Real time detection of pathogenic bacteria in veterinary microbiology using isothermal microcalorimetry - A different approach.

With today's challenges regarding antibiotic resistance and the importance of the implementation of prudent use of antibiotics, fast and reliable diagnostic tools for bacterial infections and subsequent antimicrobial susceptibility testing are of utmost relevance. Isothermal microcalorimetry (IMC) is a broadly applicable method, with which metabolic heat flow in reproducing bacteria can be measured in real time. To the best of the authors' knowledge, this is the first report on examination of 124 urine samples from feline and canine urinary tract infection with an IMC-based prototype instrument. A concentration-dependent time of peak heat flow by dilution series with Escherichia coli and Enterococcus faecalis reference strains demonstrated the general good performance of the prototype for detection of these bacteria. With diagnostic culture being set as a gold standard, the diagnostic sensitivity of IMC compared to bacteriological culture was 80 %, the diagnostic specificity was 97 %. With a Cohens' kappa value (κ) of 0.80, the two methods show good concordance. The results from our study demonstrate that the IMC technology is suitable to allow reliable, but much faster detection of bacteria than conventional culture, especially for Escherichia coli. Thus, implementing IMC technology could markedly speed up the bacteriological diagnostic process in veterinary medicine.

Stannous Source in Toothpastes Leads to Differences in Their Antimicrobial Efficacy.

PURPOSE: The aim of this in-vitro study was to investigate the antimicrobial efficacy of identical experimental toothpastes with different stannous sources. MATERIALS AND METHODS: Streptococcus mutans biofilms were grown on protein-coated glass disks in static conditions for 24 h and thereafter exposed to toothpaste slurries or physiological saline (negative control; n = 15) for 30 s. Four experimental toothpastes were applied in this study, containing either stannous chloride (SnCl2; B: 3500 ppm Sn2+, and D: 3600 ppm Sn2+) or stannous fluoride (SnF2; C: 3500 ppm Sn2+, and E: 3600 ppm Sn2+). Marketed toothpaste meridol® (A: 3300 ppm SnF2) served as control. All five toothpastes contained amine fluoride (AmF). The biofilms were placed on agar surface and their metabolic activity was assessed by isothermal microcalorimetry over 96 h. The heat flow data was analysed for growth rate and lag time using grofit package in software R. Additionally, reduction of active biofilm compared to untreated control was calculated. RESULTS: All toothpastes significantly prolong the lag time of treated biofilms in comparison to negative control (p < 0.05). Toothpastes containing SnF2 (C and E) prolonged the lag time statistically significantly compared to toothpastes containing SnCl2 (B and D) (p < 0.05). The maximum growth rate was statistically significantly reduced by all tested toothpastes compared to the untreated control group (p < 0.05). Toothpastes containing SnF2 (A, C and E) reached 59.9 ± 7.8, 61.9 ± 7.7, and 55.6 ± 7.0% reduction of active biofilm, respectively. Thus, they exhibit statistically significantly better results than toothpastes B (52.9 ± 9.9%) and D (44.7 ± 7.6%). Toothpaste D, which contains a slightly higher concentration of Sn2+, was the least effective in reducing active biofilm. CONCLUSION: The toothpastes containing SnF2 combined with AmF had the highest antimicrobial efficacy in this study.

Antimicrobial and mechanical assessment of cellulose-based thermoformable material for invisible dental braces with natural essential oils protecting from biofilm formation.

Controlling biofilm formation in the oral cavity during orthodontic treatments is crucial. Therefore, antimicrobial surfaces for invisible dental appliances are of interest to both therapists and patients. Here we present a cellulose-based thermoformable material used for invisible braces that can be loaded with essential oils (EOs) having antibacterial and antifungal properties. We hypothesize that this material can absorb and release EOs, thus providing an antimicrobial effect without compromising the safety and mechanical properties necessary for dental invisible braces. Conventional microbiology and isothermal microcalorimetry analyses revealed that the thermoformable material loaded with essential oils significantly delayed the biofilm formation of oral streptococci (S. mutans and S. mitis) under static conditions (p < 0.05) and while simulating saliva flow (p < 0.05). In addition, cytotoxicity tests (ISO 10993-5), revealed that the loaded material is well tolerated by human gingival fibroblasts. Finally, the loading with antibacterial agents did not significantly alter the mechanical properties and stability of the material (initial force (p = 0.916); initial stress (p = 0.465)). Compared to gold-standard clear aligner materials, this material offers a reliable transmission of forces for orthodontic treatments. Moreover, this approach exhibits the potential for acting as an oral drug delivery platform for multiple compounds.

