Evaluation of Hepatic Glucose and Palmitic Acid Metabolism in Rodents on High-Fat Diet Using Deuterium Metabolic Imaging.

BACKGROUND: One of the main features of several metabolic disorders is dysregulation of hepatic glucose and lipid metabolism. Deuterium metabolic imaging (DMI) allows for assessing the uptake and breakdown of 2H-labeled substrates, giving specific insight into nutrient processing in healthy and diseased organs. Thus, DMI could be a useful approach for analyzing the differences in liver metabolism of healthy and diseased subjects to gain a deeper understanding of the alterations related to metabolic disorders. PURPOSE: Evaluating the feasibility of DMI as a tool for the assessment of metabolic differences in rodents with healthy and fatty livers (FLs). STUDY TYPE: Animal Model. POPULATION: 18 male Sprague Dawley rats on standard (SD, n = 9, healthy) and high-fat diet (HFD, n = 9, FL disease). FIELD STRENGTH/SEQUENCE: Phase-encoded 1D pulse-acquire sequence and anatomy co-registered phase-encoded 3D pulse-acquire chemical shift imaging for 2H at 9.4T. ASSESSMENT: Localized and nonlocalized liver spectroscopy was applied at eight time points over 104 minutes post injection. The obtained spectra were preprocessed and quantified using jMRUI (v7.0) and the resulting amplitudes translated to absolute concentration (mM) according to the 2H natural abundance water peak. STATISTICAL TESTS: Two-way repeated measures ANOVA were employed to assess between-group differences, with statistical significance at P < 0.05. RESULTS: DMI measurements demonstrated no significant difference (P = 0.98) in the uptake of [6,6'-2H2]glucose between healthy and impaired animals (AUCSD = 1966.0 ± 151.5 mM - minutes vs. AUCHFD = 2027.0 ± 167.6 mM·minutes). In the diseased group, the intrahepatic uptake of palmitic acid d-31 was higher (AUCHFD = 57.4 ± 17.0 mM·minutes, AUCSD = 33.3 ± 10.5 mM·minutes), but without statistical significance owing to substantial in-group variation (P = 0.73). DATA CONCLUSION: DMI revealed higher concentrations of palmitic acid in rats with FL disease and no difference in hepatic glucose concentration between healthy and impaired animals. Thus, DMI appears to be a useful tool for evaluating metabolism in rodents with FL disease. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY: Stage 3.

A Fully Automated Synthesis of 14-(R,S)-[18F]fluoro-6-thia-heptadecanoic Acid ([18F]FTHA) on the Elixys Radiosynthesizer.

14-(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA) is a radiocompound for imaging the fatty acid circulation by positron emission tomography. A revived interest in imaging of lipid metabolism led us to a constant tracer production over three years, initially using a conventional vessel-based synthesizer and later transitioning to the cassette-based Elixys synthesizer. On the Elixys module, the radiochemical yield of [18F]FTHA could be increased by more than two times, reaching 13.01 ± 5.63% at the end of the synthesis, while maintaining necessary quality control results.

Fecal microbiota transplantation promotes reduction of antimicrobial resistance by strain replacement.

Multidrug-resistant organism (MDRO) colonization is a fundamental challenge in antimicrobial resistance. Limited studies have shown that fecal microbiota transplantation (FMT) can reduce MDRO colonization, but its mechanisms are poorly understood. We conducted a randomized, controlled trial of FMT for MDRO decolonization in renal transplant recipients called PREMIX (NCT02922816). Eleven participants were enrolled and randomized 1:1 to FMT or an observation period followed by delayed FMT if stool cultures were MDRO positive at day 36. Participants who were MDRO positive after one FMT were treated with a second FMT. At last visit, eight of nine patients who completed all treatments were MDRO culture negative. FMT-treated participants had longer time to recurrent MDRO infection versus PREMIX-eligible controls who were not treated with FMT. Key taxa (Akkermansia muciniphila, Alistipes putredinis, Phocaeicola dorei, Phascolarctobacterium faecium, Alistipes species, Mesosutterella massiliensis, Barnesiella intestinihominis, and Faecalibacterium prausnitzii) from the single feces donor used in the study that engrafted in recipients and metabolites such as short-chain fatty acids and bile acids in FMT-responding participants uncovered leads for rational microbiome therapeutic and diagnostic development. Metagenomic analyses revealed a previously unobserved mechanism of MDRO eradication by conspecific strain competition in an FMT-treated subset. Susceptible Enterobacterales strains that replaced baseline extended-spectrum ß-lactamase-producing strains were not detectable in donor microbiota manufactured as FMT doses but in one case were detectable in the recipient before FMT. These data suggest that FMT may provide a path to exploit strain competition to reduce MDRO colonization.

