Identification of microRNA biomarkers simultaneously expressed in circulating extracellular vesicles and atherosclerotic plaques.

Background: Atherosclerosis is a widespread disorder of the cardiovascular system. The early detection of plaques by circulating biomarkers is highly clinically relevant to prevent the occurrence of major complications such as stroke or heart attacks. It is known that extracellular vesicles (EVs) are important in intercellular communication in atherosclerotic disorders and carry many components of their cells of origin, including microRNAs (miRNAs). In this study, we test the assumption that miRNAs present in material acquired from plaques in patients undergoing surgery for atherosclerotic carotid artery stenosis are also expressed in circulating EVs obtained from the identical patients. This would allow the adoption of a liquid biopsy approach for the detection of plaques. Methods: We studied 22 surgical patients with atherosclerotic carotid arterial stenosis and 28 healthy controls. EVs were isolated from serum by precipitation. miRNA expression profiles of serum-derived EVs were obtained by small RNA sequencing and in plaque material simultaneously acquired from patients. A comparative analysis was performed to identify circulating atherosclerosis-associated miRNAs that are also detectable in plaques. Results: Seven miRNAs were found to be differentially regulated in patient serum compared with the serum of healthy controls. Of these, miR-193b-5p, miR-193a-5p, and miR-125a-3p were significantly upregulated in patients compared with that in healthy controls and present in both, circulating EVs and plaque material. An overrepresentation analysis of experimentally validated mRNA targets revealed an increased regulation of inflammation and vascular growth factors, key players in atherosclerosis and plaque formation. Conclusion: Our findings suggest that circulating EVs reflect plaque development in patients with symptomatic carotid artery stenosis, which can serve as biomarker candidates for detecting the presence of atherosclerotic plaques.

The proteome of bacterial membrane vesicles in Escherichia coli-a time course comparison study in two different media.

Introduction: Bacteria inhabit the in- and outside of the human body, such as skin, gut or the oral cavity where they play an innoxious, beneficial or even pathogenic role. It is well known that bacteria can secrete membrane vesicles (MVs) like eukaryotic cells with extracellular vesicles (EVs). Several studies indicate that bacterial membrane vesicles (bMVs) play a crucial role in microbiome-host interactions. However, the composition of such bMVs and their functionality under different culture conditions are still largely unknown. Methods: To gain a better insight into bMVs, we investigated the composition and functionality of E. coli (DSM 105380) bMVs from the culture media Lysogeny broth (LB) and RPMI 1640 throughout the different phases of growth (lag-, log- and stationary-phase). bMVs from three time points (8 h, 54 h, and 168 h) and two media (LB and RPMI 1640) were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis (NTA), cryogenic electron microscopy (Cryo-EM), conventional transmission electron microscopy (TEM) and mass spectrometry-based proteomics (LC-MS/MS). Furthermore, we examined pro-inflammatory cytokines IL-1ß and IL-8 in the human monocyte cell line THP-1 upon bMV treatment. Results: Particle numbers increased with inoculation periods. The bMV morphologies in Cryo-EM/TEM were similar at each time point and condition. Using proteomics, we identified 140 proteins, such as the common bMV markers OmpA and GroEL, present in bMVs isolated from both media and at all time points. Additionally, we were able to detect growth-condition-specific proteins. Treatment of THP-1 cells with bMVs of all six groups lead to significantly high IL-1ß and IL-8 expressions. Conclusion: Our study showed that the choice of medium and the duration of culturing significantly influence both E. coli bMV numbers and protein composition. Our TEM/Cryo-EM results demonstrated the presence of intact E. coli bMVs. Common E. coli proteins, including OmpA, GroEL, and ribosome proteins, can consistently be identified across all six tested growth conditions. Furthermore, our functional assays imply that bMVs isolated from the six groups retain their function and result in comparable cytokine induction.

The Impact of Age on the Post-operative Outcomes in Patients Undergoing Resection for Oesophageal and Gastric Cancer.