Neurometabolic changes in a rat pup model of type C hepatic encephalopathy depend on age at liver disease onset.

Chronic liver disease (CLD) is a serious condition where various toxins present in the blood affect the brain leading to type C hepatic encephalopathy (HE). Both adults and children are impacted, while children may display unique vulnerabilities depending on the affected window of brain development.We aimed to use the advantages of high field proton Magnetic Resonance Spectroscopy (1H MRS) to study longitudinally the neurometabolic and behavioural effects of Bile Duct Ligation (animal model of CLD-induced type C HE) on rats at post-natal day 15 (p15) to get closer to neonatal onset liver disease. Furthermore, we compared two sets of animals (p15 and p21-previously published) to evaluate whether the brain responds differently to CLD according to age onset.We showed for the first time that when CLD was acquired at p15, the rats presented the typical signs of CLD, i.e. rise in plasma bilirubin and ammonium, and developed the characteristic brain metabolic changes associated with type C HE (e.g. glutamine increase and osmolytes decrease). When compared to rats that acquired CLD at p21, p15 rats did not show any significant difference in plasma biochemistry, but displayed a delayed increase in brain glutamine and decrease in total-choline. The changes in neurotransmitters were milder than in p21 rats. Moreover, p15 rats showed an earlier increase in brain lactate and a different antioxidant response. These findings offer tentative pointers as to which neurodevelopmental processes may be impacted and raise the question of whether similar changes might exist in humans but are missed owing to 1H MRS methodological limitations in field strength of clinical magnet.

Enabling direct microcalorimetric measurement of metabolic activity and exothermic reactions onto microfluidic platforms via heat flux sensor integration.

All biological processes use or produce heat. Traditional microcalorimeters have been utilized to study the metabolic heat output of living organisms and heat production of exothermic chemical processes. Current advances in microfabrication have made possible the miniaturization of commercial microcalorimeters, resulting in a few studies on the metabolic activity of cells at the microscale in microfluidic chips. Here we present a new, versatile, and robust microcalorimetric differential design based on the integration of heat flux sensors on top of microfluidic channels. We show the design, modeling, calibration, and experimental verification of this system by utilizing Escherichia coli growth and the exothermic base catalyzed hydrolysis of methyl paraben as use cases. The system consists of a Polydimethylsiloxane based flow-through microfluidic chip with two 46 µl chambers and two integrated heat flux sensors. The differential compensation of thermal power measurements allows for the measurement of bacterial growth with a limit of detection of 1707 W/m3, corresponding to 0.021OD (2 ∙ 107 bacteria). We also extracted the thermal power of a single Escherichia coli of between 1.3 and 4.5 pW, comparable to values measured by industrial microcalorimeters. Our system opens the possibility for expanding already existing microfluidic systems, such as drug testing lab-on-chip platforms, with measurements of metabolic changes of cell populations in form of heat output, without modifying the analyte and minimal interference with the microfluidic channel itself.

AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function.

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Evaluation of antibacterial properties of fluoride-containing mouth rinses differing in their acidic compound using a Streptococcus mutans biofilm.

This in vitro study assessed the antibacterial effect on Streptococcus mutans biofilms of mouth rinses with 700 ppm F- (derived from NaF) that differed only in their acid compounds (malic (A), citric (B), tartaric (C), fumaric (D), hydrochloric (E), phosphoric (F), and lactic (G) acid) used to adjust pH. S. mutans (ATCC 25175) was grown for 22 h at 37°C, harvested, resuspended in simulated body fluid and biofilm formation followed for 24 h at 37°C. Thereafter, biofilms were treated with experimental rinses for 30 s, and placed in TAM48 isothermal microcalorimeter at 37°C for 72 h. Applying Gompertz growth model parameters lag time and growth rate were determined from heatflow curves; additionally, reduction of active biofilms was calculated. Moreover, samples were live/dead stained and analyzed by confocal scanning microscopy. All mouth rinses were showing statistically significant lag time and reduction of active biofilm (p<0.05, A 19.1+/-2.3h and 58.5+/-7.7%, B 15.5+/-1.1h and 41.9+/-5.3%, C 17.6+/-1.9h and 53.1+/-7.5%, D 18.4+/-2.4h and 55.8+/-8.8%, E 20.2+/-3.3h and 61.5+/-10.0%, F 20.2+/-3.0h and 61.6+/-9.3%, and G 18.3+/-2.5h and 55.3+/-8.9%). Interestingly, there were no differences found between the treated groups (p>0.05, A 0.064+/-0.004 1/h, B 0.063+/-0.005 1/h, C 0.065+/-0.004 1/h, D 0.067+/-0.004 1/h, E 0.066+/-0.006 1/h, F 0.067+/-0.004 1/h, G 0.066+/-0.006 1/h) for the maximum growth rate. Vitality staining supported these findings.. The present investigation demonstrates that the type of acid compounds used to produce the rinses did not show any negative effect on the antimicrobial properties of the tested products as all of them exhibited a similar efficacy against S. mutans biofilms.

