A prospective pilot study of gut microbiome in cerebral vasospasm and delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

A recent systematic review indicated that gut-microbiota-brain axis contributes to growth and rupture of intracranial aneurysms. However, gaps were detected in the role of intestinal microbiome in cerebral vasospasm (CVS) after aneurysmal subarachnoid hemorrhage (aSAH). This is the first pilot study aiming to test study feasibility and identify differences in gut microbiota between subjects with and without CVS following aSAH. A prospective nested case-control pilot study with 1:1 matching was conducted recruiting subjects with aSAH: cases with CVS; and controls without CVS based on the clinical picture and structured bedside transcranial Doppler (TCD). Fecal samples for microbiota analyses by means of 16S rRNA gene amplicon sequencing were collected within the first 96 h after ictus. Operational taxonomic unit tables were constructed, diversity metrics calculated, phylogenetic trees built, and differential abundance analysis (DAA) performed. At baseline, the groups did not differ significantly in basic demographic and aneurysm-related characteristics (p > 0.05). Alpha-diversity (richness and Shannon Index) was significantly reduced in cases of middle cerebral artery (MCA) vasospasm (p < 0.05). In DAA, relative abundance of genus Acidaminococcus was associated with MCA vasospasm (p = 0.00013). Two butyrate-producing genera, Intestinimonas and Butyricimonas, as well as [Clostridium] innocuum group had the strongest negative correlation with the mean blood flow velocity in anterior cerebral arteries (p < 0.01; rho = - 0.63; - 0.57, and - 0.57, respectively). In total, 16 gut microbial genera were identified to correlate with TCD parameters, and two intestinal genera correlated with outcome upon discharge. In this pilot study, we prove study feasibility and present the first preliminary evidence of gut microbiome signature associating with CVS as a significant cause of stroke in subjects with aSAH.

Gut microbiome dynamics and predictive value in hospitalized COVID-19 patients: a comparative analysis of shallow and deep shotgun sequencing.

The COVID-19 pandemic caused by SARS-CoV-2 has led to a wide range of clinical presentations, with respiratory symptoms being common. However, emerging evidence suggests that the gastrointestinal (GI) tract is also affected, with angiotensin-converting enzyme 2, a key receptor for SARS-CoV-2, abundantly expressed in the ileum and colon. The virus has been detected in GI tissues and fecal samples, even in cases with negative results of the reverse transcription polymerase chain reaction in the respiratory tract. GI symptoms have been associated with an increased risk of ICU admission and mortality. The gut microbiome, a complex ecosystem of around 40 trillion bacteria, plays a crucial role in immunological and metabolic pathways. Dysbiosis of the gut microbiota, characterized by a loss of beneficial microbes and decreased microbial diversity, has been observed in COVID-19 patients, potentially contributing to disease severity. We conducted a comprehensive gut microbiome study in 204 hospitalized COVID-19 patients using both shallow and deep shotgun sequencing methods. We aimed to track microbiota composition changes induced by hospitalization, link these alterations to clinical procedures (antibiotics administration) and outcomes (ICU referral, survival), and assess the predictive potential of the gut microbiome for COVID-19 prognosis. Shallow shotgun sequencing was evaluated as a cost-effective diagnostic alternative for clinical settings. Our study demonstrated the diverse effects of various combinations of clinical parameters, microbiome profiles, and patient metadata on the precision of outcome prognostication in patients. It indicates that microbiological data possesses greater reliability in forecasting patient outcomes when contrasted with clinical data or metadata. Furthermore, we established that shallow shotgun sequencing presents a viable and cost-effective diagnostic alternative to deep sequencing within clinical environments.

Insights into the Mechanisms of Action of Akkermansia muciniphila in the Treatment of Non-Communicable Diseases.

This comprehensive review delineates the extensive roles of Akkermansia muciniphila in various health domains, spanning from metabolic and inflammatory diseases to neurodegenerative disorders. A. muciniphila, known for its ability to reside in the mucous layer of the intestine, plays a pivotal role in maintaining gut integrity and interacting with host metabolic processes. Its influence extends to modulating immune responses and potentially easing symptoms across several non-communicable diseases, including obesity, diabetes, inflammatory bowel disease, and cancer. Recent studies highlight its capacity to interact with the gut-brain axis, suggesting a possible impact on neuropsychiatric conditions. Despite the promising therapeutic potential of A. muciniphila highlighted in animal and preliminary human studies, challenges remain in its practical application due to stability and cultivation issues. However, the development of pasteurized forms and synthetic mediums offers new avenues for its use in clinical settings, as recognized by regulatory bodies like the European Food Safety Authority. This narrative review serves as a crucial resource for understanding the broad implications of A. muciniphila across different health conditions and its potential integration into therapeutic strategies.

