A novel mouse model carrying a gene trap insertion into the Hmgxb4 gene locus to examine Hmgxb4 expression in vivo.

HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.

Mechanism of 2'-fucosyllactose degradation by human-associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'-FL. In culture, we found growth of two species, A. muciniphila MucT and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila. We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont.IMPORTANCEAkkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Mutations in the promoter region of methionine transporter gene metM (Rv3253c) confer para-aminosalicylic acid (PAS) resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is a significant global public health threat. Despite the long-standing use of para-aminosalicylic acid (PAS) as a second-line anti-TB drug, its resistance mechanism remains unclear. In this study, we isolated 90 mutants of PAS-resistant Mycobacterium tuberculosis (MTB) H37Ra in 7H11 solid medium and performed whole-genome sequencing, gene overexpression, transcription level comparison and amino acid level determination in MTB, and promoter activity by ß-galactosidase assays in Mycobacterium smegmatis to elucidate the mechanism of PAS resistance. Herein, we found that 47 of 90 (52.2%) PAS-resistant mutants had nine different mutations in the intergenic region of metM (Rv3253c) and Rv3254. Beta-galactosidase assays confirmed that mutations increased promoter activity only for metM but not Rv3254. Interestingly, overexpression of MetM or its M. smegmatis homolog (MSMEI_1796) either by its promoter in metM's direction or by exogenous expression in MTB induced PAS resistance in a methionine-dependent manner. Therefore, drug susceptibility results for the metM promoter mutants can be misleading when using standard 7H10 or 7H9 medium, which lacks methionine. At the metabolism level, PAS treatment led to higher intracellular methionine levels in the mutants than the wild type, antagonizing PAS and conferring resistance. Furthermore, 12 different mutations in the metM promoter were identified in clinical MTB strains. In summary, we found a novel mechanism of PAS resistance in MTB. Mutations in the metM (Rv3253c) promoter upregulate metM transcription and elevate intracellular methionine, which antagonize PAS. Our findings shed new light on the mechanism of PAS resistance in MTB and highlight issues with the current PAS susceptibility culture medium.IMPORTANCEAlthough para-aminosalicylic acid (PAS) has been used to treat TB for more than 70 years, the understanding of PAS resistance mechanisms is still vague, living gaps in our ability to predict resistance and apply PAS effectively in clinical practice. This study aimed to address this knowledge gap by inducing in vitro PAS resistance in Mycobacterium tuberculosis (MTB) using 7H11 medium and discovering a new PAS resistance mechanism. Our research revealed that spontaneous mutations occurring in the promoter region of the methionine transporting gene, metM, can upregulate the expression of metM, resulting in increased intracellular transport of methionine and consequently high-level resistance of Mycobacterium tuberculosis to PAS. Notably, this resistance phenotype cannot be observed when using the commonly recommended 7H10 medium, possibly due to the lack of additional methionine supply compared with that when using the 7H11 medium. Mutations on the regulatory region of metM were also found in some clinical MTB strains. These findings may have important implications for the unexplained PAS resistance observed in clinical settings and provide insight into the failures of PAS treatment. Additionally, they underscore the importance of considering the choice of culture media when conducting drug susceptibility testing for MTB.

Aquimarina aquimarini sp. nov. and Aquimarina spinulae sp. nov., novel bacterial species with versatile natural product biosynthesis potential isolated from marine sponges.

