Construction of a hepatocellular carcinoma high-risk population rating scale and independent predictors' assessment.

BACKGROUND: With increasing mortality and incidence, hepatocellular carcinoma (HCC) has become a major public health problem. The early diagnosis of HCC can improve its prognosis. The aim of this study was to identify potential risk factors related to HCC development and to establish a high-risk population rating scale. METHODS: A total of 853 patients with chronic hepatitis B (CHB) were enrolled in this study, including 403 patients with HCC as the case group and others as the control group. Their demographic and clinical characteristics were compared and the independent risk factors for HCC were assessed. Then, the optimal cutoff levels of these factors were analyzed by the receiver operating characteristic (ROC) method. A high-risk population rating scale was constructed based on the factors and then evaluated in the modeling population. RESULTS: The factors that presented statistically significant differences between the two groups included age, smoking, alcohol abuse, body mass index, triglyceride, high‒density lipoprotein cholesterol, aspartate transaminase, alanine transaminase, fasting plasma glucose, creatinine and uric acid. The ROC curve showed that the cutoff score for the HCC high risk population was 5 (AUC=0.74, P<0.001) and the Hosmer‒Lemeshow analysis showed that the fitting effect of this rating scale was good (P = 0.294). CONCLUSIONS: The integration of these factors can contribute to a prognostic score for the risk of HCC development, which offered certain clinical practicability.

Development and evaluation of a CRISPR-Cas13a system-based diagnostic for hepatitis E virus.

OBJECTIVES: Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV RNA detection is the gold standard for HEV infection diagnosis and PCR methods are commonly used but are usually time-consuming and expensive, resulting in low detection efficiency and coverage, especially in low-income areas. Here, we developed a simpler and more accessible HEV RNA detection method based on CRISPR-Cas13a system. METHODS: A total of 265 samples of different types and sources, including 89 positive samples and 176 negative samples, were enrolled for evaluations. The sensitivity and specificity of the Cas13a-crRNA detection system were evaluated. The World Health Organization reference panel for HEV genotypes was used to evaluate the capability for detecting different HEV genotypes. The validity of the assay was compared with RT-qPCR. RESULTS: The 95 % limits of detection (LOD) of Cas13a-crRNA-based fluorescence assay and strip assay were 12.5 and 200 IU/mL, respectively. They did not show cross-reactivity with samples positive for hepatitis A virus, hepatitis B virus, hepatitis C virus, coxsackievirus A16, rotavirus, enterovirus 71, norovirus or enteropathic Escherichia coli. Different HEV genotypes (HEV1-4) can be detected by the assay. Compared to RT-qPCR, the positive predictive agreements of Cas13a-crRNA-based fluorescence and strip assay were 98.9 % (95 % CI: 93.9-99.8 %) and 91.0 % (95 % CI: 83.3-95.4 %), respectively. The negative predictive agreements were both 100 % (95 % CI: 97.8-100 %). CONCLUSIONS: In conclusion, we established a rapid and convenient HEV RNA detection method with good sensitivity and specificity based on CRISPR-Cas13a system, providing a new option for HEV infection diagnosis.

Establishment and Characterization of the First Chinese National Standard for Nucleic Acid Amplification Technology Assays for Hepatitis E Virus Nucleic Acid Detection.

The detection of hepatitis E virus (HEV) RNA is the gold standard for HEV infection diagnosis. In order to address the quality control requirements for HEV RNA detection kits within China, we aimed to establish the first Chinese national standard for HEV RNA detection through a collaborative study. The candidate standard was quantified using digital PCR (dPCR). A total of five laboratories were invited to determine the estimated mean value of this national standard relative to the World Health Organization International Standard (WHO IS). Additionally, four commercial kits were used to assess the applicability of the candidate standard. The stability was determined by freeze-thaw cycles and storage at 37 °C, 25 °C and 4 °C. The estimated mean value of this national standard relative to the WHO IS was 5.67 log10 IU/mL. Two out of the four commercial kits can detect as low as the estimated limit of detection (LOD). The degradation rates of samples in the stability study ranged from 4% to 19%. In conclusion, we have established the first Chinese national standard for HEV nucleic acid detection against WHO IS, which can be employed to evaluate the quality of HEV RNA detection kits.

Improving the effectiveness of watershed environmental management-dynamic coordination through government pollution control and resident participation.

