluorescent assay kit to monitor Zavicefta, an effective antibiotic composed of avibactam (AVI) and ceftazidime (CFZ) to treat carbapenem-resistant gram-negative bacteria infections. AVI can emit fluorescence, but CFZ cannot. To enable simultaneous measurement of both in one kit, we designed molecularly imprinted polymer (MIP) modified quantum dots (QDs) for CFZ determination. MIPs have received significant attention as an artificial antibody due to their exceptional specificity for various targets, particularly drugs with small molecular weight. Under the excitation wavelength of 350 nm, the detection process involves a decrease in QDs' fluorescence signal at 600 nm owing to the "gate effect" between MIP and CFZ and the internal filtration effect between CFZ and QDs. Simultaneously, a fluorescence emission characteristic peak at 420 nm for AVI emerges. In addition, to simplify the operation procedure and improve determination throughput, the detection agents were incorporated into a hydrogel and placed in a 96-well plate, enabling concurrent quantification of AVI and CFZ within the respective range of 80-1000 µM and 1-1000 µM. The developed assay kit successfully determined AVI and CFZ in human serums and therapeutic drug monitoring in a live rabbit model. Recoveries of AVI and CFZ were 92.7-114%, with relative standard deviations below 6.0%. Moreover, a smartphone was employed to read the fluorescence signals, which was beneficial for cost reduction and out-of-lab analysis. This study will deliver a pragmatic resolution to developing high-throughput assay kits for drug determination.