This study describes two Gram-negative, flexirubin-producing, biofilm-forming, motile-by-gliding and rod-shaped bacteria, isolated from the marine sponges Ircinia variabilis and Sarcotragus spinosulus collected off the coast of Algarve, Portugal. Both strains, designated Aq135T and Aq349T, were classified into the genus Aquimarina by means of 16S rRNA gene sequencing. We then performed phylogenetic, phylogenomic and biochemical analyses to determine whether these strains represent novel Aquimarina species. Whereas the closest 16S rRNA gene relatives to strain Aq135T were Aquimarina macrocephali JAMB N27T (97.8 %) and Aquimarina sediminis w01T (97.1 %), strain Aq349T was more closely related to Aquimarina megaterium XH134T (99.2 %) and Aquimarina atlantica 22II-S11-z7T (98.1 %). Both strains showed genome-wide average nucleotide identity scores below the species level cut-off (95 %) with all Aquimarina type strains with publicly available genomes, including their closest relatives. Digital DNA-DNA hybridization further suggested a novel species status for both strains since values lower than 70 % hybridization level with other Aquimarina type strains were obtained. Strains Aq135T and Aq349T grew from 4 to 30°C and with between 1-5 % (w/v) NaCl in marine broth. The most abundant fatty acids were iso-C17 : 03-OH and iso-C15 : 0 and the only respiratory quinone was MK-6. Strain Aq135T was catalase-positive and ß-galactosidase-negative, while Aq349T was catalase-negative and ß-galactosidase-positive. These strains hold unique sets of secondary metabolite biosynthetic gene clusters and are known to produce the peptide antibiotics aquimarins (Aq135T) and the trans-AT polyketide cuniculene (Aq349T), respectively. Based on the polyphasic approach employed in this study, we propose the novel species names Aquimarina aquimarini sp. nov. (type strain Aq135T=DSM 115833T=UCCCB 169T=ATCC TSD-360T) and Aquimarina spinulae sp. nov. (type strain Aq349T=DSM 115834T=UCCCB 170T=ATCC TSD-361T).

Identification of GM1-Ganglioside Secondary Accumulation in Fibroblasts from Neuropathic Gaucher Patients and Effect of a Trivalent Trihydroxypiperidine Iminosugar Compound on Its Storage Reduction.

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the ß-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on ß-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD.

Changes in gut microbiota and lactose intolerance symptoms before and after daily lactose supplementation in individuals with the lactase nonpersistent genotype.

BACKGROUND: Approximately 70%-100% of the Asian adult population is lactase nonpersistent (LNP). The literature shows that many individuals with the LNP-genotype can consume ≤12 g of lactose without experiencing gastrointestinal discomfort. Repetitive consumption of lactose may reduce intolerance symptoms via adaptation of the gut microbiota. OBJECTIVE: This study aimed to assess the effects of daily consumption of incremental lactose doses on microbiota composition and function, and intolerance symptoms. METHODS: Twenty-five healthy adults of Asian origin, carrying the LNP-genotype were included in this 12-wk before and after intervention trial. Participants consumed gradually increasing lactose doses from 3 to 6 g to 12 g twice daily, each daily dose of 6 g, 12 g, or 24 g being provided for 4 consecutive weeks. Participants handed-in repeated stool samples and underwent a 25 g lactose challenge hydrogen breath test (HBT) before and after the 12-wk intervention. Daily gastrointestinal symptoms and total symptom scores (TSSs) during the lactose challenge were recorded. RESULTS: A significant increase from 5.5% ± 7.6% to 10.4% ± 9.6% was observed in Bifidobacterium relative abundance after the intervention (P = 0.009), accompanied by a 2-fold increase (570 ± 269 U/g; P < 0.001) in fecal ß-galactosidase activity compared with baseline (272 ± 158 U/g). A 1.5-fold decrease (incremental area under the curve; P = 0.01) in expired hydrogen was observed during the second HBT (38 ± 35 ppm·min), compared with the baseline HBT (57 ± 38 ppm·min). There was a nonsignificant decrease in TSS (10.6 ± 8.3 before compared with 8.1 ± 7.2 after intervention; P = 0.09). Daily consumption of lactose was well tolerated, with mild to no gastrointestinal complaints reported during the intervention. CONCLUSIONS: Increased levels of Bifidobacterium indicate an adaptation of the gut microbiota upon repetitive consumption of incremental doses of lactose, which was well tolerated as demonstrated by reduced expired hydrogen concentrations during the second 25-g lactose HBT. Bifidobacteria metabolize lactose without gas production thereby potentially reducing intestinal gas formation in the gut of individuals with the LNP-genotype. This increased lactose tolerance possibly lifts the necessity to remove nutrient-rich dairy foods completely from the diet. The trial is registered at the International Clinical Trials Registry Platform: NL9516. The effect of dietary lactose in lactase nonpersistent individuals on gut microbiota.

Sialidase NEU3 action on GM1 ganglioside is neuroprotective in GM1 gangliosidosis.

