A versatile Cas12k-based genetic engineering toolkit (C12KGET) for metabolic engineering in genetic manipulation-deprived strains.

The genetic modification of microorganisms is conducive to the selection of high-yield producers of high-value-added chemicals, but a lack of genetic tools hinders the industrialization of most wild species. Therefore, it is crucial to develop host-independent gene editing tools that can be used for genetic manipulation-deprived strains. The Tn7-like transposon from Scytonema hofmanni has been shown to mediate homologous recombination-independent genomic integration after heterologous expression in Escherichia coli, but the integration efficiency of heterologous sequences larger than 5 kb remains suboptimal. Here, we constructed a versatile Cas12k-based genetic engineering toolkit (C12KGET) that can achieve genomic integration of fragments up to 10 kb in size with up to 100% efficiency in challenging strains. Using C12KGET, we achieved the first example of highly efficient genome editing in Sinorhizobium meliloti, which successfully solved the problem that industrial strains are difficult to genetically modify, and increased vitamin B12 production by 25%. In addition, Cas12k can be directly used for transcriptional regulation of genes with up to 92% efficiency due to its naturally inactivated nuclease domain. The C12KGET established in this study is a versatile and efficient marker-free tool for gene integration as well as transcriptional regulation that can be used for challenging strains with underdeveloped genetic toolkits.

A biosensor for S100B detection based on PSS-MA-GoldMag-LFIA in early clinical diagnosis of brain damage.

S100B is an essential biomarker in the early diagnosis and treatment monitoring of brain injury. However, the traditional clinical diagnostic assay for S100B detection requires a complex operation or large equipment, which limits its application for rapid point-of-care tests (POCT). This study aimed to establish a lateral-flow immunoassay (LFIA) strip test system for S100B determination. PSS-MA-GoldMag nanoparticles were conjugated with anti-S100B antibodies as probes. Using this antibody-nanoparticle composite, an LFIA system based on magnetic quantification was established for S100B detection. For the evaluation of the performance of this LFIA system in clinical practice, 216 clinical samples were assayed using the LFIA test system and a commercial ECLI kit. Using the LFIA system, reliable results could be obtained in 30 min with a detection limit of 0.05 ng mL-1. The coefficient of variation (CV) was <13.8% and <14.03% for intra- and inter-assay precision, respectively. The recoveries were between 95.1 and 107.3%. The relative deviation of the interference experiments was <10%. In the analysis of clinical samples, the result indicated that the sera level of S100B in the detection group did not correlate with gender (p = 0.564 > 0.05) or age (p = 0.083 > 0.05). There is a good correlation between the novel method and the Elecsys®, with a determination coefficient of R2 0.9566, p > 0.05. The Bland-Altman analysis between the two ways shows that the 95% confidence bands between the two methods in measuring S100B were -0.27 ng mL-1 to +0.29 ng mL-1 with a mean difference of +0.006 ng mL-1. These results indicated that the novel LFIA system could be a simple, rapid, convenient, and accurate method for S100B determination.

Fabrication of Supramolecular System Derived from Poly ß-cyclodextrin Coupling Quinoline Dderivative and Its Fluorescence Sensing of Zinc Ion in Pure Water Environment.

Cyclodextrin (CD) is an important guest material owing to the water solubility and biocompatibility. In the paper, an organic small molecule was synthesized. According to supramolecular self-assembly, the organic molecule was bounded to the cavity of Poly ß-cyclodextrin, which was characterized by IR, SEM and TEM et al. After self-assembly interaction, the morphology has changed obviously comparing with precursors. Simultaneously, the supramolecular self-assembly complex exhibited good water solubility. Moreover, By Gaussian calculation, the high binding activity between organic molecule and cyclodextrin was confirmed. By fluorescence investigation, the supramolecular system showed high fluorescence sensing activity for Zn2+ in pure water environment, which could track the dynamic change of Zn2+ in organisms. In addition, the supramolecular system exhibited low cytotoxicity. The work provided an interesting pathway for constructing water-soluble and low cytotoxic fluorescence sensor for Zn2+.

