Serum DNA Research Articles

CLINICAL AND PARACLINICAL CRITERIA FOR LIKELYHOOD OF INFECTION AND DEVELOPMENT OF CONGENITAL PNEUMONIA IN NEWBORN CHILDREN WITH CHLAMYDIAL INFECTION

Introduction. Issues of the prevention of intrauterine diseases, and, in particular, congenital pneumonia, in newborns with Chlamydia infection, is still remaining unsolved due to the insufficiently developed criteria for diagnosis of the infection and identification of this pathology progression. The aim of this study is to develop clinical, laboratory and instrumental factors of the Chlamydia infection and the development of congenital pneumonia in newborns. Materials and methods. We examined 77 newborns (38 (49.3%) premature children, 39 (50.7%) full-term children). In the main group 54 newborns were diagnosed to have with congenital pneumonia, on them 25 (46.3%) premature and 29 (53.7%) full-term children. The control group included 23 "conditionally healthy" newborns, of the 13 (56.5%) premature and 10 (43.5%) full-term children. The diagnosis of congenital pneumonia in newborns with Chlamydia infection was confirmed based on the anamnesis, clinical criteria, risk factors over the ante-, intra- and postnatal periods, pathological changes detected on neurosonography, disorders of the intestinal microbiocenosis. Specific diagnosis was performed by the method of polymerase chain reaction with the detection of Chlamydia DNA in the blood serum of newborns. Results and discussion. Comprehensive examination of newborns with congenital pneumonia demonstrated that all newborns in this group were born to mothers infected with Chlamydia, who had burdened obstetric and gynaecological history, complicated pregnancy (due to the presence of a large percentage of extragenital and genital pathology and pathological course of childbirth of pregnant women). It was found that ventricular dilatation, cysts of vascular plexuses and leukomalacia, movement disorders and organic lesions of the central nervous system occurred only in children with congenital pneumonia. It has been demonstrated that the severity of congenital pneumonia in the examined newborns is determined by the contamination of the body with pathogenic flora (Pseudomonas aeruginosa, St. aureus, Klebsiella, Candida). Conclusion. Applying a set of diagnostic criteria for early detection of congenital pneumonia and its progression in children with Chlamydia infection enables to verify the diagnosis and timely prescribe etiotropic therapy in the first days of newborns’ life.

Levels of Circulating DNA in Blood Serum and DNA Damage in Leukocytes of Healthy Donors of Different Genders and Ages

Levels of Circulating DNA in Blood Serum and DNA Damage in Leukocytes of Healthy Donors of Different Genders and Ages

PTEN mediates serum deprivation-induced cytotoxicity in H9c2 cells via the PI3K/AKT signaling pathway

The pathogenesis of acute myocardial infarction (AMI) is associated with cardiomyocyte necrosis and apoptosis. Numerous studies have determined the regulatory effects of Phosphatase and tensin homolog (PTEN) cell proliferation and apoptosis in other cell types. However, the potential role of PTEN in cardiomyocyte is unclear. In this study, we used H9c2 cells cultured under serum deprivation to simulate the apoptosis process of myocardial infarction. Small interference RNA (siRNA) of PTEN was used to knock down the expression of PTEN. Cell viability was determined by CCK-8. Cell proliferation was examined by Edu staining, and the protein expression was analyzed by Western blot. We also evaluated the generation of ROS, the degree of DNA damage, and cell apoptosis using immunofluorescence assay. As a result, we observed that serum deprivation in H9c2 cells increased PTEN expression. Functionally, the PTEN knockdown experiment using siRNA inhibited serum deprivation-induced cell apoptosis, ROS production, and DNA damage, whereas increased cell proliferation. All these effects could be reversed by phosphatidylinositol 3-kinase (PI3K) inhibitor, which indicated the PI3K/protein kinase B (AKT) might be the critical component of the PTEN effects during serum deficiency. In conclusion, our study indicated the role of the PTEN/PI3K/AKT pathway in serum deprivation-induced cytotoxicity in H9c2 cells.

