Kainate receptors modulate the microstructure of synchrony during dentate gyrus epileptiform activity.

Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. In TLE, recurrent mossy fiber (rMF) sprouting from dentate gyrus granule cells (DGCs) forms an aberrant epileptogenic network between dentate granule cells (DGCs) that operates via ectopically expressed kainate receptors (KARs). It was previously shown that KARs expressed at the rMF-DGC synapses play a prominent role in epileptiform network events in TLE. However, it is not well understood how KARs influence neuronal network dynamics and contribute to the generation of epileptiform network activity in the dentate gyrus. To address this question, we monitored the activity of DGCs using single-cell resolution calcium imaging performed in a reliable in vitro model of TLE. Under our experimental conditions, the most prominent DGC activity patterns were interictal-like epileptiform network events, which were correlated with high levels of neuronal synchronization. The pharmacological blockade of KARs reduced the frequency as well as the number of neurons involved in these events, without altering their spatiotemporal dynamics. Analysis of the microstructure of synchrony showed that blockade of KARs diminished the fraction of neurons forming the main functional cluster. Therefore, we propose that KARs act as modulators in the epileptic network by facilitating the recruitment of neurons into coactive cell assemblies, thereby contributing to the occurrence of epileptiform network events.

GluK2 Is a Target for Gene Therapy in Drug-Resistant Temporal Lobe Epilepsy.

OBJECTIVE: Temporal lobe epilepsy (TLE) is characterized by recurrent seizures generated in the limbic system, particularly in the hippocampus. In TLE, recurrent mossy fiber sprouting from dentate gyrus granule cells (DGCs) crea an aberrant epileptogenic network between DGCs which operates via ectopically expressed GluK2/GluK5-containing kainate receptors (KARs). TLE patients are often resistant to anti-seizure medications and suffer significant comorbidities; hence, there is an urgent need for novel therapies. Previously, we have shown that GluK2 knockout mice are protected from seizures. This study aims at providing evidence that downregulating KARs in the hippocampus using gene therapy reduces chronic epileptic discharges in TLE. METHODS: We combined molecular biology and electrophysiology in rodent models of TLE and in hippocampal slices surgically resected from patients with drug-resistant TLE. RESULTS: Here, we confirmed the translational potential of KAR suppression using a non-selective KAR antagonist that markedly attenuated interictal-like epileptiform discharges (IEDs) in TLE patient-derived hippocampal slices. An adeno-associated virus (AAV) serotype-9 vector expressing anti-grik2 miRNA was engineered to specifically downregulate GluK2 expression. Direct delivery of AAV9-anti grik2 miRNA into the hippocampus of TLE mice led to a marked reduction in seizure activity. Transduction of TLE patient hippocampal slices reduced levels of GluK2 protein and, most importantly, significantly reduced IEDs. INTERPRETATION: Our gene silencing strategy to knock down aberrant GluK2 expression demonstrates inhibition of chronic seizure in a mouse TLE model and IEDs in cultured slices derived from TLE patients. These results provide proof-of-concept for a gene therapy approach targeting GluK2 KARs for drug-resistant TLE patients. ANN NEUROL 2023;94:745-761.

A Novel Highly Efficient Device for Growing Micro-Aerophilic Microorganisms.

This work describes a novel, simple and cost-effective culture system, named the Micro-Oxygenated Culture Device (MOCD), designed to grow microorganisms under particularly challenging oxygenation conditions. Two microaerophilic magnetotactic bacteria, a freshwater Magnetospirillum gryphiswaldense strain MSR-1 and a marine Magnetospira sp. strain QH-2, were used as biological models to prove the efficiency of the MOCD and to evaluate its specifications. Using the MOCD, growth rates of MSR-1 and QH-2 increased by four and twofold, respectively, when compared to traditional growing techniques using simple bottles. Oxystat-bioreactors have been typically used and specifically designed to control low dissolved oxygen concentrations, however, the MOCD, which is far less sophisticated was proven to be as efficient for both MSR-1 and QH-2 cultures with regard to growth rate, and even better for MSR-1 when looking at cell yield (70% increase). The MOCD enables a wide range of oxygenation conditions to be studied, including different O2-gradients. This makes it an innovative and ingenious culture device that opens up new parameters for growing microaerobic microorganisms.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima Part II: modeling and experimental approaches for hydrogen production.

BACKGROUND: Thermotoga maritima is a hyperthermophilic bacterium known to produce hydrogen from a large variety of substrates. The aim of the present study is to propose a mathematical model incorporating kinetics of growth, consumption of substrates, product formations, and inhibition by hydrogen in order to predict hydrogen production depending on defined culture conditions. RESULTS: Our mathematical model, incorporating data concerning growth, substrates, and products, was developed to predict hydrogen production from batch fermentations of the hyperthermophilic bacterium, T. maritima. It includes the inhibition by hydrogen and the liquid-to-gas mass transfer of H2, CO2, and H2S. Most kinetic parameters of the model were obtained from batch experiments without any fitting. The mathematical model is adequate for glucose, yeast extract, and thiosulfate concentrations ranging from 2.5 to 20 mmol/L, 0.2-0.5 g/L, or 0.01-0.06 mmol/L, respectively, corresponding to one of these compounds being the growth-limiting factor of T. maritima. When glucose, yeast extract, and thiosulfate concentrations are all higher than these ranges, the model overestimates all the variables. In the window of the model validity, predictions of the model show that the combination of both variables (increase in limiting factor concentration and in inlet gas stream) leads up to a twofold increase of the maximum H2-specific productivity with the lowest inhibition. CONCLUSIONS: A mathematical model predicting H2 production in T. maritima was successfully designed and confirmed in this study. However, it shows the limit of validity of such mathematical models. Their limit of applicability must take into account the range of validity in which the parameters were established.