To improve the effectiveness of envir onmental management of watersheds and improve the environmental management mechanism of cross-administrative watersheds, we develop a neoliberal framework for action using incentives, examine the cooperative strategies of local governments in watershed treatment and people-oriented environmental protection under central government subsidies, and analyze the cost effectiveness of multiple strategies in a dynamic perspective, and we have the following important findings: (1) Compared to vertical ecological compensation, the introduction of horizontal cost-sharing contracts is more effective in enhancing inter-local cooperative environmental governance. (2) When the marginal benefit of the downstream local government is greater than half of the upstream marginal benefit, the upstream local government's pollution control investment and the effect of pollution control are improved, and the Pareto improvement of the environmental governance benefit of the watershed is realized, i.e., the cost-sharing contract driven by the downstream can achieve a win-win situation for both environmental and government governance benefits. (3) When the marginal benefit of downstream environmental advocacy is between 0.5 and 1.5 times the marginal benefit of upstream government, the cost-sharing contract is more effective in improving downstream benefits. Conversely, when the marginal benefit of downstream is greater than 1.5 times, the marginal benefit of upstream, the more effective the cost-sharing contract is in improving the marginal benefit of downstream. The results of the study provide useful insights for the government to develop reasonable pollution management cooperation mechanisms to improve environmental management performance and thus enhance the sustainable development of the watershed.

DeepGpgs: a novel deep learning framework for predicting arginine methylation sites combined with Gaussian prior and gated self-attention mechanism.

Protein arginine methylation is an important posttranslational modification (PTM) associated with protein functional diversity and pathological conditions including cancer. Identification of methylation binding sites facilitates a better understanding of the molecular function of proteins. Recent developments in the field of deep neural networks have led to a proliferation of deep learning-based methylation identification studies because of their fast and accurate prediction. In this paper, we propose DeepGpgs, an advanced deep learning model incorporating Gaussian prior and gated attention mechanism. We introduce a residual network channel to extract the evolutionary information of proteins. Then we combine the adaptive embedding with bidirectional long short-term memory networks to form a context-shared encoder layer. A gated multi-head attention mechanism is followed to obtain the global information about the sequence. A Gaussian prior is injected into the sequence to assist in predicting PTMs. We also propose a weighted joint loss function to alleviate the false negative problem. We empirically show that DeepGpgs improves Matthews correlation coefficient by 6.3% on the arginine methylation independent test set compared with the existing state-of-the-art methylation site prediction methods. Furthermore, DeepGpgs has good robustness in phosphorylation site prediction of SARS-CoV-2, which indicates that DeepGpgs has good transferability and the potential to be extended to other modification sites prediction. The open-source code and data of the DeepGpgs can be obtained from https://github.com/saizhou1/DeepGpgs.

Does Water Matter? The Impact of Water Vulnerability on Corporate Financial Performance.

This study aims to understand the potential relationship between water vulnerability and corporate financial performance for listed companies in China. Studies have argued that water risk has begun to affect the sustainability of firms, but few studies have included water conditions in the research framework to examine whether and how water conditions have a direct impact on firms. In addition, studies on environment governance have emphasized the impact of government environmental regulation on firms. This study focuses on both regulation and government investments that have been previously neglected. Using a sample of Chinese listed companies from 2016 to 2020, this paper uses pooled cross-sectional regressions with year and industry fixed effects to examine the effects of water vulnerability on corporate financial performance and analyze the mechanism of government water governance (which can be divided into water regulation and water investment) on the relationship between water vulnerability and corporate financial performance. This study finds that water vulnerability could negatively impact corporate financial performance, and water regulation can intensify but water investment couldn't significantly relieve the negative impact. The relationships above differ between SOEs and non-SOEs and water-intensive and non-water-intensive industries.