The mediation role of SOCS3 methylation in the effect of serum testosterone on type 2 diabetes.

Previous studies reported that testosterone and DNA methylation of suppressor of cytokine signaling-3 (SOCS3) were associated with type 2 diabetes (T2D). Testosterone affects SOCS3 gene expression. Therefore, we aimed to investigate how the SOCS3 methylation mediates the relationship between testosterone and T2D among Chinese rural adults. A case-control study comprised 365 T2D patients and 651 controls was conducted. Liquid chromatography-tandem mass spectrometry and MethylTarget were used to determine the levels of serum testosterone and DNA methylation of SOCS3 gene, respectively. The odds ratio (OR) of testosterone or SOCS3 methylation for T2D was calculated using logistic regression models, and β value of testosterone for SOCS3 methylation was evaluated by linear regression models. Furthermore, through mediation analysis the mediating effect of SOCS3 methylation on the association of testosterone with T2D was estimated. After adjusting for multiple variables, the protective effect of testosterone on T2D was found in men (OR=0.61, 95% confidence interval [CI]: 0.47-0.80), and the methylation of Chr17:76356190 or Chr17:76356199 was negatively related to T2D in both men and women. Moreover, testosterone was positively associated with Chr17:76356190 methylation in men and Chr17:76356199 methylation in women (both P < .05). The mediation analysis showed that the Chr17:76356190 methylation partly mediated effect of testosterone on T2D in men was approximately 8.2%. High levels of serum testosterone in men and Chr17:76356190 and Chr17:76356199 (SOCS3) methylation were related to a lower prevalent T2D. In addition, Chr17:76356190 methylation partially mediated the effect of testosterone on T2D in Chinese rural men.

Use of Corneas from Hepatitis B Positive Donors for Corneal Transplantation

Background: In most of the world, corneas from hepatitis B surface antigen (HBsAg)-positive donors have been considered unsuitable for transplantation because of assumed risk of disease transmission. Methods: In Taiwan, an HBV endemic area, corneas from HBsAg-positive donors can be allocated to recipients who are seropositive for HBsAg or antibody against HBsAg (anti-HBs). Between January, 2015 and December, 2019, we evaluated HBV DNA in serum and corneo-scleral rims retrieved from HBsAg-positive and HBsAg-negative donors. We assessed donor serum HBsAg, anti-HBs, hepatitis B core antibody (anti-HBc) and HBV DNA to predict the presence of their corneo-scleral HBV DNA. We conducted a look-back study of recipients transplanted with corneo-scleral HBV DNA-positive donor grafts. Their HBV serological profiles were examined to identify possible post-transplantation hepatitis B transmission. Findings: Forty-five and 125 corneal grafts from 28 HBsAg-positive and 85 HBsAg-negative donors, respectively, were included. The donor corneo-scleral rim HBV DNA-positive rate was significantly greater in HBsAg-positive donor grafts (80·0%) compared to that of the HBsAg-negative grafts (6·4%, [ P <0·001]). Eight grafts harvested from HBsAg-negative, but anti-HBc-positive donors carried HBV DNA. The donor anti-HBc-positivity provides the highest sensitivity (1·00) and negative predictive value (1·00) in predicting the existence of corneo-scleral HBV DNA. A total 28 out of 40 (70·0%) recipients transplanted with HBV DNA-positive grafts participated in the look-back study, and none developed clinical disease or seroconversion to indicate post-transplantation hepatitis B. InterpretationHBV DNA was detected on corneas from HBsAg-positive and HBsAg-negative, anti-HBc-positive donors. However, the risk of post-transplantation hepatitis B is small in an endemic area. Allocation of corneas from HBsAg-positive or anti-HBc-positive donors to HBsAg-positive or anti-HBs-positive recipients may be a safe and effective way to expand corneal donor pools in hepatitis B endemic areas of the world. Funding: Ministry of Health and Welfare, Taiwan (TORSC-1031006, 1041130, 1051220, 1061030, 1071030) Declaration of Interest: All of the authors have no competing interests. Ethical Approval: The corneo-scleral HBV DNA study was approved by National Taiwan University Hospital Research Ethics Committee (NTUH REC) (No. 201702010RINC).Donor pre-mortem serologies of HBsAg, anti-HBs, and anti-HBc, as well as donor corneal endothelial cell densities were retrieved from the electronic databases of the National Eye Bank of Taiwan.