A Case Report of a Novel Isovaleryl-CoA Dehydrogenase Gene Mutation in a Chinese Family with Isovaleric Acidemia.

BACKGROUND: Isovaleric acidemia (IVA) is a rare autosomal-recessive metabolic disorder caused by a genetic deficiency of isovaleryl-CoA dehydrogenase (IVD). Deficiency of IVD leads to the accumulation of organic acids; however, the genotype-phenotype relationship has not been well established. METHODS: Two brothers with acute neonatal IVA in a Chinese family were reported, and their clinical manifestations and examination were described. MS/MS and GCMS were used to perform organic acid analysis of blood samples and urine samples, and the patient's blood was sequenced by NGS and Sanger sequencing of the ivd gene. RESULTS: Sequence analysis of the ivd gene identified compound heterozygous mutations in the patient, the c.250T>C (p.W84R) missense mutation (novel) and the c.466-3_466-2 delCAinsGG splicing mutation, which were inherited from their parents. Various bioinformatics prediction algorithms suggest that the p.W84R missense mutation may destabilize the IVD monomer and reduce its ability to bind to substrates. CONCLUSIONS: Both the clinical and genetic features of this family will help us to further expand the knowledge of IVA.

Visual Detection of ACE I/D Polymorphism Using T-ARMS-PCR Combined with a Lateral Flow Assay and Its Clinical Application.

Insertions/deletions (indels) variations have been recognized as a promising marker for the development of various diseases. However, methods used for the genotyping of indels in studies were tedious, complicated, and required sophisticated or expensive instruments, as well as complex data analysis, which makes it difficult to meet the demand of point of care testing. Herein, we presented a fast and accurate biosensor (T-ARMS-PCR-LFA) by the combination of tetra-primer amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) and GoldMag lateral flow assay (LFA) for visual genotyping of ACE I/D polymorphism. ACE I/D can be distinguished by employing four primers in one PCR reaction, and genotyping results were presented by the visual inspection of colors on the nitrocellulose membrane of LFA strips within 5 min. And 50 of the human genomic DNA samples were used for the detection of ACE I/D to further validate the accuracy of the T-ARMS-PCR-LFA system. As a demonstration, we showed that ACE I/D could be genotyped using a low amount of DNA sample (25 ng) with an accuracy of 100%, without complicated operation steps and data analysis, which is better than that of the conventional method (agarose gel electrophoresis analysis after common PCR). In conclusion, the biosensor is highly applicable for genotyping specific large indel variants in clinical practices, which enables rapid clinical decision-making, improves the management of disease diagnosis, and facilitates personalized medicine.

Construction of an in vivo NIR fluorescent probe for revealing the correlation between inflammation and mitochondrial hydrogen sulfide and viscosity.

As an indispensable part of immune response, inflammation plays a critical role in the occurring and advancing of many diseases. It is reported that the emergence of inflammation can be indicated by the change of intracellular viscosity and overexpression of the hydrogen sulfide (H2S) in mitochondria. So far, elucidating the relationship between inflammation and the above parameters remains a challenge due to the lack of validated analytical tools. Herein, a novel dual-function NIR fluorescent probe CMQT with excellent biological compatibility and mitochondrial targeting ability is designed and synthesized by using 7-diethylaminocoumarin and 4-ethylphenolate quinoline acetate through twistable vinyl bonds. With the functional probe, enhanced fluorescent signals at 570 nm and 721 nm are produced in the presence of H2S and changes of viscosity. The CMQT can be applied in living cells and zebrafish, which reveals the increases of mitochondrial H2S and viscosity generated by inflammatory response through dual-channel fluorescence imaging mode. The in vivo dual-functional probe serves as an efficient tool for imaging analysis of H2S and viscosity, and has profound implications for the early diagnosis of inflammatory diseases.

Natriuretic Peptides-New Targets for Neurocontrol of Blood Pressure via Baroreflex Afferent Pathway.

