Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.

Mild hypophosphatasia may be twice as prevalent as previously estimated: an effective clinical algorithm to detect undiagnosed cases.

OBJECTIVES: Since the prevalence of hypophosphatasia (HPP), a rare genetic disease, seems to be underestimated in clinical practice, in this study, a new diagnostic algorithm to identify missed cases of HPP was developed and implemented. METHODS: Analytical determinations recorded in the Clinical Analysis Unit of the Hospital Universitario Clínico San Cecilio in the period June 2018 - December 2020 were reviewed. A new clinical algorithm to detect HPP-misdiagnosed cases was used including the following steps: confirmation of persistent hypophosphatasemia, exclusion of secondary causes of hypophosphatasemia, determination of serum pyridoxal-5'-phosphate (PLP) and genetic study of ALPL gene. RESULTS: Twenty-four subjects were selected to participate in the study and genetic testing was carried out in 20 of them following clinical algorithm criteria. Eighty percent of patients was misdiagnosed with HPP following the current standard clinical practice. Extrapolating these results to the current Spanish population means that there could be up to 27,177 cases of undiagnosed HPP in Spain. In addition, we found a substantial proportion of HPP patients affected by other comorbidities, such as autoimmune diseases (∼40 %). CONCLUSIONS: This new algorithm was effective in detecting previously undiagnosed cases of HPP, which appears to be twice as prevalent as previously estimated for the European population. In the near future, our algorithm could be globally applied routinely in clinical practice to minimize the underdiagnosis of HPP. Additionally, some relevant findings, such as the high prevalence of autoimmune diseases in HPP-affected patients, should be investigated to better characterize this disorder.

Non-Classical Effects of FGF23: Molecular and Clinical Features.

This article reviews the role of fibroblast growth factor 23 (FGF23) protein in phosphate metabolism, highlighting its regulation of vitamin D, parathyroid hormone, and bone metabolism. Although it was traditionally thought that phosphate-calcium homeostasis was controlled exclusively by parathyroid hormone (PTH) and calcitriol, pathophysiological studies revealed the influence of FGF23. This protein, expressed mainly in bone, inhibits the renal reabsorption of phosphate and calcitriol formation, mediated by the α-klotho co-receptor. In addition to its role in phosphate metabolism, FGF23 exhibits pleiotropic effects in non-renal systems such as the cardiovascular, immune, and metabolic systems, including the regulation of gene expression and cardiac fibrosis. Although it has been proposed as a biomarker and therapeutic target, the inhibition of FGF23 poses challenges due to its potential side effects. However, the approval of drugs such as burosumab represents a milestone in the treatment of FGF23-related diseases.

Exploring the role of osteoglycin in type 2 diabetes: implications for insulin resistance and vascular pathophysiology.

Osteoglycin, a fundamental proteoglycan within the vascular extracellular matrix, is expressed in vascular smooth muscle cells (VSMCs). Type 2 diabetes (T2D) is associated with cardiovascular disease (CVD) but the role of osteoglycin in the development of CVD is controversial to date. Therefore, our aims are to determine and compare the level of osteoglycin in T2D patients with/without CVD versus control subjects both at serum and vascular tissue and to analyze in vitro role of osteoglycin in VSMCs under calcified conditions. For this, serum osteoglycin levels were determined by enzyme-linked immunosorbent assay (ELISA) in 117 controls and 129 patients with T2D (46 with CVD and 83 without CVD), revealing a significant increase in patients with T2D compared with controls. Osteoglycin level was not an estimator of CVD but correlated with markers of insulin resistance (triglycerides and triglycerides/high-density lipoprotein cholesterol index) in patients with T2D. At the vascular level, osteoglycin expression was assessed by RT-qPCR and immunohistochemistry, and no significant differences were observed between calcified arteries from patients with T2D and noncalcified arteries from controls. In vitro experiments using VSMCs (mock and overexpressing osteoglycin) under calcifying conditions were performed to analyze the osteoglycin function. The overexpression of osteoglycin in VMSCs under calcifying conditions revealed an increase of cell proliferation without effect on apoptosis and an upregulation of the expression of autotaxin (ATX) involved in inflammatory processes. In conclusion, osteoglycin could play a role in glycemic homeostasis, being a potential biomarker of insulin resistance in patients with T2D. Furthermore, osteoglycin could indirectly participate in the development of atherosclerosis through its regulatory effect on ATX and by proliferating VSMCs.NEW & NOTEWORTHY This study uncovers an increase of serum osteoglycin levels in patients with type 2 diabetes, which does not appear to be associated with the development of atherosclerosis, but rather with insulin resistance in this population. Overexpression of osteoglycin increased proliferation and upregulated the expression of autotaxin in vascular smooth muscle cells within calcified environments. Osteoglycin could be a biomarker of insulin resistance for type 2 diabetes and could be indirectly involved in the development of atherosclerosis.

