Comparison of Various Extraction Approaches for Optimized Preparation of Intracellular Metabolites from Human Mesenchymal Stem Cells and Fibroblasts for NMR-Based Study.

Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell's physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol-chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol-chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples.

Host genetic variants associated with COVID-19 reconsidered in a Slovak cohort.

We present the results of an association study involving hospitalized coronavirus disease 2019 (COVID-19) patients with a clinical background during the 3rd pandemic wave of COVID-19 in Slovakia. Seventeen single nucleotide variants (SNVs) in the eleven most relevant genes, according to the COVID-19 Host Genetics Initiative, were investigated. Our study confirms the validity of the influence of LZTFL1 and 2'-5'-oligoadenylate synthetase (OAS)1/OAS3 genetic variants on the severity of COVID-19. For two LZTFL1 SNVs in complete linkage disequilibrium, rs17713054 and rs73064425, the odds ratios of baseline allelic associations and logistic regressions (LR) adjusted for age and sex ranged in the four tested designs from 2.04 to 2.41 and from 2.05 to 3.98, respectively. The OAS1/OAS3 haplotype 'gttg' carrying a functional allele G of splice-acceptor variant rs10774671 manifested its protective function in the Delta pandemic wave. Significant baseline allelic associations of two DPP9 variants in all tested designs and two IFNAR2 variants in the Omicron pandemic wave were not confirmed by adjusted LR. Nevertheless, adjusted LR showed significant associations of NOTCH4 rs3131294 and TYK2 rs2304256 variants with severity of COVID-19. Hospitalized patients' reported comorbidities were not correlated with genetic variants, except for obesity, smoking (IFNAR2), and hypertension (NOTCH4). The results of our study suggest that host genetic variations have an impact on the severity and duration of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the differences in allelic associations between pandemic waves, they support the hypothesis that every new SARS-CoV-2 variant may modify the host immune response by reconfiguring involved pathways.

How to Treat Today? Oral and Facial Cancer-Associated Venous Thromboembolism.

The exact incidence of cancer-associated venous thromboembolism (CA-VTE) in patients with oral and facial cancer (OFC) is not exactly known, and this risk is empirically considered to be low. However, this suggestion may result in disease underdiagnosis, prolong the initiation of adequate therapy, and consecutively increase CA-VTE-related morbidity and mortality. In addition, there might be specific clinical problems in the treatment of CA-VTE in patients with oral and facial cancer, such as swallowing difficulties, that might limit the possibilities of oral anticoagulation. Finally, there are limited data regarding the optimal treatment of CA-VTE in patients with oral and facial cancer, and this includes data on novel therapeutic strategies, including the use of direct oral anticoagulants. This article reviews current data on the optimal treatment strategy for CA-VTE in patients with OFC.

Multiple idiopathic external cervical root resorption in patient treated continuously with denosumab: a case report.

BACKGROUND: External root resorption is an irreversible loss of dental hard tissue as a result of odontoclastic action. Multiple external cervical root resorptions in permanent teeth are rare. The exact cause of external cervical root resorption is unclear. It is currently well established that RANK/RANKL signaling is essential for osteoclastogenesis and osteoclast-mediated bone resorption. Denosumab is an anti-RANKL antibody used for the treatment of postmenopausal osteoporosis. RANK/RANKL pathway suppression by denosumab is expected to suppress the activity of clastic cells responsible for hard tissue resorption involving both osteoclasts and odontoclasts. CASE PRESENTATION: This case report demonstrates aggressive and generalized idiopathic external cervical root resorption that started and advanced during ongoing antiresorptive therapy with the human monoclonal RANKL-blocking antibody denosumab without discontinuation of therapy in a 74-year-old female patient treated for postmenopausal osteoporosis. The extent of resorptive defects was too large and progressively led to fractures of the teeth. The number of teeth involved and the extend of destruction excluded conservative treatment. The affected teeth had to be extracted for functional prosthetic reconstruction. CONCLUSIONS: This finding suggests that treatment with denosumab may be associated with severe and aggressive odontoclastic resorption of multiple dental roots despite an adequate inhibitory effect on osteoclasts in the treatment of osteoporosis. The RANKL-independent pathways of clastic cell formation are likely to be involved in this pathological process.

Improving lip aesthetics in the face profile after treatment of class II, division 2 malocclusion.