Natriuretic peptides (NPs) induce vasodilation, natriuresis, and diuresis, counteract the renin-angiotensin-aldosterone system and autonomic nervous system, and are key regulators of cardiovascular volume and pressure homeostasis. Baroreflex afferent pathway is an important reflex loop in the neuroregulation of blood pressure (BP), including nodose ganglion (NG) and nucleus tractus solitarius (NTS). Dysfunction of baroreflex would lead to various hypertensions. Here, we carried out functional experiments to explore the effects of NPs on baroreflex afferent function. Under physiological and hypertensive condition (high-fructose drinking-induced hypertension, HFD), BP was reduced by NPs through NG microinjection and baroreflex sensitivity (BRS) was enhanced via acute intravenous NPs injection. These anti-hypertensive effects were more obvious in female rats with the higher expression of NPs and its receptor A/B (NPRA/NPRB) and lower expression of its receptor C (NPRC). However, these effects were not as obvious as those in HFD rats compared with the same gender control group, which is likely to be explained by the abnormal expression of NPs and NPRs in the hypertensive condition. Our data provide additional evidence showing that NPs play a crucial role in neurocontrol of BP regulation via baroreflex afferent function and may be potential targets for clinical management of metabolic-related hypertension.

Metabolic Study of Tetra-PEG-Based Hydrogel after Pelvic Implantation in Rats.

In vivo metabolism of polyethylene glycol (PEG) hydrogels has rarely been studied. In this study, we prepared a chemically crosslinked hydrogel formulation using 14C-labeled tetra-armed poly (ethylene glycol) succinimidyl succinate (Tetra-PEG-SS) and 3H-labeled crosslinking agent for implantation into the pelvis of Sprague-Dawley (SD) rats. This radioactive labeling technique was used to investigate the radioactivity excretion rates in of feces and urine, the blood exposure time curve, and the radioactivity recovery rate in each tissue over time. We showed that the primary excretion route of the hydrogel was via urine (3H: about 86.4%, 14C: about 90.0%), with fewer portion through feces (3H: about 6.922%, 14C: about 8.16%). The hydrogel metabolites exhibited the highest distribution in the kidney, followed by the jejunal contents; The 3H and 14C radioactivity exposures in the remaining tissues were low. We also showed that the 3H and 14C radioactivity recovery rates in the blood were usually low (<0.10% g−1 at 12 h after implantation), even though, in theory, the hydrogel could be absorbed into the blood through the adjacent tissues. By using a combination of HPLC-MS/MS and offline radioactivity counting method, we established that the tetra-PEG-based hydrogel was mainly metabolized to lower-order PEG polymers and other low-molecular-weight substances in vivo.

A Novel PCR Method for Detecting ACE Gene Insertion/Deletion Polymorphisms and its Clinical Application.

BACKGROUND: Angiotensin-converting enzyme (ACE) plays a major role in blood pressure regulation and cardiovascular homeostasis. The wide distribution and multifunctional properties of ACE suggest it's involvement in various pathophysiological conditions. RESULTS: In this study, a novel visual detection method for ACE I/D polymorphisms was designed by integrating direct PCR without the need for DNA extraction using gold magnetic nanoparticles (GMNPs)-based lateral flow assay (LFA) biosensor. The entire detection procedure could enable the genotyping of clinical samples in about 80 min. The detection limit was 0.75 ng and results could be obtained in 5 min using the LFA device. Three hundred peripheral blood samples were analyzed using the direct PCR-LFA system and then verified by sequencing to determine accuracy and repeatability. A clinical preliminary study was then performed to analyze a total of 633 clinical samples. CONCLUSIONS: After grouping based on age, we found a significant difference between the genotypes and the age of patients in the CHD group. The introduction of this method into clinical practice may be helpful for the diagnosis of diseases caused by large fragment gene insertions/deletions.

One-step determination of deletion mutation based on loop-mediated isothermal amplification.

