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Ceramides are bioactive lipids that have an important role in many cellular functions such as apoptosis and inflammation. During the past decade emerging clinical data have shown that ceramides are not only of great biochemical interest but may also have diagnostic utility. Ceramides have shown independent predictive value for negative cardiovascular outcomes as well as for the onset of type 2 diabetes. Based on abundant published data, risk score using the concentrations of circulating ceramides have been developed and adapted for routine clinical practice. Currently serum ceramides are used clinically as efficient risk stratifiers for primary and secondary prevention of atherosclerotic cardiovascular disease (CVD). A direct cause-effect relationship between CVD and ceramide has not been established to date. As ceramide-specific medications are being developed, conventional strategies such as lipid lowering agents and lifestyle interventions can be used to reduce overall risk. Ceramides can identify a very high-risk coronary heart disease category of patients in need for more intense medical attention, specifically those patients at higher risk as highlighted in the 2019 European Society of Cardiology guidelines for stable chronic coronary syndrome patients. In addition, the ceramide risk score may be used as a decision-making tool in primary prevention patients with moderate CVD risk. Finally, the ceramide risk score may have a unique utility as a motivational tool to increase patient's adherence to medical therapy and lifestyle changes., (Copyright © 2020 Hilvo, Vasile, Donato, Hurme and Laaksonen.)

Objective: Ceramides are molecular lipids implicated in apoptosis, inflammation, obesity, and insulin resistance. An earlier study reported that ceramides were associated with fatal outcome among patients with coronary heart disease. Here, we examined whether ceramides are associated with major adverse cardiovascular events (MACEs) among apparently healthy individuals., Approach and Results: FINRISK 2002 is a population-based risk factor survey, which recruited men and women aged 25 to 74 years. The cohort was followed up until the end of 2014. We quantified 4 circulating ceramides, Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1), in 8101 serum samples by a targeted liquid chromatography-tandem mass spectrometry assay. Primary outcome of interest was incident MACE (n=813). Secondary analyses were performed for MACE death (n=116) without previous nonfatal MACE and for recurrent MACE (n=226) among survivors of a previous incident MACE. We used Cox proportional hazard models adjusted for the Framingham covariates to determine the association of ceramides with the outcomes. Of the ceramide species, Cer(d18:1/18:0) had the strongest association with incident MACE and the highest unadjusted hazard ratio of 1.31 (95% confidence interval, 1.21-1.41), which remained significant at 1.21 (95% confidence interval, 1.11-1.33) after Framingham risk factor adjustments. The hazard ratios were generally stronger for recurrent and fatal events than for first events. Clinical net reclassification improvement was 7.5% (P=6.9×10 - 5 ) for Cer(d18:1/18:0)., Conclusions: Distinct serum ceramides are associated with the risk of incident MACE in apparently healthy individuals. These results should encourage more detailed analyses of ceramides in cardiovascular pathobiology and suggest new biomarkers of MACE risk., (© 2016 The Authors.)