Hydrogen production by the hyperthermophilic bacterium Thermotoga maritima part I: effects of sulfured nutriments, with thiosulfate as model, on hydrogen production and growth.

BACKGROUND: Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H2/glucose yields. However, to develop economically sustainable bio-processes, the culture medium formulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen. RESULTS: Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glucose 60 mmol L-1 and yeast extract 1 g L-1), the cellular growth was limited by thiosulfate concentrations lower than 0.06 mmol L-1. Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 3617 mg mmol-1. In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysaccharides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium significantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production. CONCLUSIONS: The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present at low concentrations (about 0.3 mmol g-1 of cells), and (2) as both sulfur source and detoxifying agent for H2 when thiosulfate is present at higher concentrations and, when, simultaneously, the pH2 is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosulfate in the culture medium.

A eukaryotic-like sulfiredoxin involved in oxidative stress responses and in the reduction of the sulfinic form of 2-Cys peroxiredoxin in the cyanobacterium Anabaena PCC 7120.

The overoxidation of 2-Cys peroxiredoxins (Prxs) into a sulfinic form was thought to be an irreversible protein inactivation process until sulfiredoxins (Srxs) were discovered. These are enzymes occurring among eukaryotes, which are able to reduce sulfinylated Prxs. Although Prxs are present in the three domains of life, their reduction by Srxs has been described only in eukaryotes so far. Here it was established that the cyanobacterium Anabaena PCC 7120 has a Srx homologue (SrxA), which is able to specifically reduce the sulfinic form of the 2-Cys Prx (PrxA) both in vivo and in vitro. A mutant lacking the srxA gene was found to be more sensitive than the wild type to oxidative stress. Sulfiredoxin homologues are restricted to the cyanobacterial and eukaryotic genomes sequenced so far. The present phylogenetic analysis of Srx and 2-Cys Prx sequences showed a pattern of coevolution of the enzyme and its substrate that must have involved an ancient gene transfer between ancestors of Cyanobacteria and Eukaryotes, followed by a more recent transfer from Cyanobacteria to Plantae through the chloroplastic endosymbiosis. This is the first functional characterization of a Srx enzyme in a prokaryotic organism.

NtcA regulates patA expression in Anabaena sp. strain PCC 7120.

patA expression is induced 3 to 6 h after nitrogen step-down. We establish that the transcription of patA is under the positive control of NtcA. The patA promoter region shows two conserved NtcA-binding boxes. These NtcA-binding sites and their interaction with NtcA are key elements for patA expression in heterocysts.

Transcriptional regulation of the Clostridium cellulolyticum cip-cel operon: a complex mechanism involving a catabolite-responsive element.

The cip-cel cluster of genes plays an important role in the catabolism of the substrate cellulose by Clostridium cellulolyticum. It encodes several key components of the cellulosomes, including the scaffolding protein CipC and the major cellulase Cel48F. All the genes of this cluster display linked transcription, focusing attention on the promoter upstream from the first gene, cipC. We analyzed the regulation of the cipC promoter using a transcriptional fusion approach. A single promoter is located between nucleotides -671 and -643 with respect to the ATG start codon, and the large mRNA leader sequence is processed at position -194. A catabolite-responsive element (CRE) 414 nucleotides downstream from the transcriptional start site has been shown to be involved in regulating this operon by a carbon catabolite repression mechanism. This CRE is thought to bind a CcpA-like regulator complexed with a P-Ser-Crh-like protein. Sequences surrounding the promoter sequence may also be involved in direct (sequence-dependent DNA curvature) or indirect (unknown regulator binding) regulation.

Transcriptional analysis of the cip-cel gene cluster from Clostridium cellulolyticum.

Twelve genes encoding key components of Clostridium cellulolyticum cellulosomes are clustered. Among them, the first, second, and fifth genes encode the assembly factor CipC and the two major cellulases Cel48F and Cel9E, respectively. Cellulolytic clones were selected from the noncellulolytic cipC insertional mutant trans-complemented with a cipC expression vector, in which one homologous recombination event between the 3' end of the chromosomal cipC gene and the plasmidic cipC gene has restored the cluster continuity. The absence of the enzymes encoded by the cluster in the cipC mutant was thus only due to a strong polar effect, indicating that all genes were transcriptionally linked. Two large transcripts were detected in cellulose-grown cells by Northern hybridization: a 14-kb messenger which carries the cipC-cel48F-cel8C-cel9G-cel9E coding sequences and, in a smaller amount, a 12-kb messenger which carries the genes located in the 3' part of the cluster. Four smaller transcripts were found in large amounts: a cipC-cel48F bicistronic one and three monocistronic ones, cipC, cel48F, and cel9E. The cipC-cel48F and cel48F messengers were shown to be stable. Analysis by reverse transcription-PCR suggested transcriptional linkage of all of the open reading frames. The production of a primary very large transcript covering the entire cluster was hypothesized. Primer extension analysis has identified two putative transcriptional start sites located 638/637 and 194 nucleotides upstream of the cipC translational start. The processing of the primary transcript would lead to the production of several secondary messengers displaying different stabilities, contributing to fine tuning of expression of individual genes of the operon.