The present study has undertaken the isolation of marine yeasts from mangrove sediment samples and their ability to produce alkaline protease enzymes. A total of 14 yeast isolates were recovered on yeast-malt agar (YMA) and yeast extract peptone dextrose (YEPD) agar medium. After screening for proteolytic activity on skim milk agar, marine yeast isolate, AKB-1 exhibited a hydrolysis zone of 18 mm. Optimal conditions for the enzyme production from yeast isolate AKB-1 were at 30 °C, pH 8, fructose as carbon source, potassium nitrate as nitrogen source, and 25% saline concentration. Under the optimal conditions, the protease enzyme activity of the isolate AKB-1 was observed to be 978 IU/mL. The structural and functional analysis was carried out through FTIR and HPLC analysis for the extracted protease enzyme. Furthermore, the enzyme produced was partially purified by solvent extraction using ethyl acetate and ammonium sulfate precipitation (3.4-fold) followed by dialysis (56.8-fold). The molecular weight of the purified enzyme was observed to be around 60 kDa using SDS-PAGE. The extracted protein showed good antibacterial activity against six different clinical bacterial pathogens and the highest against Bacillus cereus (16 ± 0.5 mm). The extracted protease enzyme was revealed to remove blood stains from cloth within 20 min of application similar to the commercial detergent. The marine yeast isolate was further identified as Candida orthopsilosis AKB-1 (Accession number KY348766) through 18S rRNA sequencing, and a phylogenetic tree was generated.
- MeSH
- Anti-Bacterial Agents pharmacology metabolism chemistry isolation & purification MeSH
- Bacillus cereus drug effects MeSH
- Bacterial Proteins * chemistry pharmacology metabolism isolation & purification MeSH
- Candida * enzymology isolation & purification genetics classification MeSH
- Endopeptidases * chemistry metabolism isolation & purification pharmacology MeSH
- Phylogeny MeSH
- Geologic Sediments microbiology MeSH
- Hydrogen-Ion Concentration MeSH
- Culture Media chemistry MeSH
- Microbial Sensitivity Tests MeSH
- Molecular Weight MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
Vitamin B12, cobalamin, is indispensable for humans owing to its participation in two biochemical reactions: the conversion of l-methylmalonyl coenzyme A to succinyl coenzyme A, and the formation of methionine by methylation of homocysteine. Eukaryotes, encompassing plants, fungi, animals and humans, do not synthesise vitamin B12, in contrast to prokaryotes. Humans must consume it in their diet. The most important sources include meat, milk and dairy products, fish, shellfish and eggs. Due to this, vegetarians are at risk to develop a vitamin B12 deficiency and it is recommended that they consume fortified food. Vitamin B12 behaves differently to most vitamins of the B complex in several aspects, e.g. it is more stable, has a very specific mechanism of absorption and is stored in large amounts in the organism. This review summarises all its biological aspects (including its structure and natural sources as well as its stability in food, pharmacokinetics and physiological function) as well as causes, symptoms, diagnosis (with a summary of analytical methods for its measurement), prevention and treatment of its deficiency, and its pharmacological use and potential toxicity.
- MeSH
- Diet, Vegetarian MeSH
- Diet MeSH
- Food, Fortified MeSH
- Humans MeSH
- Vitamin B 12 Deficiency * diagnosis prevention & control drug therapy etiology MeSH
- Vitamin B 12 * pharmacokinetics chemistry metabolism therapeutic use physiology adverse effects administration & dosage pharmacology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
The honeybee (Apis mellifera) is a key pollinator critical to global agriculture, facing threats from various stressors, including the ectoparasitic Varroa mite (Varroa destructor). Previous studies have identified shared bacteria between Varroa mites and honeybees, yet it remains unclear if these bacteria assemble similarly in both species. This study builds on existing knowledge by investigating co-occurrence patterns in the microbiomes of both Varroa mites and honeybees, shedding light on potential interactions. Leveraging 16S rRNA datasets, we conducted co-occurrence network analyses, explored Core Association Networks (CAN) and assess network robustness. Comparative network analyses revealed structural differences between honeybee and mite microbiomes, along with shared core features and microbial motifs. The mite network exhibited lower robustness, suggesting less resistance to taxa extension compared to honeybees. Furthermore, analyses of predicted functional profiling and taxa contribution revealed that common central pathways in the metabolic networks have different taxa contributing to Varroa mites and honeybee microbiomes. The results show that while both microbial systems exhibit functional redundancy, in which different taxa contribute to the functional stability and resilience of the ecosystem, there is evidence for niche specialization resulting in unique contributions to specific pathways in each part of this host-parasite system. The specificity of taxa contribution to key pathways offers targeted approaches to Varroa microbiome management and preserving honeybee microbiome. Our findings provide valuable insights into microbial interactions, aiding farmers and beekeepers in maintaining healthy and resilient bee colonies amid increasing Varroa mite infestations.