A Comprehensive Review for the Surveillance of Human Pathogenic Microorganisms in Shellfish.

Bivalve molluscan shellfish have been consumed for centuries. Being filter feeders, they may bioaccumulate some microorganisms present in coastal water, either naturally or through the discharge of human or animal sewage. Despite regulations set up to avoid microbiological contamination in shellfish, human outbreaks still occur. After providing an overview showing their implication in disease, this review aims to highlight the diversity of the bacteria or enteric viruses detected in shellfish species, including emerging pathogens. After a critical discussion of the available methods and their limitations, we address the interest of technological developments using genomics to anticipate the emergence of pathogens. In the coming years, further research needs to be performed and methods need to be developed in order to design the future of surveillance and to help risk assessment studies, with the ultimate objective of protecting consumers and enhancing the microbial safety of bivalve molluscan shellfish as a healthy food.

Novel approach using [18F]FTHA-PET and de novo synthesized VLDL for assessment of FFA metabolism in a rat model of diet induced NAFLD.

BACKGROUND AND AIMS: The worldwide prevalence of Non-alcoholic Fatty Liver Disease (NAFLD) raises concerns about associated risk factors, such as obesity and type 2 Diabetes Mellitus, for leading causes of disability and death. Besides Magnetic Resonance Imaging (MRI) and Spectroscopy (MRS), functional imaging with Positron Emission Tomography (PET) could contribute to a deeper understanding of the pathophysiology of NAFLD. Here we describe a novel approach using the PET tracer [18F]FTHA, which is an analog of long-chain free fatty acids (FFA) and is taken up by tissues to enter mitochondria or to be incorporated into complex lipids for further export as very-low-density lipoprotein (VLDL). METHODS: Male Sprague Dawley rats, after 6 weeks on a high-fat diet (HFD), were used as a model of diet induced NAFLD, while a standard diet (SD) served as a control group. Liver fat was estimated by MR spectroscopy at a 9.4 T system for phenotyping. To measure hepatic FFA uptake, rats underwent 60 min dynamic [18F]FTHA-PET scans after unrestricted access to food (HFD: n = 6; SD: n = 6) or overnight (≤16h) fasting (HFD: n = 6; SD: n = 5). FFA removal was assessed from incorporated 18F-residual in de novo synthesized VLDL out of plasma. RESULTS: MRS of the liver confirmed the presence of NAFLD (>5.6% fat). Under non-fasting conditions, hepatic [18F]FTHA uptake was significantly increased in NAFLD: SUVmean (p = 0.03) within [0; 60] min interval, SUVmean (p = 0.01) and SUVmax (p = 0.03) within [30; 60] min interval. SUVs for hepatic uptake under fasting conditions were not significantly different between the groups. Analysis of FFA removal demonstrated elevated values of 18F-residue in the VLDL plasma fraction of the healthy group compared to the NAFLD (p = 0.0569). CONCLUSION: Our novel approach for assessing FFA metabolism using [18F]FTHA demonstrated differences in the hepatic FFA uptake and FFA incorporation into VLDL between healthy and NAFLD rats. [18F]FTHA-PET could be used to study metabolic disturbances involved in the progression of NAFLD.

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway.

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa.

Impact of genetic variants within serotonin turnover enzymes on human cerebral monoamine oxidase A in vivo.

