Profiling the impact of the promoters on CRISPR-Cas12a system in human cells.

The plasmid vector platform is the most commonly used vector for the expression of the versatile CRISPR-Cas technique and the promoter is a crucial element for the expression vector, thus profiling the impact of the promoters on CRISPR editors provides the basic information for the gene-editing toolkits and can be a guideline for its design. Herein, we made a parallel comparison among four commonly used promoters (CAG, ~ 1700 bp; EF1a core, ~ 210 bp; CMV, ~ 500 bp; and PGK, ~ 500 bp) in CRISPR-Cas12a system in mammalian cells to explore the impact of promoters on this powerful tool. We found that without badly damaging targeting specificity, the CAG promoter-driving Cas12a editor exhibited the most active (efficiency takes as 100%, specificity index = ~ 75%) in genomic cleavage, multiplex editing, transcriptional activation, and base editing, followed by promoter CMV (efficiency = 70 ~ 90% (vs CAG), specificity index = ~ 78%), and then EF1a core and PGK (both efficiency = 40-60%, vs CAG) but with higher specificity (specificity index = ~ 84% and ~ 82%, respectively). Therefore, CAG is recommended in the CRISPR-Cas12a system for the applications that need a robust editing activity but without size limitation, CMV mostly can be an alternative for CAG when requiring a smaller space, EF1a is similar to PGK with relatively high specificity, but has a smaller size, thus is more suitable for in vivo therapeutic applications. The data outlined the properties of the widely used promoters in the CRISPR-Cas12a system, which can be a guide for its applications and can be a useful resource for the gene-editing field.

Evaluation of characteristic metabolites of aromatic amino acids in patients with HIV infection at different stages of disease.

BACKGROUND: Acquired immune deficiency syndrome (AIDS), human immunodeficiency virus (HIV) infection, and antiretroviral therapy are usually associated with metabolic disorders. Screening for biomarkers to evaluate the progression of metabolic disorders is important for the diagnosis and treatment of HIV infection. This study aimed to establish and validate a method to quantify serum aromatic amino acid (AAA) metabolites as biomarkers of metabolic disorders in patients with HIV. METHODS: The AAAs and metabolites were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Pearson's correlation, heatmap, and receiver operating characteristic curve analyses were used for statistical analysis. RESULTS: Under optimal detection conditions, the lower limits of phenylalanine (Phe), tryptophan (Trp), kynurenine (Kyn), tyrosine, phenylacetylglutamine (PAGln), and 5-hydroxytryptamine quantification reached 0.02, 0.02, 0.01, 0.02, 0.01, and 0.002 µg/ml, respectively, and the precision of intra- and inter-day was stay below 10.30%. Serum samples were stable for at least 6 months when stored at -80°C. The inter-group differences and associations between the biomarkers exhibited a particular volatility trend in PAGln, Trp, and Kyn metabolism in HIV-infected patients with metabolic syndrome. CONCLUSIONS: The developed method can be used for rapid and sensitive quantification of the AAA metabolism profile in vivo to further appraise the process of HIV infection, evaluate intervening measures, conduct mechanistic investigations, and further study the utility of PAGln, a characteristic metabolite of AAA, as a biomarker of HIV infection coupled with metabolic syndrome.

Combination of engineering the substrate and Ca2+ binding domains of heparinase I to improve the catalytic activity.

Heparinase I (EC 4.2.2.7), is an enzyme that cleaves heparin, showing great potential for eco-friendly production of low molecular weight heparin (LMWH). However, owing to its poor catalytic activity and thermal stability, the industrial application of heparinase I has been severely hindered. To improve the catalytic activity, we proposed to engineer both the substrate and Ca2+ binding domains of heparinase I. Several heparinases I from different organisms were selected for multiple sequence alignment and molecular docking to screen the key residues in the binding domain. Nine single-point mutations were selected to enhance the catalytic activity of heparinase I. Among them, T250D was the most highly active one, whereas mutations around Ca2+ binding domain yielded two active mutants. Mutant D152S/R244K/T250D with significantly increased catalytic activity was obtained by combined mutation. The catalytic efficiency of the mutant was 118,875.8 min-1·µM-1, which was improved 5.26 times. Molecular modeling revealed that the improved activity and stability of the mutants were probably attributed to the formation of new hydrogen bonds. The highly active mutant had great potential applications in industry and the strategy could be used to improve the performance of other enzymes.

