Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.
- MeSH
- buněčný cyklus * MeSH
- konformace proteinů MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie metody MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- proteiny chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- proteiny MeSH
Syphilis is a multistage sexually transmitted disease caused by Treponema pallidum ssp. pallidum (TPA). This study analyzed clinical samples collected from patients with a diagnosed syphilis infection from 2004-2022, isolated in the Czech Republic. Mucocutaneous swab samples (n = 543) from 543 patients were analyzed, and from these samples, 80.11 % (n = 435) were PCR positive, and 19.89 % (n = 108) were PCR negative for TPA DNA. Swabs were more often positive when collected from syphilis patients in the primary and secondary stages, compared to the latent or unknown stage. There was no significant difference in PCR positivity between the primary and secondary stages (p = 0.099). In IgM-positive patients, a statistically significant association with PCR-positivity was found in samples from seropositive (p = 0.033) and serodiscrepant (RPR negative) patients (p = 0.0006). When assessing our laboratory-defined cases of syphilis, the RPR, IgM, and PCR tests were similarly effective (within the range of 80.1-86.1 %). However, parallel testing with these methods was even more effective, i.e., RPR + PCR was 96.1 % effective and RPR + IgM + PCR was 97.8 % effective. A combination of RPR + PCR, or a combination of all three tests (RPR, IgM, and PCR) can therefore be used to reliably detect active syphilis cases, including reinfections. Our findings show that the reverse algorithm for detecting syphilis could be substantially improved by adding IgM and PCR testing.
- Klíčová slova
- Nontreponemal tests, PCR syphilis detection, RPR, Syphilis, Syphilis diagnostics, Syphilis serology, Treponemal tests,
- Publikační typ
- časopisecké články MeSH
The intestine hosts the largest immune system and peripheral nervous system in the human body. The gut‒brain axis orchestrates communication between the central and enteric nervous systems, playing a pivotal role in regulating overall body function and intestinal homeostasis. Here, using a human three-dimensional in vitro culture model, we investigated the effects of serotonin, a neuromodulator produced in the gut, on immune cell and intestinal tissue interactions. Serotonin attenuated the tumor necrosis factor-induced proinflammatory response, mostly by affecting the expression of chemokines. Serotonin affected the phenotype and distribution of tissue-migrating monocytes, without direct contact with the cells, by remodeling the intestinal tissue. Collectively, our results show that serotonin plays a crucial role in communication among gut-brain axis components and regulates monocyte migration and plasticity, thereby contributing to gut homeostasis and the progression of inflammation. In vivo studies focused on the role of neuromodulators in gut inflammation have shown controversial results, highlighting the importance of human experimental models. Moreover, our results emphasize the importance of human health research in human cell-based models and suggest that the serotonin signaling pathway is a new therapeutic target for inflammatory bowel disease.
- MeSH
- lidé MeSH
- monocyty metabolismus imunologie MeSH
- pohyb buněk MeSH
- serotonin * metabolismus MeSH
- signální transdukce MeSH
- střevní sliznice * metabolismus patologie MeSH
- TNF-alfa metabolismus MeSH
- zánět metabolismus patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- serotonin * MeSH
- TNF-alfa MeSH
BACKGROUND: Treponema pallidum subspecies pertenue (TPE) is the causative agent of human and nonhuman primate (NHP) yaws infection. The discovery of yaws bacterium in wild populations of NHPs opened the question of transmission mechanisms within NHPs, and this work aims to take a closer look at the transmission of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our study determined eleven whole TPE genomes from NHP isolates collected from three national parks in Tanzania: Lake Manyara National Park (NP), Serengeti NP, and Ruaha NP. The bacteria were isolated from four species of NHPs: Chlorocebus pygerythrus (vervet monkey), Cercopithecus mitis (blue monkey), Papio anubis (olive baboon), and Papio cynocephalus (yellow baboon). Combined with previously generated genomes of TPE originating from NHPs in Tanzania (n = 11), 22 whole-genome TPE sequences have now been analyzed. Out of 231 possible combinations of genome-to-genome comparisons, five revealed an unexpectedly high degree of genetic similarity in samples collected from different NHP species, consistent with inter-species transmission of TPE among NHPs. We estimated a substitution rate of TPE of NHP origin, ranging between 1.77 × 10-7 and 3.43 × 10-7 per genomic site per year. CONCLUSIONS/SIGNIFICANCE: The model estimations predicted that the inter-species transmission happened recently, within decades, roughly in an order of magnitude shorter time compared to time needed for the natural diversification of all tested TPE of Tanzanian NHP origin. Moreover, the geographical separation of the sampling sites (NPs) does not preclude TPE transmission between and within NHP species.