The novel sterilization device: the prototype testing.

Currently, there are numerous methods that can be used to neutralize pathogens (i.e., devices, tools, or protective clothing), but the sterilizing agent must be selected so that it does not damage or change the properties of the material to which it is applied. Dry sterilization with hydrogen peroxide gas (VHP) in combination with UV-C radiation is well described and effective method of sterilization. This paper presents the design, construction, and analysis of a novel model of sterilization device. Verification of the sterilization process was performed, using classical microbiological methods and flow cytometry, on samples containing Geobacillus stearothermophilus spores, Bacillus subtilis spores, Escherichia coli, and Candida albicans. Flow cytometry results were in line with the standardized microbiological tests and confirmed the effectiveness of the sterilization process. It was also determined that mobile sterilization stations represent a valuable solution when dedicated to public institutions and businesses in the tourism sector, sports & fitness industry, or other types of services, e.g., cosmetic services. A key feature of this solution is the ability to adapt the device within specific constraints to the user's needs.

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted ß-d-galactosidase from Arthrobacter sp. 32cB.

ArthßDG is a dimeric, cold-adapted ß-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthßDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthßDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthßDG_E441Q/LACs and ArthßDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthßDG_E441Q/LACd, ArthßDG/ONPG (ligands bound in deep mode), and galactose ArthßDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 ßDGs, in ArthßDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

Biochemical Characterization of a Novel Monospecific Endo-ß-1,4-Glucanase Belonging to GH Family 5 From a Rhizosphere Metagenomic Library.

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-ß-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modeling analysis indicated that CelRH5 is related to endo-ß-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum, and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC), and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5-8.5. Highest activity was observed at 40°C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30°C. The presence of the final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+, and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulfate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications.

Structural studies of a cold-adapted dimeric ß-D-galactosidase from Paracoccus sp. 32d.

The crystal structure of a novel dimeric ß-D-galactosidase from Paracoccus sp. 32d (ParßDG) was solved in space group P212121 at a resolution of 2.4 Šby molecular replacement with multiple models using the BALBES software. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric ß-D-galactosidases from Escherichia coli and Arthrobacter sp. C2-2, respectively. It is the second known structure of a cold-active GH2 ß-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted ß-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical ß-D-galactosidase, the crystal structure in complex with galactose (ParßDG-Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric ß-D-galactosidase reveals significant differences in comparison to other ß-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 in ParßDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

[Immune system and influenza virus]. / Uklad odpornosciowy a wirus grypy

Influenza viruses are a significant cause of respiratory infections, causing 3-5 million clinical infections and 250-500 thousand deaths per year. Infections caused by the influenza virus induce a host immune response at the non-specific and specific level (defined as natural and acquired), which leads to limitation of virus replication. Moreover the elements of immunological memory are induced so that they can protect against subsequent infection by the influenza virus. However, there is still no effective way for the total elimination of this virus, and the only effective method to combat this pathogen appears to be vaccination, which through immune system activation greatly limits its spread. The present paper presents the immune reaction at different levels in response to the influenza virus after entering the body and the mechanisms of the influenza virus for avoiding reactions of the immune system, which correspond to its high variability at the molecular level. Moreover, in this paper we describe various methods of stimulating the organism's immune systems with different generations of vaccines and their effectiveness in the fight against this pathogen.

Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with ß-glucosidase, ß-fucosidase and ß-galactosidase activities.

BACKGROUND: Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds. RESULTS: A bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and ß-linked oligosaccharides, and had remarkably high ß-galactosidase, ß-glucosidase and ß-fucosidase activities. Because of the lack of information about the usefulness of ß-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for ß-glucosidase or ß-galactosidase. The BglMKg had maximal ß-galactosidase and ß-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for ß-galactosidase activity was 6.5, whereas the optimum pH for ß-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed ß-glucosidase substrates than ß-galactosidase ones. CONCLUSIONS: BglMKg is a small, monomeric, cold-active ß-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.

A study on the interaction of rhodamine B with methylthioadenosine phosphorylase protein sourced from an Antarctic soil metagenomic library.

The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.

A novel cold-active ß-D-galactosidase from the Paracoccus sp. 32d--gene cloning, purification and characterization.

BACKGROUND: ß-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing ß-D-galactose residues in ß-D-galactosides. Cold-active ß-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic ß-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis. RESULTS: A genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active ß-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the ß-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-ß-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C. CONCLUSIONS: A novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active ß-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active ß-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d ß-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.