3-methylglutaconic aciduria (3-MGCA) is a biochemical finding in a diverse group of inherited metabolic disorders. Conditions manifesting 3-MGCA are classified into two major categories, primary and secondary. Primary 3-MGCAs involve two inherited enzymatic deficiencies affecting leucine catabolism, whereas secondary 3-MGCAs comprise a larger heterogeneous group of conditions that have in common compromised mitochondrial energy metabolism. Here, we report 3-MGCA in two siblings presenting with sensorineural hearing loss and neurological abnormalities associated with a novel, homozygous missense variant (c.1999C>G, p.Leu667Val) in the YME1L1 gene which encodes a mitochondrial ATP-dependent metalloprotease. We show that the identified variant results in compromised YME1L1 function, as evidenced by abnormal proteolytic processing of substrate proteins, such as OPA1 and PRELID1. Consistent with the aberrant processing of the mitochondrial fusion protein OPA1, we demonstrate enhanced mitochondrial fission and fragmentation of the mitochondrial network in patient-derived fibroblasts. Furthermore, our results indicate that YME1L1L667V is associated with attenuated activity of rate-limiting Krebs cycle enzymes and reduced mitochondrial respiration, which may explain the build-up of 3-methylglutaconic and 3-methylglutaric acid due to the diversion of acetyl-CoA, not efficiently processed in the Krebs cycle, towards the formation of 3-methylglutaconyl-CoA, the precursor of these metabolites. In summary, our findings classify YME1L1 deficiency as a new type of secondary 3-MGCA, thus expanding the genetic landscape and facilitating the diagnosis of inherited metabolic disorders featuring this biochemical phenotype.
- Klíčová slova
- 3‐methylglutaconic aciduria, YME1L1, inherited metabolic disorders, mitochondrial disorders, mitochondrial dysfunction, mitochondrial fragmentation,
- MeSH
- dítě MeSH
- fibroblasty metabolismus MeSH
- glutaráty MeSH
- lidé MeSH
- metaloendopeptidasy * genetika metabolismus MeSH
- missense mutace MeSH
- mitochondriální dynamika MeSH
- mitochondriální proteiny * genetika MeSH
- mitochondrie metabolismus MeSH
- percepční nedoslýchavost genetika MeSH
- sourozenci MeSH
- vrozené poruchy metabolismu * genetika MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- Názvy látek
- 3-methylglutaconic acid MeSH Prohlížeč
- glutaráty MeSH
- metaloendopeptidasy * MeSH
- mitochondriální proteiny * MeSH
Cellular processes such as tissue regeneration, inflammation, and migration require the proteolysis of the extracellular matrix and the proteolytic activation of signaling molecules. A widely used and accessible technique for studying these processes is gelatin zymography, particularly for investigating matrix metalloproteinases (MMPs), though it is not limited to them. This method is favored for its simplicity, low cost, and robustness. Despite certain limitations, it remains a preferred approach for the initial investigation of complex samples.Here, we present a protocol applicable to various sample sources, including proteases from human cell lines and bacteria isolated from chronic wounds. We also explore changes in protease activity within exudates from human chronic wounds, a challenging analysis for more complex techniques. Additionally, we emphasize the potential to extend the basic protocol to study the conditions under which proteases are active.
- Klíčová slova
- Bacterial protease, Chronic wound, Exudate, Gelatin zymography, MMPs, Protease,
- MeSH
- chronická nemoc MeSH
- elektroforéza MeSH
- enzymatické testy * metody MeSH
- lidé MeSH
- matrixové metaloproteinasy * metabolismus MeSH
- rány a poranění * enzymologie MeSH
- želatina * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- matrixové metaloproteinasy * MeSH
- želatina * MeSH
Masticatory muscle hypertrophy (MMH) is a rare clinical phenomenon of uncertain etiology, characterized by a soft swelling near the angle of the jaw. This abnormal enlargement of the masseter muscle can alter the facial profile, leading to aesthetic concerns. Moreover, MMH may also have significant functional repercussions, including pain in the masseter region, often associated with temporomandibular disorders, fatigue, and discomfort during mastication. Non-conservative approaches offer an effective and minimally invasive solution by inducing localized muscle relaxation and reducing hypertrophy. Botulinum neurotoxin type A (BoNT/A) represents a therapeutic option for managing MMH, considering that injections can effectively reduce the masseter muscle volume, improving both facial aesthetics and related symptoms. Currently, the standard non-surgical management of MMH is BoNT/A injections, although consensus on the average dosage has not been definitely reached; on the other hand, there are data available in the literature about the injection technique of BoNT/A for lower face contouring. Therefore, the present comprehensive review aimed at exploring in detail the role of BoNT/A in the treatment of masseter muscle hypertrophy, describing its mechanism of action, the administration protocols, the clinical effects, and any side effects.