Trends in Hepatocellular Carcinoma Incidence and Risk Among Persons With HIV in the US and Canada, 1996-2015

People with HIV (PWH) are often coinfected with hepatitis B virus (HBV) and/or hepatitis C virus (HCV), leading to increased risk of developing hepatocellular carcinoma (HCC), but few cohort studies have had sufficient power to describe the trends of HCC incidence and risk among PWH in the combination antiretroviral therapy (cART) era. To determine the temporal trends of HCC incidence rates (IRs) and to compare rates by risk factors among PWH in the cART era. This cohort study used data from the North American AIDS Cohort Collaboration on Research and Design (NA-ACCORD) study, which was conducted between 1996 and 2015. NA-ACCORD pooled individual-level data from 22 HIV clinical and interval cohorts of PWH in the US and Canada. PWH aged 18 years or older with available CD4 cell counts and HIV RNA data were enrolled. Data analyses were completed in March 2020. HBV infection was defined as detection of either HBV surface antigen, HBV e antigen, or HBV DNA in serum or plasma any time during observation. HCV infection was defined by detection of anti-HCV seropositivity, HCV RNA, or detectable genotype in serum or plasma at any time under observation. HCC diagnoses were identified on the basis of review of medical records or cancer registry linkage. Of 109 283 PWH with 723 441 person-years of follow-up, the median (interquartile range) age at baseline was 43 (36-51) years, 93 017 (85.1%) were male, 44 752 (40.9%) were White, 44 322 (40.6%) were Black, 21 343 (19.5%) had HCV coinfection, 6348 (5.8%) had HBV coinfection, and 2082 (1.9%) had triple infection; 451 individuals received a diagnosis of HCC by 2015. Between the early (1996-2000) and modern (2006-2015) cART eras, the crude HCC IR increased from 0.28 to 0.75 case per 1000 person-years. HCC IRs remained constant among HIV-monoinfected persons or those coinfected with HBV, but from 1996 to 2015, IRs increased among PWH coinfected with HCV (from 0.34 cases/1000 person-years in 1996 to 2.39 cases/1000 person-years in 2015) or those with triple infection (from 0.65 cases/1000 person-years in 1996 to 4.49 cases/1000 person-years in 2015). Recent HIV RNA levels greater than or equal to 500 copies/mL (IR ratio, 1.8; 95% CI, 1.4-2.4) and CD4 cell counts less than or equal to 500 cells/μL (IR ratio, 1.3; 95% CI, 1.0-1.6) were associated with higher HCC risk in the modern cART era. People who injected drugs had higher HCC risk compared with men who had sex with men (IR ratio, 2.0; 95% CI, 1.3-2.9), adjusted for HBV-HCV coinfection. HCC rates among PWH increased significantly over time from 1996 to 2015. PWH coinfected with viral hepatitis, those with higher HIV RNA levels or lower CD4 cell counts, and those who inject drugs had higher HCC risk.

Circulating Cell-Free DNA Methylation Profiles in the Early Detection of Ovarian Cancer: A Scoping Review of the Literature.

