Extending the clinical spectrum of X-linked Tonne-Kalscheuer syndrome (TOKAS): new insights from the fetal perspective.

INTRODUCTION: Tonne-Kalscheuer syndrome (TOKAS) is a recessive X-linked multiple congenital anomaly disorder caused by RLIM variations. Of the 41 patients reported, only 7 antenatal cases were described. METHOD: After the antenatal diagnosis of TOKAS by exome analysis in a family followed for over 35 years because of multiple congenital anomalies in five male fetuses, a call for collaboration was made, resulting in a cohort of 11 previously unpublished cases. RESULTS: We present a TOKAS antenatal cohort, describing 11 new cases in 6 French families. We report a high frequency of diaphragmatic hernia (9 of 11), differences in sex development (10 of 11) and various visceral malformations. We report some recurrent dysmorphic features, but also pontocerebellar hypoplasia, pre-auricular skin tags and olfactory bulb abnormalities previously unreported in the literature. Although no clear genotype-phenotype correlation has yet emerged, we show that a recurrent p.(Arg611Cys) variant accounts for 66% of fetal TOKAS cases. We also report two new likely pathogenic variants in RLIM, outside of the two previously known mutational hotspots. CONCLUSION: Overall, we present the first fetal cohort of TOKAS, describe the clinical features that made it a recognisable syndrome at fetopathological examination, and extend the phenotypical spectrum and the known genotype of this rare disorder.

Enterotype-Dependent Probiotic-Mediated Changes in the Male Rat Intestinal Microbiome In Vivo and In Vitro.

Beneficial properties of lactic acid bacteria have been known long ago, but particular interest in probiotics has arisen in the last two decades due to the understanding of the important role of intestinal microflora in human life. Thus, the ability of probiotics to support healthy homeostasis of gut microbiomes has received particular attention. Here, we evaluated the effect of a probiotic consisting of Bifidobacterium longum and Lacticaseibacillus paracasei on the gut microbiome of male rats, assessed their persistence in the fecal biota, and compared probiotic-mediated changes in vitro and in vivo. As expected, microbiomes of two enterotypes were identified in the feces of 21 animals, and it turned out that even a single dose of the probiotic altered the microbial composition. Upon repeated administration, the E1 biota temporarily acquired properties of the E2 type. Being highly sensitive to the intervention of probiotic bacteria at the phylum and genus levels, the fecal microbiomes retained the identity of their enterotypes when transferred to a medium optimized for gut bacteria. For the E2 biota, even similarities between probiotic-mediated reactions in vitro and in vivo were detected. Therefore, fecal-derived microbial communities are proposed as model consortia to optimize the response of resident bacteria to various agents.

Exoproteomic Study and Transcriptional Responses of Laccase and Ligninolytic Peroxidase Genes of White-Rot Fungus Trametes hirsuta LE-BIN 072 Grown in the Presence of Monolignol-Related Phenolic Compounds.

Being an abundant renewable source of aromatic compounds, lignin is an important component of future bio-based economy. Currently, biotechnological processing of lignin through low molecular weight compounds is one of the conceptually promising ways for its valorization. To obtain lignin fragments suitable for further inclusion into microbial metabolism, it is proposed to use a ligninolytic system of white-rot fungi, which mainly comprises laccases and peroxidases. However, laccase and peroxidase genes are almost always represented by many non-allelic copies that form multigene families within the genome of white-rot fungi, and the contributions of exact family members to the overall process of lignin degradation has not yet been determined. In this article, the response of the Trametes hirsuta LE-BIN 072 ligninolytic system to the presence of various monolignol-related phenolic compounds (veratryl alcohol, p-coumaric acid, vanillic acid, and syringic acid) in culture media was monitored at the level of gene transcription and protein secretion. By showing which isozymes contribute to the overall functioning of the ligninolytic system of the T. hirsuta LE-BIN 072, the data obtained in this study will greatly contribute to the possible application of this fungus and its ligninolytic enzymes in lignin depolymerization processes.

