Desorption Electrospray Ionization Mass Spectrometry Imaging of Powder-Treated Fingermarks on Forensic Gelatin Lifters and its Application for Separating Overlapping Fingermarks.

Fingermarks are frequently collected at crime scenes by using gelatin lifters for preservation and transport of the marks to a forensic laboratory for inspection. The gelatin lifters preserve both the imprint of the fingermark pattern necessary for identification purposes and the chemical residue of the mark potentially useful for profiling the person who left the fingermark. The fingermark patterns are traditionally recorded using photography/optical imaging, but methods for chemical analysis of fingermark residues on gelatin lifters are scarce. Here we report the first method for the chemical analysis of fingermarks on gelatin lifters using desorption electrospray ionization mass spectrometry (DESI-MS) imaging. The imaging can be done directly on the gelatin support without any sample preparation, supporting immediate operational use of the method for fingermarks collected at crime scenes. Operational use of the method is further supported by successful chemical imaging of fingermarks enhanced by traditional dusting with forensic powders and lifted off different surfaces (glass, stainless steel, painted aluminum, polystyrene, cardboard, and plastic) as well as fingermarks lifted multiple times. We also demonstrate that the present method can be used to visually separate natural overlapping powder-treated fingermarks, and the chemical composition of the fingermarks can be analyzed on the gelatin support by DESI-MS/MS. The presented method has potential for integration into the traditional workflow for fingermark analysis, and will allow more fingermarks collected at crime scenes to be evaluated both visually and chemically.

Circulating 3-hydroxy butyrate predicts mortality in patients with chronic heart failure with reduced ejection fraction.

AIMS: In patients with chronic heart failure with reduced ejection fraction (HFrEF), myocardial ketone metabolism is increased and short-term treatment with the ketone body 3-hydroxy butyrate (3-OHB) has beneficial haemodynamic effects. In patients with HFrEF, we investigated whether the level of circulating 3-OHB predicted all-cause mortality and sought to identify correlations between patient characteristics and circulating 3-OHB levels. METHODS AND RESULTS: We conducted a cohort study in 218 patients with HFrEF. Plasma 3-OHB levels were measured using high-performance liquid chromatography tandem mass spectrometry. Data on all-cause mortality were obtained by reviewing the patients' medical records, which are linked to the national 'Central Person Registry' that registers the timing of all deaths in the country. Mean left ventricular ejection fraction was 35 ± 8.6%, mean age was 67 ± 10 years, 54% were New York Heart Association II, and 27% had type 2 diabetes mellitus. Median follow-up time was 7.3 (interquartile range 6.3-8.4) years. We observed large variations in 3-OHB levels between patients (median 59 µM, range: 14-694 µM). Patients with 3-OHB levels above the median displayed a markedly increased risk of death compared with those with low levels {hazard ratio [HR]: 2.1 [95% confidence interval (CI): 1.3-3.5], P = 0.003}. In a multivariate analysis, 3-OHB predicted mortality independently of known chronic heart failure risk factors [HR: 1.004 (95% CI: 1.001-1.007), P = 0.02] and with a similar statistical strength as N-terminal pro-brain natriuretic peptide (NT-proBNP) [HR: 1.0005 (95% CI: 1.000-1.001), P = 0.02]. For every 100 µmol increase in plasma 3-OHB, the hazard of death increased by 49%. The following factors significantly predicted 3-OHB levels in the univariate analysis: free fatty acids (FFAs) [ß: 238 (95% CI: 185-292), P < 0.0001], age [ß: 2.43 (95% CI: 1.14-3.72), P < 0.0001], plasma insulin {ß: -0.28 [95% CI: -0.54-(-0.02)], P = 0.036}, body mass index {ß: -3.15 [95% CI: -5.26-(-0.05)], P = 0.046}, diabetes [ß: 44.49 (95% CI: 14.84-74.14), P = 0.003], glycosylated haemoglobin [ß: 1.92 (95% CI: 0.24-3.59), P = 0.025], New York Heart Association class [ß: 26.86 (95% CI: 5.99-47.72), P = 0.012], and NT-proBNP [ß: 0.03 (95% CI: 0.01-0.04), P = 0.001]. In a multivariate analysis, only FFAs predicted 3-OHB levels [ß: 216 (95% CI: 165-268), P > 0.001]. CONCLUSIONS: In patients with HFrEF, circulating 3-OHB was a strong predictor of all-cause mortality independently of NT-proBNP. Circulating FFAs were the best predictor of 3-OHB levels.

Enantiomer-Specific Cardiovascular Effects of the Ketone Body 3-Hydroxybutyrate.