GM1 gangliosidosis is a neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes lysosomal ß-galactosidase. The enzyme deficiency blocks GM1 ganglioside catabolism, leading to accumulation of GM1 ganglioside and asialo-GM1 ganglioside (GA1 glycolipid) in brain. This disease can present in varying degrees of severity, with the level of residual ß-galactosidase activity primarily determining the clinical course. Glb1 null mouse models, which completely lack ß-galactosidase expression, exhibit a less severe form of the disease than expected from the comparable deficiency in humans, suggesting a potential species difference in the GM1 ganglioside degradation pathway. We hypothesized this difference may involve the sialidase NEU3, which acts on GM1 ganglioside to produce GA1 glycolipid. To test this hypothesis, we generated Glb1/Neu3 double KO (DKO) mice. These mice had a significantly shorter lifespan, increased neurodegeneration, and more severe ataxia than Glb1 KO mice. Glb1/Neu3 DKO mouse brains exhibited an increased GM1 ganglioside to GA1 glycolipid ratio compared with Glb1 KO mice, indicating that NEU3 mediated GM1 ganglioside to GA1 glycolipid conversion in Glb1 KO mice. The expression of genes associated with neuroinflammation and glial responses were enhanced in Glb1/Neu3 DKO mice compared with Glb1 KO mice. Mouse NEU3 more efficiently converted GM1 ganglioside to GA1 glycolipid than human NEU3 did. Our findings highlight NEU3's role in ameliorating the consequences of Glb1 deletion in mice, provide insights into NEU3's differential effects between mice and humans in GM1 gangliosidosis, and offer a potential therapeutic approach for reducing toxic GM1 ganglioside accumulation in GM1 gangliosidosis patients.

Dual 6Pß-Galactosidase/6Pß-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving ß-galactosidase (ß-gal) and 6Pß-galactosidase (6Pß-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pß-gal gene, the 11 GH1 family proteins, in which their 6Pß-glucosidase (6Pß-glc) activity was experimentally demonstrated,, were assayed for 6Pß-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pß-gal activity. The sequence comparison of this dual 6Pß-gal/6Pß-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pß-gal/6Pß-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pß-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pß-gal activity with possible relevance for lactose maldigestion management.

Efficient secreted expression of natural intracellular ß-galactosidase from Bacillus aryabhattai via non-classical protein secretion pathway in Bacillus subtilis.

In this study, the natural intracellular ß-galactosidase (lacZBa) from Bacillus aryabhattai was expressed extracellularly in Bacillus subtilis. Sec and Tat signal peptides from different secretion pathways were incorporated to facilitate extracellular secretion of lacZBa, resulting in a yield of only 0.54 U/mL. Interestingly, it was discovered that lacZBa could be efficiently expressed and secreted in B. subtilis via a non-classical secretory pathway without the need for a signal peptide. The extracellular activity and secretion ratio were 5.3 U/mL and 65 %, respectively. Compared to E. coli, the expression of lacZBa in B. subtilis resulted in increased acid resistance and higher pH stability and thermostability, with a 1.7-fold increase in half-life at 50 °C and pH 6.0. Additionally, we combined single-factor experiments and response surface methodology to enhance extracellular expression of ß-galactosidase in shake-flasks. The resulting optimal medium contained 4.46 % glucose, 1.47 % corn steep liquor, 1.5 % beef extract, 0.82 % CaCl2, and 0.1 % MgSO4. Under optimal conditions, the yield of extracellularly secreted ß-galactosidase at the shake flask level was 17.41 U/mL, representing a 32.2-fold increase in initial extracellular enzyme activity. This study represents the first successful report of natural intracellular ß-galactosidase being expressed through the non-classical secretory pathway in B. subtilis.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

GH2 family ß-galactosidases evolution using degenerate oligonucleotide gene shuffling.