The Fate and Functionality of Alien tRNA Fragments in Culturing Medium and Cells of Escherichia coli.

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria.

Comparative Analysis of Peniophora lycii and Trametes hirsuta Exoproteomes Demonstrates "Shades of Gray" in the Concept of White-Rotting Fungi.

White-rot basidiomycete fungi are a unique group of organisms that evolved an unprecedented arsenal of extracellular enzymes for an efficient degradation of all components of wood such as cellulose, hemicelluloses and lignin. The exoproteomes of white-rot fungi represent a natural enzymatic toolbox for white biotechnology. Currently, only exoproteomes of a narrow taxonomic group of white-rot fungi-fungi belonging to the Polyporales order-are extensively studied. In this article, two white-rot fungi, Peniophora lycii LE-BIN 2142 from the Russulales order and Trametes hirsuta LE-BIN 072 from the Polyporales order, were compared and contrasted in terms of their enzymatic machinery used for degradation of different types of wood substrates-alder, birch and pine sawdust. Our findings suggested that the studied fungi use extremely different enzymatic systems for the degradation of carbohydrates and lignin. While T. hirsuta LE-BIN 072 behaved as a typical white-rot fungus, P. lycii LE-BIN 2142 demonstrated substantial peculiarities. Instead of using cellulolytic and hemicellulolytic hydrolytic enzymes, P. lycii LE-BIN 2142 primarily relies on oxidative polysaccharide-degrading enzymes such as LPMO and GMC oxidoreductase. Moreover, exoproteomes of P. lycii LE-BIN 2142 completely lacked ligninolytic peroxidases, a well-known marker of white-rot fungi, but instead contained several laccase isozymes and previously uncharacterized FAD-binding domain-containing proteins.

Exoproteome Analysis of Antagonistic Interactions between the Probiotic Bacteria Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F and Multidrug Resistant Strain of Klebsiella pneumonia.

The expansion of multiple drug resistant (MDR) strains of Klebsiella pneumoniae presents an immense threat for public health. Annually, this microorganism causes thousands of lethal nosocomial infections worldwide. Currently, it has been shown that certain strains of lactic acid bacteria (LAB) can efficiently inhibit growth of K. pneumoniae and the formation of its biofilms; however, the active principle of such action remains unknown. In the current article, the growth inhibition of MDR K. pneumoniae by two LAB-Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F-is demonstrated, and the nature of this inhibition studied at the level of exoproteome. This article shows that the exoproteomes of studied LAB contains both classically and non-classically secreted proteins. While for L. reuteri LR1 the substantial portion of classically secreted proteins was presented by cell-wall-degrading enzymes, for L. rhamnosus F only one out of four classically secreted proteins was presented by cell-wall hydrolase. Non-classically secreted proteins of both LAB were primarily metabolic enzymes, for some of which a possible moonlighting functioning was proposed. These results contribute to knowledge regarding antagonistic interaction between LAB and pathogenic and opportunistic microorganisms and set new perspectives for the use of LAB to control the spread of these microorganisms.

Laccases with Variable Properties from Different Strains of Steccherinum ochraceum: Does Glycosylation Matter?

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and characterized seven laccases from different strains of Steccherinum ochraceum obtained from regions of central Russia. Although all seven laccases had the same primary sequences, there was a little variation in their molecular weights and thermostabilities. Moreover, statistically significant differences in laccases' catalytic parameters of oxidation of phenolic substrates and ABTS were observed. After the deglycosylation of four selected laccases by Endo H and PNGase F, their affinities to pyrocatechol and ABTS became the same, suggesting a substantial role of N-linked glycosylation in moderation of enzymatic properties of laccases.

Elucidation of the crystal structure of Coriolopsis caperata laccase: restoration of the structure and activity of the native enzyme from the T2-depleted form by copper ions.