Simple SummaryThere are limited non-invasive methods for detecting epithelial ovarian cancer despite early detection and treatment dramatically increasing survival. As alterations in serum or plasma cell-free (cf)DNA methylation occur early in cancer development, they are promising biomarkers for ovarian cancer. Our literature review includes 18 studies depicting a wide array of gene targets and techniques. The data suggest a good performance of these cfDNA methylation tests, with accuracies up to 91% in detecting ovarian cancer in serum or plasma.Epithelial ovarian cancer is the most lethal gynecologic malignancy and has few reliable non-invasive tests for early detection or diagnosis. Recent advances in genomic techniques have bolstered the utility of cell-free DNA (cfDNA) evaluation from peripheral blood as a viable cancer biomarker. For multiple reasons, comparing alterations in DNA methylation is particularly advantageous over other molecular assays. We performed a literature review for studies exploring cfDNA methylation in serum and plasma for the early diagnosis of ovarian cancer. The data suggest that serum/plasma cfDNA methylation tests have strong diagnostic accuracies for ovarian cancer (median 85%, range 40–91%). Moreover, there is improved diagnostic performance if multiple genes are used and if the assays are designed to compare detection of ovarian cancer with benign pelvic masses. We further highlight the vast array of possible gene targets and techniques, and a need to include more earlier-stage ovarian cancer samples in test development. Overall, we show the promise of cfDNA methylation analysis in the development of a viable diagnostic biomarker for ovarian cancer.

Detection and analysis of blood donors seropositive for syphilis.

The increasing incidence of syphilis worldwide has called attention to the risk of transmission by transfusion. To determine the prevalence of active syphilis in blood donors and characterise the serological profile of syphilis-positive donors. Samples positive for Treponema pallidum using the chemiluminescent microparticle immunoassay (CMIA) during blood donor screening from 2017 to 2018 were tested by the Venereal Disease Research Laboratory (VDRL) non-treponemal test and for anti-T. pallidum IgM by ELISA (Immunoassay Enzyme test for detection of IgM antibodies). The INNO-LIA Syphilis test (Line Immuno Assay solid test for confirmation antibodies to Treponema pallidum) was performed as a confirmatory test on samples that were positive on ELISA-IgM but negative on VDRL. ELISA-IgM (+) samples were also tested for T. pallidum DNA in sera by real-time polymerase chain reaction (PCR). Of 248 542 samples screened, 1679 (0.67%) were positive for syphilis by CMIA. Further analysis was performed on 1144 (68.1%) of these samples. Of those tested, 16% were ELISA IgM(+)/VDRL(+), 16.5% were ELISA IgM(-)/VDRL(+), 4.1% were ELISA IgM(+)/VDRL(-), and 63.4% were ELISA IgM (-)/VDRL(-). The INNO-LIA Syphilis test results were 33 (3%) positive, 2 (0.2%) undetermined and 12 (1%) negative. Of the 230 EIA-IgM(+) samples (20.1%), 5 (2.2%) were PCR positive. The prevalence of active syphilis in 2017 and 2018 was 0.1% and 0.07%, respectively, and overall prevalence of serologic markers for syphilis was highest among male, unmarried, 25-34-year-olds with a high school education and who were first-time donors. There is a risk of transfusion-transmitted syphilis in blood banks that exclusively use the VDRL test for donor screening, as is currently the situation in some Brazilian blood centres, as well as in other blood centres around the world.

Validation of real-time polymerase chain reaction versus conventional polymerase chain reaction for diagnosis of brucellosis in cattle sera

Background and Aim:Different polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and Methods:One hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.Results:TaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.Conclusion:A cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Evaluation of Cytotoxic T-Lymphocyte Antigen-4 (+49A/G) Gene Polymorphism in Chronic Hepatitis B Virus Infection