Variants within the monoamine oxidase A (MAO-A, MAOA) and tryptophan hydroxylase 2 (TPH2) genes, the main enzymes in cerebral serotonin (5-HT) turnover, affect risk for depression. Depressed cohorts show increased cerebral MAO-A in positron emission tomography (PET) studies. TPH2 polymorphisms might also influence brain MAO-A because availability of substrates (i.e. monoamine concentrations) were shown to affect MAO-A levels. We assessed the effect of MAOA (rs1137070, rs2064070, rs6323) and TPH2 (rs1386494, rs4570625) variants associated with risk for depression and related clinical phenomena on global MAO-A distribution volume (VT) using [11C]harmine PET in 51 participants (21 individuals with seasonal affective disorder (SAD) and 30 healthy individuals (HI)). Statistical analyses comprised general linear models with global MAO-A VT as dependent variable, genotype as independent variable and age, sex, group (individuals with SAD, HI) and season as covariates. rs1386494 genotype significantly affected global MAO-A VT after correction for age, group and sex (p < 0.05, corr.), with CC homozygotes showing 26% higher MAO-A levels. The role of rs1386494 on TPH2 function or expression is poorly understood. Our results suggest rs1386494 might have an effect on either, assuming that TPH2 and MAO-A levels are linked by their common product/substrate, 5-HT. Alternatively, rs1386494 might influence MAO-A levels via another mechanism, such as co-inheritance of other genetic variants. Our results provide insight into how genetic variants within serotonin turnover translate to the cerebral serotonin system. Clinicaltrials.gov Identifier: NCT02582398. EUDAMED Number: CIV-AT-13-01-009583.

The never-ending battle between lactic acid bacteria and their phages.

Over the past few decades, the interest in lactic acid bacteria (LAB) has been steadily growing. This is mainly due to their industrial use, their health benefits as probiotic bacteria and their ecological importance in host-related microbiota. Phage infection represents a significant risk for the production and industrial use of LAB. This created the need to study the various means of defense put in place by LAB to resist their viral enemies, as well as the countermeasures evolved by phages to overcome these defenses. In this review, we discuss defense systems that LAB employ to resist phage infections. We also describe how phages counter these mechanisms through diverse and sophisticated strategies. Furthermore, we discuss the way phage-host interactions shape each other's evolution. The recent discovery of numerous novel defense systems in other bacteria promises a new dawn for phage research in LAB.

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

Effects of bilateral sequential theta-burst stimulation on 5-HT1A receptors in the dorsolateral prefrontal cortex in treatment-resistant depression: a proof-of-concept trial.

Theta-burst stimulation (TBS) represents a brain stimulation technique effective for treatment-resistant depression (TRD) as underlined by meta-analyses. While the methodology undergoes constant refinement, bilateral stimulation of the dorsolateral prefrontal cortex (DLPFC) appears promising to restore left DLPFC hypoactivity and right hyperactivity found in depression. The post-synaptic inhibitory serotonin-1A (5-HT1A) receptor, also occurring in the DLPFC, might be involved in this mechanism of action. To test this hypothesis, we performed PET-imaging using the tracer [carbonyl-11C]WAY-100635 including arterial blood sampling before and after a three-week treatment with TBS in 11 TRD patients compared to sham stimulation (n = 8 and n = 3, respectively). Treatment groups were randomly assigned, and TBS protocol consisted of excitatory intermittent TBS to the left and inhibitory continuous TBS to the right DLPFC. A linear mixed model including group, hemisphere, time, and Hamilton Rating Scale for Depression (HAMD) score revealed a 3-way interaction effect of group, time, and HAMD on specific distribution volume (VS) of 5-HT1A receptor. While post-hoc comparisons showed no significant changes of 5-HT1A receptor VS in either group, higher 5-HT1A receptor VS after treatment correlated with greater difference in HAMD (r = -0.62). The results of this proof-of-concept trial hint towards potential effects of TBS on the distribution of the 5-HT1A receptor. Due to the small sample size, all results must, however, be regarded with caution.

ABCB1 variants and sex affect serotonin transporter occupancy in the brain.