BACKGROUND: Within our ageing population, there is an increasing number of elderly patients presenting with oesophagogastric cancer. Resection remains the mainstay of curative treatment however it has substantial morbidity. The aim of this study was to assess whether age was an independent predictor of resection related complications in our unit. METHODS: A retrospective cohort study of prospectively collated data from 2002 to 2020 of patients undergoing resection for oesophageal and gastric cancers was analysed. Patients aged over 75 and 75 and under were compared for peri-operative morbidity (via the Clavien-Dindo classification), length of stay (LOS), unplanned readmission, 30- and 90-day mortality, and use of neoadjuvant therapy. RESULTS: Data for 466 consecutive patients undergoing oesophagogastric resection (277 oesophagectomy and 189 gastrectomy) were available for analysis. 22% of patients were aged over 75 (14% (39/277) of the oesophagectomy cohort, 34% (65/189) of the gastrectomy cohort). Oesophagectomy patients over 75 were more likely to develop post-operative complications, particularly cardiac or thromboembolic, (69.2%) than those in the younger cohort (50.4%, p = 0.029). There was no difference in complication rates between the younger and older patients undergoing gastrectomy (29.0% vs. 33.9% p = 0.495). The 30- and 90-day mortality rates were 1.4% (n = 4) and 2.5% (n = 7), respectively, for the oesophagectomy cohort and 1.1% (n = 2) and 1.6% (n = 3) for the gastrectomy cohort, with no difference between age groups. CONCLUSION: In this series, we found that patients over the age of 75 were able to undergo oesophageal and gastric resection with curative intent with acceptable post-operative morbidity and mortality.

Discovering the Unclassified Suicide Cases Among Undetermined Drug Overdose Deaths Using Machine Learning Techniques.

OBJECTIVE: The Centers for Disease Control and Prevention (CDC) monitor accidental and intentional deaths to answer questions that are critical for the development of effective prevention and resource allocation. CDC's National Violent Death Reporting System (NVDRS) is a major innovation in surveillance linking individual-level data from multiple sources. However, suicide underreporting is common, particularly from drug overdose deaths. This study sought to assess machine learning (ML) techniques in quantifying drug overdose suicide underreporting rates. METHODS: Clinical, sociodemographic, toxicological, and proximal stressor data on overdose decedents (n = 2,665) were extracted from Utah's NVDRS from 2012 to 2015. The existing well-determined cases were used to train and test our ML models. We assessed and compared multiple machine learning methods including Logistic Regression, Random Forest Classifier, Support Vector Machines, and Artificial Neural Networks. We applied a majority voting methodology to classify undetermined drug overdose deaths. RESULTS: Overdose suicide rates were estimated to be underreported by 33% across all years, increasing yearly from 29% in 2012 to 37% in 2015. The overall test accuracies for all models ranged from 92.3% to 94.6%. CONCLUSIONS: This research identifies a cost-effective, replicable, and expandable ML-based methodology to estimate the true rates of suicide which may be partially masked during the opioid epidemic.

[Pulmonary embolism in patients with chronic obstructive pulmonary disease exacerbations of unknown origin: clinical characteristics and risk factors].

OBJECTIVE: To evaluate the prevalence of pulmonary embolism(PE) in patients with chronic obstructive pulmonary disease (COPD) exacerbations of unknown origin and to explore the risk factors associated with PE. METHODS: A total of 208 consecutive patients with COPD were referred to this hospital for severe exacerbations of unknown origin. Their age was 50 - 82 years, with a mean of (62 ± 12) years. All patients were examined within 48 h of admission by CT pulmonary angiography (CTPA) and lower extremity ultrasonography. The patients were classified as PE positive (positive results on CTPA) or PE negative (negative results on CTPA). Arterial blood gas, the levels of D-dimer and ET-1 were measured in all the patients. Differences between groups were analyzed using a two-tailed unpaired t test for normally distributed variables and a Mann-Whitney u test for non-normally distributed variables. Qualitative data were assessed using chi-square test, and risk factors were analyzed using logistic regression analysis. RESULTS: The frequency of PE was 33% in this series of 208 consecutive patients with COPD referred for exacerbations of unknown origin. There were differences between PE positive and PE negative groups in the following factors (χ(2) = 4.32 - 6.79, mean P < 0.05): immobilization ≥ 7 days 21.7% (15/208) vs 13.7% (19/208); difference in circumference of lower limbs ≥ 1 cm 34.8% (24/208) vs 15.1% (21/208); deep venous thrombosis (DVT) 37.7% (26/208) vs 12.2% (17/208); syncope 11.6% (8/208) vs 0.06% (9/208); S(I)Q(III)T(III) syndrome 11.6% (8/208) vs 0.04% (5/208); decrease in PaCO2 ≥ 5 mm Hg (1 mm Hg = 0.133 kPa) 27.5% (19/208) vs 9.3% (13/208). Plasma D-dimer and ET-1 levels were significantly higher in patients with PE as compared to patients without PE. D-dimer levels were (760 ± 152) µg/L and (253 ± 56) µg/L (Z = -2.946, P < 0.01); ET-1 levels were 5.4 ng/L (1.6 - 6.9 ng/L) and 1.8 ng/L (1.3 - 4.8 ng/L), Z = -2.532, P < 0.01. Risk factors identified by logistic regression analysis included immobilization ≥ 7 days (P < 0.05, OR = 3.24, 95%CI = 1.56 - 4.98), difference in circumference of lower limbs ≥ 1 cm (P < 0.05, OR = 2.56, 95%CI = 1.48 - 3.93), and deep venous thrombosis (DVT) (P < 0.05, OR = 2.31, 95%CI = 1.23 - 3.58). CONCLUSIONS: This study showed a 33% prevalence of PE in patients with COPD who were hospitalized for severe exacerbations of unknown origin. Immobilization ≥ 7 days, difference in circumference of lower limbs ≥ 1 cm, and DVT were risk factors for PE in this group of patients.