Laccases are members of a large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of dioxygen to water. A new laccase was isolated from the basidiomycete Coriolopsis caperata strain 0677 and its amino-acid sequence was determined. According to its physicochemical properties and spectroscopic features, the laccase from C. caperata is a high redox-potential blue laccase. Attempts to crystallize the native enzyme were unsuccessful. The copper type 2-depleted (T2D) laccase was prepared and crystallized. The structure of T2D laccase from C. caperata was solved at 1.6 Šresolution, and attempts to reconstruct the T2 copper centre were performed using Cu(+) and Cu(2+) ions. The structure of T2D+Cu(+) laccase was solved at 1.89 Šresolution. It was shown that the T2D+Cu(+) laccase structure contained four copper ions in the active site. Reconstruction could not be achieved when the T2D laccase crystals were treated with CuSO4.

Hepatitis C virus of subtype 2l in Marseille, southeastern France.

The rate of eradication of chronic hepatitis C considerably increases with direct-acting antiviral agents, particularly hepatitis C virus (HCV) polymerase inhibitors. While implementing full-length HCV NS5B polymerase sequencing in our clinical microbiology laboratory, we identified atypical HCV sequences, classified as subtype 2l, from 2 patients. HCV-2l NS5B polymerase sequences were detected from 5 and 14 additional patients by screening our laboratory hepatitis virus sequence database and the NCBI GenBank sequence database. Phylogenetic analyses show unambiguously that all HCV-2l sequences are clustered apart from HCV 2 non-l sequences, which compose a second cluster. Mean (±SD) nucleotide identity between near full-length NS5B fragments of subtype 2l was 93.4 ± 0.8% (range: 92.4-95.1). Of note, all HCV-2l sequences obtained in our laboratory and in other centers were from serum samples collected in France. Analysis of the HCV-2l NS5B polymerase amino acid sequences at 30 positions critical for interaction with or resistance to HCV polymerase inhibitors showed specific patterns.

Changes in Composition of Some Bioactive Molecules upon Inclusion of Lacticaseibacillus paracasei Probiotic Strains into a Standard Yogurt Starter Culture.

Incorporation of probiotic Lacticaseibacillus paracasei into a standard yogurt starter culture can drastically improve its health promoting properties. However, besides being an advantage in itself, the incorporation of a new probiotic strain can significantly affect the overall composition of fermented milk. In this article, the effect of incorporation of the L. paracasei probiotic strains (KF1 and MA3) into several standard yogurt starter cultures (consisting of the following strains: Streptococcus thermophilus 16t and either Lactobacillus delbrueckii Lb100 or L. delbrueckii Lb200) was investigated. Such parameters as the degree of proteolysis, antioxidant activity, ACE-inhibitory activity, content of organic acids, profile of FAs and profile of volatile organic compounds were measured, and the influence of the starter culture composition on these parameters was described. It was demonstrated that, at least in the case of the studied strains, yogurt with L. paracasei had an advantage over the standard yogurt in terms of the content of acetoin, acetic acid, butyric acid and conjugated linoleic acid. Moreover, the incorporation of L. paracasei KF1 significantly improved the hypotensive properties of the resulting yogurt. Thus, the presented study provides insight into the bioactive molecules of probiotic yogurt and may be useful for both academia and industry in the development of new dairy-based functional products.

Biochemical and Genomic Characterization of Two New Strains of Lacticaseibacillus paracasei Isolated from the Traditional Corn-Based Beverage of South Africa, Mahewu, and Their Comparison with Strains Isolated from Kefir Grains.

