In vitro measurement of enzymatic markers as a tool to detect mouse cardiomyocytes injury

Primary cultures of cardiomyocytes represent a useful model for analyzing cardiac cell biology as well as pathogenesis of several cardiovascular disorders. Our aim was to standardize protocols for determining the damage of cardiac cells cultured in vitro by measuring the creatine kinase and its cardiac isotype and lactate dehydrogenase activities in the supernatants of mice cardiomyocytes submitted to different protocols of cell lysis. Our data showed that due to its higher specificity, the cardiac isotype creatine kinase was the most sensitive as compared to the others studied enzymatic markers, and can be used to monitor and evaluate cardiac damage in in vitro assays.     

Novel γ-secretase enzyme modulators directly target presenilin protein

γ-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid β-peptide (Aβ(42)). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the β-amyloid precursor protein by an intramembrane cleavage of γ-secretase. Because Aβ(42) is widely believed to trigger AD pathogenesis, γ-secretase is a key AD drug target. Unlike inhibitors of the enzyme, γ-secretase modulators (GSMs) selectively lower Aβ(42) without interfering with the physiological function of γ-secretase. The molecular target(s) of GSMs and hence the mechanism of GSM action are not established. Here we demonstrate by using a biotinylated photocross-linkable derivative of highly potent novel second generation GSMs that γ-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of γ-secretase, but not to other γ-secretase subunits. Binding was differentially competed by GSMs of diverse structural classes, indicating the existence of overlapping/multiple GSM binding sites or allosteric alteration of the photoprobe binding site. The β-amyloid precursor protein C-terminal fragment previously implicated as the GSM binding site was not targeted by the compound. The identification of presenilin as the molecular target of GSMs directly establishes allosteric modulation of enzyme activity as a mechanism of GSM action and may contribute to the development of therapeutically active GSMs for the treatment of AD.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

Constitutive delivery of bovine beta-lactoglobulin to the digestive tracts of gnotobiotic mice by engineered Lactobacillus casei

The gut microbiota is critical for maturation of the immune system. Recent evidence suggests that early establishment of lactobacilli in the intestinal microbiota, during neonatal colonization or by probiotic supplementation, could prevent the development of allergic disorders. Postnatal maturation of the gut immune system with allergen-producing lactobacilli colonizing the digestive tract could then affect the development of further allergic sensitization. In this paper, we describe construction of a recombinant Lactobacillus casei strain that can constitutively deliver bovine beta-lactoglobulin (BLG), a major cow's milk allergen, to the guts of gnotobiotic mice. The blg gene was inserted into the L. casei chromosome downstream of an endogenous promoter. BLG production was improved by fusing the propeptide LEISSTCDA (LEISS) to the BLG mature moiety. This led to a 10-fold increase in LEISS-BLG production compared to the production obtained without the propeptide and also led to enhanced secretion corresponding to 5% of the total production. After inoculation into germfree C3H/HeN mice, the genetic stability of the recombinant strain and in vivo BLG production were confirmed for at least 10 weeks. BLG stimulation of spleen cells from mice monoassociated with the BLG-producing lactobacilli induced secretion of the Th1 cytokine gamma interferon and, to a lesser extent, the Th2 cytokine interleukin-5. No BLG-specific immunoglobulin G1 (IgG1), IgG2a, or IgA was detected in sera or in fecal samples. These results suggest that gut colonization with allergen-producing lactobacilli could provide a useful model for studying the modulation of allergic disorders.     

Microbial Community Development in a Dynamic Gut Model Is Reproducible,Colon Region Specific,and Selective for Bacteroidetes and Clostridium Cluster IX

