Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• farmakoterapie COVID-19 MeSH
• HEK293 buňky MeSH
• inhibitory proteas farmakologie   chemie   MeSH
• koronavirové proteasy 3C * metabolismus   antagonisté a inhibitory   chemie   genetika   MeSH
• lidé MeSH
• mutace MeSH
• objevování léků * metody   MeSH
• proteolýza MeSH
• SARS-CoV-2 * enzymologie   účinky léků   metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cervical cancer (CC) is the fourth most common malignant tumor in women worldwide. Detecting different biomarkers together on single cells by novel method mass cytometry could contribute to more precise screening. Liquid-based cytology (LBC) cervical samples were collected (N = 53) from women categorized as normal and precancerous lesions. Human papillomavirus was genotyped by polymerase chain reaction, while simultaneous examination of the expression of 29 proteins was done by mass cytometry (CyTOF). Differences in cluster abundances were assessed with Spearman's rank correlation as well as high dimensional data analysis (t-SNE, FlowSOM). Cytokeratin (ITGA6, Ck5, Ck10/13, Ck14, Ck7) expression patterns allowed determining the presence of different cells in the cervical epithelium. FlowSOM analysis enabled to phenotype cervical cells in five different metaclusters and find new markers that could be important in CC screening. The markers Ck18, Ck18, and CD63 (Metacluster 3) showed significantly increasing associated with severity of the precancerous lesions (Spearman rank correlation rho 0.304, p = 0.0271), while CD71, KLF4, LRIG1, E-cadherin, Nanog and p53 (Metacluster 1) decreased with severity of the precancerous lesions (Spearman rank correlation rho -0.401, p = 0.0029). Other metaclusters did not show significant correlation, but metacluster 2 (Ck17, MCM, MMP7, CD29, E-cadherin, Nanog, p53) showed higher abundance in low- and high-grade intraepithelial lesion cases. CyTOF appears feasible and should be considered when examining novel biomarkers on cervical LBC samples. This study enabled us to characterize different cells in the cervical epithelium and find markers and populations that could distinguish precancerous lesions.

• MeSH
• cervix uteri patologie   metabolismus   MeSH
• dospělí MeSH
• dysplazie děložního hrdla diagnóza   patologie   MeSH
• infekce papilomavirem patologie   diagnóza   virologie   MeSH
• Krüppel-like faktor 4 * MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové biomarkery * genetika   metabolismus   MeSH
• nádory děložního čípku * diagnóza   patologie   genetika   MeSH
• prekancerózy * patologie   diagnóza   MeSH
• průtoková cytometrie * metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Allodiploid hybrid species, Aspergillus latus, belonging to section Nidulantes, is a hybrid of A. spinulosporus and an unknown species closely related to A. quadrilineatus and A. sublatus. This hybrid has often been misidentified as the species in section Nidulantes, such as A. nidulans, A. spinulosporus, A. sublatus, or other cryptic species. Aspergillus latus has not been reported in Japan as well as Asia so far. In this study, we screened 23 clinical strains identified as A. spinulosporus isolated in Japan from 2012 to 2023 and found seven A. latus strains. To characterize the A. latus strains, we conducted comprehensive phenotyping including morphological observation, whole genome sequences, and phylogenetic analysis based on calmodulin (CaM) gene. In addition, we conducted antifungal susceptibility testing for A. latus strains. As a result, the morphological characters of A. latus were more similar to those of A. spinulosporus compared to A. sublatus. However, the ascospore of A. latus differed from that of A. spinulosporus. Phylogenetic analysis revealed that different CaM alleles from the same isolate clustered separately with A. spinulosporus and A. sublatus, consistent with its hybrid origin. Furthermore, A. latus strains showed reduced susceptibility to caspofungin and amphotericin B compared to A. spinulosporus, while they were susceptible to azoles. Our results suggest that A. latus has been a causative pathogen of aspergillosis in Japan since 2013.