Lacticaseibacillus paracasei (formerly Lactobacillus paracasei) is a nomadic lactic acid bacterium (LAB) that inhabits a wide variety of ecological niches, from fermented foodstuffs to host-associated microenvironments. Many of the isolated L. paracasei strains have been used as single-strain probiotics or as part of a symbiotic consortium within formulations. The present study contributes to the exploration of different strains of L. paracasei derived from non-conventional isolation sources-the South African traditional fermented drink mahewu (strains MA2 and MA3) and kefir grains (strains KF1 and ABK). The performed microbiological, biochemical and genomic comparative analyses of the studied strains demonstrated correlation between properties of the strains and their isolation source, which suggests the presence of at least partial strain adaptation to the isolation environments. Additionally, for the studied strains, antagonistic activities against common pathogens and against each other were observed, and the ability to release bioactive peptides with antioxidant and angiotensin I-converting enzyme inhibitory (ACE-I) properties during milk fermentation was investigated. The obtained results may be useful for a deeper understanding of the nomadic lifestyle of L. paracasei and for the development of new starter cultures and probiotic preparations based on this LAB in the future.

In Vitro and In Vivo Antihypertensive Effect of Milk Fermented with Different Strains of Common Starter Lactic Acid Bacteria.

Currently, functional dairy products pave a promising way for the prophylaxis of essential hypertension, and the search for new strains capable of producing such products is a constant challenge for scientists around the world. In this study, the antihypertensive properties of milk fermented with several strains of traditional yogurt starters (Lactobacillus delbrueckii strains Lb100 and Lb200; Lactococcus lactis strains dlA, AM1 and MA1; Streptococcus thermophilus strains 159 and 16t) and one strain of non-conventional probiotic starter (Lacticaseibacillus paracasei ABK) were assessed. The in vitro assessment using angiotensin-converting enzyme inhibition assay was performed for all fermentation products, and the best performed products were tested in vivo using Spontaneously Hypertensive Rat (SHR) animal model. In addition, for the best performed products the fatty acid (FA) composition and FA-related nutritional indices were determined. As a result, the milk fermented with two strains (Lb. delbrueckii LB100 and Lc. lactis AM1) demonstrated significant antihypertensive effect during both in vitro and in vivo experiments. Moreover, the milk fermented with Lb. delbrueckii Lb100 demonstrated significantly better FA-related nutritional indexes and lowered total cholesterol in SHRs upon regular consumption. The obtained results can be used in the future to develop new starter cultures producing effective functional antihypertensive dairy products.

Fungal Laccases: The Forefront of Enzymes for Sustainability.

Enzymatic catalysis is one of the main pillars of sustainability for industrial production. Enzyme application allows minimization of the use of toxic solvents and to valorize the agro-industrial residues through reuse. In addition, they are safe and energy efficient. Nonetheless, their use in biotechnological processes is still hindered by the cost, stability, and low rate of recycling and reuse. Among the many industrial enzymes, fungal laccases (LCs) are perfect candidates to serve as a biotechnological tool as they are outstanding, versatile catalytic oxidants, only requiring molecular oxygen to function. LCs are able to degrade phenolic components of lignin, allowing them to efficiently reuse the lignocellulosic biomass for the production of enzymes, bioactive compounds, or clean energy, while minimizing the use of chemicals. Therefore, this review aims to give an overview of fungal LC, a promising green and sustainable enzyme, its mechanism of action, advantages, disadvantages, and solutions for its use as a tool to reduce the environmental and economic impact of industrial processes with a particular insight on the reuse of agro-wastes.

Suppression of Escherichia coli Growth Dynamics via RNAs Secreted by Competing Bacteria.

With the discovery of secreted RNAs, it has become apparent that the biological role of regulatory oligonucleotides likely goes beyond the borders of individual cells. However, the mechanisms of their action are still comprehended only in general terms and mainly for eukaryotic microRNAs, which can interfere with mRNAs even in distant recipient cells. It has recently become clear that bacterial cells lacking interference systems can also respond to eukaryotic microRNAs that have targets in their genomes. However, the question of whether bacteria can perceive information transmitted by oligonucleotides secreted by other prokaryotes remained open. Here we evaluated the fraction of short RNAs secreted by Escherichia coli during individual and mixed growth with Rhodospirillum rubrum or Prevotella copri, and found that in the presence of other bacteria E. coli tends to excrete oligonucleotides homologous to alien genomes. Based on this observation, we selected four RNAs secreted by either R. rubrum or P. copri, together with one E. coli-specific oligonucleotide. Both fragments of R. rubrum 23S-RNA suppressed the growth of E. coli. Of the two fragments secreted by P. copri, one abolished the stimulatory effect of E. coli RNA derived from the 3'-UTR of ProA mRNA, while the other inhibited bacterial growth only in the double-stranded state with complementary RNA. The ability of two RNAs secreted by cohabiting bacteria to enter E. coli cells was demonstrated using confocal microscopy. Since selected E. coli-specific RNA also affected the growth of this bacterium, we conclude that bacterial RNAs can participate in inter- and intraspecies signaling.