Aims: The aim was to study the prognostic value of plasma ceramides (Cer) as cardiovascular death (CV death) markers in three independent coronary artery disease (CAD) cohorts., Methods and Results: Corogene study is a prospective Finnish cohort including stable CAD patients (n = 160). Multiple lipid biomarkers and C-reactive protein were measured in addition to plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and Cer(d18:1/24:1). Subsequently, the association between high-risk ceramides and CV mortality was investigated in the prospective Special Program University Medicine-Inflammation in Acute Coronary Syndromes (SPUM-ACS) cohort (n = 1637), conducted in four Swiss university hospitals. Finally, the results were validated in Bergen Coronary Angiography Cohort (BECAC), a prospective Norwegian cohort study of stable CAD patients. Ceramides, especially when used in ratios, were significantly associated with CV death in all studies, independent of other lipid markers and C-reactive protein. Adjusted odds ratios per standard deviation for the Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio were 4.49 (95% CI, 2.24-8.98), 1.64 (1.29-2.08), and 1.77 (1.41-2.23) in the Corogene, SPUM-ACS, and BECAC studies, respectively. The Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio improved the predictive value of the GRACE score (net reclassification improvement, NRI = 0.17 and ΔAUC = 0.09) in ACS and the predictive value of the Marschner score in stable CAD (NRI = 0.15 and ΔAUC = 0.02)., Conclusions: Distinct plasma ceramide ratios are significant predictors of CV death both in patients with stable CAD and ACS, over and above currently used lipid markers. This may improve the identification of high-risk patients in need of more aggressive therapeutic interventions., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Context: Coronary artery disease (CAD) is among the leading causes of mortality and morbidity worldwide. Traditional risk markers explain only a proportion of total cardiovascular risk. Thus, development and improvement of early diagnostic strategies and targeted initiation of preventive measures would be of great benefit., Objective: We aimed to identify molecular lipids that are associated with fatal outcome of CAD patients. Furthermore, the effect of different lipid-lowering drugs on novel risk lipids was evaluated., Methods: Serum samples of 445 CAD subjects participating in a long-term follow-up of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study were analyzed. In addition, samples obtained from a separate randomized parallel three-group study of subjects treated with simvastatin (n=24), ezetimibe (n=24), or their combination (n=24) were studied. Furthermore, samples from the LURIC participants with a loss-of-function mutation (R46L) in the PCSK9 gene (n=19) were analyzed and compared with major allele carriers (n=868)., Results: Distinct ceramide species were significantly associated with the fatal outcome of CAD patients. Simvastatin lowered plasma ceramides broadly by about 25%, but no changes in ceramides were observed in the ezetimibe group. PCSK9 deficiency was significantly associated (-13%) with lowered low-density lipoprotein cholesterol accompanied by a significant 20% reduction in CAD outcome risk-related ceramides., Conclusions: These data suggest that distinct ceramides associate significantly with CAD outcome independently of traditional risk factors and that the mechanism of lipid lowering is important.

Objectives: Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) has been proposed to be a potential new therapeutic target for treatment of hypercholesterolaemia. However, little is known about the effects of PCSK9 inhibition on the lipidome., Methods: We performed molecular lipidomic analyses of plasma samples obtained from PCSK9-deficient mice, and serum of human carriers of a loss-of-function variant in the PCSK9 gene (R46L)., Results: In both mouse and man, PCSK9 deficiency caused a decrease in several cholesteryl esters (CE) and short fatty acid chain containing sphingolipid species such as CE 16:0, glucosyl/galactosylceramide (Glc/GalCer) d18:1/16:0, and lactosylceramide (LacCer) d18:1/16:0. In mice, the changes in lipid concentrations were most prominent when animals were given regular chow diet. In man, a number of molecular lipid species was shown to decrease significantly even when LDL-cholesterol was non-significantly reduced by 10% only. Western diet attenuated the lipid lowering potency of PCSK9 deficiency in mice., Conclusions: Plasma molecular lipid species may be utilized for characterizing novel compounds inhibiting PCSK9 and as sensitive efficacy markers of the PCSK9 inhibition., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Objective: Although relationships between smoking and cardiovascular diseases (CVD), and between CVD and lipids are established, the direct impact of smoking on lipidomes is not well understood. We investigated the effect of mainstream cigarette smoke (CS) exposure on plasma, liver, and aorta molecular lipid profiles, and liver transcriptome in the ApoE(-/-) mouse, a well-established mouse model for human atherogenesis., Methods: Plasma, liver, and aorta samples from ApoE(-/-) mice exposed to CS or fresh air (sham) for six months were extracted for lipids using robotic-assisted method and analyzed by mass spectrometry. Gene expression in the liver was obtained on microarrays. Development of atherosclerosis in the aorta was further assessed by plaque size in the aortic arch and lipoprotein concentration in plasma and plaque., Results: CS increased most lipid classes and molecular lipid species. In plasma, free cholesterol, ceramides, cerebrosides, and most phospholipids were increased in CS-exposed mice. In the liver, several lipid species including free and esterified cholesterol, triacylglycerols, phospholipids, sphingomyelins, and ceramides were elevated. In the aorta, more than 2-fold higher cholesteryl ester (CE), lysophosphatidylcholine, and glucosyl/galactosylceramide levels were seen. Moreover, CS exposure induced a significant decrease in several plasma CE and phosphatidylcholine species that contained polyunsaturated fatty acids. Genes involved in amino acid and lipid metabolism showed perturbed transcription profiles in the liver., Conclusion: We have quantified some of the molecular changes that accompany the increase of plaque size that is accelerated by CS exposure in the aortae of ApoE(-/-) mice. These results suggest that specific changes in the lipidome and transcriptome, for example in ceramide and polyunsaturated fatty acid species, may be associated with atherosclerosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Lipids, abundant constituents of both the vascular plaque and lipoproteins, play a pivotal role in atherosclerosis. Mass spectrometry-based analysis of lipids, called lipidomics, presents a number of opportunities not only for understanding the cellular processes in health and disease but also in enabling personalized medicine. Lipidomics in its most advanced form is able to quantify hundreds of different molecular lipid species with various structural and functional roles. Unraveling this complexity will improve our understanding of diseases such as atherosclerosis at a level of detail not attainable with classical analytical methods. Improved patient selection, biomarkers for gauging treatment efficacy and safety, and translational models will be facilitated by the lipidomic deliverables. Importantly, lipid-based biomarkers and targets should lead the way as we progress toward more specialized therapeutics.