- MeSH
- Bacteria * classification genetics isolation & purification MeSH
- Microbiota * MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Varroidae * microbiology MeSH
- Bees microbiology parasitology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Purine de novo purine synthesis involves 10 reactions catalysed by six enzymes, including phosphoribosylformyglycinamidine synthase (PFAS). To date, genetic defects of three of these enzymes, namely ATIC, ADSL and PAICS, have been characterised in humans. Here, we report for the first time two individuals with PFAS deficiency. Probands were identified through metabolic and genetic screening of neurologically impaired individuals. The pathogenicity of the variants was established by structural and functional studies. Probands C1 and C2 presented with prematurity, short stature, recurrent seizures and mild neurological impairment. C1 had elevated urinary levels of formylglycineamide riboside (FGAr) and bi-allelic PFAS variants encoding the NP_036525.1:p.Arg811Trp substitution and the NP_036525.1:p.Glu228_Ser230 in-frame deletion. C2 is a 20-year-old female with a homozygous NP_036525.1:p.Asn264Lys substitution. These amino acid changes are predicted to affect the structural stability of PFAS. Accordingly, C1 skin fibroblasts showed decreased PFAS content and activity, with impaired purinosome formation that was restored by transfection with pTagBFP_PFAS_wt. The enzymatic activities of the corresponding recombinant mutant PFAS proteins were also reduced, and none of them, after transfection, corrected the elevated FGAR/r levels in PFAS-deficient HeLa cells. While genetic defects in purine de novo synthesis are typically considered in patients with severe neurological impairment, these disorders, especially PFAS deficiency, should also be considered in milder phenotypes.
- MeSH
- Humans MeSH
- Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor * genetics deficiency metabolism MeSH
- Young Adult MeSH
- Mutation MeSH
- Purine-Pyrimidine Metabolism, Inborn Errors * genetics MeSH
- Child, Preschool MeSH
- Purines * biosynthesis MeSH
- Check Tag
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
In this study, simple oil-in-water emulsions (O/W) and multiple O/W/O emulsions were employed as carriers for a curcumin delivery system. The stability of emulsions was evaluated using DSC (differential scanning calorimetry), accompanied by particle size measurement by DLS (dynamic light scattering) and rheological analysis. The amount of freezable water (Wfs) in O/W emulsion was determined to be 80.4%, while that in O/W/O emulsion was 23.7%. Multiple emulsions had a more complex structure than simple emulsions, being characterized by higher stability with predominant loss modulus over storage modulus (G" > G'). The mean surface diameter for O/W emulsion was 198.7 ± 9.8 nm, being approximately two times lower than that for multiple emulsions. Curcumin in vitro digestibility was observed for both emulsions and, additionally, the digestibility of fresh and dried curcuma root powders was investigated. Multiple emulsions were found to be a superior matrix for curcumin delivery, with higher stability and emulsion digestibility of 50.6% for the stomach and small intestine. In vitro digestion of dried curcuma powders and curcuma root samples was monitored by HPLC (high-performance liquid chromatography). The DMD (dry matter digestibility) for dried curcuma powders ranged between 52.9% to 78.8%, and for fresh curcuma (KF) was 95.5%.
Alveolární echinokokóza (AE) je vzácné, ale závažné parazitární onemocnění jater, které často napodobuje maligní ložiska. V této kazuistice prezentujeme případ 76letého pacienta, u něhož byla AE diagnostikována náhodně při vyšetření pro renální insuficienci. Ultrazvukové vyšetření odhalilo mnohočetná hyperechogenní ložiska v játrech. Následné CT a MR jater zobrazily mnohočetná ložiska nepravidelných okrajů bez typické vaskularizace, největší o velikosti 71 × 38 mm. Laboratorní výsledky ukázaly zvýšené jaterní enzymy, CRP a renální insuficienci. Core cut biopsie jater potvrdila přítomnost pseudocystických struktur. Vzorky byly následně zaslány do Národní referenční laboratoře pro tkáňové helmintózy, která diagnózu AE potvrdila. Sérologické vyšetření bylo provedeno až po bioptickém vyšetření a potvrdilo přítomnost protilátek proti Echinococcus multilocularis. Pacient byl odeslán do infekční ambulance, kde byla zahájena léčba albendazolem v dávce 800 mg denně. Po rehydrataci a úpravě léčby došlo ke stabilizaci renálních funkcí a při kontrolních zobrazovacích vyšetřeních byl nález stacionární. Tento případ zdůrazňuje nutnost zařazení AE do diferenciální diagnostiky ložiskových procesů jater, zejména u pacientů bez onkologické anamnézy.