- MeSH
- Cercopithecus aethiops MeSH
- Cercopithecus mikrobiologie MeSH
- frambézie * mikrobiologie přenos MeSH
- fylogeneze * MeSH
- genom bakteriální MeSH
- lidé MeSH
- nemoci opic mikrobiologie přenos MeSH
- Papio anubis mikrobiologie MeSH
- Papio cynocephalus mikrobiologie genetika MeSH
- primáti mikrobiologie MeSH
- sekvenování celého genomu * MeSH
- Treponema pallidum genetika izolace a purifikace klasifikace MeSH
- Treponema MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Tanzanie MeSH
Taylorella equigenitalis is the causative agent of sexually transmitted contagious equine metritis. Infections manifest as cervicitis, vaginitis and endometritis and cause temporary infertility and miscarriages of mares. While previous studies have analyzed this organism for various parameters, the evolutionary dynamics of this pathogen, including the emergence of antibiotic resistance, remains unresolved. The aim of this study was to isolate contemporary strains, determine their genome sequences, evaluate their antibiotic resistance and compare them with other strains. We determined nine complete whole genome sequences of T. equigenitalis strains, mainly from samples collected from Kladruber horses in the Czech Republic. While T. equigenitalis strains from Kladruby isolated between 1982 and 2018 were inhibited by streptomycin, contemporary strains were found to be resistant to streptomycin, suggesting the recent emergence of this mutation. In addition, we used the collection dates of Kladruber horse strains to estimate the genome substitution rate, which resulted in a scaled mean evolutionary rate of 6.9×10-7 substitutions per site per year. Analysis with other available T. equigenitalis genome sequences (n = 18) revealed similarity of the Czech T. equigenitalis genomes with the Austrian T. equigenitalis genome, and molecular dating suggested a common ancestor of all analyzed T. equigenitalis strains from 1.5-2.6 thousand years ago, dating to the first centuries A.D. Our study revealed a recently emerged streptomycin resistance in T. equigenitalis strains from Kladruber horses, emphasizing the need for antibiotic surveillance and alternative treatments. Additionally, our findings provided insights into the pathogen's evolution rate, which is important for understanding its evolution and preparing preventive strategies.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- fylogeneze MeSH
- genom bakteriální * genetika MeSH
- koně mikrobiologie MeSH
- molekulární evoluce MeSH
- nemoci koní * mikrobiologie MeSH
- sekvenování celého genomu * MeSH
- Taylorella equigenitalis * genetika MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- antibakteriální látky MeSH
FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.
- Klíčová slova
- Fibroblast growth factor 21, Protein engineering, Protein stabilization,
- Publikační typ
- časopisecké články MeSH
The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential.
- Klíčová slova
- Aminophylline, Arrhythmogenic effects, Atomic force microscopy, iPSC, Biomechanical properties, Cardiomyocytes, Drug cardiotoxicity, HESC, Pulmonary drug screening, Salbutamol,
- MeSH
- albuterol * farmakologie MeSH
- aminofylin * farmakologie MeSH
- bronchodilatancia farmakologie MeSH
- buněčné linie MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- lidé MeSH
- mikroskopie atomárních sil MeSH
- pluripotentní kmenové buňky účinky léků cytologie MeSH
- srdeční arytmie * farmakoterapie MeSH
- srdeční frekvence účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- albuterol * MeSH
- aminofylin * MeSH
- bronchodilatancia MeSH
BACKGROUND AND OBJECTIVES: Meningioma, the most common primary intracranial tumor, presents challenges in surgical treatment because of varying tissue stiffness. This study explores the molecular background of meningioma stiffness, a critical factor in surgical planning and prognosis, focusing on the utility of microRNAs (miRNAs) as diagnostic biomarkers of tissue stiffness. METHODS: Patients with meningiomas treated surgically at the University Hospital Brno were included in this study. Total RNA, isolated from tumor tissue samples, underwent quality control and small RNA sequencing to analyze miRNA expression. Differentially expressed miRNAs were identified, and their association with tumor stiffness was assessed. RESULTS: This study identified specific miRNAs differentially expressed in meningiomas with different stiffness levels. Key miRNAs, such as miR-31-5p and miR-34b-5p, showed significant upregulation in stiffer meningiomas. These findings were validated using reverse transcription-quantitative polymerase chain reaction, revealing a potential link between miRNA expression and tumor consistency. The expression of miR-31-5p was most notably associated with the stiffness of the tumor tissue (sensitivity = 71% and specificity = 83%). CONCLUSION: This research highlights the potential of miRNAs as biomarkers for determining meningioma tissue stiffness. Identifying specific miRNAs associated with tumor consistency could improve preoperative planning and patient prognosis. These findings pave the way for further exploration of miRNAs in the clinical assessment of meningiomas.