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Intervertebral disc disease (IDD) is a debilitating spine condition that can be caused by intervertebral disc (IVD) damage which progresses towards IVD degeneration and dysfunction. Recently, human pluripotent stem cells (hPSCs) were recognized as a valuable resource for cell-based regenerative medicine in skeletal diseases. Therefore, adult somatic cells reprogrammed into human induced pluripotent stem cells (hiPSCs) represent an attractive cell source for the derivation of notochordal-like cells (NCs) as a first step towards the development of a regenerative therapy for IDD. Utilizing a differentiation method involving treatment with a four-factor cocktail targeting the BMP, FGF, retinoic acid, and Wnt signaling pathways, we differentiate CRISPR/Cas9-generated mCherry-reporter knock-in hiPSCs into notochordal-like cells. Comprehensive analysis of transcriptomic changes throughout the differentiation process identified regulation of histone methylation as a pivotal driver facilitating the differentiation of hiPSCs into notochordal-like cells. We further provide evidence that specific inhibition of histone demethylases KDM2A and KDM7A/B enhanced the lineage commitment of hiPSCs towards notochordal-like cells. Our results suggest that inhibition of KDMs could be leveraged to alter the epigenetic landscape of hiPSCs to control notochord-specific gene expression. Thus, our study highlights the importance of epigenetic regulators in stem cell-based regenerative approaches for the treatment of disc degeneration.

Background Osteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment’s osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA. Methods We examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1β and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays. Results The transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1β stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1β stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes. Conclusions Our results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA.

Elaf Alkuwaiti,1 Rand Alshubaili,1 Nada AlZahrani,1 Sarah Khusheim,1 Raghad AlMunif,1 Nawal Alharbi,1 Muhammad Nazir2 1College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia; 2Department of Preventive Dental Science, College of Dentistry Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi ArabiaCorrespondence: Muhammad Nazir, Tel +966-543579615, Email manazir@iau.edu.saPurpose: The purpose of study was to investigate dental anxiety (DA) and its relationship with oral health problems, dental visits, and socio-demographic factors among physicians.Patients and Methods: This cross-sectional study was conducted on physicians working in Dhahran, Khobar, Dammam, and Qatif cities of the kingdom of Saudi Arabia. The study included physicians (general practitioners, residents, specialists, and consultants) working in the public and private sectors. Modified Dental Anxiety Scale and World Health Organization’s Oral Health Questionnaire for Adults were used to evaluate DA, oral health problems, and dental attendance.Results: The study included data from 355 participants with a mean age of 40.13 ± 10.45 years. There were 57.2% of non-Saudi and 42.8% of Saudi participants in the study. Bad dental experience in the previous dental visit was reported by 40% of participants, which was significantly related to DA (P = 0.002). Only 9.60% of participants had no DA, whereas 41.10% demonstrated low DA, 23.4% moderate DA, 18.9% high DA, and 7% extreme DA. Common oral problems included tooth sensitivity (65.40%), tooth cavities (45.90%), bleeding gums (43.10%), and bad breath (36.90%). More than half of participants (58.3%) visited the dentist during the last year and dental pain was the most common reason for dental visits (31.3%). Saudi participants demonstrated significantly increased DA than non-Saudis (P = 0.019). DA was significantly related to tooth sensitivity (P = 0.001), tooth cavities (P = 0.002), dry mouth (P = 0.044), and bad breath (P = 0.005). The participants with difficulty in biting foods (P > 0.001) and feeling embarrassed due to the appearance of teeth (P < 0.001) demonstrated significantly higher DA.Conclusion: This sample of physicians showed a high prevalence of DA, oral problems, and dental visits due to pain. DA was significantly related to physicians’ negative dental experience, tooth sensitivity, dental decay, dry mouth, and bad breath.Keywords: physicians, dental anxiety, dental attendance, oral health