Torpedoes in the cerebellar vermis in essential tremor cases vs. controls.

The study of the postmortem changes in essential tremor (ET) is in its infancy, although recent evidence points to a central role of the cerebellum, where Purkinje cell axonal swellings ("torpedoes") are significantly more common in ET than control brains. Yet, all existing studies have been confined to the cerebellar hemispheres, and whether there is a more widely distributed cerebellar problem is presently unknown. Our aims were to address whether: (1) ET cases have greater numbers of torpedoes in the vermis than controls, (2) there a correlation between the extent of vermal torpedo pathology and hemispheric torpedo pathology, and (3) vermal torpedo pathology is correlated with clinical features of the disease. A parasagittal neocerebellar block and a vermal block were harvested from 24 ET and 10 control brains. Paraffin sections (7 µm) were stained with Luxol fast blue/hematoxylin and eosin, and torpedoes were quantified. All torpedo counts were corrected for Purkinje cell layer length. Vermal corrected torpedo count (VermTc) was higher in ET cases than controls (7.1±6.8 [median, 4.3] vs. 2.6±2.5 [median, 2]), p=0.002). The VermTc and the hemispheric corrected torpedo count (HemTc) were correlated with one another (Spearman's r=0.54, p=0.002). ET cases with neck, voice, and jaw tremors had the highest VermTc (p=0.046). The abundance of torpedoes in the ET brain is not confined to the ponto- or neocerebellum but is more broadly distributed, also involving the spino- or paleocerebellum. These data further confirm the central role of the cerebellum in the underlying pathophysiology of this common neurological disorder.

Long-term AZT exposure alters the metabolic capacity of cultured human lymphoblastoid cells.

The antiretroviral efficacy of 3'-azido-3'-deoxythymidine (AZT) is dependent upon intracellular mono-, di-, and triphosphorylation and incorporation into DNA in place of thymidine. Thymidine kinase 1 (TK-1) catalyzes the first step of this pathway. MOLT-3, human lymphoblastoid cells, were exposed to AZT continuously for 14 passages (P(1)-P(14)) and cultured for an additional 14 passages (P(15)-P(28)) without AZT. Progressive and irreversible depletion of the enzymatically active form of the TK-1 24-kDa monomer with loss of active protein was demonstrated during P(1)-P(5) of AZT exposure. From P(15) to P(28), both the 24- and the 48-kDa forms of TK-1 were undetectable and a tetrameric 96-kDa form was present. AZT-DNA incorporation was observed with values of 150, 133, and 108 molecules of AZT/10(6) nucleotides at the 10 microM plasma-equivalent AZT dose at P(1), P(5), and P(14), respectively. An exposure-related increase in the frequency of micronuclei (MN) was observed in cells exposed to either 10 or 800 microM AZT during P(1)-P(14). Analysis of the cell cycle profile revealed an accumulation of S-phase cells and a decrease in G(1)-phase cells during exposure to 800 microM AZT for 14 passages. When MOLT-3 cells were grown in AZT-free media (P(15)-P(29)), there was a reduction in AZT-DNA incorporation and MN formation; however, TK-1 depletion and the persistence of S-phase delay were unchanged. These data suggest that in addition to known mutagenic mechanisms, cells may become resistant to AZT partially through inactivation of TK-1 and through modulation of cell cycle components.

Centrosome amplification induced by the antiretroviral nucleoside reverse transcriptase inhibitors lamivudine, stavudine, and didanosine.