Dynamic, multicompartment in vitro gastrointestinal simulators are often used to monitor gut microbial dynamics and activity. These reactors need to harbor a microbial community that is stable upon inoculation, colon region specific, and relevant to in vivo conditions. Together with the reproducibility of the colonization process, these criteria are often overlooked when the modulatory properties from different treatments are compared. We therefore investigated the microbial colonization process in two identical simulators of the human intestinal microbial ecosystem (SHIME), simultaneously inoculated with the same human fecal microbiota with a high-resolution phylogenetic microarray: the human intestinal tract chip (HITChip). Following inoculation of the in vitro colon compartments, microbial community composition reached steady state after 2 weeks, whereas 3 weeks were required to reach functional stability. This dynamic colonization process was reproducible in both SHIME units and resulted in highly diverse microbial communities which were colon region specific, with the proximal regions harboring saccharolytic microbes (e.g., Bacteroides spp. and Eubacterium spp.) and the distal regions harboring mucin-degrading microbes (e.g., Akkermansia spp.). Importantly, the shift from an in vivo to an in vitro environment resulted in an increased Bacteroidetes/Firmicutes ratio, whereas Clostridium cluster IX (propionate producers) was enriched compared to clusters IV and XIVa (butyrate producers). This was supported by proportionally higher in vitro propionate concentrations. In conclusion, high-resolution analysis of in vitro-cultured gut microbiota offers new insight on the microbial colonization process and indicates the importance of digestive parameters that may be crucial in the development of new in vitro models.The human gastrointestinal tract harbors a complex microbial ecosystem with a coding capacity exceeding that of the host genome by a factor of 100 (13). These gut microbes play a determining role in host health by converting otherwise indigestible compounds (14, 19), protecting against gut epithelial cell injury (46), regulating host fat storage (49), and inducing immunity (20, 48). Modulation of the composition and metabolic activity of these microbes to improve host health attracts a lot of attention and is referred to as gastrointestinal resource management (15, 37). Such new strategies are often evaluated during human trials or in vivo studies of animals associated with conventional or human microbiota (50).Despite the physiological relevance, in vivo experimental setups are inherently associated with some drawbacks. First, apart from fecal analyses over time, most in vivo data are derived from endpoint measurements, thereby limiting the dynamic monitoring of the gut microbiota. Second, troublesome sampling of different gut regions makes it difficult to locate the effects of a treatment. For mechanistic reasons, a third drawback of an in vivo approach is the inability to focus solely on gut microbial activity, because there is always a host involved. For these reasons, different types of in vitro systems have been developed, ranging from simple nonstirred batch cultures without pH control (44) to more complex continuous models involving pH-controlled single (55) or dynamic multicompartment (2, 29, 32, 34) culture systems. Other advantages are the lack of ethical constraints and a higher reproducibility due to strict control of environmental factors that can influence the microbiota, such as retention time, pH, temperature, and food intake. Therefore, in vitro methods are widely used to elucidate the mechanism behind the degradation of prebiotics (17, 52), bioactivation of polyphenols (10, 36, 38), adhesion of microbes to mucins (51), or bioavailability of environmental contaminants (53, 54).Dynamic in vitro gut models need to fulfil certain criteria before they can be used to monitor the modulating potency of specific treatments toward the microbiota. To ensure that effects are due solely to the treatment and not to the adaptation of microbes to the in vitro environment, steady-state conditions in terms of microbial community composition and metabolic activity need to be established prior to the actual start of the experiment (39). Moreover, the stabilization of this in vitro microbiota needs to be reproducible, as comparison of different treatments requires identical starting communities. Former studies assumed but never fully substantiated this requirement (17, 38). Further, in vitro microbiota need to be gut region specific, be representative for the in vivo situation, and maintain a high diversity. The potency of in vitro models thus relies on a good characterization of its microbiota. Molecular techniques, such as denaturing gradient gel electrophoresis (DGGE) (17, 39, 52), fluorescent in situ hybridization (FISH) (6), and quantitative real-time PCR (Q-PCR) (17, 29), provide useful information but do not provide direct phylogenetic information or target only a limited group of previously identified organisms, therefore limiting current knowledge. Recently, high-resolution techniques, such as microarrays (41, 42) and pyrosequencing (59), have provided access to phylogenetic and metagenomic analysis of the gut microbiota in unprecedented detail.In this study, we performed conventional metabolic analysis, applied existing molecular techniques (DGGE), and for the first time provided an in-depth phylogenetic analysis on the simulator of the human intestinal microbial ecosystem (SHIME) in vitro microbiota using the recently developed human intestinal tract chip (HITChip) microarray (41, 42). We evaluated the microbial colonization process in two parallel in vitro simulators (Twin-SHIME) simultaneously inoculated with the same human fecal microbiota. The aims of this study were (i) to determine when the microbial community composition and metabolic activity reach steady-state conditions, (ii) to assess the reproducibility of the stabilization process in two identical in vitro simulators, (iii) to obtain a high-resolution characterization of the colon region specificity of the residing communities, and (iv) to evaluate how the in vivo fecal inoculum changes to the in vitro colon microbial communities.     

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O₂, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O₂ permeability. High flow rates of 520 μL min⁻¹ prevented biofilm sloughing in 1 m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O₂ concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5 m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8 g m⁻² and, thus, a successful CBR scale-up by a factor of 25. Practical application : Cyanobacteria use light energy to upgrade CO₂, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O₂ accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.     

Novel 5′ Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

We reported that iron influx drives the translational expression of the neuronal amyloid precursor protein (APP), which has a role in iron efflux. This is via a classic release of repressor interaction of APP mRNA with iron-regulatory protein-1 (IRP1) whereas IRP2 controls the mRNAs encoding the L- and H-subunits of the iron storage protein, ferritin. Here, we identified thirteen potent APP translation blockers that acted selectively towards the uniquely configured iron-responsive element (IRE) RNA stem loop in the 5′ untranslated region (UTR) of APP mRNA. These agents were 10-fold less inhibitory of 5′UTR sequences of the related prion protein (PrP) mRNA. Western blotting confirmed that the ‘ninth’ small molecule in the series selectively reduced neural APP production in SH-SY5Y cells at picomolar concentrations without affecting viability or the expression of α-synuclein and ferritin. APP blocker-9 (JTR-009), a benzimidazole, reduced the production of toxic Aβ in SH-SY5Y neuronal cells to a greater extent than other well tolerated APP 5′UTR-directed translation blockers, including posiphen, that were shown to limit amyloid burden in mouse models of Alzheimer''s disease (AD). RNA binding assays demonstrated that JTR-009 operated by preventing IRP1 from binding to the IRE in APP mRNA, while maintaining IRP1 interaction with the H-ferritin IRE RNA stem loop. Thus, JTR-009 constitutively repressed translation driven by APP 5′UTR sequences. Calcein staining showed that JTR-009 did not indirectly change iron uptake in neuronal cells suggesting a direct interaction with the APP 5′UTR. These studies provide key data to develop small molecules that selectively reduce neural APP and Aβ production at 10-fold lower concentrations than related previously characterized translation blockers. Our data evidenced a novel therapeutic strategy of potential impact for people with trisomy of the APP gene on chromosome 21, which is a phenotype long associated with Down syndrome (DS) that can also cause familial Alzheimer''s disease.