• MeSH
• antifungální látky farmakologie   MeSH
• Aspergillus * genetika   klasifikace   izolace a purifikace   účinky léků   MeSH
• aspergilóza * mikrobiologie   epidemiologie   MeSH
• fylogeneze MeSH
• kalmodulin genetika   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• sekvenování celého genomu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Japonsko MeSH

V kohortových studiích měli muži s diabetes mellitus 2. typu (DM2) konzistentně 2krát vyšší frekvenci nízké hladiny testosteronu než muži bez diabetu. Metabolický syndrom a diabetes mellitus 2. typu vedou k nižší hladině testosteronu u mužů zejména útlumem hypotalamické sekrece gonadoliberinu a zvýšenou konverzí testosteronu na estrogen. Hypogonadismus potom sám o sobě zhoršuje obezitu a inzulinovou rezistenci. Za hypogonadismus je podle současných doporučení považován stav, kdy jsou zároveň přítomny typické příznaky hypogonadismu a současně snížená hladina testosteronu (pod 12 nmol/l). Dle doporučených postupů je vhodné aktivně pátrat po hypogonadismu u mužů s DM2 a v případě jeho potvrzení zahájit vhodnou léčbu. Ke screeningu příznaků hypogonadismu se mezinárodně používá Aging Males’ Symptoms (AMS) dotazník, který je cenným nástrojem pro hodnocení kvality života a symptomů souvisejících se zdravím u stárnoucích mužů. Na základě výsledků a hladiny testosteronu se rozhodujeme o léčbě, která při nízkých hladinách testosteronu je substituční. Dotazník nám také pomáhá prolomit komunikační bariéru na téma sexuálního života pacienta a případné erektilní dysfunkce, kterou lze v řadě případů úspěšně léčit.

In cohort studies, men with type 2 diabetes mellitus (T2DM) consistently had twice the frequency of low testosterone levels compared to men without diabetes. Metabolic syndrome and type 2 diabetes mellitus lead to lower testosterone levels primarily by suppressing hypothalamic gonadoliberin secretion and by increasing the conversion of testosterone to oestrogen. Hypogonadism itself then worsens obesity and insulin resistance. According to current recommendations, hypogonadism is defined as the presence of typical hypogonadal symptoms together with a reduced testosterone level (below 12 nmol/l). It is advisable to actively screen for hypogonadism in men with T2DM and, if confirmed, initiate appropriate treatment. The Aging Males’ Symptoms (AMS) questionnaire is used internationally to screen for symptoms of hypogonadism; it is a valuable tool for assessing quality of life and health-related symptoms in aging men. Based on the questionnaire results and testosterone levels, treatment decisions are made; in cases of low testosterone levels, testosterone replacement therapy is indicated. The questionnaire also helps break down communication barriers regarding the patient’s sexual life and potential erectile dysfunction, which can often be successfully treated.

• MeSH
• diabetes mellitus 2. typu MeSH
• erektilní dysfunkce MeSH
• hypogonadismus * diagnóza   farmakoterapie   patofyziologie   MeSH
• lidé MeSH
• obezita * komplikace   MeSH
• průzkumy a dotazníky MeSH
• testosteron krev   nedostatek   terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH

Nucleus pulposus cells (NPC) are important for the development of intervertebral disc degeneration (IVDD). miR-4478 can aggravate IVDD, but whether it can aggravate IVDD by regulating ferroptosis in NPC remains unclear. The optimal level of ferroptosis activator RSL3 for eliciting ferroptosis in NPC was screened by Western blot and CCK-8 assay. The targeting relationship between miR-4478 and its potential target solute carrier family 7 member 11 (SLC7A11) was explored based on dual luciferase assay. On this basis, IVDD models were constructed. After over-expression or knockdown of miR-4478 or SLC7A11, CCK-8 and calcein-AM/PI assays were employed to evaluate cell damage. Flow cytometry, Western blot and Prussian blue staining were employed to evaluate oxidation and ferroptosis levels, and histopathological staining was applied to evaluate the intervertebral disc tissue injury degree. The optimal concentration of RSL3 was 1 μM. Under these conditions, miR-4478 or SLC7A11 can be effectively over-expressed or knocked down after transfection. Knockdown of miR-4478 can improve the survival rate of NPC, the level of Fe2+ ions, improve the pathological damage of intervertebral disc structure, reduce iron deposition in tissues, and significantly reduce expression of reactive oxygen species (ROS) and ferroptosis-related protein. The levels of antioxidant enzymes were significantly increased. When miR-4478 was over-expressed, the above phenomenon was reversed. On this basis, after SLC7A11 was over-expressed, the effect of miR-4478 up-regulation was weakened, and NPC ferroptosis was improved. miR-4478 can target SLC7A11 to promote NPC damage, peroxide accumulation and iron metabolism disorders, leading to ferroptosis, thereby inducing IVDD.

• MeSH
• degenerace meziobratlové ploténky * metabolismus   genetika   patologie   MeSH
• ferroptóza * genetika   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mikro RNA * metabolismus   genetika   MeSH
• nucleus pulposus * metabolismus   patologie   cytologie   MeSH
• potkani Sprague-Dawley MeSH
• transportní systém aminokyselin y+ * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články 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
• acetylcholinesterasa metabolismus   MeSH
• Alzheimerova nemoc * farmakoterapie   metabolismus   MeSH
• biologická dostupnost MeSH
• butyrylcholinesterasa metabolismus   MeSH
• cholinesterasové inhibitory * farmakologie   chemie   chemická syntéza   MeSH
• hematoencefalická bariéra metabolismus   MeSH
• jaterní mikrozomy metabolismus   MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• myši MeSH
• receptory N-methyl-D-aspartátu * antagonisté a inhibitory   metabolismus   MeSH
• takrin * farmakologie   chemie   chemická syntéza   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Macrocyclic inhibitors have emerged as a privileged scaffold in medicinal chemistry, offering enhanced selectivity, stability, and pharmacokinetic profiles compared to their linear counterparts. Here, we describe a novel, on-resin macrocyclization strategy for the synthesis of potent inhibitors targeting the secreted protease Major Aspartyl Peptidase 1 in Cryptococcus neoformans, a pathogen responsible for life-threatening fungal infections. By employing diverse aliphatic linkers and statine-based transition-state mimics, we constructed a focused library of 624 macrocyclic compounds. Screening identified several subnanomolar inhibitors with desirable pharmacokinetic and antifungal properties. Lead compound 25 exhibited a Ki of 180 pM, significant selectivity against host proteases, and potent antifungal activity in culture. The streamlined synthetic approach not only yielded drug-like macrocycles with potential in antifungal therapy but also provided insights into structure-activity relationships that can inform broader applications of macrocyclization in drug discovery.

• MeSH
• antifungální látky * farmakologie   chemie   chemická syntéza   farmakokinetika   MeSH
• Cryptococcus neoformans * účinky léků   enzymologie   MeSH
• inhibitory proteas * farmakologie   chemie   chemická syntéza   farmakokinetika   MeSH
• lidé MeSH
• makrocyklické sloučeniny * farmakologie   chemie   chemická syntéza   farmakokinetika   MeSH
• mikrobiální testy citlivosti MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The Takeda G protein-coupled receptor 5 (TGR5), also known as GPBAR1 (G protein-coupled bile acid receptor), is a membrane-type bile acid receptor that regulates blood glucose levels and energy expenditure. These essential functions make TGR5 a promising target for the treatment of type 2 diabetes and metabolic disorders. Currently, most research on developing TGR5 agonists focuses on modifying the structure of bile acids, which are the endogenous ligands of TGR5. However, TGR5 agonists with nonsteroidal structures have not been widely explored. This study aimed at discovering new TGR5 agonists using bile acid derivatives as a basis for a computational approach. We applied a combination of pharmacophore-based, molecular docking, and molecular dynamic (MD) simulation to identify potential compounds as new TGR5 agonists. Through pharmacophore screening and molecular docking, we identified 41 candidate compounds. From these, five candidates were selected based on criteria including pharmacophore features, a docking score of less than 9.2 kcal/mol, and similarity in essential interaction patterns with a reference ligand. Biological assays of the five hits confirmed that Hit-3 activates TGR5 similarly to the bile acid control. This was supported by MD simulation results, which indicated that a hydrogen bond interaction with Tyr240 is involved in TGR5 activation. Hit-3 (CSC089939231) represents a new nonsteroidal lead that can be further optimized to design potent TGR5 agonists.