Background: Chronic hepatitis B (CHB) infection is associated with the depletion of T cells, resulting in weak or absent virus specific T cells reactivity, which is described as ‘exhaustion’. This exhaustion is characterized by impaired cytokine production and sustained expression of multiple coinhibitory molecules. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is one of many coinhibitory molecules that can attenuate T cell activation by inhibiting stimulation and transmitting inhibitory signals to T cells. Objective: To explore the effect of CTLA-4+49A/G single nucleotide polymorphism (SNP) on the progression CHB in Iraqi patients. Methods: Blood serum and genomic DNA was isolated from 90 patients with CHB. Tetra-Primer Amplification Refractory System-Polymerase Chain Reaction (ARMS-PCR) was used for amplification and genotyping of CTLA-4 gene using specific primers, and plasma hepatitis B virus (HBV) viral load was investigated by real time PCR, in addition to estimate the hepatitis B e antigen (HBeAg) and anti-HBe by enzyme-linked immunosorbent assay (ELISA). Results: AA genotype was more frequent among uncomplicated than complicated CHB (44.83% versus 28.12%) with a significant difference (OR= 0.315, 95%CI=1.0-0.99, p= 0.048). Conclusion: These data strongly suggested the persistence role of CTLA-4+49 polymorphism against HBV among Iraqi patients. Keywords: CTLA 4, SNP, ARMS-PCR Citation: Mahdi YS, Kadhim HS. Evaluation of cytotoxic T-lymphocyte Antigen-4 (+49A/G) gene polymorphism in chronic hepatitis B virus infection. Iraqi JMS. 2020; 18(2): 101-109. doi: 10.22578/IJMS.18.2.3

DNA methylation biomarkers in peripheral blood of patients with head and neck squamous cell carcinomas. A systematic review.

BackgroundHead and neck squamous cell carcinomas (HNSCC) are often diagnosed in advanced stages. In search of new diagnostic tools, focus has shifted towards the biological properties of the HNSCC, and the number of different biomarkers under investigation is rapidly growing.ObjectivesThe objective was to review the current literature regarding aberrantly methylated DNA found in peripheral blood plasma or serum in patients with HNSCC and to evaluate the diagnostic accuracy of these changes.MethodsThe inclusion criteria were clinical studies involving patients with verified HNSCC that reported findings of aberrantly methylated DNA in peripheral blood serum or plasma. We systematically searched PubMed, OVID Embase and Cochrane Library. In addition to the search, we performed forward and backward chaining in references and Web of Science. The protocol was registered in PROSPERO: CRD42019135406. Two authors independently extracted data. The quality and the risk of bias of the included studies were assessed by the QUADAS-2 tool.ResultsA total of 1,743 studies were found eligible for screening, while ultimately seven studies were included. All studies were found to have methodological weaknesses, mainly concerning patient selection bias. The best individual marker of HNSCC was Septin 9 in plasma with a sensitivity of 57% and a specificity of 95%.ConclusionsNone of the aberrantly methylated genes found in the retrieved studies are applicable as single diagnostic markers for HNSCC and the best gene-panels still lack diagnostic accuracy. Future studies may benefit from newer sequencing techniques but validation studies with well-designed cohorts are also needed in the process of developing epigenetic based diagnostic tests for HNSCC.

Antimicrobial and antitumor activity of S-methyl dithiocarbazate Schiff base zinc(II) complexes

Schiff bases (SB) obtained from S-methyl dithiocarbazate and aromatic aldehydes: salicylaldehyde (H2L1), o-vanillin (H2L2), pyridoxal (H2L3) and 2,6-diformyl-4-methylphenol (H3L4), and their corresponding Zn(II)-complexes (1–4), are synthesized. All compounds are characterized by elemental analyses, infrared, UV–Vis, nuclear magnetic resonance spectroscopy and mass spectrometry. The structures of H2L2 and [Zn2(L1)2(H2O)(DMF)] (1a) (DMF = dimethylformamide) are solved by single crystal X-ray diffraction. The SB coordinates the metal center through the Ophenolate, Nimine and Sthiolate atoms. The radical scavenging activity is tested using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, with all ligand precursors showing IC50 values ~40 μM. Cytotoxicity studies with several tumor cell lines (PC-3, MCF-7 and Caco-2) as well as a non-tumoral cell line (NHDF) are reported. Interestingly, 1 has relevant and selective antiproliferative effect against Caco-2 cells (IC50 = 9.1 μM). Their antimicrobial activity is evaluated in five bacterial strains (Klebsiella pneumoniae, Acinetobacter baumannii, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus) and two yeast strains (Candida albicans and Candida tropicalis) with some compounds showing bacteriostatic and fungicidal activity. The minimal inhibitory concentration (MIC90) of HnL against Mycobacterium tuberculosis is also reported, with H2L2 and H3L4 showing very high activity (MIC90 < 0.6 μg/mL). The ability of the compounds to bind bovine serum albumin (BSA) and DNA is evaluated for H3L4 and [Zn2(L4)(CH3COO)] (4), both showing high binding constants to BSA (ca. 106 M−1) and ability to bind DNA. Overall, the reported compounds show relevant antitumor and antimicrobial properties, our data indicating they may be promising compounds in several fields of medicinal chemistry.

