Volume 30 (2022): Edizione 4 (October 2022)

Cadmium (Cd) is known as a pollutant source in recent years with the increase in industrialization. Algae have secondary metabolites with high biological activity, used for pharmaceutical agents. The liver and kidney are the primary organs involved in the elimination of systemic cadmium and the main targets of cadmium toxicity. In the previous research, it was determined the ameliorative effects of the extract obtained from Ulva rigida in the liver tissue of rats induced by cadmium. 35 female Wistar rats between 225-240 g were used. The subjects were injected subcutaneously with 1 mg/kg cadmium chloride (CdCl₂) four times a week for four weeks. The study was carried out by groups as control (G1), Cadmium group (1 mg/kg CdCl₂-G2), Algae group (100 mg/kg-G3), Cd+algae group (1 mg/kg CdCl₂+50 mg/kg algae extract-G4) and Cd+algae group (1 mg/kg CdCl₂+100 mg/kg algae extract -G5). The subjects were sacrificed by cervical dislocation. Liver tissue and cardiac blood were collected. It was determined that oxidative stress with iNOS, inflammation and apoptosis with TNF-α increased with cadmium induction, while there was a statistically significant decrease in the groups that were given algae extract. In addition, biochemical changes in SOD, CAT and MDA values were found to be significant (p<0.05). As a result, it was determined that algae extract could play a protective role with its antioxidant and antiapoptotic properties in experimentally induced cadmium toxicity in rats.

Introduction: Hypertension and diabetes mellitus affect a large number of patients and can significantly influence their life expectancy. Changes in metabolic and oxidative stress parameters are common in these pathologies, contributing to associated complications. The aim of the study was assessment of relationship between laboratory parameters and their role in evaluation of cardiovascular risk, and possible gender-related differences in the protective factors.

Material and methods: Blood samples were collected from hypertensive patients with/without diabetes mellitus admitted to the Cardiovascular Rehabilitation Clinic in Tîrgu Mureș and controls without these pathologies. Biochemical analyses were performed on Konelab analyzer (glycemia, lipid profile, kidney function tests, zinc, hsCRP). Oxidative stress markers, such as serum malondialdehyde (MDA), oxidized (GSSG) and reduced glutathione (GSH) were evaluated using an HPLC-UV/VIS technique at GEP UMPhST. Statistical analysis was performed by GraphPad InStat3.

Results: Mean age of hypertensive patients (n=131) was 69.44 ± 9.02 years, 45.8% males, 31.3% being diabetics. 74.1% of the studied patients had zinc deficiency, 19.8% presented slightly elevated hsCRP. The control group included 24 nonhypertensive/nondiabetic patients of similar age. Average GSH was significantly lower (p=0.0002) in hypertensive patients, 1.89 ± 0.82 µg/ml, compared to the control group (3.23 ± 0.49 µg/ml), and no correlation could be observed between GSH and MDA values. GSH concentration was significantly higher in males (p=0.0395) and HDL-cholesterol significantly higher in females (p=0.0132). A negative correlation was observed between serum triglyceride and HDL-cholesterol concentration.

Conclusions: Gender differences are present in the level of protective factors against cardiovascular diseases, while oxidative stress is intensified in hypertensive/diabetic patients.

Background: Diabetes is a common chronic disease which has caused a great burden on families and society. The treatment of diabetes has always been a hotspot. This study aimed to explore the effect and mechanism of miR-30d-5pon inflammation of high glucose-impaired human keloid fibroblasts (HKF).

Methods: Differently-expressed miRNAs were predicted by bioinformatics methods. Exosomes were observed by transmission electron microscope. Exosome particle sizes were measured by NanoSight. Western Blot was used to detect the expression of CD81, CD63, CD9, and Calnexin. QRT-PCR was used to detect the expression of miR-30d-5p, IL-1β, TNF-α, VEGF, FGF21, NRF2, and HO-1. The levels of IL-1β, TNF-α, IL-6, IL-10, and TGF-β were determined by ELISA. Cell apoptosis and CD86, CD206 positive cells were detected by flow cytometry.

Results: Tori formula could promote the secretion of endothelial progenitor cell (EPCs) exosomes. EPCs exosomes and miR-30d-5p could stimulate the proliferation of HKF impaired by high glucose and the expression of IL-10 and TGF-β. MiR-30d-5p inhibited the proliferation of M1 macrophages and the expression of IL-1β and TNF-α. It could also promote the proliferation of M2 macrophages and the expression of CCL17 and CCL22. Moreover, miR-30d-5p stimulated the expression of VEGF, FGF21, NRF2, and HO-1, as well as suppressed the expression of IL-1β, TNF-α, and IL-6. MiR-30d-5p also restrained the apoptosis of impaired HKF. Conclusion: This study confirmed that miR-30d-5p could promote the M1/M2 polarization and inhibit the inflammatory response of impaired HKF, which provided a certain idea and direction for treating diabetes.