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The increase in syphilis rates worldwide necessitates development of a vaccine with global efficacy. We aimed to explore Treponema pallidum subspecies pallidum (TPA) molecular epidemiology essential for vaccine research by analysing clinical data and specimens from early syphilis patients using whole-genome sequencing (WGS) and publicly available WGS data. METHODS: In this multicentre, cross-sectional, molecular epidemiology study, we enrolled patients with primary, secondary, or early latent syphilis from clinics in China, Colombia, Malawi, and the USA between Nov 28, 2019, and May 27, 2022. Participants aged 18 years or older with laboratory confirmation of syphilis by direct detection methods or serological testing, or both, were included. Patients were excluded from enrolment if they were unwilling or unable to give informed consent, did not understand the study purpose or nature of their participation, or received antibiotics active against syphilis in the past 30 days. TPA detection and WGS were conducted on lesion swabs, skin biopsies, skin scrapings, whole blood, or rabbit-passaged isolates. We compared our WGS data to publicly available genomes and analysed TPA populations to identify mutations associated with lineage and geography. FINDINGS: We screened 2802 patients and enrolled 233 participants, of whom 77 (33%) had primary syphilis, 154 (66%) had secondary syphilis, and two (1%) had early latent syphilis. The median age of participants was 28 years (IQR 22-35); 154 (66%) participants were cisgender men, 77 (33%) were cisgender women, and two (1%) were transgender women. Of the cisgender men, 66 (43%) identified as gay, bisexual, or other sexuality. Among all participants, 56 (24%) had HIV co-infection. WGS data from 113 participants showed a predominance of SS14-lineage strains with geographical clustering. Phylogenomic analyses confirmed that Nichols-lineage strains were more genetically diverse than SS14-lineage strains and clustered into more distinct subclades. Differences in single nucleotide variants (SNVs) were evident by TPA lineage and geography. Mapping of highly differentiated SNVs to three-dimensional protein models showed population-specific substitutions, some in outer membrane proteins (OMPs) of interest. INTERPRETATION: Our study substantiates the global diversity of TPA strains. Additional analyses to explore TPA OMP variability within strains is vital for vaccine development and understanding syphilis pathogenesis on a population level. FUNDING: US National Institutes of Health National Institute for Allergy and Infectious Disease, the Bill & Melinda Gates Foundation, Connecticut Children's, and the Czech Republic National Institute of Virology and Bacteriology.
- MeSH
- bakteriální vakcíny imunologie aplikace a dávkování MeSH
- dospělí MeSH
- fylogeneze MeSH
- genetická variace genetika MeSH
- genom bakteriální MeSH
- genomika MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- molekulární epidemiologie * MeSH
- průřezové studie MeSH
- sekvenování celého genomu * MeSH
- syfilis * epidemiologie mikrobiologie MeSH
- Treponema pallidum * genetika imunologie MeSH
- Treponema MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- multicentrická studie MeSH
- Geografické názvy
- Spojené státy americké epidemiologie MeSH
- Názvy látek
- bakteriální vakcíny MeSH
The treponemes infecting lagomorphs include Treponema paraluisleporidarum ecovar Cuniculus (TPeC) and ecovar Lepus (TPeL), infecting rabbits and hares, respectively. In this study, we described the first complete genome sequence of TPeL, isolate V3603-13, from an infected mountain hare (Lepus timidus) in Sweden. In addition, we determined 99.0% of the genome sequence of isolate V246-08 (also from an infected mountain hare, Sweden) and 31.7% of the genome sequence of isolate Z27 A77/78 (from a European hare, Lepus europeaus, The Netherlands). The TPeL V3603-13 genome had considerable gene synteny with the TPeC Cuniculi A genome and with the human pathogen T. pallidum, which causes syphilis (ssp. pallidum, TPA), yaws (ssp. pertenue, TPE) and endemic syphilis (ssp. endemicum, TEN). Compared to the TPeC Cuniculi A genome, TPeL V3603-13 contained four insertions and 11 deletions longer than three nucleotides (ranging between 6 and2,932 nts). In addition, there were 25 additional indels, from one to three nucleotides long, altogether spanning 36 nts. The number of single nucleotide variants (SNVs) between TPeC Cuniculi A and TPeL V3603-13 were represented by 309 nucleotide differences. Major proteome coding differences between TPeL and TPeC were found in the tpr gene family, and (predicted) genes coding for outer membrane proteins, suggesting that these components are essential for host adaptation in lagomorph syphilis. The phylogeny revealed that the TPeL sample from the European brown hare was more distantly related to TPeC Cuniculi A than V3603-13 and V246-08.
- MeSH
- fylogeneze * MeSH
- genom bakteriální MeSH
- králíci MeSH
- syfilis * mikrobiologie MeSH
- Treponema * genetika izolace a purifikace MeSH
- zajíci * mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