• MeSH
• lidé MeSH
• ligandy MeSH
• molekulární struktura MeSH
• objevování léků MeSH
• receptory spřažené s G-proteiny * agonisté   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• simulace molekulového dockingu * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• žlučové kyseliny a soli chemie   metabolismus   farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that these inhibitors restrict an essential catalytic motion by extending an α helix over a conserved glycine hinge. SMURF1 levels are increased in pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevented BMPR2 ubiquitylation, normalized bone morphogenetic protein (BMP) signaling, restored pulmonary vascular cell homeostasis, and reversed pathology in established experimental PAH. Leveraging this deep mechanistic understanding, we undertook an in silico machine-learning-based screen to identify inhibitors of the prototypic HECT E6AP and confirmed glycine-hinge-dependent allosteric activity in vitro. Inhibiting HECTs and other glycine-hinge proteins opens a new druggable space.

• MeSH
• alosterická regulace účinky léků   MeSH
• lidé MeSH
• myši MeSH
• plicní arteriální hypertenze farmakoterapie   MeSH
• receptory morfogenetických kostních proteinů typu II MeSH
• signální transdukce účinky léků   MeSH
• ubikvitinace účinky léků   MeSH
• ubikvitinligasy * antagonisté a inhibitory   metabolismus   chemie   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Data on the presentation of Autosomal Dominant Polycystic Kidney Disease (ADPKD) in children have been based on small/regional cohorts and practices regarding both asymptomatic screening in minors and genetic testing differ greatly between countries. To provide a global perspective, we analyzed over 2100 children and adolescents with ADPKD from 32 countries in six World Health Organization regions: 1060 children from the multi-national ADPedKD registry were compared to 269 pediatric patients from the United Kingdom (RaDaR) and 825 from the European Rare Kidney Disease Registry (ERKReg). Asymptomatic family screening was a common mode of presentation (48% in ADPedKD, 62% in ERKReg) with broad international variability (19%-75%), but fairly stable temporal trends in both registries with no correlation to genetic testing. The national rates of genetic testing varied and correlated significantly with healthcare expenditure (odds ratio 1.030 per 100 United States Dollars/capita/year, in the ERKReg cohort), with little variation over time. Diagnosis due to prenatal abnormalities was more common than anticipated at 14% increasing steadily from 2000 onward in both registries. Realistically, a high proportion of children were diagnosed with ADPKD by active screening, underlining that families affected by ADPKD have a high need for counselling on the complex issues around presymptomatic diagnosis. Regional variations in rate of genetic testing appeared to be driven by economic factors. However, large differences in rate of active screening were not correlated to healthcare spending and probably reflect the influence of different of cultural, legal and ethical frameworks on families and clinicians in different healthcare systems.

• MeSH
• dítě MeSH
• genetické testování statistika a číselné údaje   ekonomika   MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• polycystické ledviny autozomálně dominantní * diagnóza   epidemiologie   genetika   ekonomika   MeSH
• předškolní dítě MeSH
• prenatální diagnóza MeSH
• registrace MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• srovnávací studie MeSH
• Geografické názvy
• Evropa MeSH
• Spojené království MeSH