HighlightsImproved catalytic activity of heparinase I by engineering the binding domains of substrate and Ca²⁺.The mutant D152S/R244K/T250D showed the highest catalytic performance.The increased hydrogen bonds attribute to the increased activity.

Towards High Accuracy Pedestrian Detection on Edge GPUs.

Despite the rapid development of pedestrian detection algorithms, the balance between detection accuracy and efficiency is still far from being achieved due to edge GPUs (low computing power) limiting the parameters of the model. To address this issue, we propose the YOLOv4-TP-Tiny based on the YOLOv4 model, which mainly includes two modules, two-dimensional attention (TA) and pedestrian-based feature extraction (PFM). First, we integrate the TA mechanism into the backbone network, which increases the attention of the network to the visible area of pedestrians and improves the accuracy of pedestrian detection. Then, the PFM is used to replace the original spatial pyramid pooling (SPP) structure in the YOLOv4 to obtain the YOLOv4-TP algorithm, which can adapt to different sizes of people to obtain higher detection accuracy. To maintain detection speed, we replaced the normal convolution with a ghost network with a TA mechanism, resulting in more feature maps with fewer parameters. We constructed a one-way multi-scale feature fusion structure to replace the down-sampling process, thereby reducing network parameters to obtain the YOLOv4-TP-Tiny model. The experimental results show that the YOLOv4-TP-tiny has 58.3% AP and 31 FPS in the winder person pedestrian dataset. With the same hardware conditions and dataset, the AP of the YOLOv4-tiny is 55.9%, and the FPS is 29.

Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Single-cell sequencing reveals the immune landscape of breast cancer patients with brain metastasis.

BACKGROUND: Breast cancer has the highest incidence rate of cancer worldwide, and brain metastases (BrM) are among the most malignant cases. While some patients have benefited from immune checkpoint inhibitors (ICIs), the complex anatomical structure of the brain and the heterogeneity of metastatic tumors have made it difficult to characterize the tumor immune microenvironment (TME) of metastatic tumors. METHODS: To address this, we used single-cell RNA sequencing (scRNA-seq) to analyze immune cells in the cerebrospinal fluid (CSF) of BrM patients with breast cancer, thereby providing a comprehensive view of the immune microenvironment landscape of BrM. RESULTS: Based on canonical marker genes, we identified nine cell types, and further identified their subtypes through differential expression gene (DEG) analysis. We compared the changes in cells and functions in the immune microenvironment of patients with different prognoses. Our analysis revealed a series of genes that promote tumor immune function (CCR5, LYZ, IGKC, MS4A1, etc.) and inhibit tumor immune function (SCGB2A2, CD24, etc.). CONCLUSIONS: The scRNA-seq in CSF provides a noninvasive method to describe the TME of breast cancer patients and guide immunotherapy.

Cloning, Expression, and Characterization of a Highly Stable Heparinase I from Bacteroides xylanisolvens.

