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Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury. • Oxygen-glucose deprivation/oxygen-glucose resupply impaired autophagy degradation. • Oxygen-glucose deprivation/oxygen-glucose resupply caused lysosomal dysfunction. • Autophagic clearance dysfunction was one of the causes of endothelial injury. • CTSD knockdown mimicked these effects on autophagy-lysosomal function. • CTSD restoration protected endothelial cells against autophagy-lysosomal dysfunction. [ABSTRACT FROM AUTHOR]

Objective: Adenosine deaminase (ADA) plays a major role in maintaining metabolic homeostasis via catalysis of hydrolytic deamination of adenosine to inosine. The ADA1 isoenzyme of ADA is an analyte tested in clinical laboratories; however, lack of quality control (QC) material in terms of enzyme homogeneity, stability, and coverage of the clinically relevant analytical measurement range (AMR), poses a challenge for adequate monitoring of this analyte. The aim of this study was to address the need for manufacture of QC material through recombinant expression of catalytically active ADA1 in eukaryotic cells ( Pichia pastoris GS115)., Methods: The coding region of ADA1 gene was amplified by PCR and ligated into plasmid pPICZαA, followed by transfer into P. pastoris using electroporation. Recombinant ADA1 produced by P. pastoris was purified using a Ni-NTA resin column, yielding 5 mL of purified ADA1 with an activity of 4200.6 U/L. Purified ADA1 protein was added to human donor serum as the appropriate matrix for QC materials preparation., Results: One hundred vials of lyophilized ADA1 were prepared at clinically significant concentrations at 41.6 U/L and 115.5 U/L (50 vials each). Both concentrations were homogenous and stable at room temperature (RT, 22-24°C) for at least 7 d, at 4°C for 3 months, and at -20°C for 12 months. Reconstituted aliquots of QC material were found to be stable at -20°C for up to 60 d and should be used within 8 h or 48 h when stored at RT or 4°C, respectively., Conclusion: Success of this ADA1 expression system presents a potential solution to increase production options available to clinical laboratories., (Copyright © 2022 by the Association of Clinical Scientists, Inc.)

Background: Traditional Chinese medicine Smilax is the rhizome of liliaceous plant Smilax china L., which is used to treat pelvic inflammatory disease and anxieties., Purpose: To investigate the mechanism of anti-inflammatory activity of the extract from Smilax china L. (ES)., Methods: The components of ES were identified by UPLC-QTOF-MS/MS. The anti-inflammatory activities were evaluated in xylene-induced ear oedema and egg white-induced plantar swelling test. Cell viability was examined by CCK-8 assay. The inflammatory mediators, proinflammatory cytokines, and MAPK and NF- κ B signals in LPS-stimulated THP-1 cells were determined using ELISA, real-time PCR, and Western blot, respectively., Results: 20 compounds of ES were confirmed by comparing with the reference substance. ES displayed more prominent anti-inflammatory activity than the positive control "Jin Gang Teng" capsule in the in vivo acute inflammatory model. ES suppressed the expression of PGE 2 and 6-Keot-PGF 1 α , and the ratio of IC 50 (COX-1)/IC 50 (COX-2) of ES was 3.15, which indicated that ES could selectively inhibit COX-2. ES dose-dependently (12.5, 25, and 50 mg/L) decreased the production and mRNA levels of proinflammatory cytokines IL-1 β , IL-6, and TNF- α . Furthermore, ES significantly decreased LPS-induced phosphorylation of p38, JNK, ERK1/2, and p65, inhibiting the expression of IKK α and the degradation of I κ B α ., Conclusion: The results suggested that ES could selectively inhibit the activity of COX-2, and the anti-inflammatory effect of ES was associated with the inhibition of IL-1 β , IL-6, and TNF- α via negative regulation of MAPK and NF- κ B signaling pathways in LPS-induced THP-1 cells., Competing Interests: The authors declare that they have no conflicts of interest regarding the publication of this paper., (Copyright © 2021 Siyi Jiang et al.)

Objective: To screen potential plasma protein biomarkers for the progression of cervical precancerous lesions into cervical carcinoma and analyze their functions., Methods: Plasma samples obtained from healthy control subjects, patients with low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical cancer (CC), and patients with CC after treatment were enriched for low-abundance proteins for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The MS data of the samples were analyzed using Discoverer 2.2 software, and the differential proteins (peptide coverage ≥20%, unique peptides≥2) were screened by comparison of LSIL, HSIL and CC groups against the control group followed by verification using target proteomics technology. Protein function enrichment and coexpression analyses were carried out to explore the role of the differentially expressed proteins as potential biomarkers and their pathological mechanisms., Results: Compared with the control group, both LSIL group and HSIL group showed 9 differential proteins; 5 differentially expressed proteins were identified in CC group. The proteins ORM2 and HPR showed obvious differential expressions in LSIL and HSIL groups compared with the control group, and could serve as potential biomarkers for the progression of cervical carcinoma. The expression of F9 increased consistently with the lesion progression from LSIL to HSIL and CC, suggesting its value as a potential biomarker for the progression of cervical cancer. CFI and AFM protein levels were obviously decreased in treated patients with CC compared with the patients before treatment, indicating their predictive value for the therapeutic efficacy. Protein function enrichment analysis showed that all these differentially expressed proteins were associated with the complement system and the coagulation cascades pathway., Conclusions: We identified 5 new protein biomarkers (F9, CFI, AFM, HPR, and ORM2) for cervical precancerous lesions and for prognostic evaluation of CC, and combined detection of these biomarkers may help in the evaluation of the development and progression of CC and also in improving the diagnostic sensitivity and specificity of cervical lesions.