Preservation of exopolymeric substances in estuarine sediments.

The surface of intertidal estuarine sediments is covered with diatom biofilms excreting exopolymeric substances (EPSs) through photosynthesis. These EPSs are highly reactive and increase sediment cohesiveness notably through organo-mineral interactions. In most sedimentary environments, EPSs are partly to fully degraded by heterotrophic bacteria in the uppermost millimeters of the sediment and so they are thought to be virtually absent deeper in the sedimentary column. Here, we present the first evidence of the preservation of EPSs and EPS-mineral aggregates in a 6-m-long sedimentary core obtained from an estuarine point bar in the Gironde Estuary. EPSs were extracted from 18 depth intervals along the core, and their physicochemical properties were characterized by (i) wet chemical assays to measure the concentrations of polysaccharides and proteins, and EPS deprotonation of functional groups, (ii) acid-base titrations, and (iii) Fourier transform infrared spectroscopy. EPS-sediment complexes were also imaged using cryo-scanning electron microscopy. EPS results were analyzed in the context of sediment properties including facies, grain size, and total organic carbon, and of metabolic and enzymatic activities. Our results showed a predictable decrease in EPS concentrations (proteins and polysaccharides) and reactivity from the surface biofilm to a depth of 0.5 m, possibly linked to heterotrophic degradation. Concentrations remained relatively low down to ca. 4.3 m deep. Surprisingly, at that depth EPSs abundance was comparable to the surface and showed a downward decrease to 6.08 m. cryo-scanning electron microscopy (Cryo-SEM) showed that the EPS complexes with sediment were abundant at all studied depth and potentially protected EPSs from degradation. EPS composition did not change substantially from the surface to the bottom of the core. EPS concentrations and acidity were anti-correlated with metabolic activity, but showed no statistical correlation with grain size, TOC, depth or enzymatic activity. Maximum EPS concentrations were found at the top of tide-dominated sedimentary sequences, and very low concentrations were found in river flood-dominated sedimentary sequences. Based on this observation, we propose a scenario where biofilm development and EPS production are maximal when (i) the point bar and the intertidal areas were the most extensive, i.e., tide-dominated sequences and (ii) the tide-dominated deposit were succeeded by rapid burial beneath sediments, potentially decreasing the probability of encounter between bacterial cells and EPSs.

Saliva profiling with differential scanning calorimetry: A feasibility study with ex vivo samples.

Differential scanning calorimetry (DSC) has been used widely to study various biomarkers from blood, less is known about the protein profiles from saliva. The aim of the study was to investigate the use DSC in order to detect saliva thermal profiles and determine the most appropriate sampling procedure to collect and process saliva. Saliva was collected from 25 healthy young individuals and processed using different protocols based on centrifugation and filtering. The most effective protocol was centrifugation at 5000g for 10 min at 4°C followed by filtration through Millex 0.45 µm filter. Prepared samples were transferred to 3 mL calorimetric ampoules and then loaded into TAM48 calibrated to 30°C until analysis. DSC scans were recorded from 30°C to 90°C at a scan rate of 1°C/h with a pre-conditioning the samples to starting temperature for 1 h. The results show that the peak distribution of protein melting points was clearly bimodal, and the majority of peaks appeared between 40-50°C. Another set of peaks is visible between 65°C- 75°C. Additionally, the peak amplitude and area under the peak are less affected by the concentration of protein in the sample than by the individual differences between people. In conclusion, the study shows that with right preparation of the samples, there is a possibility to have thermograms of salivary proteins that show peaks in similar temperature regions between different healthy volunteers.

A Method to Determine the Efficacy of a Commercial Phage Preparation against Uropathogens in Urine and Artificial Urine Determined by Isothermal Microcalorimetry.