OBJECTIVES: To improve the biochemical characteristics of the GH2 family ß-galactosidases using a family shuffling method based on degenerate oligonucleotide gene shuffling. RESULTS: Four ß-galactosidase genes from the genus Alteromonas were divided into 14 gene segments, and each included the homologous sequence in the adjacent segments. The gene segments were regenerated into complete ß-galactosidase genes and amplified by PCR. The obtained chimeric genes were cloned into a plasmid and screened for ß-galactosidase activity. Approximately 320 positive clones were observed on the screening plate, of which nine sequenced genes were chimera. Additionally, the M22 and M250 mutants were expressed, purified, and characterized. The optimal temperature and substrate specificity of the recombinant M22 and M250 were consistent with those of the wild-type enzymes. The catalytic efficiency of recombinant M22 enzyme was higher than that of the wild-type enzymes, and the recombinant M250 displayed weak transglycosylation activity. CONCLUSIONS: The chimeric genes of GH2 ß-galactosidase were obtained using a controlled family shuffling that will provide an enzyme evolutionary method to obtain the ß-galactosidases with excellent characteristics for laboratory and industrial purposes.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of ß-Galactosidase in Pichia pastoris.

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of ß-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Reduction of senescence-associated secretory phenotype and exosome-shuttled miRNAs by Haritaki fruit extract in senescent dermal fibroblasts.

OBJECTIVE: Skin ageing is linked to the accumulation of senescent cells and a "senescence-associated secretory phenotype" (SASP). SASP factors include chemokines, cytokines, and small extracellular vesicles (EVs) containing miRNAs. We characterized SASP profile markers in normal human dermal fibroblasts (HDFs) and evaluated the effect of Haritaki fruit extract on these senescence markers. METHODS: Senescence was induced in HDFs by ionizing radiation (X ray), followed by 14 days of culture. Parallel incubations included fibroblasts treated for 12 days with 10 or 100 µg/mL Haritaki (a standardized extract of Terminalia chebula fruit). Senescence was assessed on Day 14 according to cell morphology, ß-galactosidase activity, RT-qPCR measurement of SASP genes, as well as semi-quantitative (RT-qPCR) expression of miRNAs contained in EVs isolated from the medium. The size and distribution of EVs were measured by Nanoparticle Tracking Analysis. RESULTS: Human dermal fibroblasts exhibited a senescent phenotype 14 days after ionizing-radiation, demonstrated by a flattened and irregular shape, increased ß-galactosidase activity and over-expression of SASP genes. CSF3, CXCL1, IL1ß, IL6 and IL8 genes were increased by 1492%, 1041%, 343%, 478%, 2960% and 293%, respectively. The cell cycle inhibitor, CDKN1A, was increased by 357%, while COL1A1, was decreased by 56% and MMP1 was increased by 293%. NTA analysis of the EVs size distribution indicated a mix of exosomes (45-100 nm) and microvesicles (100-405 nm). miRNA expression in EVs was increased in senescent fibroblasts. miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p and miR 186-5p were increased in senescent HDF by 4.17-, 2.43-, 1.17-, 2.01, 12.5-fold, respectively. Incubation of senescent fibroblasts with Haritaki extract strongly decreased SASP mRNA levels and miRNA expression in EVs. CONCLUSION: Haritaki strongly reduced SASP expression and EV-shuttled miRNAs in senescent fibroblasts. These results indicate that Haritaki has strong senomorphic properties and may be a promising ingredient for the development of new anti-ageing dermo-cosmetic products by inhibiting deleterious effects of senescent cells.