Bacteria are ubiquitous colonizers of various environments and host organisms, and they are therefore often subjected to drastic temperature alterations. Temperature alterations set demands on these colonizers, in that the bacteria need to readjust their biochemical constitution and physiology in order to survive and resume growth at the new temperature. Furthermore, temperature alteration is also a main factor determining the expression or repression of bacterial virulence functions. To cope with temperature variation, bacteria have devices for sensing temperature alterations and a means of translating this sensory event into a pragmatic gene response. While such regulatory cascades may ultimately be complicated, it appears that they contain primary sensor machinery at the top of the cascade. The functional core of such machinery is usually that of a temperature-induced conformational or physico-chemical change in the central constituents of the cell. In a sense, a bacterium can use structural alterations in its biomolecules as the primary thermometers or thermostats.

Interaction of internalin with E-cadherin promotes entry of Listeria monocytogenes into human epithelial cells. This process requires actin cytoskeleton rearrangements. Here we show, by using a series of stably transfected cell lines expressing E-cadherin variants, that the ectodomain of E-cadherin is sufficient for bacterial adherence and that the intracytoplasmic domain is required for entry. The critical cytoplasmic region was further mapped to the beta-catenin binding domain. Because beta-catenin is known to interact with alpha-catenin, which binds to actin, we generated a fusion molecule consisting of the ectodomain of E-cadherin and the actin binding site of alpha-catenin. Cells expressing this chimera were as permissive as E-cadherin-expressing cells. In agreement with these data, alpha- and beta-catenins as well as E-cadherin clustered and colocalized at the entry site, where F-actin then accumulated. Taken together, these results reveal that E-cadherin, via beta- and alpha-catenins, can trigger dynamic events of actin polymerization and membrane extensions culminating in bacterial uptake.

Many bacterial gene regulatory circuits are controlled by temperature. Temperature-mediated regulation occurs at the level of transcription and translation. Supercoiling, changes in mRNA conformation and protein conformation are all implicated in thermosensing. Bacterial virulence functions are often temperature regulated and thus many an example of thermoregulation comes from pathogenic organisms. H-NS is at the crossroads of regulation in many such systems. mRNA melting has also been shown to act as a thermosensing mechanism in various contexts. Proteins can also act as temperature sensors as exemplified by the gene regulator TlpA in Salmonella typhimurium.