Alveolar echinococcosis (AE) is a rare but serious parasitic liver disease that often mimics malignant lesions. We present the case of a 76-year-old patient in whom AE was incidentally diagnosed during examination for renal insufficiency. Abdominal ultrasound revealed multiple hyperechogenic liver lesions. Subsequent MRI showed multiple irregularly bordered lesions without typical vascularization, the largest measuring 71 × 38 mm. Laboratory tests revealed elevated liver enzymes, CRP, and renal insufficiency. A core-cut liver biopsy confirmed the presence of pseudocystic structures. Samples were sent to the National Reference Laboratory for Tissue Helminthiases, which confirmed the diagnosis of AE. Serological testing was performed after the biopsy confirming the presence of antibodies against Echinococcus multilocularis. The patient was referred to an infectious disease clinic, where treatment with albendazole at a dose of 800 mg daily was initiated. After rehydration and adjustment of therapy, renal function stabilized, and follow-up imaging showed stable findings. This case highlights the need to include AE in the differential diagnosis of hepatic lesions, particularly in patients without an oncological history.
- MeSH
- Albendazole pharmacology therapeutic use MeSH
- Biopsy MeSH
- Diagnostic Imaging methods MeSH
- Echinococcosis, Hepatic * diagnosis drug therapy MeSH
- Liver diagnostic imaging pathology MeSH
- Humans MeSH
- Aged MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Aged MeSH
- Publication type
- Case Reports MeSH
Posterior fossa syndrome (PFS) is a serious postoperative complication that primarily affects children following resection of posterior fossa tumors. Although its complex pathophysiology, involving disruption of cerebellar structures and the dentato-thalamo-cortical pathway, is increasingly being elucidated, effective treatments remain limited. This perspective explores acetyl-DL-leucine (ADLL) and its active L-enantiomer, N-acetyl-L-leucine (NALL), as promising therapeutic candidates for PFS. Emerging mechanistic, preclinical, and clinical evidence suggests that both compounds might alleviate PFS symptoms through neuroprotective and neurorestorative mechanisms, including neuronal membrane stabilization, metabolic enhancement, antioxidant and anti-inflammatory effects, and dopaminergic modulation. NALL, which has greater neurotherapeutic potential than ADLL, might particularly support recovery through its multimodal effects on neuronal function, thereby enhancing perioperative resilience. Further translational research into these acetylated leucine analogues is warranted.
- MeSH
- Infratentorial Neoplasms * surgery MeSH
- Leucine * analogs & derivatives therapeutic use pharmacology MeSH
- Humans MeSH
- Neuroprotective Agents * pharmacology therapeutic use MeSH
- Postoperative Complications * drug therapy MeSH
- Syndrome MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Nedd4-2 E3 ligase regulates Na+ homeostasis by ubiquitinating various channels and membrane transporters, including the epithelial sodium channel ENaC. In turn, Nedd4-2 dysregulation leads to various conditions, including electrolytic imbalance, respiratory distress, hypertension, and kidney diseases. However, Nedd4-2 regulation remains mostly unclear. The present study aims at elucidating Nedd4-2 regulation by structurally characterizing Nedd4-2 and its complexes using several biophysical techniques. Our cryo-EM reconstruction shows that the C2 domain blocks the E2-binding surface of the HECT domain. This blockage, ubiquitin-binding exosite masking by the WW1 domain, catalytic C922 blockage and HECT domain stabilization provide the structural basis for Nedd4-2 autoinhibition. Furthermore, Ca2+-dependent C2 membrane binding disrupts C2/HECT interactions, but not Ca2+ alone, whereas 14-3-3 protein binds to a flexible region of Nedd4-2 containing the WW2 and WW3 domains, thereby inhibiting its catalytic activity and membrane binding. Overall, our data provide key mechanistic insights into Nedd4-2 regulation toward fostering the development of strategies targeting Nedd4-2 function.