Introduction: Thalassemia could interfere with some assays for haemoglobin A 1c (HbA 1c ) measurement, therefore, it is useful to be able to screen for thalassemia while measuring HbA 1c . We used Capillarys 2 Flex Piercing (Capillarys 2FP) HbA 1c programme to simultaneously measure HbA 1c and screen for thalassemia., Materials and Methods: Samples from 498 normal controls and 175 thalassemia patients were analysed by Capillarys 2FP HbA 1c programme (Sebia, France). For method comparison, HbA 1c was quantified by Premier Hb9210 (Trinity Biotech, Ireland) in 98 thalassaemia patients samples. For verification, HbA 1c from eight thalassaemia patients was confirmed by IFCC reference method., Results: Among 98 thalassaemia samples, Capillarys 2FP did not provide an HbA 1c result in three samples with HbH due to the overlapping of HbBart's with HbA 1c fraction; for the remaining 95 thalassaemia samples, Bland-Altman plot showed 0.00 ± 0.35% absolute bias between two systems, and a significant positive bias above 7% was observed only in two HbH samples. The HbA 1c values obtained by Capillarys 2FP were consistent with the IFCC targets (relative bias below ± 6%) in all of the eight samples tested by both methods. For screening samples with alpha (α-) thalassaemia silent/trait or beta (β-) thalassemia trait, the optimal HbA 2 cut-off values were ≤ 2.2% and > 2.8%, respectively., Conclusions: Our results demonstrated the Capillarys 2FP HbA 1c system could report an accurate HbA 1c value in thalassemia silent/trait, and HbA 2 value (≤ 2.2% for α-thalassaemia silent/trait and > 2.8% for β-thalassemia trait) and abnormal bands (HbH and/or HbBart's for HbH disease, HbF for β-thalassemia) may provide valuable information for screening., Competing Interests: Potential conflict of interest: None declared.

Haemoglobinopathies may interfere with the haemoglobin A 1c (HbA 1c ) measurement, leading to incorrect diagnosis and inappropriate treatment. It is essential that HbA 1c assays are capable of identifying haemoglobinopathies. We report two cases of haemoglobin New York (HbNY) discovered through HbA 1c analysis using capillary electrophoresis (Capillarys 2 Flex Piercing [C2FP], Sebia). We used these samples to evaluate the ability of three other HbA 1c assays to identify this variant: ion-exchange high-performance liquid chromatography (Variant II Turbo [VII-T], Bio-Rad); boronate affinity high-performance liquid chromatography (Ultra 2 , Trinity Biotech) and immunoassay (Cobas c501 Tina-quant Generation 3, Roche Diagnostics). Each method was used for HbA 1c assay of in samples from two cases of heterozygous haemoglobinopathy: β 0 -thalassemia/HbNY (Case 1) and HbA/NY (Case 2). Only the C2FP system detected HbNY (an additional peak appeared between HbA 1c and HbA 0 ). Clinical laboratories should be aware of the limitations of their HbA 1c assay methods especially in geographic areas, where haemoglobinopathy prevalence is high.

Introduction: Haemoglobin A 1c (HbA 1c ) is widely used in the management of diabetes. Therefore, the reliability and comparability among different analytical methods for its detection have become very important., Materials and Methods: A comparative evaluation of the analytical performances (precision, linearity, accuracy, method comparison, and interferences including bilirubin, triglyceride, cholesterol, labile HbA 1c (LA 1c ), vitamin C, aspirin, fetal haemoglobin (HbF), and haemoglobin E (Hb E)) were performed on Capillarys 2 Flex Piercing (Capillarys 2FP) (Sebia, France), Tosoh HLC-723 G8 (Tosoh G8) (Tosoh, Japan), Premier Hb9210 (Trinity Biotech, Ireland) and Roche Cobas c501 (Roche c501) (Roche Diagnostics, Germany)., Results: A good precision was shown at both low and high HbA 1c levels on all four systems, with all individual CVs below 2% (IFCC units) or 1.5% (NGSP units). Linearity analysis for each analyzer had achieved a good correlation coefficient (R 2 > 0.99) over the entire range tested. The analytical bias of the four systems against the IFCC targets was less than ± 6% (NGSP units), indicating a good accuracy. Method comparison showed a great correlation and agreement between methods. Very high levels of triglycerides and cholesterol (≥ 15.28 and ≥ 8.72 mmol/L, respectively) led to falsely low HbA 1c concentrations on Roche c501. Elevated HbF induced false HbA 1c detection on Capillarys 2FP (> 10%), Tosoh G8 (> 30%), Premier Hb9210 (> 15%), and Roche c501 (> 5%). On Tosoh G8, HbE induced an extra peak on chromatogram, and significantly lower results were reported., Conclusions: The four HbA 1c methods commonly used with commercial analyzers showed a good reliability and comparability, although some interference may falsely alter the result., Competing Interests: Conflicts of Interest: None declared.