Heparinase I (Hep I), which specifically degrades heparin to oligosaccharide or unsaturated disaccharide, has an important role in the production of low molecular weight heparin (LMWH). However, low productivity and stability of heparinase I hinders its applications. Here, a novel heparinase I (BxHep-I) was cloned from Bacteroides xylanisolvens and overexpressed in soluble form in Escherichia coli. The expression conditions of BxHep-I were optimized for an activity of 7144 U/L. BxHep-I had a specific activity of 57.6 U/mg at the optimal temperature and pH of 30 °C and pH 7.5, with the Km and Vmax of 0.79 mg/mL and 124.58 U/mg, respectively. BxHep-I catalytic activity could be enhanced by Ca2+ and Mg2+, while strongly inhibited by Zn2+ and Co2+. Purified BxHep-I displayed an outstanding thermostability with half-lives of 597 and 158 min at 30 and 37 °C, respectively, which are the highest half-lives ever reported for heparinases I. After storage at 4 °C for one week, BxHep-I retained 73% of its initial activity. Molecular docking revealed that the amino acids Asn25, Gln27, Arg88, Lys116, His156, Arg161, Gln228, Tyr356, Lys358, and Tyr362 form 13 hydrogen bonds with the substrate heparin disaccharides in the substrate binding domain and are mainly involved in the substrate binding of BxHep-I. These results suggest that the BxHep-I with high stability could be a candidate catalyst for the industrial production of LMWH.

Bioinformatics Analysis of WRKY Family Genes in Flax (Linum usitatissimum).

WRKY gene family is one of the largest transcription factor families involved in various physiological processes of plants. Flax (Linum usitatissimum) is an important stem fiber crop, and it is also an economically important crop in natural fiber and textile industries around the world. In this study, 105 WRKY genes were obtained by screening the whole genome of flax. There were 26 in group I, 68 in group II, 8 in group III and 3 in group UN. The characteristics of the WRKY motif and gene structure in each group are similar. The promoter sequence of WRKY genes includes photoresponsive elements, core regulatory elements and 12 cis-acting elements under abiotic stress. Similar to A. thaliana and Compositae plants, WRKY genes are evenly distributed on each chromosome, with segmental and tandem repeated events, which play a major role in the evolution of WRKY genes. The flax WRKY gene family is mainly concentrated in group I and group II. This study is mainly based on genome-wide information to classify and analyze the flax WRKY gene family, laying a foundation for further understanding the role of WRKY transcription factors in species evolution and functional analysis.

Bibliometrics research on radiomics of lung cancer.

BACKGROUND: Lung cancer is currently the most commonly diagnosed malignant tumor worldwide. Exploring ways to improve the accuracy and timeliness of diagnosis has important clinical significance. Radiomics transforms images into high-dimensional data, and uses deep learning and artificial intelligence to improve the accuracy and efficiency of disease diagnosis. There is an increasing amount of research on radiomics in the diagnosis of lung cancer. This study analyzes the relevant literature in the Science Citation Index Expanded (SCI-E) database to understand the current research status and future development direction of lung cancer radiomics. METHODS: This study is based on the SCI-E database. The first search formula is topic = Lung cancer OR Lung neoplasms (#1), the second search formula is topic = Radiomics (#2), and the third search formula is #1 and #2, that is, literature that meets both the first and second search results. CiteSpace software was used to analyze lung cancer radiomics from the annual distribution of articles, countries, institutions, journals, and authors and keywords. HistCite software was used to visualize the citation chronology of the lung cancer radiomics literature, and Pajek software was used to analyze the main path of the citation chronology. RESULTS: There were a total of 749 publications, of which most were original articles (529, 70.63%) and reviews (109, 14.55%). The citation frequency is 21,676 times, the h-index is 66, and the average number of citations per publication is 28.94. The research mainly comes from the United States of America, China and other countries. The research institutions are mainly medical centers such as Moffitt Cancer Center, Maastricht University and Harvard Medical School. The authors are also mainly from these institutions. The literature was published in many related journals, mainly imaging and oncology journals. Keyword analysis shows that in recent years, research has focused on deep learning and artificial intelligence. CONCLUSIONS: The field of lung cancer radiomics is developing rapidly, and the main focuses of research are deep learning and artificial intelligence.

Circ_0046599 Promotes the Development of Hepatocellular Carcinoma by Regulating the miR-1258/RPN2 Network.