- Klíčová slova
- botulinum toxin type A, lower face contouring, masseter muscle hypertrophy,
- MeSH
- botulotoxiny typu A * terapeutické užití aplikace a dávkování MeSH
- hypertrofie farmakoterapie MeSH
- lidé MeSH
- musculus masseter * účinky léků patologie abnormality MeSH
- nervosvalové látky terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- botulotoxiny typu A * MeSH
- nervosvalové látky MeSH
UNLABELLED: Patients with dry eye syndrome form a significant proportion of those treated in everyday ophthalmology practice. Diabetes mellitus is a major risk factor for the development of dry eye syndrome. Changes in tear film homeostasis, chronic inflammation and subsequent corneal nerve fiber pathology play a key role in its progression. The aim of this study was to describe the status of modern biomarkers of ocular surface damage in patients with type 1 diabetes and asses their utility in early diagnosis of dry eye syndrome. MATERIAL AND METHODS: In total the pilot study included 19 patients with type 1 diabetes (T1D) and 15 patients in the control group. All patients underwent a detailed ocular surface examination, sample collection for matrix metalloproteinase-9 (MMP-9) laboratory analysis and epithelial HLA-DR expression evaluation, and in-vivo corneal confocal microscopy. RESULTS: T1D patients showed statistically significantly reduced corneal nerve fiber length (p = 0.0482). The differences between the groups in terms of osmolarity, corneal sensitivity, Oxford score, tear break-up time and MMP-9 level were not statistically significant (p = 0.8272, p = 0.6029, p = 0.3507, p = 0.7561 and p = 0.0826 respectively). HLA-DR expression was examined in 10 T1D patients and 8 patients in the control group. Both groups showed minimal or no expression (p > 0.9999). CONCLUSION: The previously published literature supports our finding of corneal nerve fiber length reduction in T1D patients compared to controls. However, we did not find any significant changes in standard or modern ocular surface markers (MMP-9 levels, HLA-DR expression) measured in patients with dry eye syndrome.
- Klíčová slova
- Confocal microscopy, HLA-DR, MMP-9, biomarker, cardiovascular disease, diabetes mellitus, dry eye disease,
- MeSH
- biologické markery * analýza metabolismus MeSH
- časná diagnóza * MeSH
- diabetes mellitus 1. typu * komplikace metabolismus MeSH
- dospělí MeSH
- HLA-DR antigeny metabolismus analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- matrixová metaloproteinasa 9 * metabolismus analýza MeSH
- pilotní projekty MeSH
- rohovka metabolismus patologie MeSH
- slzy metabolismus MeSH
- syndromy suchého oka * diagnóza metabolismus etiologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- biologické markery * MeSH
- HLA-DR antigeny MeSH
- matrixová metaloproteinasa 9 * MeSH
Antibiotic-resistant strains of Staphylococcus aureus pose a significant threat in healthcare, demanding urgent therapeutic solutions. Combining bacteriophages with conventional antibiotics, an innovative approach termed phage-antibiotic synergy, presents a promising treatment avenue. However, to enable new treatment strategies, there is a pressing need for methods to assess their efficacy reliably and rapidly. Here, we introduce a novel approach for real-time monitoring of pathogen lysis dynamics employing the piezoelectric quartz crystal microbalance (QCM) with dissipation (QCM-D) technique. The sensor, a QCM chip modified with the bacterium S. aureus RN4220 ΔtarM, was utilized to monitor the activity of the enzyme lysostaphin and the phage P68 as model lytic agents. Unlike conventional QCM solely measuring resonance frequency changes, our study demonstrates that dissipation monitoring enables differentiation of bacterial growth and lysis caused by cell-attached lytic agents. Compared to reference turbidimetry measurements, our results reveal distinct alterations in the growth curve of the bacteria adhered to the sensor, characterized by a delayed lag phase. Furthermore, the dissipation signal analysis facilitated the precise real-time monitoring of phage-mediated lysis. Finally, the QCM-D biosensor was employed to evaluate the synergistic effect of subinhibitory concentrations of the antibiotic amoxicillin with the bacteriophage P68, enabling monitoring of the lysis of P68-resistant wild-type strain S. aureus RN4220. Our findings suggest that this synergy also impedes the formation of bacterial aggregates, the precursors of biofilm formation. Overall, this method brings new insights into phage-antibiotic synergy, underpinning it as a promising strategy against antibiotic-resistant bacterial strains with broad implications for treatment and prevention.
