The biosynthesis of the lincosamide antibiotics lincomycin A and celesticetin involves the pyridoxal-5'-phosphate (PLP)-dependent enzymes LmbF and CcbF, which are responsible for bifurcation of the biosynthetic pathways. Despite recognizing the same S-glycosyl-L-cysteine structure of the substrates, LmbF catalyses thiol formation through β-elimination, whereas CcbF produces S-acetaldehyde through decarboxylation-coupled oxidative deamination. The structural basis for the diversification mechanism remains largely unexplored. Here we conduct structure-function analyses of LmbF and CcbF. X-ray crystal structures, docking and molecular dynamics simulations reveal that active-site aromatic residues play important roles in controlling the substrate binding mode and the reaction outcome. Furthermore, the reaction selectivity and oxygen-utilization of LmbF and CcbF were rationally engineered through structure- and calculation-based mutagenesis. Thus, the catalytic function of CcbF was switched to that of LmbF, and, remarkably, both LmbF and CcbF variants gained the oxidative-amidation activity to produce an unnatural S-acetamide derivative of lincosamide.
The forskolin-induced swelling assay (FIS) in patient-derived intestinal organoids (PDIOs), used to determine in vitro responsiveness to elexacaftor/tezacaftor/ivacaftor (ETI), showed variability in swelling among PDIOs obtained from people with CF (pwCF) carrying the same F508del/F508del CFTR genotype. We aimed to characterise the effect of ETI on the transcriptional activity of PDIOs-derived cells to understand the intracellular processes triggered by ETI and the differences in treatment response. Six high- and six low-responding PDIOs to ETI, derived from F508del/F508del pwCF, were incubated with or without ETI for 2 to 6 h. Gene expression was assessed using 3'-mRNA sequencing and modelled using negative binomial models. Incubation with ETI resulted in a significant upregulation of several biological processes: mostly related to chemokines and signalling, chemotaxis, and tissue development processes. No changes were observed in abundance of the CFTR transcripts or in CFTR-related gene sets and pathways. The genes and pathways associated with ETI did not overlap with those whose expression changed with time only. PDIOs with a high FIS response did not significantly differ in any interpretable gene from the FIS-low organoids. The changes in the PDIOs gene expression upon the exposure to ETI cannot explain differences in the magnitude of PDIOs FIS-measured response to ETI. In conclusion, on incubation with ETI, genes of the CFTR-related pathways do not change their transcriptional activity; instead, overexpression was observed in genes of inflammatory-like cytokine response and receptor activation pathways.
- MeSH
- Chloride Channel Agonists therapeutic use pharmacology MeSH
- Aminophenols * therapeutic use pharmacology MeSH
- Benzodioxoles * therapeutic use pharmacology MeSH
- Quinolones * pharmacology therapeutic use MeSH
- Cystic Fibrosis * genetics drug therapy MeSH
- Drug Combinations MeSH
- Indoles * pharmacology MeSH
- Humans MeSH
- Organoids * metabolism MeSH
- Cystic Fibrosis Transmembrane Conductance Regulator genetics MeSH
- Pyrazoles * pharmacology MeSH
- Pyridines pharmacology MeSH
- Pyrrolidines pharmacology MeSH
- Pyrroles pharmacology MeSH
- Gene Expression Profiling methods MeSH
- Intestines drug effects MeSH
- Transcriptome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
BACKGROUND: Many ulcerative colitis (UC) patients require the use of second-line agents after the failure of anti-TNF therapy. RESEARCH DESIGN AND METHODS: We conducted a multicenter, retrospective study including 683 chronically active, moderate-to-severe UC patients who failed first-line anti-TNFs. The rate of treatment persistence and colectomy-free survival was assessed up to 3 years after the initiation of second-line therapy. Predictors for colectomy and persistence were investigated. RESULTS: After the failure of the first-line anti-TNF, ustekinumab had superior persistence and colectomy-free survival rates compared to tofacitinib (p = 0.05; p = 0.001) and vedolizumab (p = 0.02; p = 0.05), but significant difference was only found in persistence rates in comparison with anti-TNFs (p < 0.001). Regardless of the number of prior anti-TNFs, significantly higher persistence (p = 0.05) and colectomy-free survival rates (p = 0.01) were observed over 2 years with ustekinumab than with vedolizumab or tofacitinib, whereas ustekinumab's superiority over tofacitinib seemed to disappear by the third year. Hypoalbuminaemia (p = 0.002) and shorter disease duration at second-line initiation (p = 0.03) increased, while concomitant immunomodulators (p = 0.05) reduced the risk for colectomy. Shorter disease duration (p = 0.01) and primary non-response to the previously used anti-TNF (p < 0.001) negatively influenced persistence with second-line non-TNF-targeted agents. CONCLUSION: After first-line anti-TNF failure, switching to a non-anti-TNF agent is worth considering in moderate-to-severe UC.