Deletion mutation has been proved as the important factor for occurrence and development of disease, especially those with cancer. With the popularity of precision medicine, the individual cancer therapeutic strategy has highlighted the requirement to develop a straightforward and competent strategy for deletion mutation determination. Hence, the present study is dedicated to develop a one-step assay to identify deletion mutation with sequence specificity for clinical practice. Taking advantage of loop-mediated isothermal amplification, an ultrasensitive and rapid deletion mutation determination method is established, which allow as low as 30 copies or 0.1% target variants under strong interferential background can be accurately distinguished in 30 min dispensing with professional operation and complex data interpretation. As a demonstration, the epidermal growth factor receptor p.E746-A750del, a crucial factor for the susceptibility of tyrosine kinase inhibitor in non-small-cell lung cancer treatment, has been accurately identified by this method with both cell lines and real clinical samples. By tailor-made primer set, this method can be extended for other deletion mutants, making it a molecular diagnostic tool and could be readily adapted for cancer diagnosis, therapy and prognosis in point of care diagnostic test scenario.

Lateral flow immunoassay based on gold magnetic nanoparticles for the protein quantitative detection: Prostate-specific antigen.

Point-of-care testing (POCT) demands for rapidly obtaining test results by means of portable analytical instruments and auxiliary reagents at the sampling site. It's important for tumor marker to be recognized and detected in early clinical diagnosis. Many studies focused on producing small portable devices that would allow fast, accurate, and on-site detection. This study aimed to report a magnetic quantitative lateral flow immunoassay (LFIA) system based on poly (acrylic acid) (PAA)-modified gold magnetic nanoparticles (PGMNs) for detecting prostate-specific antigen (PSA) qualitatively and quantitatively. The result was easily achievable with a portable magnetic reader within 15 min. Under optimal conditions, as low as 0.17 ng/mL PSA could be detected. The method was validated using a well-established Solin electrochemiluminescence immunoassay and showed high consistency in detecting 84 serum samples (R2 = 0.98). The quantitative LFIA based on PGMNs established in this study was proven to be rapid, accurate, sensitive, and inexpensive. As a POCT, it can be potentially developed for the quantitative diagnosis of other disease-related protein biomarkers.

A site-specific DNA methylation biosensor for both visual and magnetic determination based on lateral flow assay.

Tumorigenesis driven by abnormal DNA methylation has highlighted the need to develop a portable, rapid and sensitive strategy for accurate methylation detection with a specific cancer-prognostic gene, which caters to the popularization of precision medicine. In this study, a site-specific biosensor for both visual and magnetic DNA methylation determination has been established based on lateral flow assay. By introducing digoxin- and biotin-labeled primers into PCR, the amplicons can be recognized and captured by gold magnetic nanoparticles (GMNPs) in this biosensor. Working as a signal probe, the optical property of GMNPs allows the amplicons to be interpreted with naked eyes avoiding any complex equipment and cumbersome operation after PCR. Moreover, by virtue of the magnetic property of GMNP, the signal can be explained and recorded by a magnetometer in clinical practice. The introduction of tailor-made primer sets makes it possible to accurately distinguish 0.1% methylated variants in the presence of numerous unmethylated variants as strong interferential background and vice versa at target cytosine-guanine dinucleotide. A distinct signal can be observed with as low as 0.01 pg variants for both visual and magnetic analyses. As a significant tumor suppressor gene, the promoter methylation status of miR-34a is accurately determined with not only cell lines but also with clinical samples, which demonstrates the great potential of this biosensor for cancer diagnosis and prognosis.

Application of different types of CRISPR/Cas-based systems in bacteria.

As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.

Designing enhanced and ratiometric probes for detecting OCl- based on substituents influencing the fluorescence of HBT and their application in strips and bioimaging.

Nitro groups with a strong electron-withdrawing effect can powerfully influence the fluorescence of fluorophores. In this work, through adjusting the nitro group at the HBT fluorophore to construct a phenylhydrazone structure, two probes (HBTN and HBTH) were synthesized to detect OCl-. Consequently, HBTN with the nitro group quenched the fluorescence of HBT and HBTH without the nitro group causing a redshift of the fluorescence, which resulted in enhanced and ratiometric fluorescence signal changes during the detection process. Among them, HBTN shows good ability to selectively detect OCl- in the concentration range of 0-14 µM with the detection limit of 2.06 nM. Based on HBTN, a portable test strip for detecting OCl- was made for the convenient quantification of the OCl- concentration with a spectrophotometer, and exogenous and endogenous OCl- was successfully imaged in cells.