District Bagh is a biodiversity rich area of Azad Jammu and Kashmir. However, the exploration of bryoflora has been neglected due to many reasons. The current research project is the first attempt to explore moss flora of the area. As part of the ongoing studies, five different sites were surveyed during March to June, 2020. Collected species were identified by comparing with different bryophytes floras. The taxonomic studies of the species was carried out by using stereomicroscope, compound microscope and photomicroscope. Antibacterial activity of three different solvent extracts (ethanol, methanol and water) was evaluated against Acromobacter xyloxoxidans (Gram-negative) and Bacillus licheniformis (Gram-positive) bacteria by using disc diffusion method. A total of eight species were collected between 1510-1980m altitudes. The results of antibacterial activity showed that different moss extract retained different impact on the growth of bacteria. It has been find out that ethanol extract exhibited highest potency as compared to other solvents against both bacterial strains. In general, Gram- negative bacteria showed more resistance as compared to Gram- positive bacteria. The aqueous extract of most of the species was inactive against both tested bacterial strains. All extracts of Phelonotis fontana, Tortula sabulata and Grimmia montana showed positive results against both bacterial, whereas maximum susceptibility was demonstrated by Erythrodontum julaceum. Present study will open fresh avenue to use these identified moss species for the biochemical characterization.

Preclinical models of osteochondral defects (OCDs) are fundamental test beds to evaluate treatment modalities before clinical translation. To increase the rigor and reproducibility of translational science for a robust "go or no‐go," we evaluated disease progression and pain phenotypes within the whole joint for two OCD rat models with same defect size (1.5 x 0.8 mm) placed either in the trochlea or medial condyle of femur. Remarkably, we only found subtle transitory changes to gaits of rats with trochlear defect without any discernible effect to allodynia. At 8‐weeks post‐surgery, anatomical evaluations of joint showed early signs of osteoarthritis with EPIC‐microCT. For the trochlear defect, cartilage attenuation was increased in trochlear, medial, and lateral compartments of the femur. For condylar defect, increased cartilage attenuation was isolated to the medial condyle of the femur. Further, the medial ossicle showed signs of deterioration as indicated with decreased bone mineral density and increased bone surface area to volume ratio. Thus, OCD in a weight‐bearing region of the femur gave rise to more advanced osteoarthritis phenotype within a unilateral joint compartment. Subchondral bone remodeling was evident in both models without any indication of closure of the articular cartilage surface. We conclude that rat OCD, placed in the trochlear or condylar region of the femur, leads to differing severity of osteoarthritis progression. As found herein, repair of the defect with fibrous tissue and subchondral bone is insufficient to alleviate onset of osteoarthritis. Future therapies using rat OCD model should address joint osteoarthritis in addition to repair itself. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted mesenchymal-like progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. Our molecular tracing data demonstrated that dysregulation of epigenetic and metabolic factors seen in OA chondrocytes relative to healthy chondrocytes persisted following iPSC reprogramming and differentiation toward mesenchymal progenitors. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.

Abstract Background Intervertebral disc (IVD) degeneration is strongly associated with low back pain and is highly prevalent in the elderly population. Hallmarks of IVD degeneration include cell loss and extracellular matrix degradation. The PH domain leucine‐rich‐repeats protein phosphatase (PHLPP1) is highly expressed in diseased cartilaginous tissues where it is linked to extracellular matrix degradation. This study explored the ability of PHLPP1 deficiency to protect against age‐related spontaneous IVD degeneration. Methods Lumbar IVDs of global Phlpp1 knockout (KO) and wildtype (WT) mice were collected at 5 months (young) and 20 months (aged). Picrosirius red–alcian blue staining (PR‐AB) was performed to examine IVD structure and histological score. The expression of aggrecan, ADAMTS5, KRT19, FOXO1 and FOXO3 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human nucleus pulposus (NP) samples were obtained from patients diagnosed with IVD degeneration. PHLPP1 knockdown in human degenerated NP cells was conducted using small interfering RNA (siRNA) transfection. The expression of PHLPP1 regulated downstream targets was analyzed via immunoblot and real time quantitative PCR. Results Histological analysis showed that Phlpp1 KO decreased the prevalence and severity of age‐related IVD degeneration. The deficiency of PHLPP1 promoted the increased expression of NP phenotypic marker KRT19, aggrecan and FOXO1, and decreased levels of ADMATS5 and cell apoptosis in the NP of aged mice. In degenerated human NP cells, PHLPP1 knockdown induced FOXO1 protein levels while FOXO1 inhibition offset the beneficial effects of PHLPP1 knockdown on KRT19 gene and protein expression. Conclusions Our findings indicate that Phlpp1 deficiency protected against NP phenotypic changes, extracellular matrix degradation, and cell apoptosis in the process of IVD degeneration, probably through FOXO1 activation, making PHLPP1 a promising therapeutic target for treating IVD degeneration.