BACKGROUND: Many studies have confirmed that circular RNAs (circRNAs) play a key role in the biological progression of cancers. However, the function of a novel circRNA, circ_0046599, in hepatocellular carcinoma (HCC) progression has not been explored. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of circ_0046599, microRNA (miR)-1258 and Ribophorin II (RPN2). Subcellular fractionation location assay was used to localize circ_0046599 in HCC cells. The circular characteristic of circ_0046599 was verified using Ribonuclease R (RNase R) digestion assay. Besides, cell counting kit 8 (CCK8) assay, colony formation assay, wound healing assay and transwell assay were used to detect cell proliferation, migration and invasion, respectively. The lactate production and glucose level were determined by Lactate and Glucose Assay Kits. Furthermore, the protein levels of glycolysis, metastasis and proliferation-related marker proteins, as well as RPN2 were tested by Western blot (WB) analysis. Moreover, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to confirm the interactions among circ_0046599, miR-1258 and RPN2. In addition, mice xenograft models were applied to observe the effect of circ_0046599 silencing on HCC tumor growth in vivo. RESULTS: Circ_0046599 was highly expressed in HCC tissues and cells, and its knockdown could suppress HCC cell proliferation, migration, invasion and glycolysis process. MiR-1258 could be targeted by circ_0046599, and its inhibitor could invert the suppressing effect of circ_0046599 knockdown on HCC progression. Further, RPN2 was a target of miR-1258. Overexpressed RPN2 could reverse the inhibiting effect of miR-1258 overexpression on HCC progression. Also, knockdown of circ_0046599 could restrain HCC tumor growth in vivo. CONCLUSION: Our results provided new evidence that circ_0046599 could promote the progression of HCC by increasing RPN2 expression via sponging miR-1258.

Paraoxonase 3 gene polymorphisms are associated with occupational noise-induced deafness: A matched case-control study from China.

Chronic exposure to noise is a detrimental environmental factor that can contribute to occupational noise-induced deafness (ONID) in industrial workers. ONID is caused by both environmental and genetic factors, and negatively impacts workers and manufacturing industries in China. Polymorphisms in the paraoxonase 2 gene (PON2) is associated with noise-induced hearing loss, and PON3 expression may modulate oxidative stress in cells and tissues by reducing the levels of reactive oxygen species, which are prominent in ONID. We conducted a matched case-control study to investigate whether PON3 polymorphisms and activity were associated with susceptibility to ONID. We genotyped PON3 single nucleotide polymorphisms (SNPs) using Sanger sequencing and measured the plasma PON3 activity using enzyme-linked immunosorbent assay. Conditional logistic regression models were fitted to evaluate the potential risk factors of ONID. A total of 300 subjects were included (n = 150 ONID and n = 150 control cases) from October 2017 to October 2019. We identified two types of genotypes for the PON3 SNPs. The independent risk factors for ONID were genotype CT and allele C with Odd's ratio (OR) = 2.12 (95% confidence interval [CI]: 1.18-3.84) and OR = 1.68 (95% CI: 1.06-2.66) for SNP rs11767787; AG and allele A with OR = 2.09 (95% CI: 1.25-3.47) and OR = 1.87 (95% CI: 1.19-2.93) for SNP rs13226149; and CT and allele T with OR = 2.59 (95% CI: 1.44-4.67) and OR = 1.95 (95% CI: 1.22-3.14) for SNP rs17882539, respectively. Furthermore, the plasma PON3 level (> 1504 U/L) was observed to be a protective factor associated with the lowest level of ONID (less than 991 U/L) after adjusting for confounding factors (OR = 0.27, 95% CI: 0.13-0.54). In conclusion, the PON3 polymorphisms rs11767787, rs13226149, and rs17882539 and plasma PON3 activity are associated with susceptibility to ONID in the Chinese population.

MicroRNA-550a is associated with muscle system conferring poorer survival for esophageal cancer.