Novel utilization of the coiled-coil motif is presented that enables TlpA, an autoregulatory repressor protein in Salmonella, to sense temperature shifts directly and thereby to modulate the extent of transcription repression. Salmonella cells shifted to higher temperatures, such as those encountered at host entry, showed derepressed tlpA activity. tlpA::lacZ fusions indicated that the promoter itself is insensitive to thermal shifts and that transcription control was exerted by the autorepressor TlpA only. In vitro studies with highly purified TlpA showed concentration and temperature dependence for both fully folded conformation and function, indicating that the thermosensing in TlpA is based on monomer-to-coiled-coil equilibrium.

Although quite common in the eukaryotic cell, bacterial proteins with an extensive coiled-coil domain are still relatively rare. One of the few thus far documented examples, TlpA from Salmonella typhimurium, is characterized by a remarkably long (250 amino acids) alpha-helical coiled-coil domain. Herein, we demonstrate that TlpA is a novel sequence-specific DNA-binding protein. Several tlpA deletion mutants have been constructed, and their corresponding protein products were purified and tested for DNA binding. Two of the mutant proteins were shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins wre shown to be deficient in DNA binding. Both mutants were analyzed by circular dichroism and electron microscopy, supporting the notion that mutant proteins were largely intact despite lacking the amino acid residues necessary for DNA binding. In vivo studies with transcriptional tlpA-lacZ fusions demonstrated that TlpA acts as a repressor. Using the repressor phenotype as a readout, the chain exchange previously described in vitro could also be confirmed in vivo. We believe the coiled-coil domain acts not only as a dimerization interface but could also serve a role as a flexible modulator of the protein-DNA interaction.

The expression of Salmonella enterica spv virulence genes was studied in serovariants Dublin and Typhimurium using Western blotting (immunoblotting), spv-lacZ operon fusions and Northern blotting. The SpvA protein was detected in immunoblots from stationary phase cultures of Dublin but not from the corresponding cultures of Typhimurium. Transcriptional measurements, using a spvA-lacZ operon fusion, indicated 8-10 times higher spvA transcription in Dublin. In an isogenic Escherichia coli chromosomal background, virulence plasmids from various Dublin strains systematically had a significantly higher induction level of the spvA-lacZ operon fusion than virulence plasmids from Typhimurium strains. The cloned spvR transcriptional activator gene of Dublin strain 2229 was found to activate both spvR-lacZ and spvA-lacZ operon fusions, as well as to raise spv mRNA levels in E. coli TG1. In contrast, the corresponding cloned gene of Typhimurium strain SL2965 possessed a lower induction potential and required higher spvR gene dosage for activation. A comparison of the nucleotide sequences of spvR genes from two Dublin and four Typhimurium strains revealed conserved, serovariant-associated basepair substitutions. Our results indicate that the spv virulence gene cluster possesses different functional alleles of the regulator gene spvR. This finding has important consequences for comparative studies of regulation and virulence in different serovariants of Salmonella.

An open, non-comparative clinical trial of cefaclor in adults with acute exacerbations of chronic bronchitis (54 patients) or pneumonia (24 patients) is reported. The dosage of cefaclor used was either 250 mg or 500 mg taken orally three times daily. Clinical cure was obtained in 39 of 42 (93%) of patients on the lower dose and 32 of 33 (97%) on the higher dose. Side effects were minimal and the antibiotic was very well tolerated. Microbiological evaluation was possible in 32 of 75 (43%) of patients in whom potential pathogens were identified before treatment. Microbiological 'cure' was achieved in 17 of 32 (53%), the majority of whom had received the higher dose. The lack of correlation between the clinical and microbiological results cast further doubts on the value of the standard sputum culture methods in the diagnosis and management of lower respiratory tract infection. Cefaclor is useful in the management of acute lower respiratory tract infections by virtue of its excellent clinical efficacy and safety.