A cathodic photoelectrochemical biosensor based on CRISPR/Cas12a trans-cleavage mediated p-n heterojunction quenching mode for microRNA determination.

In this study, a cathodic photoelectrochemical (PEC) bioanalysis for sensitive determination of microRNA (miRNA) has been constructed based on CRISPR/Cas12a trans-cleavage mediated [(C6)2Ir(dcbpy)]+PF6- (C6 represents coumarin-6 and dcbpy represents 4,4'-dicarboxyl-2,2'-bipyridine)-sensitized NiO photocathode and p-n heterojunction quenching mode. The [(C6)2Ir(dcbpy)]+PF6--sensitized NiO photocathode exhibits a stable and dramatically improved photocurrent signal due to highly effective photosensitization of [(C6)2Ir(dcbpy)]+ PF6-. Then Bi2S3 quantum dots (Bi2S3 QDs) is captured on the photocathode, resulting in markedly quenching of the photocurrent. When target miRNA is specifically recognized by the hairpin DNA to stimulate the trans-cleavage activity of CRISPR/Cas12a, leading to the leave of the Bi2S3 QDs. The photocurrent is gradually recovered with the increasing target concentration. Thus, the quantitative signal response to target is achieved. Benefiting from excellent performance of NiO photocathode, intense quenching effect of p-n heterojunction and accurate recognition ability of CRISPR/Cas12a, the cathodic PEC biosensor shows a wider linear range over 0.1 fM-10 nM, with a low detection limit of 36 aM. Also, the biosensor exhibits satisfying stability and selectivity.

Structure‒function‒pathogenicity analysis of C-terminal myocilin missense variants based on experiments and 3D models.

MYOC is a common pathogenic gene for primary open-angle glaucoma and encodes the protein named myocilin. Multiple MYOC variations have been found, with different clinical significance. However, the pathogenesis of glaucoma induced by MYOC mutations has not been fully clarified. Here, we analyze the molecular and cellular biological differences caused by multiple variant myocilins, including protein secretion characteristics, structural changes, subcellular localization, cellular autophagic activity and oxidative stress. Denaturing and nondenaturing electrophoresis showed myocilin to be a secreted protein with the tendency to self-oligomerize. The full-length myocilin and its C-terminal cleavage fragment are secreted. Secretion analysis of 23 variant myocilins indicated that secretion defects are closely related to the pathogenicity of MYOC variants. Structural analysis showed that the alteration of steric clash is associated with the secretion characteristics and pathogenicity of myocilin variants. Immunocytochemistry results demonstrated that mutated myocilins are retained in the endoplasmic reticulum and disrupt autophagy. MTT assay, MitoTracker staining, and DCFH-DA staining showed increased oxidative injury in cells expressing MYOC mutants. Taken together, MYOC mutations are able to induce cell dysfunction via secretion defects and intracellular accumulation resulting from steric clash alterations.

Clinical and imaging features preoperative evaluation of histological grade and microvascular infiltration of hepatocellular carcinoma.

BACKGROUND: To predict the histological grade and microvascular invasion (MVI) in patients with HCC. METHODS: A retrospective analysis was conducted on 175 patients who underwent MRI enhancement scanning (from September 2016.9 to October 2020). They were divided into MVI positive, MVI negative, Grade-high and Grade-low groups. RESULTS: The AFP of 175 HCC patients distributed in MVI positive and negative groups, Grade-low and Grade-high groups were statistically significant (P = 0.002 and 0.03, respectively). Multiple HCC lesions were more common in MVI positive and Grade-high groups. Correspondingly, more single lesions were found in MVI negative and Grade-low groups (P = 0.005 and 0.019, respectively). Capsule on MRI was more common in MVI negative and Grade-high groups, and the difference was statistically significant (P = 0.02 and 0.011, respectively). There were statistical differences in the distribution of three MRI signs: artistic rim enhancement, artistic peripheral enhancement, and tumor margin between MVI positive and MVI negative groups (P = 0.001, < 0.001, and < 0.001, respectively). Tumor hypointensity on HBP was significantly different between MVI positive and negative groups (P < 0.001). CONCLUSIONS: Our research shows that preoperative enhanced imaging can be used to predict MVI and tumor differentiation grade of HCC. The prognosis of MVI-negative group was better than that of MVI-positive group.

Streptomyces akebiae sp. nov., a novel actinomycete isolated from rhizosphere soil of Akebia trifoliate.