Strategies to personalize psychopharmacological treatment promise to improve efficacy and tolerability. We measured serotonin transporter occupancy immediately after infusion of the widely prescribed P-glycoprotein substrate citalopram and assessed to what extent variants of the ABCB1 gene affect drug target engagement in the brain in vivo. A total of 79 participants (39 female) including 31 patients with major depression and 48 healthy volunteers underwent two PET/MRI scans with the tracer [11C]DASB and placebo-controlled infusion of citalopram (8 mg) in a cross-over design. We tested the effect of six ABCB1 single nucleotide polymorphisms and found lower SERT occupancy in ABCB1 rs2235015 minor allele carriers (n = 26, MAF = 0.18) compared to major allele homozygotes (t73 = 2.73, pFWE < 0.05) as well as in men compared to women (t73 = 3.33, pFWE < 0.05). These effects were robust to correction for citalopram plasma concentration, age and diagnosis. From occupancy we derived the ratio of occupied to unoccupied SERT, because in theory this measure is equal to the product of drug affinity and concentration at target sites. A model combining genotype with basic clinical variables, predicted that, at the same dosage, occupied to unoccupied SERT ratio was -14.48 ± 5.38% lower in rs2235015 minor allele carriers, +19.10 ± 6.95% higher in women, -4.83 ± 2.70% lower per 10 kg bodyweight, and -2.68 ± 3.07% lower per 10 years of age. Our results support the exploration of clinical algorithms with adjustment of initial citalopram dosing and highlight the potential of imaging-genetics for precision pharmacotherapy in psychiatry.

Phage-host interactions as a driver of population dynamics during wine fermentation: Betting on underdogs.

Winemaking is a complex process in which numerous microorganisms, mainly yeasts and lactic acid bacteria (LAB), play important roles. After alcoholic fermentation (AF), most wines undergo malolactic fermentation (MLF) to improve their organoleptic properties and microbiological stability. Oenococcus oeni is mainly responsible for this crucial process where L-malic acid (MA) in wine converts to softer L-lactic acid. The bacterium is better adapted to the limiting conditions imposed by the wine matrix and performs MLF under regular winemaking conditions, especially in wines with a pH below 3.5. Traditionally, this process has been conducted by the natural microbiota present within the winery. However, the start, duration and qualitative impact of spontaneous MLF are unpredictable, which prompts winemakers to use pure starter cultures of selected bacteria to promote a more reliable, simple, fast and efficient fermentation. Yet, their use does not always ensure a problem-free fermentation. Spontaneous initiation of the process may prove very difficult or does not occur at all. Such difficulties arise from a combination of factors found in some wines upon the completion of AF (high ethanol concentration, low temperature and pH, low nutrient concentrations, presence of free and bound SO2). Alongside these well documented facts, research has also provided evidence that negative interactions between O. oeni and other biological entities such as yeasts may also impact MLF. Another insufficiently described, but highly significant factor inhibiting bacterial growth is connected to the presence of bacteriophages of O. oeni which are frequently associated to musts and wines. The purpose of this review is to summarize the current knowledge about the phage life cycles and possible impacts on the trajectory of the microbiota during winemaking.

Autoradiography on deparaffinized tissue sections - A feasibility study with 68Ga-labeled PET-tracers.

Tissue available for retrospective research questions is often already paraffin-embedded for better preservation. However, in vitro autoradiography (AURA) is normally performed on cryopreserved tissue sections. We hypothesized a) that it would also be feasible with deparaffinized tissue sections, enabling the use of human paraffin-embedded tissue for in vitro AURA and b) that the results would be comparable to those obtained with corresponding cryosections. For that purpose, the clinically relevant oncological targets CXCR4, SSTR and PSMA were evaluated. In vitro AURA on deparaffinized tissue sections was feasible, but only with the two receptor ligands [68Ga]Ga-PentixaFor and [68Ga]Ga-DOTANOC. [68Ga]Ga-PSMA-11 did not show any binding on deparaffinized tissue sections, suggesting that native tissue is required for an interaction between this inhibitor and the enzyme.

A truncated anti-CRISPR protein prevents spacer acquisition but not interference.

CRISPR-Cas systems in prokaryotic cells provide an adaptive immunity against invading nucleic acids. For example, phage infection leads to addition of new immunity (spacer acquisition) and DNA cleavage (interference) in the bacterial model species Streptococcus thermophilus, which primarily relies on Cas9-containing CRISPR-Cas systems. Phages can counteract this defense system through mutations in the targeted protospacers or by encoding anti-CRISPR proteins (ACRs) that block Cas9 interference activity. Here, we show that S. thermophilus can block ACR-containing phages when the CRISPR immunity specifically targets the acr gene. This in turn selects for phage mutants carrying a deletion within the acr gene. Remarkably, a truncated acrIIA allele, found in a wild-type virulent streptococcal phage, does not block the interference activity of Cas9 but still prevents the acquisition of new immunities, thereby providing an example of an ACR specifically inhibiting spacer acquisition.