Evaluation of factors contributing to variability of qualitative and quantitative proficiency testing for SARS-CoV-2 nucleic acid detection.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to unprecedented social and economic disruption. Many nucleic acid testing (NAT) laboratories in China have been established to control the epidemic better. This proficiency testing (PT) aims to evaluate the participants' performance in qualitative and quantitative SARS-CoV-2 NAT and to explore the factors that contribute to differences in detection capabilities. Two different concentrations of RNA samples (A, B) were used for quantitative PT. Pseudovirus samples D, E (different concentrations) and negative sample (F) were used for qualitative PT. 50 data sets were reported for qualitative PT, of which 74.00% were entirely correct for all samples. Forty-two laboratories participated in the quantitative PT. 37 submitted all gene results, of which only 56.76% were satisfactory. For qualitative detection, it is suggested that laboratories should strengthen personnel training, select qualified detection kits, and reduce cross-contamination to improve detection accuracy. For quantitative detection, the results of the reverse transcription digital PCR (RT-dPCR) method were more comparable and reliable than those of reverse transcription quantitative PCR (RT-qPCR). The copy number concentration of ORF1ab and N in samples A and B scattered in 85, 223, 50, and 106 folds, respectively. The differences in the quantitative result of RT-qPCR was mainly caused by the non-standard use of reference materials and the lack of personnel operating skills. Comparing the satisfaction of participants participating in both quantitative and qualitative proficiency testing, 95.65% of the laboratories with satisfactory quantitative results also judged the qualitative results correctly, while 85.71% of the laboratories with unsatisfactory quantitative results were also unsatisfied with their qualitative judgments. Therefore, the quantitative ability is the basis of qualitative judgment. Overall, participants from hospitals reported more satisfactory results than those from enterprises and universities. Therefore, surveillance, daily qualitiy control and standardized operating procedures should be strengthened to improve the capability of SARS-CoV-2 NAT.

Economic benefit evaluation of water resources allocation in transboundary basins based on particle swarm optimization algorithm and cooperative game model-A case study of Lancang-Mekong River Basin.

The present work aims to find the optimal solution of Nash Equilibrium (NE) in the traditional Game Theory (GT) applied to water resources allocation. Innovatively, this paper introduces Particle Swarm Optimization (PSO) into GT to propose a cooperative game model to solve the NE problem. Firstly, the basic theory of the PSO algorithm and cooperative game model is described. Secondly, the PSO-based cooperative game model is explained. Finally, the PSO-based cooperative game model is compared with the Genetic Algorithm (GA) to test the performance. Besides taking the countries in Lancang Mekong River Basin as the research object, this paper discusses each country's water consumption and economic benefits under different cooperation patterns. Then, a series of improvement measures and suggestions are put forward accordingly. The results show that the average server occupancy time of the PSO-based cooperative game model is 78.46% lower than that of GA, and the average waiting time is 79.24% lower than that of the GA. Thus, the model reported here has higher computational efficiency and excellent performance than the GA and is more suitable for the current study. In addition, the multi-country cooperation mode can obtain more economic benefits than the independent water resource development mode. This model can quickly find the optimal combination of 16 cooperation modes and has guiding significance for maximizing the benefits of cross-border water Resource Utilization. This research can provide necessary technical support to solve the possible contradictions and conflicts between cross-border river basin countries and build harmonious international relations.

Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses.

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

Multicenter assessment of shotgun metagenomics for pathogen detection.

BACKGROUND: Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. METHODS: A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. FINDINGS: Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. INTERPRETATION: Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. FUNDING: This work was supported by National Science and Technology Major Project of China (2018ZX10102001).

Computed Tomography Image Texture under Feature Extraction Algorithm in the Diagnosis of Effect of Specific Nursing Intervention on Mycoplasma Pneumonia in Children.

To evaluate the effect of specific nursing intervention in children with mycoplasma pneumonia (MP), a feature extraction algorithm based on gray level co-occurrence matrix (GLCM) was proposed and combined with computed tomography (CT) image texture features. Then, 98 children with MP were rolled into the observation group with 49 cases (specific nursing) and the control group with 49 cases (routine nursing). CT images based on feature extraction algorithm of optimized GLCM were used to examine the children before and after nursing intervention, and the recovery of the two groups of children was discussed. The results showed that the proportion of lung texture increase, rope shadow, ground glass shadow, atelectasis, and pleural effusion in the observation group (24.11%, 3.86%, 8.53%, 15.03%, and 3.74%) was significantly lower than that in the control group (28.53%, 10.23%, 13.34%, 21.15%, and 8.13%) after nursing (P < 0.05). There were no significant differences in the proportion of small patchy shadows, large patchy consolidation shadows, and bronchiectasis between the observation group and the control group (P > 0.05). In the course of nursing intervention, in the observation group, the disappearance time of cough, normal temperature, disappearance time of lung rales, and absorption time of lung shadow (2.15 ± 0.86 days, 4.81 ± 1.14 days, 3.64 ± 0.55 days, and 5.96 ± 0.62 days) were significantly shorter than those in the control group (2.87 ± 0.95 days, 3.95 ± 1.06 days, 4.51 ± 1.02 days, and 8.14 ± 1.35 days) (P < 0.05). After nursing intervention, the proportion of satisfaction and total satisfaction in the experimental group (67.08% and 28.66%) was significantly higher than that in the control group (40.21% and 47.39%), while the proportion of dissatisfaction (4.26%) was significantly lower than that in the control group (12.4%) (P < 0.05). To sum up, specific nursing intervention was more beneficial to improve the progress of characterization recovery and the overall recovery effect of children with MP relative to conventional nursing. CT image based on feature extraction algorithm of optimized GLCM was of good adoption value in the diagnosis and treatment of MP in children.