Background Esophageal cancer (ESCA) is one of the most common cancers in the digestive tract. Approximately 300000 people on an average die of ESCA per year worldwide. The determination of key microRNAs for the prognosis of ESCA is of indispensable significance in the clinical treatment. Methods The differentially expressed microRNAs were screened by analyzing The Cancer Genome Atlas (TCGA) database. By using the survival data of the database, we analyzed correlation between patients' survival time and miR-550a expression levels. Differential expression analysis and gene set enrichment analysis were performed using the targeted data. Results It was found that patients with high miR-550a expression levels had shorter survival time. Data mining and signal pathway enrichment analysis of TCGA database showed that abnormal miR-550a expressions affected the recurrence of tumors by the muscle system regulation. Conclusions Through the proposed investigation, miR-550a is found to be a potential biomarker as well as non-coding therapeutic target for esophagus cancer. These results suggest that miR-550a may serve as a therapeutic target and predictor for ESCA survival.

Long non-coding RNA LOC285194 functions as a tumor suppressor by targeting p53 in non-small cell lung cancer.

Recently, LOC285194 has shown a potential tumor-suppressor function in several types of human cancers, but its function in non-small cell lung cancer (NSCLC) remains unknown. This study intended to investigate the biological role of LOC285194 and its clinical significance in NSCLC. LOC285194 was detected by qRT-PCR, and its correlation with clinicopathological features of NSCLC was analyzed. The expression of LOC285194 was knocked down or ectopically expressed in lung cancer cells (A549 and H1299) and tumor cell growth, migration and invasion in vitro were investigated. In addition, the interaction of LOC285194 and target proteins was assessed by RNA pulldown and RNA immunoprecipitation in vitro. The results revealed that the expression of LOC285194 was significantly lower in tumor tissues when compared with the corresponding non-tumor tissues (P<0.001). Its expression was correlated with the tumor size (P=0.027). Kaplan-Meier analysis revealed that patients with lower LOC285194 expression had worse disease-free survival and overall survival rates (P<0.05). RNA protein interaction analysis revealed that p53 was the direct binding target of LOC285194 in NSCLC. Bioinformatics analyses suggested that depletion of LOC285149 could affect its antitumor function through the KRAS/BRAF/SMEK pathway. Our findings indicated that LOC285194 was a novel non-coding prognostic indicator and contributed to tumor suppression by targeting p53 in NSCLC, suggesting that it may be a non-coding target for NSCLC gene therapy.

miR-193b availability is antagonized by LncRNA-SNHG7 for FAIM2-induced tumour progression in non-small cell lung cancer.

OBJECTIVES: Long non-coding RNAs have identified to involve into the tumour cell proliferation, apoptosis and metastasis. We previously found that up-regulated LncRNA-SNHG7 (SNHG7) positively correlated to the Fas apoptosis inhibitory molecule 2 (FAIM2) in lung cancer cells with unclear mechanism. METHODS: Non-small cell lung cancer (NSCLC) and relative normal tissues (n = 25) were collected. The SNHG7 expression and function in NSCLC was determined. The SNHG7-miR 193b-FAIM2 network was analysed in vitro and vivo. RESULTS: We reported that oncogene SNHG7 predicted a poor clinical outcome and functioned as competitive endogenous RNA (ceRNA) antagonized microRNA-193b (miR-193b) to up-regulate the FAIM2 level in NSCLC. Bioinformatic analysis predicted that SNHG7 harboured miR-193b-binding sites, and we found decreased miR-193b levels in NSCLC tissues when compared to relative normal tissues. Luciferase assays indicated that overexpression of miR-193b inhibited the Ruc expression of plasmid with miR-193b-binding sites of SNHG7 in a dose-dependent manner. Ectopically expressed SNHG7 also as a molecular sponge sequestered endogenous miR-193b. Besides, FAIM2 was found to be directly targeted by miR-193b. The restoration of miR-193b levels in NSCLC cell lines A549 and H125 suppressed the expression of FAIM2 and related tumour proliferation, metastasis and induced apoptosis. However, forced expression of SNHG7 could down-regulate miR-193b to elevate the FAIM2 level of tumour cells, leading to impaired miR-193b/FAIM2-induced tumour progression. Knockdown of SNHG7 in vivo significantly delayed the tumour growth with decreased tumour volume, which accompanied with enhanced miR-193b expression and reduced FAIM2 levels. CONCLUSION: The results indicated that miR-193b is indispensible for the ceRNA role of SNHG7 in FAIM2-supported tumourigenesis of lung cancer.