Simultaneous radiomethylation of [11C]harmine and [11C]DASB and kinetic modeling approach for serotonergic brain imaging in the same individual.

Simultaneous characterization of pathologies by multi-tracer positron emission tomography (PET) is among the most promising applications in nuclear medicine. Aim of this work was the simultaneous production of two PET-tracers in one module and test the relevance for human application. [11C]harmine and [11C]DASB were concurrently synthesized in a 'two-in-one-pot' reaction in quality for application. Dual-tracer protocol was simulated using 16 single PET scans in different orders of tracer application separated by different time intervals. Volume of distribution was calculated for single- and dual-tracer measurements using Logan's plot and arterial input function in 13 brain regions. The 'two-in-one-pot' reaction yielded equivalent amounts of both radiotracers with comparable molar activities. The simulations of the dual-tracer application were comparable to the single bolus injections in 13 brain regions, when [11C]harmine was applied first and [11C]DASB second, with an injection time interval of 45 min (rxy = 0.90). Our study shows the successful simultaneous dual-tracer production leading to decreased radiation burden and costs. The simulation of dual subject injection to quantify the monoamine oxidase-A and serotonin transporter distribution proved its high potential. Multi-tracer imaging may drive more sophisticated study designs and diminish the day-to-day differences in the same individual as well as increase PET scanner efficiency.

Deciphering metformin action in obese mice: A critical re-evaluation of established protocols.

BACKGROUND AND PURPOSE: Despite extensive efforts and a plethora of suggested targets and pathways, the mechanism via which metformin lowers blood glucose remains obscure. Obstacles that hamper progress in understanding metformin action include unexplained discrepancies between preclinical models and patients. PROCEDURES: We treated obese male C57BL/6J mice fed high fat diet with metformin provided in the form of a single dose, daily intraperitoneal injections, admixture to drinking water, or continuous infusion via intraperitoneal minipumps. RESULTS: The results suggest several superimposed components, via which metformin acts on blood glucose. These include (i) marked glucose lowering shortly after dosing, which fades rapidly with the decrease in metformin concentrations in plasma and liver, but could, at least to a major extent, rely on the mechanism also accounting for metformin's therapeutic action in humans; (ii) indirect action via reduced weight gain, which might be responsible for glucose lowering observed in many previous rodent studies; and (iii) deterioration of glucose homeostasis by prolonged treatment that can be unmasked by avoidance of dosing shortly before measuring blood glucose in combination with exclusion of weight-related actions via restricted feeding of the control mice. CONCLUSIONS: Our work raises the question whether elucidation of metformin's anti-diabetic mechanism(s) in rodent experiments may in the past have been hampered by failure to mimic clinical circumstances, as caused by insufficient consideration of pharmacokinetics and multiplicity of involved actions.

Phage Encounters Recorded in CRISPR Arrays in the Genus Oenococcus.

The Oenococcus genus comprises four recognized species, and members have been found in different types of beverages, including wine, kefir, cider and kombucha. In this work, we implemented two complementary strategies to assess whether oenococcal hosts of different species and habitats were connected through their bacteriophages. First, we investigated the diversity of CRISPR-Cas systems using a genome-mining approach, and CRISPR-endowed strains were identified in three species. A census of the spacers from the four identified CRISPR-Cas loci showed that each spacer space was mostly dominated by species-specific sequences. Yet, we characterized a limited records of potentially recent and also ancient infections between O. kitaharae and O. sicerae and phages of O. oeni, suggesting that some related phages have interacted in diverse ways with their Oenococcus hosts over evolutionary time. Second, phage-host interaction analyses were performed experimentally with a diversified panel of phages and strains. None of the tested phages could infect strains across the species barrier. Yet, some infections occurred between phages and hosts from distinct beverages in the O. oeni species.