- MeSH
- Chronic Disease MeSH
- Adult MeSH
- Gastrointestinal Agents * therapeutic use administration & dosage MeSH
- Antibodies, Monoclonal, Humanized therapeutic use administration & dosage MeSH
- Cohort Studies MeSH
- Colectomy MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Treatment Failure MeSH
- Piperidines therapeutic use administration & dosage MeSH
- Pyrimidines therapeutic use administration & dosage MeSH
- Pyrrolidines therapeutic use administration & dosage MeSH
- Retrospective Studies MeSH
- Aged MeSH
- Severity of Illness Index MeSH
- Tumor Necrosis Factor-alpha * antagonists & inhibitors MeSH
- Colitis, Ulcerative * drug therapy surgery MeSH
- Ustekinumab therapeutic use administration & dosage MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Aged MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Comparative Study MeSH
Diftérie představuje důležité, znovu se objevující infekční onemocnění. Patří mu zvýšená pozornost ze stran lékařské komunity, a to nejen v primární péči, ale napříč všemi obory. Nejrozšířenějším původcem je bakterie Corynebacterium diphtheriae. Existují dvě formy onemocnění: kožní a respirační. Lékem první volby jsou penicilinová antibiotika. Kromě úvodního stručného popisu mikroorganismu se následující text zaměří na tři kazuistiky pacientů, u nichž bylo v kultivačním vyšetření identifikováo Corynebacterium diphtheriae jako etiologické agens. První případ je respirační, další dva kožní formy. Je popsán průběh onemocnění, nezbytnost izolace na infekčním oddělení a realizovaný terapeutický postup. Dále je uveden lokální epidemiologický přehled incidence a závěrečné zhodnocení uvedených případů a nemoci samotné.
Diphtheria is an important, re-emerging infection that requires increased attention from the medical community, not only in primary care but across all medical fields. The causative agent is the bacterium Corynebacterium diphtheriae. Two forms of the disease are distinguished: skin and respiratory. The first-line treatment consists of penicillin antibiotics. In addition to a brief initial description of the microorganism, the following text focuses on three case studies of patients in whom Corynebacterium diphtheriae was identified as the etiological agent through culture examination. The first case involves the respiratory form, while the other two concern the skin form. The course of the disease, the necessity of isolation in an infectious disease ward, and the therapeutic approach taken are described. Furthermore, a local epidemiological overview of incidence is provided, along with a final evaluation of the presented cases and the disease itself.
- MeSH
- Anti-Bacterial Agents therapeutic use MeSH
- Corynebacterium diphtheriae pathogenicity MeSH
- Diphtheria Toxin adverse effects MeSH
- Diphtheria * epidemiology drug therapy pathology prevention & control MeSH
- Clindamycin therapeutic use MeSH
- Skin Manifestations MeSH
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Check Tag
- Middle Aged MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Case Reports MeSH
The utilization of 3D printing- digital light processing (DLP) technique, for the direct fabrication of microneedles encounters the problem of drug solubility in printing resin, especially if it is predominantly composed of water. The possible solution how to ensure ideal belonging of drug and water-based printing resin is its pre-formulation in nanosuspension such as nanocrystals. This study investigates the feasibility of this approach on a resin containing nanocrystals of imiquimod (IMQ), an active used in (pre)cancerous skin conditions, well known for its problematic solubility and bioavailability. The resin blend of polyethylene glycol diacrylate and N-vinylpyrrolidone, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate as a photoinitiator, was used, mixed with IMQ nanocrystals in water. The final microneedle-patches had 36 cylindrical microneedles arranged in a square grid, measuring approximately 600 μm in height and 500 μm in diameter. They contained 5wt% IMQ, which is equivalent to a commercially available cream. The homogeneity of IMQ distribution in the matrix was higher for nanocrystals compared to usual crystalline form. The release of IMQ from the patches was determined ex vivo in natural skin and revealed a 48% increase in efficacy for nanocrystal formulations compared to the crystalline form of IMQ.
- MeSH
- Printing, Three-Dimensional * MeSH
- Administration, Cutaneous MeSH
- Imiquimod * chemistry administration & dosage MeSH
- Needles * MeSH
- Skin Absorption MeSH
- Skin metabolism MeSH
- Drug Delivery Systems instrumentation MeSH
- Microinjections instrumentation MeSH
- Nanoparticles * chemistry administration & dosage MeSH
- Polyethylene Glycols chemistry administration & dosage MeSH
- Povidone chemistry MeSH
- Solubility * MeSH
- Drug Liberation MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Spray drying and hot-melt extrusion are among the most prevalent preparation techniques used in the pharmaceutical industry to produce amorphous solid dispersions (ASDs). This study advances previous research by integrating sample production, comprehensive analytical characterization, intrinsic dissolution rate measurements, and assessments of the behavior of ASDs under elevated temperature and humidity conditions. The study focuses on indomethacin, a widely used model for poorly soluble drugs, processed with PVP K30 or HPMC E5, both commonly used polymers. The findings demonstrate that hot-melt extruded samples exhibit superior stability against recrystallization, whereas spray dried samples achieve higher intrinsic dissolution rates. Furthermore, PVP K30 significantly outperforms HPMC E5 in the co-processing of indomethacin, enhancing both the intrinsic dissolution rate and the stability.