Hypotensive and Hepatoprotective Properties of the Polysaccharide-Stabilized Foaming Composition Containing Hydrolysate of Whey Proteins.

Whey protein hydrolysates (WPHs) are one of the most promising sources of biofunctional peptides with such beneficial properties as antioxidant, antihypertensive, anti-inflammatory and others. WPHs also could be used as foaming agents for aerated products (e.g., milk shake type drinks). However, WPH alone has a bitter taste and foamed WPH should be stabilized by additional ingredients. Here, we present a composition including WPH and three polysaccharides-pumpkin pectin, sodium alginate and ι-carrageenan-used as foam stabilizers. Polysaccharide content was selected according to foaming, organoleptic antioxidant and angiotensin-I-converting enzyme inhibitory characteristics of the resulted composition. Further, the hypotensive, antioxidant and hepatoprotective properties of the composition were proved by in vivo tests performed in spontaneously hypertensive rats and Wistar rats with CCl4-induced hepatic injury.

Analytical Characterization of the Widely Consumed Commercialized Fermented Beverages from Russia (Kefir and Ryazhenka) and South Africa (Amasi and Mahewu): Potential Functional Properties and Profiles of Volatile Organic Compounds.

In this study, four commercialized indigenous fermented beverages most highly consumed in Russia (kefir and ryazhenka) and South Africa (amasi and mahewu) were analyzed for their potential health-promoting properties and flavor-forming volatile organic compounds (VOC). The analysis of antioxidant capacity demonstrated superiority of dairy-based beverages (kefir, ryazhenka and amasi) over the corn-based mahewu; however, mahewu outperformed dairy-based beverages in terms of its potential antihypertensive effect (i.e., the ability to inhibit angiotensin I converting enzyme). The fatty acid (FA) content of kefir and ryazhenka were more diverse compared to that of amasi, but included a lesser amount of branched chain FA. In terms of calculated FA nutritional indices (e.g., indices of atherogenicity and thrombogenicity), kefir and ryazhenka performed similarly and significantly better than amasi. The agreement between beverages theoretical flavor profiles, which was obtained based on the flavors of individual VOC, and consumers' flavor perception allow hypothesizing about the contribution of detected VOC to the overall products' flavor. The obtained data expand current knowledge regarding traditional fermented beverages and their values in terms of national dietary recommendations. Additionally, reported VOC profiles will promote the inclusion of traditional fermented beverages into the rations based on the flavor pairing concept (which is controversial but widely applied).

Lacticaseibacillus paracasei: Occurrence in the Human Gut Microbiota and K-Mer-Based Assessment of Intraspecies Diversity.

Alignment-free approaches employing short k-mers as barcodes for individual genomes have created a new strategy for taxonomic analysis and paved a way for high-resolution phylogeny. Here, we introduce this strategy for the Lacticaseibacillus paracasei species as a taxon requiring barcoding support for precise systematics. Using this approach for phylotyping of L. paracasei VKM B-1144 at the genus level, we identified four L. paracasei phylogroups and found that L. casei 12A belongs to one of them, rather than to the L. casei clade. Therefore, we propose to change the specification of this strain. At the genus level we found only one relative of L. paracasei VKM B-1144 among 221 genomes, complete or available in contigs, and showed that the coding potential of the genome of this "rare" strain allows its consideration as a potential probiotic component. Four sets of published metagenomes were used to assess the dependence of L. paracasei presence in the human gut microbiome on chronic diseases, dietary changes and antibiotic treatment. Only antibiotics significantly affected their presence, and strain-specific barcoding allowed the identification of the main scenarios of the adaptive response. Thus, suggesting bacteria of this species for compensatory therapy, we also propose strain-specific barcoding for selecting optimal strains for target microbiomes.