The impact of sample processing on the rapid antigen detection test for SARS-CoV-2: Virus inactivation, VTM selection, and sample preservation.

Many factors have been identified as having the ability to affect the sensitivity of rapid antigen detection (RAD) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to identify the impact of sample processing on the sensitivity of the RAD tests. We explored the effect of different inactivation methods, viral transport media (VTM) solutions, and sample preservation on the sensitivity of four RAD kits based on two SARS-CoV-2 strains. Compared with non-inactivation, heat inactivation significantly impacted the sensitivity of most RAD kits; however, ß-propiolactone inactivation only had a minor effect. Some of the VTM solutions (VTM2, MANTACC) had a significant influence on the sensitivity of the RAD kits, especially for low viral-loads samples. The detection value of RAD kits was slightly decreased, while most of them were still in the detection range with the extension of preservation time and the increase of freeze-thaw cycles. Our results showed that selecting the appropriate inactivation methods and VTM solutions is necessary during reagent development, performance evaluation, and clinical application.

Characteristics of enhanced biological phosphorus removal (EBPR) process under the combined actions of intracellular and extracellular polyphosphate.

The characteristics of enhanced biological phosphorus removal (EBPR) process under the combined actions of intracellular and extracellular polyphosphate (polyP) were investigated with the 31P Nuclear Magnetic Resonance (NMR) and the fractionation extracting the loosely-bound and tightly-bound extracellular polymer substances (i.e., LB-EPS and TB-EPS) and bacterial cells in EBPR sludge. The hydrolysis/synthesis of extracellular and intracellular polyP was a key step of the phosphate migration and transformation in EBPR sludge. The orthophosphate (orthoP) produced from the intracellular and extracellular polyP anaerobic-hydrolysis was partially accumulated in the bacterial cells and TB-EPS, and then the accumulated orthoP was main composition for these polyP aerobic-synthesis. Importantly, the anaerobic-hydrolysis enhancement of intracellular and extracellular ployP could promote EBPR sludge to absorb volatile fatty acids (VFAs) followed by being transformed into intracellular poly-hydroxy-alkanoates (PHAs). The mechanism for VFAs passing through the LB-EPS and TB-EPS should be an anion-exchange action between orthoP and VFAs. The orthoP accumulation in the TB-EPS kept an orthoP concentration gradient among the TB-EPS, LB-EPS and bulk solution, driving orthoP and VFAs migrations. The orthoP accumulation in the bacterial cells could keep an orthoP concentration difference between the cell-membrane two sides of phosphorus accumulating organisms (PAOs) to promote VFAs passing through the cell membrane considered as an anion exchange membrane. The intracellular PHAs continuously hydrolyzed accompanied with the average chain-length increases of the extracellular and intracellular polyP during the whole aerobic stage. Additionally, the energy of the extracellular polyP synthesized in situ should came from the intracellular PHAs hydrolysis.

A SARS-CoV-2 Reference Standard Quantified by Multiple Digital PCR Platforms for Quality Assessment of Molecular Tests.