Cardioprotective function of sclerostin by reducing calcium deposition, proliferation, and apoptosis in human vascular smooth muscle cells.

BACKGROUND: Sclerostin is an inhibitor of the Wnt/b-catenin pathway, which regulates bone formation, and can be expressed in vascular smooth muscle cells (VSMCs). Type 2 diabetes (T2D) is associated with an increased risk of cardiovascular disease (CVD) and increased serum and tissue expression of sclerostin. However, whether the role of sclerostin is detrimental or protective in the development of CVD is unknown. Therefore, our aims are to determine the level of sclerostin in T2D patients with/without CVD and in controls, both at serum and vascular tissue, and to analyze the role of sclerostin in VSMCs under calcified environments. METHODS: Cross-sectional study including 121 controls and 139 T2D patients with/without CVD (48/91). Sclerostin levels in serum were determined by ELISA, and sclerostin expression was analyzed by RT-qPCR and immunohistochemistry in calcified and non-calcified artery of lower limb from T2D patients (n = 7) and controls (n = 3). In vitro experiments were performed in VSMCs (mock and sclerostin overexpression) under calcifying conditions analyzing the sclerostin function by determination of calcium and phosphate concentrations, and quantification of calcium deposits by Alizarin Red. Proliferation and apoptosis were analyzed by MTT assay and flow cytometry, respectively. The regulation of the expression of genes involved in bone metabolism was determined by RT-qPCR. RESULTS: A significant increase in serum sclerostin levels in T2D patients with CVD compared to T2D patients without CVD and controls (p < 0.001) was observed. Moreover, higher circulating sclerostin levels were independently associated with CVD in T2D patients. Increased sclerostin expression was observed in calcified arteries of T2D patients compared to non-calcified arteries of controls (p = 0.003). In vitro experiments using VSMCs under calcified conditions, revealed that sclerostin overexpression reduced intracellular calcium (p = 0.001), calcium deposits (p < 0.001), cell proliferation (p < 0.001) and promoted cell survival (p = 0.015). Furthermore, sclerostin overexpression exhibited up-regulation of ALPL (p = 0.009), RUNX2 (p = 0.001) and COX2 (p = 0.003) and down-regulation of inflammatory genes, such as, IL1ß (p = 0.005), IL6 (p = 0.001) and IL8 (p = 0.003). CONCLUSIONS: Sclerostin could play a protective role in the development of atherosclerosis in T2D patients by reducing calcium deposits, decreasing proliferation and inflammation, and promoting cell survival in VSMCs under calcifying conditions. Therefore, considering the bone-vascular axis, treatment with anti-sclerostin for bone disease should be used with caution.

Analysis of the Genetic Relationship between Atherosclerosis and Non-Alcoholic Fatty Liver Disease through Biological Interaction Networks.

Non-alcoholic fatty liver disease (NAFLD) seems to have some molecular links with atherosclerosis (ATH); however, the molecular pathways which connect both pathologies remain unexplored to date. The identification of common factors is of great interest to explore some therapeutic strategies to improve the outcomes for those affected patients. Differentially expressed genes (DEGs) for NAFLD and ATH were extracted from the GSE89632 and GSE100927 datasets, and common up- and downregulated DEGs were identified. Subsequently, a protein-protein interaction (PPI) network based on the common DEGs was performed. Functional modules were identified, and the hub genes were extracted. Then, a Gene Ontology (GO) and pathway analysis of common DEGs was performed. DEGs analysis in NAFLD and ATH showed 21 genes that were regulated similarly in both pathologies. The common DEGs with high centrality scores were ADAMTS1 and CEBPA which appeared to be down- and up-regulated in both disorders, respectively. For the analysis of functional modules, two modules were identified. The first one was oriented to post-translational protein modification, where ADAMTS1 and ADAMTS4 were identified, and the second one mainly related to the immune response, where CSF3 was identified. These factors could be key proteins with an important role in the NAFLD/ATH axis.

The Contribution of Wnt Signaling to Vascular Complications in Type 2 Diabetes Mellitus.