Impact of Vitamin D and Vitamin D Receptor on Risk of Cardiovascular Diseases in Children and Adolescents with Obesity in Sichuan, China: A Cross-Sectional Study.

BACKGROUND: Previous studies have demonstrated the close relationship between vitamin D, vitamin D receptor (VDR), and obesity. Nevertheless, few studies have reported wherther the relationship among these is associated with the risk of cardiovascular diseases (CVDs) in Chinese children and adolescents. OBJECTIVE: The present study aimed to reveal the effects of obesity, serum vitamin D levels, and VDR FokI genotype on the risk of CVDs in children and adolescents in Sichuan, China. METHODS: Children and adolescents were recruited into a cross-sectional study. Serum vitamin D levels, serum lipid levels, and VDR FokI gene polymorphisms were measured in the laboratory. The selected lipid factors were used as biomarkers of CVD risk. The impact of obesity, vitamin D levels and VDR FokI genotype on CVD risk factors were investigated. RESULTS: Higher lipid levels were observed in children and adolescents in the obese group, when compared to the nonobese group. In the obese group, the C allele carriers had significantly lower concentrations of lipids, when compared to the TT genotype. C allele carriers who were vitamin D deficient had lower levels of total cholesterol (TC), triglycerides (TG), apolipoprotein B (Apo-B), total cholesterol/high-density lipoprotein cholesterol (TC/HDL-C), low-density lipoprotein cholesterol/high-density lipoprotein cholesterol (LDL-C/HDL-C), and triglycerides/high-density lipoprotein cholesterol (TG/HDL-C), when compared to those with the TT genotype in obese children and adolescents. For vitamin D-insufficient obese children and adolescents, the TC, Apo-B, and TC/HDL-C in the C allele carriers were significantly lower, when compared to those in the TT genotype in obese children and adolescents. CONCLUSION: Obese children with low vitamin D levels, who are carriers of the C allele of the FokI gene, have lower levels of several biochemical markers of CVD risk, when compared to those who were TT homozygous. Obese children and adolescents may benefit from vitamin D supplementation, terms of lowering their CVD risk, particularly when they are carriers of the C allele of the FokI gene.

Direct genotyping from whole blood using alkaline polyethylene glycol.

In order to simplify biological sample preparation to meet the demand of rapid genotyping, we improve alkaline polyethylene glycol (APEG) based on Chomczynski's procedure for the PCR-based lateral flow genotyping system, which enables the rapid and efficient direct genotyping from whole blood without DNA purification. The improved APEG has a high tolerance for extreme storage conditions. Testing whole blood with an abnormal hematological index indicates that APEG efficiency would not be influenced by pathological factors. Compared with sequencing, the accuracy of this genotyping system was 100% on testing with 200 clinical samples.

Berberine Promotes Apoptosis of Colorectal Cancer via Regulation of the Long Non-Coding RNA (lncRNA) Cancer Susceptibility Candidate 2 (CASC2)/AU-Binding Factor 1 (AUF1)/B-Cell CLL/Lymphoma 2 (Bcl-2) Axis.