- MeSH
- Cryoelectron Microscopy MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Models, Molecular MeSH
- Protein Domains MeSH
- 14-3-3 Proteins * metabolism chemistry MeSH
- Ubiquitination MeSH
- Nedd4 Ubiquitin Protein Ligases * metabolism chemistry genetics ultrastructure MeSH
- Calcium * metabolism MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Tumor suppressor p53 is a key player in the cell response to DNA damage that suffers by frequent inactivating aberrations. Some of them disturb p53 oligomerization and influence cell decision between proliferation, growth arrest and apoptosis. Active p53 resides mostly in the nucleus, degradation occurs in the cytoplasm. Acute myeloid leukemia (AML)-related mutation of NPM (NPMmut) induces massive mislocalization of p53 to the cytoplasm, which might be related to leukemia initiation. Since both proteins interact and execute their function as oligomers, we investigated the role of perturbed p53 oligomerization in the p53 mislocalization process in live cells by FLIM (fluorescence lifetime imaging microscopy), fluorescence anisotropy imaging (FAIM), fluorescence cross-correlation spectroscopy (FCCS) and immunochemical methods. On a set of fluorescently labeled p53 variants, monomeric R337G and L344P, dimeric L344A, and multimeric D352G and A353S, we correlated their cellular localization, oligomerization and interaction with NPMmut. Interplay between nuclear export signal (NES) and nuclear localization signal (NLS) of p53 was investigated as well. While NLS was found critical for the nuclear p53 localization, NES plays less significant role. We observed cytoplasmic translocation only for multimeric A353S variant with sufficient stability and strong interaction with NPMmut. Less stable multimer D352G and L344A dimer were not translocated, monomeric p53 variants always resided in the nucleus independently of the presence of NPMmut and NES intactness. Oligomeric state of NPMmut is not required for p53 translocation, which happens also in the presence of the nonoligomerizing NPMmut variant. The prominent structural and functional role of the R337 residue is shown.
- MeSH
- Leukemia, Myeloid, Acute * genetics metabolism MeSH
- Cell Nucleus metabolism MeSH
- Cytoplasm metabolism MeSH
- Nuclear Localization Signals metabolism MeSH
- Nuclear Proteins * genetics metabolism MeSH
- Humans MeSH
- Protein Multimerization MeSH
- Mutation * MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 * metabolism genetics chemistry MeSH
- Nucleophosmin MeSH
- Nuclear Export Signals MeSH
- Protein Transport MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder for which current treatments provide only symptomatic relief, primarily through cholinesterase (ChE) inhibition and N-methyl-d-aspartate receptor (NMDAR) antagonism. To improve therapeutic efficacy and safety, we designed and synthesized 16 novel tacrine derivatives modified at position 7 with various (hetero)aryl groups or deuterium substitution. Initially, in silico screening predicted favorable CNS permeability and oral bioavailability. Subsequent in vitro evaluations demonstrated significant inhibitory potency against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with derivatives 5i and 5m displaying particularly promising profiles. Metabolic stability assessed using human liver microsomes revealed enhanced stability for compound 5e, whereas 5i and 5m underwent rapid metabolism. Notably, compound 7 showed improved metabolic stability attributed to deuterium incorporation. The newly synthesized compounds were further tested for antagonistic activity on the GluN1/GluN2B subtype of NMDAR, with compound 5m exhibiting the most potent and voltage-independent inhibition. The ability of these compounds to permeate the blood-brain barrier (BBB) was confirmed through in vitro PAMPA assays. In preliminary hepatotoxicity screening (HepG2 cells), most derivatives exhibited higher cytotoxicity than tacrine, emphasizing the ongoing challenge in hepatotoxicity management. Based on its overall favorable profile, compound 5m advanced to in vivo pharmacokinetic studies in mice, demonstrating efficient CNS penetration, with brain concentrations exceeding plasma levels (brain-to-plasma ratio 2.36), indicating active transport across the BBB. These findings highlight compound 5m as a promising tacrine-based multi-target-directed ligand, supporting further preclinical development as a potential therapeutic candidate for AD.
- MeSH
- Acetylcholinesterase metabolism MeSH
- Alzheimer Disease * drug therapy metabolism MeSH
- Biological Availability MeSH
- Butyrylcholinesterase metabolism MeSH
- Cholinesterase Inhibitors * pharmacology chemistry chemical synthesis MeSH
- Blood-Brain Barrier metabolism MeSH
- Microsomes, Liver metabolism MeSH
- Humans MeSH
- Ligands MeSH
- Molecular Structure MeSH
- Mice MeSH
- Receptors, N-Methyl-D-Aspartate * antagonists & inhibitors metabolism MeSH
- Tacrine * pharmacology chemistry chemical synthesis MeSH
- Dose-Response Relationship, Drug MeSH
- Structure-Activity Relationship MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