BACKGROUND: Urinary tract infections are commonly encountered and often treated with antibiotics. However, the inappropriate use of the latter has led to the appearance of resistant strains. In this context we investigate the use of calorimetry to rapidly determine if a phage cocktail can be used as alternative to antibiotics. METHODS: We used a commercially available phage cocktail from an online pharmacy and tested it against a strain of Escherichia coli and a strain of Proteus mirabilis. We used isothermal microcalorimetry to follow the metabolic activity of the bacterial culture treated with the phage cocktail. RESULTS: Isothermal microcalorimetry was able to follow the dynamic of the bacterial metabolic activity reduction by the phage cocktail. Both pathogens were strongly inhibited; however, some regrowth was observed for E. coli in urine. CONCLUSIONS: Isothermal microcalorimetry proved to be a valuable technique when investigating the efficacy of phage cocktails against uropathogens. We foresee that isothermal microcalorimetry could be used to obtain rapid phagograms.

Bacterial extracellular polymeric substances as potential saliva substitute.

This proof-of-principle study aims to find commensal oral bacteria that can produce extracellular polymeric substances (EPS), which have similar lubrication properties to saliva and could serve as saliva substitutes. Saliva and plaque samples were collected from 21 generally healthy individuals. Primary screening was done by conventional culturing and Gram-staining; all species selected for further analysis were identified by MALDI-TOF and deposited in DSMZ. Lactobacillus gasseri (DSM32453 and DSM32455), Lactobacillus rhamnosus (DSM32452), Lactobacillus paracasei (DSM32454), and Streptococcus sanguinis (DSM32456) produced 413.6, 415.7, 431.1, 426.8, and 877.6 µg/ml of EPS, respectively. At the same time calcium dissolution could not be detected for both L. gasseri strains, minimal dissolution for the other three: S. sanguinis 0.3 mm, and 3.7 mm for L. rhamnosus and L. paracasei. There were no differences found between the EPS samples and the saliva for the effect of shear rate on the viscosity and for the effect of sliding speed on lubrication properties. In conclusion, five commensal bacterial strains have been isolated, all able to produce EPS and lead to no or to low calcium dissolution. EPS produced exhibits rheological and tribological properties comparable to human saliva. A total of four out of five selected strains are probiotic and, therefore, may exhibit additional beneficial influence within the oral cavity.

PET CMRglc mapping and 1H-MRS show altered glucose uptake and neurometabolic profiles in BDL rats.

Type C hepatic encephalopathy (HE) is a complex neuropsychiatric disorder occurring as a consequence of chronic liver disease. Alterations in energy metabolism have been suggested in type C HE, but in vivo studies on this matter remain sparse and have reported conflicting results. Here, we propose a novel preclinical 18F-FDG PET methodology to compute quantitative 3D maps of the regional cerebral metabolic rate of glucose (CMRglc) from a labelling steady-state PET image of the brain and an image-derived input function. This quantitative approach shows its strength when comparing groups of animals with divergent physiology, such as HE animals. PET CMRglc maps were registered to an atlas and the mean CMRglc from the hippocampus and the cerebellum were associated to the corresponding localized 1H MR spectroscopy acquisitions. This study provides for the first time local and quantitative information on both brain glucose uptake and neurometabolic profile alterations in a rat model of type C HE. A 2-fold lower brain glucose uptake, concomitant with an increase in brain glutamine and a decrease in the main osmolytes, was observed in the hippocampus and in the cerebellum. These novel findings are an important step towards new insights into energy metabolism in the pathophysiology of HE.

Current and potential new treatment strategies for creatine deficiency syndromes.

Creatine deficiency syndromes (CDS) are inherited metabolic disorders caused by mutations in GATM, GAMT and SLC6A8 and mainly affect central nervous system (CNS). AGAT- and GAMT-deficient patients lack the functional brain endogenous creatine (Cr) synthesis pathway but express the Cr transporter SLC6A8 at blood-brain barrier (BBB), and can thus be treated by oral supplementation of high doses of Cr. For Cr transporter deficiency (SLC6A8 deficiency or CTD), current treatment strategies benefit one-third of patients. However, as their phenotype is not completely reversed, and for the other two-thirds of CTD patients, the development of novel more effective therapies is needed. This article aims to review the current knowledge on Cr metabolism and CDS clinical aspects, highlighting their current treatment possibilities and the most recent research perspectives on CDS potential therapeutics designed, in particular, to bring new options for the treatment of CTD.