Muhammad Nazir,1 Khalid S Almulhim,2 Ziyad AlDaamah,3 Salman Bubshait,3 Mohammed Sallout,3 Salman AlGhamdi,3 Jehan Alhumaid1 1Department of Preventive Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Eastern Province, Saudi Arabia; 2Department of Restorative Dental Sciences, College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Eastern Province, Saudi Arabia; 3College of Dentistry, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Eastern Province, Saudi ArabiaCorrespondence: Muhammad Nazir Tel +966-543569615Email manazir@iau.edu.saPurpose: Dental fear is associated with the avoidance of dental visits and negative oral health outcomes. It is important to assess distribution of dental fear and preference for emergency dental treatment among adult population during COVID-19. Therefore, the purpose of this study was to evaluate dental fear and emergency dental treatment among adults in COVID-19 quarantine centers in Greater Dammam, Saudi Arabia.Materials and Methods: Male and female adults (N=606) in quarantine centers for COVID-19 participated in this cross-sectional study (June–July 2020). Dental Fear Survey was used to collect data on dental fear. The participants also provided their responses about emergency dental treatment/dental attendance patterns in addition to demographic information.Results: The mean DFS score of the sample was 39.08 ± 19.47, and 23.1% of the participants were a little fearful and 22.6% were highly fearful. About 36.8% reported that they would prefer to visit a dental office in case of dental emergency mostly for dental pain and fractured tooth during the COVID-19 pandemic. Only 10.4% of the participants reported visiting a dentist in less than 3 months, 35.8% in less than 6 months, 52.5% between 6 and 12 months and more, and 1.3% never visited the dentist. DFS scores significantly differed among participants who visited the dentist in less than 3 months (31.86 ± 14.57), less than 6 months (37.70 ± 18.57), between 6 and 12 months and more (40.78 ± 20.23), and never visited the dentist (65.75 ± 14.74) (P < 0.001). The timing of last dental visit (P < 0.001), female gender (P 0.039), and dental pain (P < 0.001) were the significant predictors of dental fear.Conclusion: Dental fear was common among adults with a predilection for the female gender. Increased dental fear was significantly related to dental pain and reduced dental attendance. Only one-third of adults were willing to perform emergency dental visits.Keywords: dental anxiety, emergency treatment, dental visits, pandemic

Intervertebral disc disease (IDD) is a debilitating spine condition that can be caused by intervertebral disc (IVD) damage which progresses towards IVD degeneration and dysfunction. Recently, human pluripotent stem cells (hPSCs) were recognized as a valuable resource for cell-based regenerative medicine in skeletal diseases. Therefore, adult somatic cells reprogrammed into human induced pluripotent stem cells (hiPSCs) represent an attractive cell source for the derivation of notochordal-like cells (NCs) as a first step towards the development of a regenerative therapy for IDD. Utilizing a differentiation method involving treatment with a four-factor cocktail targeting the BMP, FGF, retinoic acid, and Wnt signaling pathways, we differentiate CRISPR/Cas9-generated mCherry-reporter knock-in hiPSCs into notochordal-like cells. Comprehensive analysis of transcriptomic changes throughout the differentiation process identified regulation of histone methylation as a pivotal driver facilitating the differentiation of hiPSCs into notochordal-like cells. We further provide evidence that specific inhibition of histone demethylases KDM2A and KDM7A/B enhanced the lineage commitment of hiPSCs towards notochordal-like cells. Our results suggest that inhibition of KDMs could be leveraged to alter the epigenetic landscape of hiPSCs to control notochord-specific gene expression. Thus, our study highlights the importance of epigenetic regulators in stem cell-based regenerative approaches for the treatment of disc degeneration. [ABSTRACT FROM AUTHOR]