OBJECTIF: Le vieillissement cutané est lié à l'accumulation de cellules sénescentes et à un « phénotype sécrétoire associé à la sénescence ¼ (SASP). Le SASP est constitué de chimiokines, cytokines et de petites vésicules extracellulaires (VE) contenant des miARN. Nous avons caractérisé les marqueurs du SASP dans des fibroblastes dermiques humains normaux (HDF) et évalué l'effet d'un extrait de fruit d'Haritaki sur ces marqueurs de la sénescence. MÉTHODES: La sénescence a été induite dans les HDF par des rayonnements ionisants (rayons X), suivis de 14 jours de culture. Parallèlement, des HDF ont été traités pendant 12 jours avec 10 ou 100 µg/mL d'Haritaki (un extrait standardisé de fruit de Terminalia chebula). La sénescence a été évaluée au jour 14 en fonction de la morphologie cellulaire, de l'activité ß-galactosidase, de la mesure des gènes du SASP par RT-PCR, ainsi que de l'expression semi-quantitative (RT-qPCR) des miARN contenus dans les VE isolées du milieu. La taille et la distribution des VE ont été mesurées par Nanoparticle Tracking Analysis (NTA). RÉSULTATS: Les HDF ont présenté un phénotype sénescent 14 jours après le rayonnement ionisant, en effet, ils avaient une forme aplatie et irrégulière, une activité ß-galactosidase accrue et une surexpression des gènes du SASP. Les ARNm de CSF3, CXCL1, IL1ß, IL6 et IL8 ont été augmentés de 1492%, 1041%, 343%, 478%, 2960% et 293%, respectivement. L'inhibiteur du cycle cellulaire, CDKN1A, a été augmenté de 357%, tandis que le COL1A1 a diminué de 56% et la MMP1 a augmenté de 293%. L'analyse NTA de la distribution de taille des VE a montré un mélange d'exosomes (45-100 nm) et de microvésicules (100-405 nm). L'expression des miARN dans les VE a augmenté dans les fibroblastes sénescents. Les miR 29a-3p, miR 30a-3p, miR 34a-5p, miR 24a-3p et miR 186-5p ont été augmentés dans le HDF sénescent de, respectivement, 4,17-, 2,43-, 1,17-, 2,01 et 12,5- fois. L'incubation de fibroblastes sénescents avec l'extrait de Haritaki a fortement diminué les niveaux d'ARNm du SASP et l'expression de miARN dans les VE. CONCLUSION: L'extrait d'Haritaki a fortement réduit l'expression du SASP et de miARN contenus dans les VE des fibroblastes sénescents. Ces résultats indiquent que Haritaki possède de fortes propriétés sénomorphiques et pourrait être un ingrédient prometteur pour le développement de nouveaux produits dermo-cosmétiques anti-âge en inhibant les effets délétères des cellules sénescentes.

Small non-coding RNAome changes during human chondrocyte senescence as potential epigenetic targets in age-related osteoarthritis.

Chondrocyte senescence is a decisive component of age-related osteoarthritis, however, the function of small noncoding RNAs (sncRNAs) in chondrocyte senescence remains underexplored. Human hip joint cartilage chondrocytes were cultivated up to passage 4 to induce senescence. RNA samples were extracted and then analyzed using small RNA sequencing and qPCR. ß-galactosidase staining was used to detect the effect of sncRNA on chondrocyte aging. Results of small RNA sequencing showed that 279 miRNAs, 136 snoRNAs, 30 snRNAs, 102 piRNAs, and 5 rasiRNAs were differentially expressed in senescent chondrocytes. The differential expression of 150 sncRNAs was further validated by qPCR. Transfection of sncRNAs and ß-galactosidase staining were also performed to further revealed that hsa-miR-135b-5p, SNORA80B-201, and RNU5E-1-201 have the function to restrain chondrocyte senescence, while has-piR-019102 has the function to promote chondrocyte senescence. Our data suggest that sncRNAs have therapeutic potential as novel epigenetic targets in age-related osteoarthritis.

Protein phosphatase 2A activators reverse age-related behavioral changes by targeting neural cell senescence.

The contribution of cellular senescence to the behavioral changes observed in the elderly remains elusive. Here, we observed that aging is associated with a decline in protein phosphatase 2A (PP2A) activity in the brains of zebrafish and mice. Moreover, drugs activating PP2A reversed age-related behavioral changes. We developed a transgenic zebrafish model to decrease PP2A activity in the brain through knockout of the ppp2r2c gene encoding a regulatory subunit of PP2A. Mutant fish exhibited the behavioral phenotype observed in old animals and premature accumulation of neural cells positive for markers of cellular senescence, including senescence-associated ß-galactosidase, elevated levels cdkn2a/b, cdkn1a, senescence-associated secretory phenotype gene expression, and an increased level of DNA damage signaling. The behavioral and cell senescence phenotypes were reversed in mutant fish through treatment with the senolytic ABT263 or diverse PP2A activators as well as through cdkn1a or tp53 gene ablation. Senomorphic function of PP2A activators was demonstrated in mouse primary neural cells with downregulated Ppp2r2c. We conclude that PP2A reduction leads to neural cell senescence thereby contributing to age-related behavioral changes and that PP2A activators have senotherapeutic properties against deleterious behavioral effects of brain aging.