A novel actinomycete strain, designated as MG28T, was isolated from rhizosphere soil of Akebia trifoliate. The taxonomic position of the strain was investigated by using a polyphasic approach. BLAST search of the full-length 16S rRNA gene sequence of strain MG28T indicated it represented a member of the genus Streptomyces, and displayed 99.03%, 98.90%, 98.90%, 98.89%, 98.83% and less than 98.70% sequence similarities with S. phaeolivaceus GY16T, S. deccanensis KCTC 19241T, S. europaeiscabiei KACC 20186T, S. fructofermentans CGMCC 4.1593T, S. scabiei NRRL B-16523T and other species of the genus Streptomyces with validly published names, respectively. Phylogenomic analysis indicated that strain MG28T was closely related to Streptomyces deccanensis KCTC 19241T. However, the average nucleotide identity values and the digital DNA-DNA hybridization values between them indicated that strain MG28T represented a distinct species. Furthermore, strain MG28T was also distinctly differentiated from strain KCTC 19241T by morphological, physiological and biochemical characteristics. Therefore, strain MG28T (= MCCC 1K06895T = JCM 34922T) represents a novel species of the genus Streptomyces, for which the name Streptomyces akebiae sp. nov. is proposed.

[Determination of nine ginsenosides in health foods by solid extraction phase-ultra performance liquid chromatography-tandem mass spectrometry].

Ginsenosides are the main active compounds of ginseng, American ginseng and Panax notoginseng. They have certain pharmacological effects on the cardiovascular, immune and central nervous systems. Most ginsenosides are naturally classified as protopanaxatriol (PPT), protopanaxadiol (PPD), and oleanolic acid (OA) according to their aglycone skeletons. The nine main ginsenosides are Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1 and Rg2. Accurate quantification of ginsenosides is imperative because they are the characteristic components and quality evaluation indicators of health foods. A new method based on solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) was developed for the determination of the nine ginsenosides in health foods. First, the pretreatment conditions were optimized. With the aim of purifying the samples and removing impurities, SPE cartridges with different packing materials, such as Alumina-N/XAD-2 SPE Cartridge, C18 and HLB were investigated. Based on the purification efficiencies, recoveries and other factors, the Alumina-N/XAD-2 SPE cartridge composite SPE column was selected as the pretreatment purification column. The eluents were then optimized. When water was used as the eluent, the ginsenosides could remain adsorbed on the SPE column, and could not be eluted down with other water-soluble substances. By increasing the proportion of ethanol in the eluent, the ginsenoside adsorbed on the filler of the SPE column could be gradually eluted. When the proportion of ethanol in the eluent reached 70%, the ginsenosides could be completely eluted. The effects of different volumes of 70% ethanol elution solvent (5-30 mL) on the extraction efficiencies of ginsenosides were also investigated. The results showed that when the volume of the elution solvent reached 20 mL, the ginsenosides were completely eluted. Then, the chromatographic conditions and MS parameters were optimized. By examining the ionization cracking of ginsenosides, the quasi-molecular ions and corresponding fragment ions in ginsenoside primary MS were determined. After optimizing the chromatographic conditions and MS parameters, not only the sensitivity of the method was improved, but also the isomers Rb2, Rb3 and Rc with the same quasi-molecular ions and the corresponding fragment ions were completely separated. Good separation was achieved for the nine ginsenosides, thus meeting the requirements for accurate quantification. Finally, chromatographic separation was achieved on a Hypersil Gold C18 column (100 mm×2.1 mm, 1.9 µm) under linear gradient elution using a 5 mmol/L ammonium acetate solution (with 0.1% formic acid) and acetonitrile as the mobile phases. The nine ginsenosides were detected using a triple quadrupole MS detector under ESI - and multiple reaction monitoring (MRM) modes, and quantified by the external standard method. The nine ginsenosides showed a strong positive linear correlation (r 2>0.9950) in the range of 0.005-0.5 µg/mL. The sample recoveries and the corresponding relative standard deviations (RSDs) were 81.1%-114.2% and 0.4%-8.0% (n=6), respectively. Eleven batches of health foods on the market, among which six batches contained ginseng, American ginseng or Panax notoginseng ingredients, were analyzed by the developed method, and the ginsenosides were detected. The total ginsenosides contents were close to those mentioned on the label. However, the nine ginsenosides were detected in one batch of health food, whose label did not indicated ginseng, American ginseng or Panax notoginseng. The nine ginsenosides were not detected in the remaining batches of health foods.The health food extract was directly loaded and purified without any complex pretreatment. The UPLC⁃MS/MS method, not only helped shorten the analysis time, but also accurate quantification of low ginsenoside contents in complex matrix samples. The developed method is simple and rapid, with high throughput, thus being suitable for the quantitative analysis of the nine ginsenosides in health foods.

Additive value of exposure parameters for breast cancer diagnosis in digital mammography.