Relation between lignin molecular profile and fungal exo-proteome during kraft lignin modification by Trametes hirsuta LE-BIN 072.

The process of kraft lignin modification by the white-rot fungus Trametes hirsuta was investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS), and groups of systematically changing compounds were delineated. In the course of cultivation, fungus tended to degrade progressively more reduced compounds and produced more oxidized ones. However, this process was not gradual - the substantial discontinuity was observed between 6th and 10th days of cultivation. Simultaneously, the secretion of ligninolytic peroxidases by the fungus was changing in a cascade manner - new isoenzymes were added to the mixture of the already secreted ones, and once new isoenzyme appeared both its relative quantity and number of isoforms increased as cultivation proceeded. It was proposed, that the later secreted peroxidases (MnP7 and MnP1) possess higher substrate affinity for some phenolic compounds and act in more specialized manner than the early secreted ones (MnP5 and VP2).

Severe Phenotype in Patients with Large Deletions of NF1.

Complete deletion of the NF1 gene is identified in 5-10% of patients with neurofibromatosis type 1 (NF1). Several studies have previously described particularly severe forms of the disease in NF1 patients with deletion of the NF1 locus, but comprehensive descriptions of large cohorts are still missing to fully characterize this contiguous gene syndrome. NF1-deleted patients were enrolled and phenotypically characterized with a standardized questionnaire between 2005 and 2020 from a large French NF1 cohort. Statistical analyses for main NF1-associated symptoms were performed versus an NF1 reference population. A deletion of the NF1 gene was detected in 4% (139/3479) of molecularly confirmed NF1 index cases. The median age of the group at clinical investigations was 21 years old. A comprehensive clinical assessment showed that 93% (116/126) of NF1-deleted patients fulfilled the NIH criteria for NF1. More than half had café-au-lait spots, skinfold freckling, Lisch nodules, neurofibromas, neurological abnormalities, and cognitive impairment or learning disabilities. Comparison with previously described "classic" NF1 cohorts showed a significantly higher proportion of symptomatic spinal neurofibromas, dysmorphism, learning disabilities, malignancies, and skeletal and cardiovascular abnormalities in the NF1-deleted group. We described the largest NF1-deleted cohort to date and clarified the more severe phenotype observed in these patients.

Purification and Characterization of Two Novel Laccases from Peniophora lycii.

Although, currently, more than 100 laccases have been purified from basidiomycete fungi, the majority of these laccases were obtained from fungi of the Polyporales order, and only scarce data are available about the laccases from other fungi. In this article, laccase production by the white-rot basidiomycete fungus Peniophora lycii, belonging to the Russulales order, was investigated. It was shown that, under copper induction, this fungus secreted three different laccase isozymes. Two laccase isozymes-Lac5 and LacA-were purified and their corresponding nucleotide sequences were determined. Both purified laccases were relatively thermostable with periods of half-life at 70 °C of 10 and 8 min for Lac5 and LacA, respectively. The laccases demonstrated the highest activity toward ABTS (97 U·mg-1 for Lac5 and 121 U·mg-1 for LacA at pH 4.5); Lac5 demonstrated the lowest activity toward 2,6-DMP (2.5 U·mg-1 at pH 4.5), while LacA demonstrated this towards gallic acid (1.4 U·mg-1 at pH 4.5). Both Lac5 and LacA were able to efficiently decolorize such dyes as RBBR and Bromcresol Green. Additionally, phylogenetic relationships among laccases of Peniophora spp. were reconstructed, and groups of orthologous genes were determined. Based on these groups, all currently available data about laccases of Peniophora spp. were systematized.