- Klíčová slova
- Staphylococcus aureus, Antimicrobial treatment, Multidrug-resistant bacteria, Phage therapy, Phage-antibiotic synergy, Piezoelectric biosensor,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriofágy MeSH
- biosenzitivní techniky * metody MeSH
- lysostafin farmakologie MeSH
- mikrorovnovážné techniky křemenného krystalu * MeSH
- Staphylococcus aureus * účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- lysostafin MeSH
Non-healing wounds are a serious complication in diabetic patients. One of the detrimental factors contributing to limited wound healing is the accumulation of metalloproteinase-9 (MMP-9) in the wound. Selective inhibition of MMP-9 is one of the established therapeutic targets for diabetic wound healing. Here, a functional and biocompatible wound dressing is developed to enable a controlled release of a traceable vector loaded with the antisense siRNA against MMP-9 in the wound. The dressing consists of degradable polymer nanofibers embedded with a vector nanosystem - polymer-coated fluorescent nanodiamonds optimized for the binding of siRNA and colloidal stability of nanodiamond-siRNA complexes in a physiological environment. The developed dressing is tested on murine fibroblasts and also applied to wounds in a diabetic murine model to evaluate its suitability in terms of in vivo toxicity, biological efficacy, and handling. The treatment results in significant local inhibition of MMP-9 and a shortening of the wound healing time. The scar formation in treated diabetic-like mice becomes comparable with that in non-treated diabetes-free mice. Our results suggest that the application of our biocompatible dressing loaded with a non-toxic vector nanosystem is an effective and promising approach to gene therapy of non-healing wounds.
- MeSH
- aplikace lokální MeSH
- experimentální diabetes mellitus * MeSH
- hojení ran * účinky léků MeSH
- malá interferující RNA * aplikace a dávkování genetika farmakologie MeSH
- matrixová metaloproteinasa 9 metabolismus genetika MeSH
- myši MeSH
- obvazy MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- malá interferující RNA * MeSH
- matrixová metaloproteinasa 9 MeSH
Caspase-12 is a molecule whose functions are still not well understood. Although its expression has been found in various tissues, specific roles have been described in only a few cases. These include the effect of caspase-12 on murine bone cell differentiation during craniofacial development. This work focused on the development of the limbs taking place through endochondral ossification, which precedes the formation of the cartilaginous growth plate. Caspase-12 was described here for the first time in growth plate chondrocytes during physiological development. Using pharmacological inhibition, caspase-12 was found to affect chondrogenesis. Limb-derived micromass cultures showed a significantly increased area of chondrogenic nodules after caspase-12 inhibition and there were changes in gene expression, the most significant of which was the reduction of Mmp9. These data point to potential new functions of caspase-12 in chondrogenesis.
- Klíčová slova
- Caspase-12, Chondrocyte, Chondrogenesis, Differentiation, Growth plate,
- MeSH
- buněčná diferenciace MeSH
- chondrocyty * MeSH
- chondrogeneze * fyziologie MeSH
- inhibitory kaspas farmakologie MeSH
- kaspasa 12 * metabolismus genetika MeSH
- kultivované buňky MeSH
- matrixová metaloproteinasa 9 metabolismus genetika MeSH
- myši MeSH
- růstová ploténka růst a vývoj MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inhibitory kaspas MeSH
- kaspasa 12 * MeSH
- matrixová metaloproteinasa 9 MeSH
INTRODUCTION: The aging process is intricately linked to alterations in cellular and tissue structures, with the respiratory system being particularly susceptible to age-related changes. Therefore, this study aimed to profile the activity of proteases using activity-based probes in lung tissues of old and young rats, focusing on the expression levels of different, in particular cathepsins G and X and matrix Metalloproteinases (MMPs). Additionally, the impact on extracellular matrix (ECM) components, particularly fibronectin, in relation to age-related histological and ultrastructural changes in lung tissues was investigated. MATERIALS AND METHODS: Lung tissues from old and young rats were subjected to activity-based probe profiling to assess the activity of different proteases. Expression levels of cathepsins G and X were quantified, and zymography was performed to evaluate matrix metalloproteinases activity. Furthermore, ECM components, specifically fibronectin, were examined for signs of degradation in the old lung tissues compared to the young ones. Moreover, histological, immunohistochemical and ultrastructural assessments of old and young lung tissue were also conducted. RESULTS: Our results showed that the expression levels of cathepsins G and X were notably higher in old rat lung tissues in contrast to those in young rat lung tissues. Zymography analysis revealed elevated MMP activity in the old lung tissues compared to the young ones. Particularly, significant degradation of fibronectin, an essential ECM component, was observed in the old lung tissues. Numerous histological and ultrastructural alterations were observed in old lung tissues compared to young lung tissues. DISCUSSION AND CONCLUSION: The findings indicate an age-related upregulation of cathepsins G and X along with heightened MMP activity in old rat lung tissues, potentially contributing to the degradation of fibronectin within the ECM. These alterations highlight potential mechanisms underlying age-associated changes in lung tissue integrity and provide insights into protease-mediated ECM remodeling in the context of aging lungs.