lncRNA-SNHG7 promotes the proliferation, migration and invasion and inhibits apoptosis of lung cancer cells by enhancing the FAIM2 expression.

There is growing evidence that long non-coding RNAs (lncRNAs) are related to cancer development. In the present study, we found that the expression levels of lncRNA-SNHG7 mRNA and protein obviously increased in lung cancer tissues compared to adjacent non-cancerous tissues. Simultaneously, the expression levels of Fas apoptotic inhibitory molecule 2 (FAIM2) also increased in lung cancer tissues. In addition, lncRNA-SNHG7 was of positive relevance with FAIM2 in human lung cancer tissues. Silence of lncRNA­SNHG7 by siRNA repressed the level of FAIM2 protein and suppressed cell proliferation, migration and invasion and accelerated apoptosis of A594 cells in vitro. Furthermore, silence of FAIM2 by siRNA generated a phenotype similar to silence of lncRNA-SNHG7 by siRNA. Therefore, our research showed that lncRNA-SNHG7 promotes the proliferation, migration and invasion, and inhibits apoptosis of lung cancer cells by enhancing the FAIM2 expression, suggesting that lncRNA-SNHG7 as a key regulator of gene expression, may be a promising therapeutic strategy for the treatment of lung cancer. It may improve the understanding of their biogenesis and function of lung cancer and further provide the theoretical fundamental basis for cancer pathogenesis and treatment.

Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions.

Maize, the highest-yielding cereal crop worldwide, is particularly susceptible to drought during its 2- to 3-week flowering period. Many genetic engineering strategies for drought tolerance impinge on plant development, reduce maximum yield potential or do not translate from laboratory conditions to the field. We overexpressed a gene encoding a rice trehalose-6-phosphate phosphatase (TPP) in developing maize ears using a floral promoter. This reduced the concentration of trehalose-6-phosphate (T6P), a sugar signal that regulates growth and development, and increased the concentration of sucrose in ear spikelets. Overexpression of TPP increased both kernel set and harvest index. Field data at several sites and over multiple seasons showed that the engineered trait improved yields from 9% to 49% under non-drought or mild drought conditions, and from 31% to 123% under more severe drought conditions, relative to yields from nontransgenic controls.

[Retrospective study on HIV infection among blood donors in Zhejiang Province over 11-year period (1993 - 2004)].

To determine HIV prevalence among blood donors in Zhejiang Province from 1993 to 2004 years, almost 6,600, 000 blood donors information were collected and analyzed. Every sample was screened for markers of HIV-1 and HIV-2 by using commercially available ELISA kits twice, and Western blot for confirmation if positive or suspicious result appeared. The results indicated that during the study period, prevalence rate of HIV infection was 0.85/1000 donors (0.085%), with the rising tendency from 1:600000 in 1995 to 1:37500 in 2004. The prevalence of HIV infection in Zhejiang donors was significantly lower than that in donors of other provinces, prevalence of HIV infection in male was higher than that in female. In recent years, the prevalence of HIV in blood donors was obviously increased, but the difference among various populations began to reduce. It is concluded that in a low HIV prevalence area like Zhejiang province where no obvious AIDS occurred, risk for the expansion of the HIV epidemic was on the increase. Screening procedure used in transfusion services nowaday is reliable, but a comprehensive approach is required to make the blood more safe.