The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site-enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery.

Objective: To establish of blood beta hydroxybutyrate (βOHB) threshold for diagnosing type 2 diabetes ketoacidosis (DKA) and explore the relationship between βOHB levels and the severity of DKA., Methods: Correlation analysis was performed between serum βOHB and [HCO(3)] in type 2 diabetic patients admitted in the emergency department in the past year. Regression equation was used to calculate the concentration of βOHB corresponding to a [HCO(3)] level of 18.0, 15, and 10.0 mmol/L, and βOHB concentration corresponding to a [HCO(3)] level of 18.0 mmol/l was used as the DKA diagnostic threshold., Results: The serum βOHB level and [HCO3] concentration showed a good correlation (R²=0.7023, P<0.001). βOHB concentrations that corresponded to a [HCO(3)] level of 18.0, 15, and 10.0 mmol/L were 3.0, 4.70, and 7.5 mmol/L, respectively, in accordance with the severity of DKA. Combined with the blood glucose concentration ≥ 13.9 mmol/L, a blood βOHB≥3.0 mmol/L showed a sensitivity of 99%, specificity of 86%, and total effectiveness of 92.81% for diagnosing DKA., Conclusion: A serum βOHB level above 3.0 mmol/L can be used as the diagnostic threshold of DKA. βOHB can serve as an index for assessing the severity of DKA.

Objective: To explore the anti-inflammatory effect of triptolide (TPT) by regulating miR-155 in monocytes pre-stimulated by lipopolysaccharide (LPS) from rheumatoid arthritis (RA) patients., Methods: Monocytes were isolated by CD14⁺ magnetic beads from peripheral blood mononuclear cells (PBMCs) of RA and stimulated by LPS for 24 hours. The levels of tumor-necrosis factor-α (TNF-α) and interleukin 6 (IL-6) in monocytes were detected by ELISA and the expression of miR-155 was measured by real-time quantitative PCR (qRT-PCR) in monocytes before and after the treatment of TPT at different concentrations. MiR-155 mimic and negative control were respectively transfected into the LPS-stimulated monocytes by Lipofectamine(TM)2000. Twenty-four hours later, the monocytes were treated with or without TPT for another 24 hours. TNF-α and IL-6 expressions in the cell culture supernatants were detected by ELISA and the expressions of suppressor of cytokine signaling-1 (SOCS1) and Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1) were tested by Western blotting., Results: TPT suppressed the expressions of TNF-α, IL-6 and miR-155 in LPS-stimulated peripheral blood monocytes from RA patients. Over-expression of miR-155 significantly reversed the down-regulation of TNF-α and IL-6 by TPT in monocytes. TPT up-regulated the expressions of SOCS1 and SHIP-1 in monocytes, but over-expressed miR-155 antagonized the effect of TPT on SHIP-1 while the expression of SOCS1 was not affected., Conclusion: TPT suppressed the expression of miR-155 and up-regulated the release of SHIP-1, thus inhibiting the inflammatory response in the LPS-stimulated monocytes of RA patients.

Objective: To investigate the incidence of human papillomavirus type 16 (HPV16) infection in patients with cervical diseases and analyze E2 gene mutations and its correlation with cervical lesions., Methods: Specific primers were designed according to the full-length E2 gene sequence of HPV16 from the GenBank. PCR was used to analyze the incidence of HPV16 infection in the exfoliated cell samples from 379 patients with different cervical diseases positive for high-risk HPV. The polymorphisms of E2 gene of HPV16 were analyzed by high-resolution melting., Results: Of the 379 exfoliated cell samples from the cervix, 78 were positive for HPV16 infection. The positivity rates of HPV16 infection was 44.8% in patients with cervical cancer, 31.5% in patients with CIN II-III, 24.1% in patients with CIN I and 9.6% in patients with cervical inflammation. The frequencies of E2 gene 68C and 133G variations were significantly higher in patients with CIN II-III and those with cervical cancer than in those with CIN I and those with cervical inflammation (P<0.05)., Conclusions: HPV16 is an important cause of cervical cancer, and HPV16 infection rate increases with the pathological progression of the cervical lesions. The naturally occurring sequence variations of HPV16 E2 gene may cause alterations of the carcinogenic potential of the virus.