- MeSH
- extracelulární matrix metabolismus ultrastruktura MeSH
- fibronektiny * metabolismus MeSH
- kathepsin G metabolismus MeSH
- krysa rodu Rattus MeSH
- lyzozomy ultrastruktura metabolismus MeSH
- matrixové metaloproteinasy metabolismus MeSH
- plíce * ultrastruktura metabolismus MeSH
- proteasy metabolismus MeSH
- stárnutí * metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fibronektiny * MeSH
- kathepsin G MeSH
- matrixové metaloproteinasy MeSH
- proteasy MeSH
Fibroblast activation protein (FAP) has been extensively studied as a cancer biomarker for decades. Recently, small-molecule FAP inhibitors have been widely adopted as a targeting moiety of experimental theranostic radiotracers. Here we present a fast qPCR-based analytical method allowing FAP inhibition screening in a high-throughput regime. To identify clinically relevant compounds that might interfere with FAP-targeted approaches, we focused on a library of FDA-approved drugs. Using the DNA-linked Inhibitor Antibody Assay (DIANA), we tested a library of 2667 compounds within just a few hours and identified numerous FDA-approved drugs as novel FAP inhibitors. Among these, prodrugs of cephalosporin antibiotics and reverse transcriptase inhibitors, along with one elastase inhibitor, were the most potent FAP inhibitors in our dataset. In addition, by employing FAP DIANA in the quantification mode, we were able to determine FAP concentrations in human plasma samples. Together, our work expands the repertoire of FAP inhibitors, analyzes the potential interference of co-administered drugs with FAP-targeting strategies, and presents a sensitive and low-consumption ELISA alternative for FAP quantification with a detection limit of 50 pg/ml.
- Klíčová slova
- DIANA, FAP quantification, Fibroblast activation protein, High-throughput screening,
- MeSH
- cefalosporiny chemie farmakologie MeSH
- endopeptidasy * metabolismus MeSH
- knihovny malých molekul farmakologie chemie MeSH
- lidé MeSH
- membránové proteiny * antagonisté a inhibitory metabolismus MeSH
- molekulární struktura MeSH
- rychlé screeningové testy * MeSH
- schvalování léčiv MeSH
- serinové endopeptidasy * metabolismus MeSH
- Úřad Spojených států pro potraviny a léky MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- želatinasy * antagonisté a inhibitory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Spojené státy americké MeSH
- Názvy látek
- cefalosporiny MeSH
- endopeptidasy * MeSH
- fibroblast activation protein alpha MeSH Prohlížeč
- knihovny malých molekul MeSH
- membránové proteiny * MeSH
- serinové endopeptidasy * MeSH
- želatinasy * MeSH
Botulinum neurotoxins (BoNTs) and tetanus toxin (TeTX) are the deadliest biological substances that cause botulism and tetanus, respectively. Their astonishing potency and capacity to enter neurons and interfere with neurotransmitter release at presynaptic terminals have attracted much interest in experimental neurobiology and clinical research. Fused with reporter proteins or labelled with fluorophores, BoNTs and TeTX and their non-toxic fragments also offer remarkable opportunities to visualize cellular processes and functions in neurons and synaptic connections. This study presents the state-of-the-art optical probes derived from BoNTs and TeTX and discusses their applications in molecular and synaptic biology and neurodevelopmental research. It reviews the principles of the design and production of probes, revisits their applications with advantages and limitations and considers prospects for future improvements. The versatile characteristics of discussed probes and reporters make them an integral part of the expanding toolkit for molecular neuroimaging, promoting the discovery process in neurobiology and translational neurosciences.
- Klíčová slova
- Advanced biomaterials, Fluorescent probes, Fusion proteins, Molecular trafficking, Optical imaging, Retrograde transport, SNARE proteins,
- MeSH
- botulotoxiny chemie MeSH
- fluorescenční barviva chemie MeSH
- lidé MeSH
- molekulární sondy chemie MeSH
- neurony * metabolismus MeSH
- neurotoxiny * MeSH
- neurozobrazování * metody MeSH
- synapse * metabolismus MeSH
- tetanový toxin * chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- botulotoxiny MeSH
- fluorescenční barviva MeSH
- molekulární sondy MeSH
- neurotoxiny * MeSH
- tetanový toxin * MeSH