Creatine transporter-deficient rat model shows motor dysfunction, cerebellar alterations, and muscle creatine deficiency without muscle atrophy.

Creatine (Cr) is a nitrogenous organic acid and plays roles such as fast phosphate energy buffer to replenish ATP, osmolyte, antioxidant, neuromodulator, and as a compound with anabolic and ergogenic properties in muscle. Cr is taken from the diet or endogenously synthetized by the enzymes arginine:glycine amidinotransferase and guanidinoacetate methyltransferase, and specifically taken up by the transporter SLC6A8. Loss-of-function mutations in the genes encoding for the enzymes or the transporter cause creatine deficiency syndromes (CDS). CDS are characterized by brain Cr deficiency, intellectual disability with severe speech delay, behavioral troubles, epilepsy, and motor dysfunction. Among CDS, the X-linked Cr transporter deficiency (CTD) is the most prevalent with no efficient treatment so far. Different animal models of CTD show reduced brain Cr levels, cognitive deficiencies, and together they cover other traits similar to those of patients. However, motor function was poorly explored in CTD models, and some controversies in the phenotype exist in comparison with CTD patients. Our recently described Slc6a8Y389C knock-in rat model of CTD showed mild impaired motor function, morphological alterations in cerebellum, reduced muscular mass, Cr deficiency, and increased guanidinoacetate content in muscle, although no consistent signs of muscle atrophy. Our results indicate that such motor dysfunction co-occurred with both nervous and muscle dysfunctions, suggesting that muscle strength and performance as well as neuronal connectivity might be affected by this Cr deficiency in muscle and brain.

Cinnamaldehyde as antimicrobial in cellulose-based dental appliances.

AIMS: In the context of minor orthodontic intervention using clear aligner technologies, we determined antimicrobial properties of a cellulose-based material loaded with essential oils such as cinnamaldehyde. METHODS AND RESULTS: Isothermal microcalorimetry was used to assess the growth of bacterial biofilms at the interface between the tested material and the solid growth medium. The calorimetric data were analyzed using conventional growth models (Gompertz and Richards), and inhibition at 12 and 24 h was calculated. CONCLUSIONS: The tested material showed antimicrobial properties against Staphylococcus epidermidis as well as Streptococcus mutans and Streptococcus mitis clinical isolates. The inhibition was more pronounced against S. epidermidis, for which growth rate was reduced by 70% and lag phase was extended by 12 h. For S. mutans and S. mitis, the decrease in growth rate was 20% and 10%, and the lag phase increased by 2 and 6 h, respectively. SIGNIFICANCE AND IMPACT: Clear aligners for minor teeth alignment are becoming very popular. As they must be worn for at least 22 h per day for up to 40 weeks, it is important that they remain clean and do not promote caries formation or other oral infections. Therefore, introducing material with antimicrobial properties is expected to maintain oral hygiene during the aligner therapy. Here, we demonstrate the use of cinnamaldehyde for reducing microbial growth and biofilm formation on cellulose-based dental clear aligners.

Detection and Drug Susceptibility Testing of Neisseria gonorrhoeae Using Isothermal Microcalorimetry.

BACKGROUND: Gonorrhea is a frequently encountered sexually transmitted disease that results in urethritis and can further lead to pelvic inflammatory disease, infertility, and possibly disseminated gonococcal infections. Thus, it must be diagnosed promptly and accurately. In addition, drug susceptibility testing should be performed rapidly as well. Unfortunately, Neisseria gonorrhoea is a fastidious microorganism that is difficult to grow and requires culturing in an opaque medium. METHODS: Here, we used isothermal microcalorimetry (IMC) to monitor the growth and the antimicrobial susceptibility of N. gonorrhoea. RESULTS: Using IMC, concentrations of N. gonorrhoea between 2000 and 1 CFU·mL-1 were detected within 12 to 33 h. In addition, drug susceptibility could be monitored easily. CONCLUSIONS: The use of isothermal microcalorimetry provides an interesting and useful tool to detect and characterize fastidious microbes such as N. gonorrhoea that require media incompatible with optical detection conventionally used in many commercial systems.