The outbreak of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. To meet the urgent and massive demand for the screening and diagnosis of infected individuals, many in vitro diagnostic assays using nucleic acid tests (NATs) have been urgently authorized by regulators worldwide. A reference standard with a well-characterized concentration or titer is of the utmost importance for the study of limit of detection (LoD), which is a crucial feature for a diagnostic assay. Although several reference standards of plasmids or synthetic RNA have already been announced, a reference standard for inactivated virus particles with an accurate concentration is still needed to evaluate the complete procedure. Here, we performed a collaborative study to estimate the NAT-detectable units as a viral genomic equivalent quantity (GEQ) of an inactivated whole-virus SARS-CoV-2 reference standard candidate using digital PCR (dPCR) on multiple commercialized platforms. The median of the quantification results (4.6 × 105 ± 6.5 × 104 GEQ/mL) was treated as the consensus true value of GEQ of virus particles in the reference standard. This reference standard was then used to challenge the LoDs of six officially approved diagnostic assays. Our study demonstrates that an inactivated whole virus quantified by dPCR can serve as a reference standard and provides a unified solution for assay development, quality control, and regulatory surveillance.

Weight prioritized slicing based on constraint logic programming for fault localization.

Fault localization, a technique to fix and ensure the dependability of software, is rapidly becoming infeasible due to the increasing scale and complexity of multilingual programs. Compared to other fault localization techniques, slicing can directly narrow the range of the code which needed checking by abstracting a program into a reduced one by deleting irrelevant parts. Only minority slicing methods take into account the fact that the probability of different statements leading to failure is different. Moreover, no existing prioritized slicing techniques can work on multilingual programs. In this paper, we propose a new technique called weight prioritized slicing(WP-Slicing), an improved static slicing technique based on constraint logic programming, to help the programmer locate the fault quickly and precisely. WP-Slicing first converts the original program into logic facts. Then it extracts dependences from the facts, computes the static backward slice and calculates the statements' weight. Finally, WP-Slicing provides the slice in a suggested check sequence by weighted-sorting. By comparing it's slice time and locate effort with three pre-exsiting slicing techniques on five real world C projects, we prove that WP-Slicing can locate fault within less time and effort, which means WP-Slicing is more effectively.

Correlation between expression of Ki-67 and MSCT signs in different types of lung adenocarcinoma.

To investigate the correlation between the proliferating cell nuclear antigen Ki-67 and the multislice computed tomography (MSCT) signs in different subtypes of lung adenocarcinoma.Ninety-five patients with lung adenocarcinoma confirmed by surgical pathology and treated between January 2017 and December 2017 were included. MSCT was performed before the operation, and the characteristics of the high-resolution CT (HRCT) signs of the lesions were compared with the Ki-67 immunohistochemistry results.The levels of Ki-67 in the 95 lung adenocarcinoma specimens were positively correlated with the malignancy of lung adenocarcinoma. Spearman correlation coefficient was 0.615. The expression of Ki-67 was positively correlated with the nodules' diameter, density, and lobulated sign, with Spearman correlation coefficients of 0.58, 0.554, and 0.436. There was no significant correlation with spiculation and pleural retraction, with correlation coefficients of 0.319/0.381.These findings suggest that the MSCT signs of different types of lung adenocarcinoma might be associated with the expression of Ki-67. Without replacing biopsy, the imaging features of pulmonary nodules could be comprehensively analyzed to evaluate the proliferation potential of preoperative nodules, but additional studies are needed for confirmation.

HIV-1 low-level viremia affects T cell activation rather than T cell development in school-age children, adolescents, and young adults during antiretroviral therapy.

OBJECTIVES: Given the improvements in antiretroviral therapy (ART) in recent years, more pediatric HIV patients receiving ART are reaching adolescence and adulthood. This study investigated the influence of poor virological response (low-level viremia (LLV) and virological failure (VF)) on the immune system of these patients. METHODS: HIV-infected, ART-experienced pediatric patients (n=206) were enrolled in this cross-sectional study. The patients were subdivided into school-age children/early adolescents, middle adolescents, and late adolescents/young adults according to their age, and further classified into virological suppression (VS), LLV, and VF groups according to plasma viral load (pVL) measurement. Thymic output, T cells subsets, and immune activation were analyzed by flow cytometry. RESULTS: Compared with VS patients, VF patients displayed decreased CD4+ T cell counts, while LLV and VS patients had comparable CD4+ T cell counts regardless of age. Compared with VS patients, LLV and VF patients had higher percentages of CD8+HLA-DR+ and CD8+CD38high T cells, and the immune activation was positively correlated with pVL in VF and LLV patients. Thymic output levels (CD31+) and regulatory T cell subpopulations in LLV and VF patients were comparable to those in VS patients. LLV patients showed comparable percentages of T cell subsets (TN, TCM, TEMRA, and TEM) as VS patients in all age groups. CONCLUSIONS: LLV causes excessive immune activation although it does not impair T cell recovery or naïve-to-memory T cell conversion in pediatric patients living with HIV. Therefore, T cell immune activation should be monitored at the management of LLV during ART.