Vascular complications are the leading cause of morbidity and mortality among patients with type 2 diabetes mellitus (T2DM). These vascular abnormalities result in a chronic hyperglycemic state, which influences many signaling molecular pathways that initially lead to increased oxidative stress, increased inflammation, and endothelial dysfunction, leading to both microvascular and macrovascular complications. Endothelial dysfunction represents the initial stage in both types of vascular complications; it represents "mandatory damage" in the development of microvascular complications and only "introductory damage" in the development of macrovascular complications. Increasing scientific evidence has revealed an important role of the Wnt pathway in the pathophysiology of the vascular wall. It is well known that the Wnt pathway is altered in patients with T2DM. This review aims to be an update of the current literature related to the Wnt pathway molecules that are altered in patients with T2DM, which may also be the cause of damage to the vasculature. Both microvascular complications (retinopathy, nephropathy, and neuropathy) and macrovascular complications (coronary artery disease, cerebrovascular disease, and peripheral arterial disease) are analyzed. This review aims to concisely concentrate all the evidence to facilitate the view on the vascular involvement of the Wnt pathway and its components by highlighting the importance of exploring possible therapeutic strategy for patients with T2DM who develop vascular pathologies.

Erratum: Rodrigo-Mor et al. Principles of Charge Estimation Methods Using High-Frequency Current Transformer Sensors in Partial Discharge Measurements. Sensors 2020, 20, 2520.

The authors wish to make the following erratum to this paper [...].

Principles of Charge Estimation Methods Using High-Frequency Current Transformer Sensors in Partial Discharge Measurements.

This paper describes a simplified model and a generic model of high-frequency current transformer (HFCT) sensors. By analyzing the models, a universal charge estimation method based on the double time integral of the measured voltage is inferred. The method is demonstrated to be valid irrespective of HFCT sensor, assuming that its transfer function can be modelled as a combination of real zeros and poles. This paper describes the mathematical foundation of the method and its particularities when applied to measure nanosecond current pulses. In practice, the applicability of the method is subjected to the characteristics and frequency response of the sensor and the current pulse duration. Therefore, a proposal to use the double time integral or the simple time integral of the measured voltage is described depending upon the sensor response. The procedures used to obtain the respective calibration constants based on the frequency response of the HFCT sensors are explained. Two examples, one using a HFCT sensor with a broadband flat frequency response and another using a HFCT sensor with a non-flat frequency response, are presented.

A Novel Antenna for Partial Discharge Measurements in GIS Based on Magnetic Field Detection.

This paper presents a new concept for partial discharge measurements in gas insulated systems (GIS). The proposed technique uses a novel GIS magnetic antenna that measures the magnetic field produced by partial discharges (PD) propagating in GIS. The foundations of the measurement technique and the magnetic antenna design are presented together with laboratory measurements. The magnetic antenna performance and the sensitivity of the acquisition system are studied. The bandwidth of the measurement system is in the high frequency and very high frequency (HF⁻VHF) range. Laboratory experiments demonstrate the suitability of the novel magnetic antenna-based measuring system for PD in GIS for corona, surface discharges, and free moving particles in SF6.

Removal of Cu2+ and Ni2+ from Aqueous Solution using SnO2 Nanomaterial effect of: pH, Time, Temperature, interfering cations.

Tin oxide, SnO2, nanomaterial was synthesized and tested for the removal of Cu2+ and Ni2+ ions from aqueous solutions. Various parameters for the binding were investigated in batch studied, which included pH, time, temperature, and interferences. In addition, isotherm studied were performed to determine the maximum binding capacity for both Cu2+ and Ni2+ ions. The optimal binding pH determined from the effects of pH were to be at pH 5 for both the Cu2+ and Ni2+ ions. The isotherm studies were performed at temperatures of 4°C, 25 °C, and 45 °C for both the Cu2+ and Ni2+ ions and were found to follow the Langmuir isotherm model. The binding capacities for the Cu2+ ions were 2.63 mg/g, 2.95 mg/g and 3.27 mg/g at the aforementioned temperatures, respectively. Whereas the binding capacities for Ni2+ were 0.79 mg/g, 1.07 mg/g, and 1.46 mg/g at the respective temperatures. The determined thermodynamic parameters for the binding showed that the binding processes for the reactions were endothermic, as the ΔG was observed to decrease with decreasing temperatures. As well the ΔH was 28.73 kJ/mol for Cu2+ (III) and 13.37 kJ/mol for Ni2+. The ΔS was observed to be 92.65 J/mol for Cu2+ and 54.53 J/mol for Ni2+. The free energy of adsorption for the Cu2+ was determined to be 13.99 kJ/mol and the activation energy for the binding of Ni2+ was determined to be 8.09 KJ/mol. The activation energy data indicate that the reaction was occurring through chemisorption.

A Novel Approach for Partial Discharge Measurements on GIS Using HFCT Sensors.