A knock-in rat model unravels acute and chronic renal toxicity in glutaric aciduria type I.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an autosomal recessive inborn error of metabolism caused by deficiency of the mitochondrial enzyme glutaryl-CoA dehydrogenase (GCDH). The principal clinical manifestation in GA-I patients is striatal injury most often triggered by catabolic stress. Early diagnosis by newborn screening programs improved survival and reduced striatal damage in GA-I patients. However, the clinical phenotype is still evolving in the aging patient population. Evaluation of long-term outcome in GA-I patients recently identified glomerular filtration rate (GFR) decline with increasing age. We recently created the first knock-in rat model for GA-I harboring the mutation p.R411W (c.1231 C>T), corresponding to the most frequent GCDH human mutation p.R402W. In this study, we evaluated the effect of an acute metabolic stress in form of high lysine diet (HLD) on young Gcdhki/ki rats. We further studied the chronic effect of GCDH deficiency on kidney function in a longitudinal study on a cohort of Gcdhki/ki rats by repetitive 68Ga-EDTA positron emission tomography (PET) renography, biochemical and histological analyses. In young Gcdhki/ki rats exposed to HLD, we observed a GFR decline and biochemical signs of a tubulopathy. Histological analyses revealed lipophilic vacuoles, thinning of apical brush border membranes and increased numbers of mitochondria in proximal tubular (PT) cells. HLD also altered OXPHOS activities and proteome in kidneys of Gcdhki/ki rats. In the longitudinal cohort, we showed a progressive GFR decline in Gcdhki/ki rats starting at young adult age and a decline of renal clearance. Histopathological analyses in aged Gcdhki/ki rats revealed tubular dilatation, protein accumulation in PT cells and mononuclear infiltrations. These observations confirm that GA-I leads to acute and chronic renal damage. This raises questions on indication for follow-up on kidney function in GA-I patients and possible therapeutic interventions to avoid renal damage.

Probiotics combined with rifaximin influence the neurometabolic changes in a rat model of type C HE.

Type C hepatic encephalopathy (HE) is a neuropsychiatric disease caused by chronic liver disease. Management of type C HE remains an important challenge because treatment options are limited. Both the antibiotic rifaximin and probiotics have been reported to reduce the symptoms of HE, but longitudinal studies assessing their effects on brain metabolism are lacking and the molecular mechanisms underpinning their effects are not fully understood. Therefore, we evaluated in detail the effects of these different treatments on the neurometabolic changes associated with type C HE using a multimodal approach including ultra-high field in vivo 1H MRS. We analyzed longitudinally the effect of rifaximin alone or in combination with the probiotic Vivomixx on the brain metabolic profile in the hippocampus and cerebellum of bile duct ligated (BDL) rats, an established model of type C HE. Overall, while rifaximin alone appeared to induce no significant effect on the neurometabolic profile of BDL rats, its association with the probiotic resulted in more attenuated neurometabolic alterations in BDL rats followed longitudinally (i.e. a smaller increase in Gln and milder decrease in Glu and Cr levels). Given that both rifaximin and some probiotics are used in the treatment of HE, the implications of these findings may be clinically relevant.

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

Classical homocystinuria, is it safe to exercise?

Background Cystationine ß-synthase (CBS) deficiency is a genetic disorder characterized by severe hyperhomocysteinemia and thrombotic complications. In healthy individuals, physical exercise may result in a transient increase in plasma total homocysteine (tHcy) raising the possibility that exercise might be detrimental in CBS deficiency. Our main objective was to determine plasma tHcy kinetics in response to physical exercise in homocystinuria patients. Methods Six adult patients (2 males, 4 females) with homocystinuria and 6 age- and gender-matched controls completed a 30-min aerobic exercise of moderate-intensity with fixed power output (50 W for women and 100 W for men). Blood samples were drawn before, immediately, 180 min and 24 h after exercise. tHcy levels were determined by standard procedures; substrate oxidation and energy expenditure were measured using indirect calorimetry. Results Acute exercise was well tolerated and safe in patients and controls. During the exercise bout, heart rate and energy expenditure increased equally in both groups. tHcy levels were higher in patients compared to controls at all time points (p < 0.05). There was no significant effect of exercise on tHcy levels at any time point (p = 0.36). Although two patients with partial pyridoxine responsiveness presented higher homocysteine responses, their highest value remained below 55 µmol/l. Conclusions Overall metabolic responses to acute exercise were similar between homocystinuria patients and controls; specifically, exercise did not significantly change tHcy concentrations. Moderate physical exercise was well tolerated without any adverse event in our cohort of patients. Further studies are needed to identify the effects of different intensities and modes of exercise in larger cohorts of CBS patients with different levels of pyridoxine responsiveness.