AIM: The aims of our research were as follows: 1) Description of changes in the position of the upper and lower lips, as a result of the change in the position of upper and lower incisors after orthodontic treatment of malocclusion of Class II, division 2 type. 2) Determination of correlation between changes in the position of lips and incisors in the profile of the face after orthodontic treatment of malocclusion of Class II, division 2 type.MATERIALS AND METHODS: Our study analyses the documentation of 115 patients with malocclusion of Class II, division 2 type treated with a fixed orthodontic appliance in the upper and lower dental arches at the Orthodontic Department of the Clinic of Dentistry in Olomouc from January 1, 1996 to December 31, 2017. There were 78 women and 37 men aged 11 to 36 years included in the database. Cephalometric images of patients from the group taken before and after the treatment were used for the purpose of cephalometric analysis according to Kamínek (1) and Burstone's analysis (2) of soft tissues. Subsequently, all output data were processed statistically. RESULTS: While the protrusion of the incisors after treatment of malocclusion of Class II, division 2 type was 3 mm, the facial profile showed statistically significant changes in soft tissues in the ventral direction by 1 mm on average just in the area of the upper and lower lips, which means a shift corresponding to one third of teeth movement. CONCLUSION: A statistically significant change in the position of incisors, soft tissues as well as correlation between changes in hard and soft tissues in the face profile after orthodontic treatment of malocclusion of Class II, division 2 type were demonstrated. The more the incisors were inclined, the more the lips moved forward, and the patient's profile turned out to be aesthetically improved (Tab. 2, Fig. 3, Ref. 39).

Comparative Proteomic and Metabolomic Analysis of Human Osteoblasts, Differentiated from Dental Pulp Stem Cells, Hinted Crucial Signaling Pathways Promoting Osteogenesis.

Population aging has been a global trend for the last decades, which increases the pressure to develop new cell-based or drug-based therapies, including those that may cure bone diseases. To understand molecular processes that underlie bone development and turnover, we followed osteogenic differentiation of human dental pulp stem cells (DPSCs) using a specific induction medium. The differentiation process imitating in vivo osteogenesis is triggered by various signaling pathways and is associated with massive proteome and metabolome changes. Proteome was profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility-enhanced mass spectrometry. From 2667 reproducibly quantified and identified proteins, 432 were differentially abundant by strict statistic criteria. Metabolome profiling was carried out by nuclear magnetic resonance. From 27 detected metabolites, 8 were differentially accumulated. KEGG and MetaboAnalyst hinted metabolic pathways that may be involved in the osteogenic process. Enrichment analysis of differentially abundant proteins highlighted PPAR, FoxO, JAK-STAT, IL-17 signaling pathways, biosynthesis of thyroid hormones and steroids, mineral absorption, and fatty acid metabolism as processes with prominent impact on osteoinduction. In parallel, metabolomic data showed that aminoacyl-tRNA biosynthesis, as well as specific amino acids, likely promote osteodifferentiation. Targeted immunoassays validated and complemented omic results. Our data underlined the complexity of the osteogenic mechanism. Finally, we proposed promising targets for future validation in patient samples, a step toward the treatment of bone defects.

Gossypiboma mimicking recurrent mandibular tumor: case report.

Gossypiboma is not a commonly known surgical complication. It is a tumorous lesion usually caused by hemostatic material used in surgery. Such lesions are most commonly described after abdominal surgery. In this case report, the authors describe a case of a 17 year old female patient, operated for a mandible tumor. Histopathologically it was an ameloblastoma. The patient was treated lege artis, with the use of Surgicel® felt (Surgicel FibrillarTM Absorbable Hemostat). After two months, the young woman returned to clinics with a tumorous lesion at the same location. On the CT scan the lesion appeared to be a recurrence of the originally diagnosed ameloblastoma. Histopathologically, the lesion consisted of a foreign material with surrounding granulation tissue and massive inflamation. The foreign material had an atypical structure. Subsequent consultations and consensus at the clinic confirmed that it was a haemostatic foreign material with a surrounding hyper-inflammatory response mimicking a tumor, known in the literature under various names, most often as gossypiboma or textiloma.

A Comparative In Vitro Analysis of the Osteogenic Potential of Human Dental Pulp Stem Cells Using Various Differentiation Conditions.

Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.

Intracellular signaling MAPK pathway after cerebral ischemia-reperfusion injury.

The MAPK/ERK/p38 are signal transduction pathways that couple intracellular responses to the external stimuli. Contrary to ERK protein which is part of the survival route, presence of p38 could have an impact on cell injury. Tolerance induced by ischemic preconditioning (IPC) is a phenomenon of tissue adaptation, which results in increased tolerance to lethal ischemia-reperfusion injury (IRI). Paper describes changes in MAPK protein pathways after brain IPC. Ischemia was induced by 4-vessels occlusion and rats were preconditioned by sub-lethal ischemia. Western blot and immunohistochemistry identified ERK/p38 proteins in injured areas. The highest level of the pERK was detected at 24 h in IPC groups. A contrary pattern of MAPK/p38 activation was observed in this group, where the lowest level of p38 was displayed at 24 h after ischemia. This suggests that the MAPK signal transduction might have a potential role in tissues response subjected to IRI and in the phenomenon of tolerance.