Gene Editing Corrects In Vitro a G > A GLB1 Transition from a GM1 Gangliosidosis Patient.

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced ß-galactosidase (ß-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of ß-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on ß-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.

Glb1 knockout mouse model shares natural history with type II GM1 gangliosidosis patients.

GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of ß-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1-/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1-/- mice die six weeks earlier than male Glb1-/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1-/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1-/- mice as seen in some patients. In addition, Glb1-/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1-/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1-/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.

Optimization of the production conditions of tri-GOS and lactosucrose from lactose and sucrose with recombinant ß-galactosidase.

Few studies expressed the ß-galactosidase encoding gene from L. plantarum in E. coli so far. In the present study, the recombinant ß-galactosidase from L. plantarum FMNP01 was used as a catalyst in transgalactosylation to form tri-GOS and lactosucrose. In the presence of lactose and sucrose, six transfer products were formed in the transgalactosylation reaction with recombinant ß-galactosidase L.pFMNP01Gal as a catalyst. Three transfer products were tri-galacto-oligosaccharides (tri-GOS), lactosucrose, and lactulose; the other three transfer products needed to be identified further. Based on a single factor test and response surface methodological approach, the optimal transgalactosylation conditions of the production of tri-GOS and lactosucrose were determined as initial sugar concentration of 50%, lactose: sucrose ratio of 1:2, enzyme concentration of 3 U/mL, and reaction time of 6 h at 50 °C resulting in a maximum tri-GOS concentration of 47.69 ± 1.36 g/L and a maximum lactosucrose concentration of 8.18 ± 0.97 g/L.

Cloning, Expression, Purification and Characterization of the ß-galactosidase PoßGal35A from Penicillium oxalicum.

Galactosidases are industrially important enzymes that hydrolyze galactosidic bonds in carbohydrates. Identifying new galactosidases with distinct functional characteristics is of paramount importance. In this study, we report the finding of a novel ß-galactosidase PoßGal35A from the fungus Penicillium oxalicum. PoßGal35A belongs to the glycoside hydrolase family 35 (GH35), functions optimally at 70 °C and pH 5.0, and exhibits a specific high activity (191 ± 6.2 U/mg) towards pNPßgal. Ca2+, Fe3+and Ba2+ ions enhance the activity of the enzyme, whereas Cu2+ and Hg2+ significantly reduce it. This enzyme releases galactose from ß-1,3-galactan, ß-1,4-galactan, ß-1,6-galactan, as well as arabinogalactan from larchwood (LWAG). In addition, PoßGal35A acts synergistically with arabinosidase to degrade LWAG. These results suggest that PoßGal35A is a high activity exo-ß-1,3/4/6-galactanase that can be used to establish glycan blocks in glycoconjugates, and thus provides a new tool for biotechnological applications.

A GH42 ß-galactosidase from Bifidobacterium thermophilum: Biochemical characterization and synthesis of galactosyl glycerol by reverse hydrolysis.

In this study, a ß-galactosidase gene (btgal42) was first cloned from Bifidobacterium thermophilum and successfully expressed in Escherichia coli. The molecular weight of the purified BtGal42 was estimated to be 78 kDa by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 225 kDa by the size-exclusion chromatography, indicating that the native BtGal42 was a homotrimer. BtGal42 belonged to the glycosidase hydrolase family 42, exhibiting the maximum activity at pH of 7 and at temperature of 50°C. The enzyme displayed a strictly specific activity toward substrates with ß-galactosyl linkages at the nonreducing ends, of which the activity on 4-nitrophenyl-ß-d-galactopyranoside was the highest, followed by 2-nitrophenyl-ß-d-galactopyranoside and lactose. Among the tested metals and reagents, BtGal42 showed tolerance in the presence of various organic solvents. Importantly, BtGal42 exhibited a high reverse hydrolysis activity when using galactose as the donor and the di-alcohol ethylene glycol and the trialcohol glycerol as the acceptors. Under unoptimized reaction conditions, the galactosyl glycerol yield reached 62.2 g/L (galactose conversion rate, 41.2%). This study might provide a feasible method for the biosynthesis of galactosyl glycerol from low-cost glycerol and galactose, which was associated with high conversion efficiency and few byproducts.