In cultured cells, exposure to the nucleoside reverse transcriptase inhibitor (NRTI) zidovudine (AZT) induces genomic instability, cell cycle arrest, micronuclei, sister chromatid exchanges, and shortened telomeres. In previous studies, we demonstrated AZT-induced centrosome amplification (>2 centrosomes/cell). Here, we investigate centrosome amplification in cells exposed to other commonly used NRTIs. Experiments were performed using Chinese Hamster ovary (CHO) cells, and two normal human mammary epithelial cell (NHMEC) strains: M99005 and M98040, which are high and low incorporators of AZT into DNA, respectively. Cells were exposed for 24 hr to lamivudine (3TC), stavudine (d4T), didanosine (ddI), and thymidine, and stained with anti-pericentrin antibody. Dose response curves were performed to determine cytotoxicity and a lower concentration at near plasma levels and a 10 fold higher concentration were chosen for the experiments. In CHO cells, there was a concentration-dependent, significant (P < 0.05) increase in centrosome amplification for each of the NRTIs. In NHMEC strain M99005, an NRTI-induced increase (P < 0.05) in centrosome amplification was observed for the high concentrations of each NRTI and the low doses of 3TC and ddI. In NHMEC strain M98040, the high doses of ddI and d4T showed significant increases in centrosome amplification. Functional viability of amplified centrosomes was assessed by arresting microtubule nucleation with nocodazole. In cells with more than two centrosomes, the ability to recover microtubule nucleation was similar to that of unexposed cells. We conclude that centrosome amplification is a consequence of exposure to NRTIs and that cells with centrosome amplification are able to accomplish cell division.

Centrosomal amplification and aneuploidy induced by the antiretroviral drug AZT in hamster and human cells.

The centrosome directs chromosomal migration by a complex process of tubulin-chromatin binding. In this contribution centrosomal abnormalities, including centrosomal amplification, were explored in Chinese hamster ovary (CHO) and normal human mammary epithelial cells (NHMECs) exposed to the antiretroviral drug zidovudine (3'-azido-3'-deoxythymidine, AZT). Centrosomal amplification/fragmentation was observed in both cell types and kinetochore positive micronuclei were found in AZT-exposed CHO cells in correlation with dose. Normal human mammary epithelial cell (NMHEC) strain M99005, previously identified as a strain that incorporates high levels of AZT into DNA (high incorporator, HI), showed greater centrosomal amplification when compared with a second strain, NHMEC M98040, which did not incorporate AZT into DNA (low incorporator, LI). Additionally, an abnormal tubulin distribution was observed in AZT-exposed HI cells bearing multiple centrosomes. Immunofluorescent staining of human cells with Aurora A, a kinase involved in the maturation of the centrosome, confirmed the induction of centrosomal amplification and revealed multipolar mitotic figures. Flow cytometric studies revealed that cells bearing abnormal numbers of centrosomes and abnormal tubulin distribution had similar S-phase percentages suggesting that cells bearing unbalanced chromosomal segregation could divide. Therefore, AZT induces genomic instability and clastogenicity as well as alterations in proteins involved in centrosomal activation, all of which may contribute to the carcinogenic properties of this compound.

Relative mutagenic potencies of several nucleoside analogs, alone or in drug pairs, at the HPRT and TK loci of human TK6 lymphoblastoid cells.

Experiments were performed to investigate the impact of didanosine (ddI), lamivudine (3TC), and stavudine (d4T) on cell survival and mutagenicity in two reporter genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK), using a cell cloning assay for assessing the effects of individual nucleoside analogs (NRTIs)/drug combinations in human TK6 B-lymphoblastoid cells. Three-day treatments with 0, 33, 100, or 300 microM ddI, 3TC, or ddI-3TC produced positive trends for increased HPRT and TK mutant frequencies. While dose-related trends were too small to reach significance after treatments with d4T or d4T-3TC, pairwise comparisons with control cells indicated that exposure to 100 microM d4T or d4T-3TC caused significant elevations in HPRT mutants. Measurements of mutagenicity in cells exposed to d4T (or d4T-3TC) were complicated by the cytotoxicity of this NRTI. Enhanced increases in mutagenic responses to combined NRTI treatments, compared with single drug treatments, occurred as additive to synergistic effects in the HPRT gene of cells exposed to 100 microM ddI-3TC or 100 microM d4T-3TC, and in the TK gene of cells exposed to 100 or 300 microM ddI-3TC. Comparisons of these data to mutagenicity studies of other NRTIs in the same system (Meng Q et al. [2000c]: Proc Natl Acad Sci USA 97:12667-126671; Torres SM et al. [2007]: Environ Mol Mutagen) indicate that the relative mutagenic potencies for all drugs tested to date are: AZT-ddI > ddI-3TC > AZT-3TC congruent with AZT-3TC-ABC (abacavir) > AZT >/=ddI > d4T-3TC > 3TC > d4T >/= ABC. These collective data suggest that all NRTIs with antiviral activity against HIV-1 may cause host cell DNA damage and mutations, and impose a cancer risk.