Abstract Background Chondroclasts and osteoclasts have been previously identified as the cells capable of resorbing mineralized cartilage and bone matrices, respectively. While both cell types appear morphologically similar, contain comparable ultrastructural features, and express tartrate-resistant acid phosphatase (TRAP), however, no information is available about the genomic similarities and differences between osteoclasts and chondroclasts. Methods To address this question, we laser captured homogeneous populations of TRAP-positive cells that interact with bone (osteoclasts) and TRAP-positive cells that interact with mineralized cartilage (chondroclasts) on the same plane from murine femoral fracture callus sections. We then performed a global transcriptome profiling of chondroclasts and osteoclasts by utilizing a mouse genome Agilent GE 4X44K V2 microarray platform. Multiple computational approaches and interaction networks were used to analyze the transcriptomic landscape of osteoclasts and chondroclasts. Results Our systematic and comprehensive analyses using hierarchical clustering and principal component analysis (PCA) demonstrate that chondroclasts and osteoclasts are transcriptionally distinct cell populations and exhibit discrete transcriptomic signatures as revealed by multivariate analysis involving scatter plot, volcano plot, and heatmap analysis. TaqMan qPCR was used to validate the microarray results. Intriguingly, the functional enrichment and integrated network analyses revealed distinct Gene Ontology terms and molecular pathways specific to chondroclasts and osteoclasts and further suggest that subsets of metabolic genes were specific to chondroclasts. Protein-protein interaction (PPI) network analysis showed an abundance of structured networks of metabolic pathways, ATP synthesis, and proteasome pathways in chondroclasts. The regulatory network analysis using transcription factor-target gene network predicted a pool of genes including ETV6, SIRT1, and ATF1 as chondroclast-specific gene signature. Conclusions Our study provides an important genetic resource for further exploration of chondroclast function in vivo. To our knowledge, this is the first demonstration of genetic landscape of osteoclasts from chondroclasts identifying unique molecular signatures, functional clustering, and interaction network.

Abstract-Method overriding is a valuable mechanism that happens when an instance method is defined in a subclass and has the same signature and return type as an instance method in the superclass. However, in Java, if those methods are static, then instead method hiding happens, which is a programming weakness and may produce unexpected results. Static analysis is an approach in software testing that examines code to identify various programming weaknesses throughout the software development process without running it. This paper addresses the detection of method-hiding problem in Java programs. We implemented a new automated checker under the SpotBugs static analysis tool that can detect the mentioned problem. According to our results, the checker precisely detected the related issues in both custom test cases and real-world programs. [ABSTRACT FROM AUTHOR]

The study aims to develop a process design and integration of a tri-generation system for simultaneous production of electricity using renewable resources, hydrogen (H2) and green ammonia (NH3). Ammonia being a vital raw material, is gaining recognition as a sustainable fuel, particularly for heavy transport and as a significant hydrogen energy carrier. The proposed integrated plant contains an alkaline water electrolysis system for hydrogen production, which is then consumed in the Haber-Bosch process for ammonia synthesis. The electricity required for electrolysis process is generated through renewable resources, specifically wind and solar energy. The energy is stored in form of molten salt through thermal energy storage technology. The process design includes detailed material and energy balances for water electrolysis and ammonia production units. Results obtained by rigorous design of equipment such a hydrogen and oxygen separators, heat exchanger, electrolyzer for hydrogen production and ammonia synthesis reactor highlight optimized operational and design parameters. The economic feasibility assessment of the process was made through cost estimation of key components, such as separators, heat exchanger, electrolyzer, compressor and tubular reactor. The findings obtained by this study highlight the potential of this tri-generation system in providing the sustainable solution for energy and climate change crisis, specifically in countries like Pakistan where renewable energy resources are present in abundance. [ABSTRACT FROM AUTHOR]