Analysis of spread of porcine circovirus type 3 detected in Russia

Porcine circovirus type 3 (PCV-3) — a recently detected member of Circoviridae family. The virus has been identified as a possible infectious agent. Clinical signs vary from stillbirth and fetus mummification, encephalitis and myocarditis in perinatal period to periarteritis in fattening period. The PCV-3 widespread has been demonstrated in major pork development countries. Establishing of the association between presence of this pathogen and disease development makes PCV3 diagnostic studies in Russia necessary. The main goal of this investigation was to detect PCV3 DNA in blood serum, saliva and bronchioalveolar lavage and pathological material in sows and piglets of different age groups at two units at an industrial swine farm. For PCV-3 detection we used PCR method. Results of this investigation revealed that PCV3 was detected in all types of biomaterial. At the unit 1 PCV3 DNA has been detected in saliva (highes ratio of infected animals was 60% in 21- and 60–70 day-old piglets), serum (100% in 150–160-day-old piglets) and bronchioalveolar fluid (40% in 90–100-day-old piglets). It was found that at the unit 2 level of piglets PCV-3 shedding was higher (50–100%) than at the unit 1 (0–60%). Moreover, PCV3 DNA has been detected in 70% of pathological material obtained from aborted sows and in 80% of their amniotic fluid.

A “turn-on” and proximity ligation assay dependent DNA tweezer for one-step amplified fluorescent detection of DNA

A “turn-on” and proximity ligation assay dependent DNA tweezer was proposed for one-step amplified fluorescent detection of DNA. Target DNA can anneal with capture probe to form an entire long sequence. The formed long sequence can circularly open the hairpin, resulting the “turn-on” of DNA tweezers. A good linear relationship was shown from 40 pM to 20 nM with limit of detection of 10 pM. In addition, it has been successfully utilized to analysis DNA in human serum, representing a great and practical application future.

Thiamethoxam at sublethal concentrations induces histopathological, serum biochemical alterations and DNA damage in fish (Labeo rohita)

The effects of thiamethoxam neonictinoids insecticide (TMX) commonly used in different food crops and in veterinary practice were evaluated in freshwater fish exposed to sublethal concentrations for 120 h. A total of 60 freshwater fish Labeo rohita were collected from local fish rearing center and kept in different five equal groups (A–E). Fish in groups B–E were exposed to sublethal concentrations of thiamethoxam (0, 0.5, 1.0, 1.5, and 2.0 mg/L) to determine the genotoxic potential and serum biochemical effects. Results showed significantly (p<.05) increased concentrations of urea and creatinine in thiamethoxam-treated fish as compared to untreated control fish. The concentrations of liver function tests (aspartate aminotransferase, aspartate aminotransferase, and alkaline phosphatase), cardiac functions tests (cholesterol, triglycerides), and lipid peroxidation product (malondialdehyde concentrations) were significantly increased while serum total proteins and globulin significantly (p<.05) decreased in thiamethoxam exposed fish. Furthermore, blood DNA contents significantly increased in thiamethoxam treated fish. At histopathological level, fatty infiltration, congestion, disruption and degenerations of bile ducts, abnormal sinusoids, nuclear fragmentation, condensation, and eccentric nuclei were observed in liver of thiamethoxam treated fish. Histological observation of kidneys of treated fish showed edema, degeneration of renal tubules, congestion, necrosis of renal tubular cells, glomerular degeneration, and detachment of tubular epithelium. DNA damage by comet assay was significantly increased in hepatocytes, kidneys, and blood cells. In conclusion, the results of our study indicate that thiamethoxam induces blood biochemical, genotoxic, and histopathological alterations in exposed fish.