BACKGROUND Berberine, a natural isoquinoline alkaloid derived from Berberis genus plants, has been reported to have anti-cancer effects. While cell behavior can be modulated by long non-coding RNAs (lncRNAs), the contributions of lncRNAs in progression and berberine effects on colorectal cancer are largely unknown. Therefore, the present study investigated the involvement and regulatory function of lncRNA cancer susceptibility candidate 2 (CASC2) during the treatment of human colorectal cancer using berberine. MATERIAL AND METHODS Reverse transcription-quantitative PCR (RT-qPCR) was performed to detect the expression levels of lncRNA CASC2 and Bcl-2 mRNA in colorectal cancer cells. MTT assay was performed to evaluate cell viability. Flow cytometry and TUNEL assay were used to analyze the apoptosis of cancer cells. RNA immunoprecipitation (RIP) assay was done to verify the interaction between lncRNA CASC2 and (AU-binding factor 1) AUF1, or AUF1 and B-cell CLL/lymphoma 2 (Bcl-2). RESULTS Treatment with berberine suppressed cell viability of colorectal cancer by promoting apoptosis level. LncRNA CASC2 was upregulated in cells treated with berberine, and knockdown of lncRNA CASC2 reversed the berberine-induced apoptosis. In addition, anti-apoptotic gene Bcl-2 was suppressed by berberine treatment and lncRNA CASC2, inducing the pro-apoptotic effects. Moreover, lncRNA CASC2 binds to AUF1, which sequestered AUF1 from binding to Bcl-2 mRNA, thus inducing the inactivation of Bcl-2 translation. CONCLUSIONS Our study reveals that lncRNA CASC2 mediates the berberine-induced pro-apoptotic effect via inhibition of Bcl-2 expression at the post-transcriptional level.

Multiple SNPs Detection Based on Lateral Flow Assay for Phenylketonuria Diagnostic.

Single nucleotide polymorphisms (SNPs) are closely related to genetic diseases, but current SNP detection methods, such as DNA microarrays that include tedious procedures and expensive, sophisticated instruments, are unable to perform rapid SNPs detection in clinical practice, especially for those multiple SNPs related to genetic diseases. In this study, we report a sensitive, low cost, and easy-to-use point-of-care testing (POCT) system formed by combining amplification refractory mutation system (ARMS) polymerase chain reaction with gold magnetic nanoparticles (GMNPs) and lateral flow assay (LFA) noted as the ARMS-LFA system, which allow us to use a uniform condition for multiple SNPs detection simultaneously. The genotyping results can be explained by a magnetic reader automatically or through visual interpretation according to the captured GMNPs probes on the test and control lines of the LFA device. The high sensitivity (the detection limit of 0.04 pg/µL with plasmid) and specificity of this testing system were found through genotyping seven pathogenic SNPs in phenylalanine hydroxylase gene ( PAH, the etiological factor of phenylketonuria). This system can also be applied in DNA quantification with a linear range from 0.02 to 2 pg/µL of plasmid. Furthermore, this ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families including 69 individuals. The concordance rate of the genotyping results detected by the ARMS-LFA system was up to 97.8% compared with the DNA sequencing results. This method is a very promising POCT in the detection of multiple SNPs caused by genetic diseases.

Prevalence of Glucose-6-Phosphate Dehydrogenase Deficiency in Sichuan, China.

BACKGROUND: Our goals were to screen newborns and characterize the occurrence of glucose-6-phosphate dehydrogenase (G6PD) deficiency in southwestern China. Meanwhile, we would like to analyze the factors that might affect the results of neonatal dried blood spots for glucose-6-phosphate dehydrogenase screening test, to improve the clinical quality control level, effectively reduce the external factors in the process of detection. METHODS: This study involved an evaluation of G6PD data for 20,644 newborns from a universal newborn screening program. Heel prick blood specimens were collected around 72 hours after birth and were dried on filter papers. For G6PD deficiency the fluorescent spot test was employed. We studied the association between incidence of G6PD deficiency and influence factors. RESULTS: This study involved an evaluation of G6PD data for 20,644 neonatal heel prick blood samples from 10,984 males and 9,660 females. There were 503 positive results for G6PD deficiency (299 males and 204 females), and the G6PD deficiency-positive rate was estimated to be around 2.4%. The gender-specific prevalence for males was 2.7%, and for females 2.1%. Multiple factors may influence the result of the G6PD test, such as season, temperature, and specimen of indwelling time. CONCLUSIONS: This study analyzed the prevalence of G6PD deficiency in Sichuan, China. Accelerating the speed of sample delivery and ensuring availability of screening results can aid the screening and diagnosis.