OBJECTIVES: To investigate the effect of different breast lesions on exposure parameters in digital mammography and to determine whether the exposure parameters can additively improve diagnostic efficiency. METHODS: Craniocaudal view and mediolateral view full-field digital mammography images from 982 women with unilateral lesions (341 with malignant lesions, 189 with benign lesions, and 452 healthy women) obtained at Nanfang Hospital were reviewed. Differences in exposure parameters (tube voltage and load, breast thickness (BT), and average glandular dose (AGD)) between breasts were calculated. The relationships between parameter differences and lesion size were explored. A logistic regression model was used based on the AGD and BT differences, and the area under the receiver operating characteristic curve (AUC) was used to assess the performance of these parameters in differentiating malignant from benign and healthy subjects. Independently, data from 129 women (82 with malignant and 47 with benign lesions) treated at Sun Yat-sen Memorial Hospital were collected to validate the model. RESULTS: Differences in tube voltage and load, BT, and AGD between breasts were significantly greater in the malignant subjects than benign (p < 0.05) and healthy subjects (p < 0.05). The AUCs for the comparisons of malignant vs. healthy subjects, malignant vs. benign subjects, and benign vs. healthy subjects were 0.77 ± 0.02, 0.72 ± 0.02, and 0.57 ± 0.02, respectively. The model combining the exposure parameters with the BI-RADS category resulted in a higher AUC (0.910 ± 0.03) compared with physician diagnosis alone (0.820 ± 0.04) for differentiating between malignant and benign lesions. CONCLUSIONS: Exposure parameters additively improved diagnostic accuracy for breast cancer and yielded more reliable results. KEY POINTS: • Differences in kVp, mAs, BT, and AGD between breasts were significantly greater in the malignant subjects than benign and healthy subjects. • The model combining exposure parameters with the BI-RADS category resulted in a higher AUC compared with the physician's diagnosis for differentiating between malignant and benign lesions. • Exposure parameters additively improved diagnostic accuracy for breast cancer.

Cyclometalated Iridium(III) Complex-Sensitized NiO-Based-Cathodic Photoelectrochemical Platform for DNA Methyltransferase Assay.

This work reports an efficient [(C6)2Ir(dppz)]+PF6- (C6 = coumarin 6 and dppz = dipyridophenazine)-sensitized NiO photocathode and its application in photoelectrochemical (PEC) bioanalysis field for the first time. This dye-sensitized NiO photocathode was found to exhibit a markedly enhanced cathodic photocurrent. A sensitive cathodic PEC platform was proposed integrating the as-prepared photocathode with enzyme-free cascaded amplification strategies of the catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR) for DNA methyltransferase (MTase) assay. A hairpin DNA(HDam) with specific recognition site of Dam MTase in its stem was designed. The site of HDam was methylated in the presence of Dam MTase and then cut by endonuclease DpnI. The released loop fragment, as an initiator, triggered the CHA circuit and the follow-up HCR circuit, resulting in long dsDNA concatemers on the ITO electrode. Numerous [(C6)2Ir(dppz)]+PF6- were intercalated into dsDNA, and highly efficient signal amplification was realized. Benefiting from the superior iridium(III) complex-sensitized NiO photocathode and effective amplification strategy, a detection limit of 0.0028 U/mL for the determination of Dam MTase was achieved. Moreover, this work further demonstrated that these proposed tactics could be applied to screen Dam MTase activity inhibitors.

[Effect of Different Walking Number on Inflammation and Nutrition in Patients with Chronic Kidney Disease].

OBJECTIVE: To ivestigate the effect of daily walking number on clinical, inflammatory and nutritional indexes for patients with chronic kidney disease. METHODS: 90 non-dialysis patients with chronic kidney disease were selected and randomly divided equally into three groups, for groups A, B and C. 30 patients for group A were asked for the number of daily walk should less than 5 000 steps, while group B patients were asked for 5 000-9 999 steps of walk and group C for more than 10 000 steps. Basic treatments were given for each group of patients and basic information, clinical, inflammatory and nutritional datas of patients were collected. RESULTS: 87 patients with chronic kidney disease completed the study, with baseline data between group A, B, C (n=30, 29, 28) consistently. After 3 months of exercise, there were no significant changes on blood lipids, serum uric acid and parathyroid hormone (PTH) for three groups, with serum creatinine of three groups stably. However, in group C, hemoglobin, ferritin, transferrin saturation were found increased significantly (P<0.05). For nutritional indexes, increasing of albumin and prealbumin level were found in three groups, while significant differences were only found in group B and C (P<0.05) and group C increased most. There were no significant change on body mass index (BMI), upper arm skinfold thickness and SGA score in three groups. For inflammatory data, significant decrease of C-reactive protein (CRP) and interleukin-6 (IL-6) were only seen in group C (P<0.05). CONCLUSION: Walking does not increase the burden of the kidney, but can improve the nutrition and clinical indicators of patients, reduce inflammation.