Introduction: In the last 40 years, Acinetobacter baumannii has been among the bacteria known to acquire multiple mechanisms of antibiotic resistance and, as a result, it is now one of the pathogens involved in healthcare-associated infections with multidrug resistant strains. Our study aimed to assess the production of carbapenemases in carbapenem-resistant A. baumannii by means of phenotypic methods and polymerase chain reaction technique (PCR), as well as to appraise the performances of carbapenemase detection by phenotypic tests compared to the PCR approach.

Materials and Methods: We used phenotypic methods (E-test MBL, CIM, MHT, Rosco® Kit/OXA/MBL, OXA-23 K-SeT® assay) to investigate the production of carbapenemases in 43 carbapenem-resistant A. baumannii isolates, and PCR to screen for the genes bla_OXA-23, bla_OXA-24, bla_OXA-58, bla_OXA-51, bla_VIM, bla_IMP and bla_NDM.

Results: The carbapenem inactivation method (CIM) at 2 hours, CIM at 4h, OXA-23 K-SeT® assay, Rosco® Kit/OXA, and modified Hodge test (MHT) identified 26%, 63%, 65%, 81%, and 42% carbapenemase-producing isolates, respectively. The phenotypic E-test MBL detected metallo-β-lactamase (MBL) production in 79% of strains. PCR revealed blaOXA-51 in all the isolates, bla_OXA-23 in 35/43 (81%), bla_OXA-24 in 28/43 (65%), bla_VIM in 7/43 (3%) and bla_OXA-58, bla_IMP, bla_NDM were not detected.

Conclusion: Because phenotypic tests do not highlight all the carbapenemase-producing strains, their results must be interpreted with caution relative to their level of performance, and negative results should be confirmed by means of PCR.

Background: To explore the correlations of cofilin1 (CFL1) and phosphorylation level of locus serine residue at position 3 (Ser3) with the sensitivity of elderly patients with non-small cell lung cancer (NSCLC) to radiotherapy.

Methods: A total of 102 eligible patients treated from June 2013 to April 2015 were selected. The cases of complete remission and partial remission were included into radiotherapy-sensitive group (n=55), while those of stable disease and progressive disease were enrolled into radiotherapy-resistant group (n=47). Before treatment, tissues were collected to detect the expressions of CFL1 and CFL1 (phospho S3) by immunohistochemistry. The survival time and rate were recorded during follow-up.

Results: Compared with the radiotherapy-sensitive group, the radiotherapy-resistant group had advanced tumor-node-metastasis (TNM) stage and higher lymph node metastasis rate (P=0.000, 0.000). Compared with the tissues with negative CFL1 expression, the tissues with positive CFL1 expression had advanced TNM stage and higher lymph node metastasis rate (P=0.013, 0.000). The positive expression rate of CFL1 in the radiotherapy-resistant group was higher than that of the radiotherapy-sensitive group, whereas the positive expression rate of CFL1 (phospho S3) in the former was lower (P=0.000, 0.000). Lymph node metastasis, high CFL1 expression, and low CFL1 (phospho S3) expression were independent predictors for resistance to radiotherapy (P=0.001, 0.006, 0.003). In the radiotherapy-sensitive group, the patients with negative CFL1 expression and positive CFL1 (phospho S3) expression had long progression-free survival and high 5-year survival rate (P=0.000, 0.000).

Conclusion: The sensitivity to radiotherapy of elderly NSCLC patients is correlated negatively with CFL1 and positively with phosphorylation at locus Ser3. CFL1 and phosphorylation at locus Ser3 are independent predictors for sensitivity to radiotherapy.

Background: DNAJA4 (PRO1472) is a heat shock protein that has been associated with several types of cancers, including breast cancer. We aimed to reveal the protein expression, clinical outcomes, and regulatory mechanisms of DNAJA4 gene in breast cancer by employing tissue microarrays, transcriptomic datasets, and in-silico tools.

Methods: DNAJA4 protein expression and its clinical implications were evaluated by immunohistochemistry assay (normals = 32; tumors = 121). RNA-seq and DNA microarray datasets were analyzed by using breast cancer gene-expression miner (Bc-GenExMiner v4.8) to estimate the survival probabilities of breast cancer patients. DNAJA4 promoter methylation level was analyzed in clinical samples by UALCAN in-silico tool (normals = 97; tumors = 793).

Results: DNAJA4 protein expression is significantly high in clinical breast cancer samples compared to the normal samples (P = 0.016). High DNAJA4 mRNA expression is correlated with poor overall survival (OS), disease-free survival (DFS), and distant metastasis-free survival (DMFS) in breast cancer patients (P < 0.05). Mutations or copy number variations of DNAJA4 are uncommon in clinical samples. Reduced promoter methylation was observed in clinical breast cancer samples.

Conclusion: We suggest DNAJA4 expression as a new biomarker candidate for breast cancer. Promoter hypomethylation could be an important epigenetic factor in the upregulation of DNAJA4 expression in breast cancer.