BACKGROUND: The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output (CO) by 35% to 40% in healthy people and people with heart failure. The mechanisms underlying the effects of 3-OHB on myocardial contractility and loading conditions as well as the cardiovascular effects of its enantiomeric forms, D-3-OHB and L-3-OHB, remain undetermined. METHODS AND RESULTS: Three groups of 8 pigs each underwent a randomized, crossover study. The groups received 3-hour infusions of either D/L-3-OHB (racemic mixture), 100% L-3-OHB, 100% D-3-OHB, versus an isovolumic control. The animals were monitored with pulmonary artery catheter, left ventricle pressure-volume catheter, and arterial and coronary sinus blood samples. Myocardial biopsies were evaluated with high-resolution respirometry, coronary arteries with isometric myography, and myocardial kinetics with D-[11C]3-OHB and L-[11C]3-OHB positron emission tomography. All three 3-OHB infusions increased 3-OHB levels (P<0.001). D/L-3-OHB and L-3-OHB increased CO by 2.7 L/min (P<0.003). D-3-OHB increased CO nonsignificantly (P=0.2). Circulating 3-OHB levels correlated with CO for both enantiomers (P<0.001). The CO increase was mediated through arterial elastance (afterload) reduction, whereas contractility and preload were unchanged. Ex vivo, D- and L-3-OHB dilated coronary arteries equally. The mitochondrial respiratory capacity remained unaffected. The myocardial 3-OHB extraction increased only during the D- and D/L-3-OHB infusions. D-[11C]3-OHB showed rapid cardiac uptake and metabolism, whereas L-[11C]3-OHB demonstrated much slower pharmacokinetics. CONCLUSIONS: 3-OHB increased CO by reducing afterload. L-3-OHB exerted a stronger hemodynamic response than D-3-OHB due to higher circulating 3-OHB levels. There was a dissocitation between the myocardial metabolism and hemodynamic effects of the enantiomers, highlighting L-3-OHB as a potent cardiovascular agent with strong hemodynamic effects.

The HIF transcription network exerts innate antiviral activity in neurons and limits brain inflammation.

Pattern recognition receptors (PRRs) induce host defense but can also induce exacerbated inflammatory responses. This raises the question of whether other mechanisms are also involved in early host defense. Using transcriptome analysis of disrupted transcripts in herpes simplex virus (HSV)-infected cells, we find that HSV infection disrupts the hypoxia-inducible factor (HIF) transcription network in neurons and epithelial cells. Importantly, HIF activation leads to control of HSV replication. Mechanistically, HIF activation induces autophagy, which is essential for antiviral activity. HSV-2 infection in vivo leads to hypoxia in CNS neurons, and mice with neuron-specific HIF1/2α deficiency exhibit elevated viral load and augmented PRR signaling and inflammatory gene expression in the CNS after HSV-2 infection. Data from human stem cell-derived neuron and microglia cultures show that HIF also exerts antiviral and inflammation-restricting activity in human CNS cells. Collectively, the HIF transcription factor system senses virus-induced hypoxic stress to induce cell-intrinsic antiviral responses and limit inflammation.

Cholesterol-binding motifs in STING that control endoplasmic reticulum retention mediate anti-tumoral activity of cholesterol-lowering compounds.

The cGAS-STING pathway plays a crucial role in anti-tumoral responses by activating inflammation and reprogramming the tumour microenvironment. Upon activation, STING traffics from the endoplasmic reticulum (ER) to Golgi, allowing signalling complex assembly and induction of interferon and inflammatory cytokines. Here we report that cGAMP stimulation leads to a transient decline in ER cholesterol levels, mediated by Sterol O-Acyltransferase 1-dependent cholesterol esterification. This facilitates ER membrane curvature and STING trafficking to Golgi. Notably, we identify two cholesterol-binding motifs in STING and confirm their contribution to ER-retention of STING. Consequently, depletion of intracellular cholesterol levels enhances STING pathway activation upon cGAMP stimulation. In a preclinical tumour model, intratumorally administered cholesterol depletion therapy potentiated STING-dependent anti-tumoral responses, which, in combination with anti-PD-1 antibodies, promoted tumour remission. Collectively, we demonstrate that ER cholesterol sets a threshold for STING signalling through cholesterol-binding motifs in STING and we propose that this could be exploited for cancer immunotherapy.

Molecular Mechanisms of Temperature Tolerance Plasticity in an Arthropod.

How species thrive in a wide range of environments is a major focus of evolutionary biology. For many species, limited genetic diversity or gene flow among habitats means that phenotypic plasticity must play an important role in their capacity to tolerate environmental heterogeneity and to colonize new habitats. However, we have a limited understanding of the molecular components that govern plasticity in ecologically relevant phenotypes. We examined this hypothesis in a spider species (Stegodyphus dumicola) with extremely low species-wide genetic diversity that nevertheless occupies a broad range of thermal environments. We determined phenotypic responses to temperature stress in individuals from four climatic zones using common garden acclimation experiments to disentangle phenotypic plasticity from genetic adaptations. Simultaneously, we created data sets on multiple molecular modalities: the genome, the transcriptome, the methylome, the metabolome, and the bacterial microbiome to determine associations with phenotypic responses. Analyses of phenotypic and molecular associations reveal that acclimation responses in the transcriptome and metabolome correlate with patterns of phenotypic plasticity in temperature tolerance. Surprisingly, genes whose expression seemed to be involved in plasticity in temperature tolerance were generally highly methylated contradicting the idea that DNA methylation stabilizes gene expression. This suggests that the function of DNA methylation in invertebrates varies not only among species but also among genes. The bacterial microbiome was stable across the acclimation period; combined with our previous demonstrations that the microbiome is temporally stable in wild populations, this is convincing evidence that the microbiome does not facilitate plasticity in temperature tolerance. Our results suggest that population-specific variation in temperature tolerance among acclimation temperatures appears to result from the evolution of plasticity in mainly gene expression.