Multiplex Cell-Free DNA Reference Materials for Quality Control of Next-Generation Sequencing-Based In Vitro Diagnostic Tests of Colorectal Cancer Tolerance.

Background: Liquid biopsies based on next-generation sequencing (NGS) assays are confronted with more opportunities and challenges. Widespread clinical implementation of NGS-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials (RMs) which could provide full control of the process from nucleic acid extraction to test report generation. Quality control based on cell-free DNA (cfDNA) RMs is especially important for liquid biopsies. Methods: Here, we used genomic DNA from thirteen cell lines to establish four negative cfDNA RMs (N1-N4) and four multiplex cfDNA RMs (L1-L4) at serial allelic frequencies ranging from approximately 2% to 0.1%. All the cfDNA RMs were quantified and validated via both droplet digital polymerase chain reaction (ddPCR) and NGS. These RMs were distributed to eight domestic manufacturers to collaboratively evaluate the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton, and BGISEQ). Results: Each multiplex RM has eleven colorectal cancer-related mutations, including six KRAS mutations (G12S, G12C, G12D, G12A, G12V, and G13D), three NRAS mutations (G12D, Q61R, and Q61K), one PIK3CA mutation (H1047R), and one BRAF mutation (V600E). Each mutation in the cfDNA RMs was quantified and validated via both ddPCR and NGS, showing the good relevance of mutant allelic frequency. These RMs were distributed to eight domestic manufacturers for collaborative evaluation. All eight manufacturers provided similar results by domestic NGS-based cancer IVDs, except for manufacturer #5. The coefficient of variation (CV) was increased with decreasing mutant allelic frequency, and poor repetition occurred when the allelic frequency was lower than 0.5%. Conclusions: These results indicated that these cfDNA RMs would be pivotal for NGS-based cancer IVDs, especially for liquid biopsies of colorectal cancer-related mutations and would guide the further development of RMs covering more onco-related mutations.

Quality Control of Next-generation Sequencing-based In vitro Diagnostic Test for Onco-relevant Mutations Using Multiplex Reference Materials in Plasma.

Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor (EGFR) gene at serial allelic frequencies. All reference materials were quantified and validated via droplet digital polymerase chain reaction (ddPCR), and then were distributed to eight domestic manufacturers for the collaborative evaluation of the performance of several domestic NGS-based cancer IVDs covering four major NGS platforms (NextSeq, HiSeq, Ion Proton and BGISEQ). Results: All expected mutations except one at extremely low allelic frequencies were detected, despite some differences in coefficient of variation (CV) which increased with the decrease of allelic frequency (CVs ranging from 18% to 106%). It was worth noting that the CV value seemed to correlate with a particular mutation as well. The repeatability of determination of different mutations was L858R>T790M>19del. Conclusions: The results indicated our reference materials would be pivotal for quality control of NGS-based cancer IVDs and would guide the further development of reference materials covering more onco-relevant mutations.

Evaluation of Neoseiulus barkeri (Acari: Phytoseiidae) for Control of Eotetranychus kankitus (Acari: Tetranychidae).

Eotetranychus kankitus Ehara (Acari: Tetranychidae) is an important pest in Chinese citrus orchards. In the present study, we aimed to evaluate the potential of Neoseiulus barkeri (Hughes) (Acari: Phytoseiidae) for the biological control of E. kankitus. A two-sex life table of E. kankitus and N. barkeri was constructed to estimate development and fecundity. The functional response and stage-specific predation rate were analyzed to evaluate predation capacity. In addition, a timing model was used to project populations of E. kankitus with release of N. barkeri. Results showed that N. barkeri was able to develop and reproduce when fed on E. kankitus. The functional responses of N. barkeri on different stages of E. kankitus all fit the Holling II disc equation. When mixed stages of E. kankitus coexisted, N. barkeri mainly consumed larvae and nymphs. Based on the life tables and stage-specific predation rates, population projection revealed the stage structure and growth rate of N. barkeri on E. kankitus. Although E. kankitus had the higher growth rate, it was maintained at a low population level for several weeks after release of N. barkeri. The results highlighted the potential for utilizing N. barkeri as a biological control agent of E. kankitus.