Quality Control (QC) in Romania is regulated by the Order of the Minister of Health no. 1608/2022 that modifies the previous Order 1301/2007. The new version of the Order introduces a more scientific approach by requesting the laboratories to assess test performance and then elaborate an appropriate internal QC plan. The aim of this study was to demonstrate how to design a QC plan for complete blood count (CBC) in an Emergency Laboratory with continuous activity, in order to comply with the new Order 1608/2022. QC data obtained over a three-month period (April-June 2022) from the Sysmex XN-1000 instrument of the Emergency Laboratory of the County Emergency Clinical Hospital of Târgu Mureș were included. In order to establish an appropriate QC plan, two models were applied and the following parameters were calculated: the number of daily QC runs (N), the probability of false rejection (Pfr), the QC frequency (run size), and the required QC rules. White blood cells achieved high performance, while Hematocrit performance was poor. Different levels of performance were achieved for Platelets. We emphasize that, when all parameters are measured on the same instrument, QC frequency and Pfr should be adjusted in order to develop a QC plan that “fits” all the parameters of the CBC as a whole. In our Emergency Laboratory, the calculated QC plan for CBC is N=2, Pfr=0.03, multi-rule 1:3s/2:2s/R:4s, and a run size of 95 samples which is approximately the same as the number of CBCs performed during one 12-hour shift.

Background: To explore the diagnostic value of combination of exfoliative cytology with detection of tumor markers carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA), neuron specific enolase (NSE), cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) and CA15-3 for lung cancer.

Methods: A total of 256 patients were enrolled, including 164 males and 92 females aged (64.51±22.68) years old. Among them, 189 patients (100 males and 89 females) were randomly selected as Tumor group, and the remaining 67 patients were used for validation. Another 514 healthy people receiving physical examination in our hospital during the same period were selected, from which 397 cases (266 males and 131 females) were randomly selected as No Tumor group, and the remaining 117 cases were used for validation. The biochemical criteria were detected in all subjects. The diagnostic value of each index for lung cancer was analyzed using receiver operating characteristic (ROC) curves.

Results: The results of ROC curve analysis revealed that in Tumor group, the area under curve (AUC) of exfoliative cytology, CA125, CYFRA21-1, CA15-3, CEA and NSE was ≥0.7, while that of CA72-4, CA19-9, TSGF, AFP, CA242, SCCAg and CA50 was <0.7. The indices in each factor were comprehensively assessed, and then exfoliative cytology, CA125, CA15-3, CYFRA21-1, CEA and NSE were screened to establish the lung cancer prediction model. The diagnostic value was comparable between the prediction model and the combined detection of 9 indices (Z=1.682, P=0.079).

Conclusions: The lung cancer prediction model balances sensitivity and specificity without reducing the diagnostic efficiency.

Objective: The contamination of polymerase chain reaction (PCR) samples in molecular diagnostic laboratories can cause serious consequences. Internal quality control efforts are often inadequate, especially in clinical next-generation sequencing (NGS) laboratories.

Methods: In this study, we retrospectively investigated an incidence of PCR contamination and its decontamination process in a clinical laboratory. We performed a series of measures for decontamination. Taqman fluorescence quantification was carried out to determine the presence of contaminating DNA. SYBR-Green PCR was conducted to evaluate the effect of chlorine disinfectant on NGS library preparation.

Results: Through a series of elimination measures undertaken over 8 weeks, the decontamination process was verified as reliable. Almost no contamination was detected. Chlorine disinfectant should be forbidden in Illumina NGS laboratories because it may cause the failure of library preparation.

Conclusion: Our prevention and decontamination strategies could effectively eliminate PCR amplicons. Chlorine disinfectants should not be used in Illumina NGS laboratories.

Background: Depression is one of the significant problems in adults that accounts for up to five percent of cases worldwide.

Methods: Volunteers were divided into eight groups, and their serum samples were tested for FBG, carbonyl contents, IFN-γ and TNF-α. Reactive oxygen species (ROS) modified human serum albumin (HSA) (ROSHSA) was used as an antigen and levels of serum autoantibodies were estimated by direct binding and inhibition ELISA in all subjects.

Results: Significant biophysical structural modifications were observed in ROS-HSA with increased carbonyl contents compared to native-HSA (N-HSA). Significantly high levels of carbonyl content (2.68 ± 0.33 nmol/mg protein; p > 0.001) and pro-inflammatory cytokines IFN-γ (7.4 ± 0.61 pg/ml; p > 0.001) and TNF-α (1.47 ± 0.23 pg/ml; p > 0.001) were detected in serum samples from F-D-S. Similarly, a high level of autoantibodies against ROS-HSA was observed in females who were depressed and smokers (F-D-S) group (0.89 ± 0.07; p > 0.001) compared to males who were both depressed and smokers (M-D-S) (0.66 ± 0.049). Furthermore, inhibition ELISA results exhibited high recognition of serum autoantibodies from F-D-S subjects (78.6 ± 5.7 mean maximum percentage inhibition MMPI) compared to M-D-S (58.8 ± 5.2 MMPI) subjects.

Conclusion: Incoherence, long term unchecked chronic psychological stress may cause oxidation of blood proteins, which subsequently result in structural alterations of biomolecules, thus generating new-epitopes, capable of inducing autoantibodies specific for ROS-modified proteins. These autoantibodies may be a potential marker for subjects suffering from depression to understand the state of immune imbalance.