Copy number variations in primary tumor, serum and lymph node metastasis of bladder cancer patients treated with radical cystectomy

The aim of the present study was to analyze copy number variations (CNV) of multiple oncogenes and tumor suppressor genes in genomic DNA from primary tumor tissue, lymph node metastasis and cell-free DNA (cfDNA) from serum of 72 urothelial carcinoma of bladder (UCB) patients treated with radical cystectomy (RC), using multiplex ligation-dependent probe amplification (MLPA). We hypothesized that primary tumor and lymph node metastasis show similar CNV profiles, and CNV are more present in lymph node metastasis compared to primary tumor tissue. Samples from 43 (59.7%) patients could be analyzed. In total, 35 (83%), 26 (68%) and 8 (42%) patients had CNV in primary tumor, serum and lymph node metastasis, respectively. MYC, CCND1, ERBB2 and CCNE1 displayed the most frequent amplifications. In particular, CNV in ERBB2 was associated with aggressive tumor characteristics. CNV in both ERBB2 and TOP2A were risk factors for disease recurrence. The current findings show that CNV are present in various oncogenes and tumor suppressor genes in genomic DNA from primary tumor, lymph node metastasis and cfDNA from serum. CNV were more present in genomic DNA from primary tumor tissue compared to cfDNA from serum and genomic DNA from lymph node metastasis. Patients with CNV in ERBB2 and TOP2A are at increased risk for disease recurrence following RC. Further studies are necessary to validate, whether these genes may represent promising candidates for targeted-therapy.

A novel water soluble axially substituted silicon(IV) phthalocyanine bearing quaternized 4-(4-pyridinyl)phenol groups: Synthesis, characterization, photophysicochemical properties and BSA/DNA binding behavior

The novel axially bis[4-(4-pyridinyl)phenol] substituted silicon(IV) phthalocyanine (3) and its quaternized derivative (3Q) were designed and synthesized. These phthalocyanines were fully characterized by elemental analysis and different spectroscopic methods, such as FT-IR, UV–Vis, MALDI-TOF and 1H NMR. The photophysical and photochemical properties, such as electronic absorption, fluorescence emission, fluorescence lifetimes, singlet oxygen generation and photostability, of these phthalocyanines were investigated. These properties were examined in DMSO for the non-ionic phthalocyanine (3) and in both DMSO and aqueous solution for the ionic phthalocyanine (3Q). The addition of bis[4-(4-pyridinyl)phenol] groups as axial ligands showed an improvement of the photophysical and photochemical properties. In addition, a spectroscopic investigation of the binding behavior of the quaternized silicon(IV) phthalocyanine complex to bovine serum albumin (BSA) and DNA was also studied in this work.

Ratiometric Detection of DNA and Protein in Serum by a Universal Tripyridinyl RuII Complex-Encapsulated SiO2@Polydopamine Fluorescence Nanoplatform.