- MeSH
- Hypromellose Derivatives chemistry MeSH
- Chemistry, Pharmaceutical methods MeSH
- Indomethacin * chemistry MeSH
- Crystallization * MeSH
- Povidone chemistry MeSH
- Drug Compounding methods MeSH
- Solubility * MeSH
- Spray Drying * MeSH
- Drug Stability * MeSH
- Hot Melt Extrusion Technology * methods MeSH
- Drug Liberation MeSH
- Humidity MeSH
- Hot Temperature MeSH
- Desiccation methods MeSH
- Publication type
- Journal Article MeSH
- MeSH
- Chloride Channel Agonists therapeutic use MeSH
- Aminophenols * therapeutic use MeSH
- Benzodioxoles * therapeutic use MeSH
- Quinolines therapeutic use MeSH
- Quinolones * therapeutic use MeSH
- Cystic Fibrosis * drug therapy genetics MeSH
- Adult MeSH
- Exocrine Pancreatic Insufficiency drug therapy etiology genetics MeSH
- Drug Combinations MeSH
- Genotype MeSH
- Indoles * therapeutic use MeSH
- Humans MeSH
- Cystic Fibrosis Transmembrane Conductance Regulator * genetics MeSH
- Pyrazoles * therapeutic use MeSH
- Pyridines therapeutic use MeSH
- Pyrrolidines therapeutic use MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Letter MeSH
- Case Reports MeSH
- MeSH
- Colorectal Neoplasms drug therapy MeSH
- Drug Therapy, Combination MeSH
- Communication MeSH
- Quality of Life MeSH
- Humans MeSH
- Neoplasms * drug therapy MeSH
- Palliative Care methods MeSH
- Prognosis MeSH
- Pyrrolidines therapeutic use MeSH
- Truth Disclosure MeSH
- Thymine therapeutic use MeSH
- Trifluridine therapeutic use MeSH
- Physician-Patient Relations * MeSH
- Patient Participation psychology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Newspaper Article MeSH
Protein cross-linking has assumed an irreplaceable role in structural proteomics. Recently, significant efforts have been made to develop novel mass spectrometry (MS)-cleavable reagents. At present, only water-insoluble MS-cleavable cross-linkers are commercially available. However, to comprehensively analyse the various chemical and structural motifs making up proteins, it is necessary to target different protein sites with varying degrees of hydrophilicity. Here we introduce the new MS-cleavable cross-linker disulfodisuccinimidyl dibutyric urea (DSSBU), which we have developed in-house for this purpose. DSSBU contains an N-hydroxysulfosuccinimide (sulfo-NHS) reactive group, so it can serve as a water-soluble counterpart to the widely used cross-linker disuccinimidyl dibutyric urea (DSBU). To investigate the applicability of DSSBU, we compared the efficacy of four similar cross-linkers: bis[sulfosuccinimidyl] suberate (BS3), disuccinimidyl suberate (DSS), DSBU and DSSBU with bovine serum albumin. In addition, we compared the efficacy of DSBU and DSSBU with human haemoglobin. Our results demonstrate that the sulfo-NHS group ensures the superior water solubility of DSSBU and thus negates the need for organic solvents such as dimethyl sulfoxide while preserving the effectivity of urea-based MS-cleavable crosslinkers such as DSBU. Additionally, it makes it possible to target polar regions in proteins. The data gathered are available via ProteomeXchange under identifier PXD055284. SIGNIFICANCE: We have synthesized the novel protein cross-linker DSSBU, which combines sulfo-NHS ester chemistry with a mass spectrometry-cleavable urea group. This makes DSSBU a water-soluble, MS-cleavable cross-linker that reacts with amino groups. To our knowledge, it is the first cross-linker which combines all three of these characteristics. We have tested the performance of our novel cross-linker on bovine serum albumin, a model widely used by the cross-linking mass spectrometry community, and on human haemoglobin. We have comprehensively assessed the performance of DSSBU and compared its efficacy with that of three other cross-linkers in current use (BS3, DSS and DSBU). We conclude that our novel cross-linker surpasses its MS-non-cleavable analogue BS3 in performance and that its water solubility eliminates the need for organic solvents while its hydrophilicity allows for the targetting of polar regions in proteins. Therefore, it will likely become a significant addition to the portfolio of N-hydroxysuccinimide ester cross-linkers.
- MeSH
- Mass Spectrometry methods MeSH
- Humans MeSH
- Urea chemistry MeSH
- Proteomics methods MeSH
- Cross-Linking Reagents * chemistry MeSH
- Serum Albumin, Bovine chemistry MeSH
- Cattle MeSH
- Succinimides * chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Cattle MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Keywords
- studie EROS,
- MeSH
- Pulmonary Disease, Chronic Obstructive * drug therapy MeSH
- Glycopyrrolate pharmacology therapeutic use MeSH
- Budesonide, Formoterol Fumarate Drug Combination pharmacology therapeutic use MeSH
- Drug Therapy, Combination MeSH
- Humans MeSH
- Retrospective Studies MeSH
- Check Tag
- Humans MeSH