Fluorescence ratiometric biosensors are valuable tools for the accurate and sensitive prediction and diagnosis of diseases. However, seldom have fluorescence ratiometric biosensors for protein and DNA been reported because of the shortage of suitable nanoscale scaffolds. Herein, a tripyridinyl RuII complex-encapsulated SiO2@polydopamine (Ru-SiO2@PDA) nanocomposite was designed as a universal platform for fluorescence ratiometric detection of DNA and protein in serum samples. The Ru-SiO2@PDA nanocomposites have a narrow size distribution, exhibit good biosafety, and are convenient for the postmodification of biorecognition elements. Under irradiation, they can emit a stable and strong luminescence at 650 nm and simultaneously quench the fluorescence emitted from the fluorophores getting close to them. Once the capture probes such as single-stranded DNA and aptamer are assembled, the fluorophores labeled on them are then brought close to their PDA shell and quenched. However, the biorecognition behaviors change the probe's configuration and take the fluorophore far away from the PDA shell. Correspondingly, the fluorescence recovers and its ratio to the constant fluorescence reference is linear to the targets' concentration. Using a D-catalyst and thrombin as model analytes, the Ru-SiO2@PDA-based nanoplatform shows high sensitivity and good accuracy in the serum sample analysis. Regarding these attractive properties, the Ru-SiO2@PDA nanoplatform provides a new avenue for the accurate and sensitive fluorescence assay of a wide range of targets in complex systems.

CLINICAL AND INSTRUMENTAL CHARACTERISTICS OF NEUROSONOGRAPHIC CHANGES DETECTED IN DYNAMICS IN CHILDREN WITH CONGENITAL CHLAMIDIA INFECTION

Introduction. The problem of congenital chlamydial infection is still remaining relevant due to the difficulties in its diagnosis and peculiarities of the course. The aim of this study was to clarify the dynamics of the severity and nature of the affection of the central nervous system in the case of congenital chlamydial infection. Materials and methods. 103 newborns were examined. Catamnestic observations were performed during a year. Specific diagnosis was made by polymerase chain reaction with the detection of chlamydia DNA in the blood serum of newborns. The main group included 80 children; the second control group consisted of 23 healthy children. The group III involved 36 children assessed as having satisfactory health status at birth, whose mothers had Chlamydia infection. Results and discussion. Analysis of the results showed that visceral forms with congenital chlamydial pneumonia were diagnosed in almost 50% of newborns; localized forms (congenital conjunctivitis) were diagnosed in 7.5% of children. 45% of newborns of the main group had no clinical signs of the disease. Neurosonography was performed in dynamics. It demonstrated that lenticular vasculopathy, areas of increased echogenicity, thickening of the ventricular walls, both alone and in combination were found only in the children of the main group and could be regarded as markers of intrauterine infection. Neurosonographic examination during the 1 year of life of the children in the main group indicated the presence of pathological changes. In the children of the control group markers of intrauterine infection were not detected. Thus, the obtained neurosonographic data confirm the long-term and damaging effects of chlamidia infection in the children of the main group. Conclusion. Pathological changes detected by neurosonography persisted during the first year of life and correlated with the formation of stato-kinetic developmental delay, muscular dystonia syndrome, and cerebral palsy in children.

Revisiting Brucellosis in Small Ruminants of Western Border Areas in Pakistan.

Brucellosis, globally known bacterial zoonosis, is endemic to Pakistan. B. abortus in bovines, B. melitensis in small ruminants and B. canis in dogs mainly cause this disease. A total of 1821 sera (1196 from sheep and 625 from goats) from animal herds near the Pakistan–Afghanistan border were collected. In parallel testing of sera for anti-Brucella antibodies (B. abortus and B. melitensis) was carried out by RBPT and indirect ELISA. The presence of Brucella DNA in sera was tested by real-time PCR. The overall percentage of seropositive samples was 0.99 (18/1821) by both tests. All positive samples originated from Baluchistan territory which translated into 1.76% (18/1021). None of the positive sera had signals for Brucella DNA and none of sera from goats carried detectable antibodies. Both tests showed an almost perfect agreement with Kappa statistics. The flock size was found to be associated with the presence of anti-Brucella antibodies. The samples of Khyber Pakhtunkhwa (KPK) tested negative in both serological tests and hence were not processed for real-time PCR. The present study shows the presence of anti-Brucella antibodies in sheep in the Baluchistan region of Pakistan. Diagnostic services need to be improved and test and slaughter policies might be implemented for eradication of Brucella infection in these areas. Awareness about the infection is needed at the farmer’s level. Isolation and molecular biology of the isolates could help with understanding the prevailing etiology in a better way.