This paper presents a novel measuring system for partial discharge (PD) measurements in Gas Insulated Systems (GIS) using high frequency current transformers (HFCT). The system is based on the measurement of the induced PD currents in the GIS enclosure. In opposition to the existing antenna technologies that measure the radiated energy in the very high frequency/ultra-high frequency (VHF/UHF) range, the proposed system measures the PD conducted currents in the high frequency (HF) range and below. The foundation of the measurements together with a detailed explanation of the sensor installed conveniently at the bolts of the GIS spacer are presented. An experimental study on the current distribution in the GIS enclosure is described to evaluate the impact of the sensor on the measurements. Laboratory experiments have been performed that show the suitability of this method to properly measure particle discharges caused by corona, surface and free moving particle discharges in SF6. Discharges in the range of 1 to 4 pC have been properly measured. An analysis to evaluate the performance of the method is shown, in comparison to VHF/UHF antenna measurements. The potential benefits of this novel technique rely on the small attenuation of PD signals in the GIS components in the HF range and sample rate reductions. Finally, a discussion on the potential applicability of present cluster and charge calculation techniques to the proposed PD GIS measurement using HFCT is presented.

Behavioural effects of PNU-282987 and stress in an animal model of Alzheimer's disease.

BACKGROUND: Cholinergic deficits play an important role in both cognitive and behavioural alterations in Alzheimer's disease. This study was aimed at evaluating the possible therapeutic role of PNU-282987 (PNU), an α7 nicotinic cholinergic receptor agonist, and the possible effects of stress in precipitating the onset of behavioural deficits in animals with susceptibility to Alzheimer's disease. METHODS: B6C3-Tg mice with susceptibility to Alzheimer's disease and wild-type mice either with or without restraint stress received 0- or 1-mg/kg PNU. At 12 months old, mice were evaluated for activity levels, anxiety-like levels, and spatial learning and memory. RESULTS: Data did not show the effects of PNU on activity and anxiety-like behaviour. No effect of PNU on acquisition of a spatial learning task was detected, but a reversal of stress effects on retention in the Morris water maze was observed in transgenic mice. CONCLUSIONS: Further studies are needed in order to better understand the role of α7 nicotinic cholinergic receptor agonists in motor activity, anxiety, and spatial learning and memory and to develop more accurate pharmacological treatment of psychopathological diseases.

The dura causes spinal cord compression after spinal cord injury.

MR scans from 65 patients with traumatic spinal cord injury were analysed; on admission 95% had evidence of cord compression - in 26% due to the dura, and in the remaining 74% due to extradural factors. Compression due to dural factors resolved with a half-life of 8.7 days. These findings suggest that bony decompression alone may not relieve spinal cord compression in the quarter of patients in whom dural factors are significant.

Validation of the Spanish version of the multifaceted empathy test: comparison between cannabis use effects and controls in social cognition.

OBJECTIVE: While social cognition is shown to be impaired in several mental disorders, the effects of cannabis on social cognition are still not clear. Past studies have used the multifaceted empathy test (MET) to study social cognition. This study aims to test the validity of the MET Spanish version and to evaluate the effects of cannabis use on social cognition. METHODS: In total 116 participants from a Cannabis Social Club (CSC) completed the MET and the reading the mind in the eyes test (RMET) under the effects of cannabis and were compared to 86 university students (control group). Internal consistency and convergent validity were assessed. Cognitive empathy (CE) and emotional empathy (EE) were tested in both groups. RESULTS: The MET CE scale shows low internal consistency, while the EE scale shows high internal consistency. Items showed similar difficulty for both groups. Cannabis users showed deficient overall emotional recognition, with reduced scores associated with positive stimuli. Overall scores for EE were similar for both groups, but the experimental group scored lower with negative stimuli when compared to controls. CONCLUSION: This study validates the MET Spanish version for its use in future studies. Results confirmed deficient emotional recognition in cannabis users and a dampened reaction to negative stimuli for the first time.

Interobserver agreement of magnetic resonance imaging signs of osteomyelitis in pelvic pressure ulcers in patients with spinal cord injury.

OBJECTIVE: To examine the interobserver reliability of magnetic resonance imaging (MRI) signs of osteomyelitis in complex chronic pressure ulcers in patients with spinal cord injury (SCI). DESIGN: Retrospective review study. SETTING: Specialist SCI rehabilitation center. PARTICIPANTS: Adult patients with SCI and pressure ulceration investigated with MRI. INTERVENTIONS: Analysis of MRI examinations and clinical records collected over a 4-year period. Images were independently assessed by 2 experienced radiologists for osteomyelitis based on assigned predictive indicators including cortical bone erosion, soft tissue edema, deep collections, heterotopic new bone, hip effusion, and abnormal signal change of the marrow. MAIN OUTCOME MEASURES: Interobserver agreement for indicative MRI signs of osteomyelitis in complex pressure ulcers. RESULTS: Thirty-seven patients underwent 41 MRI scans. Concordance for marrow edema was 71% on both short tau inversion recovery and T1-weighted sequences, and for cortical erosion was 85%. CONCLUSIONS: For the assessment of pelvic osteomyelitis related to pressure ulcers, the T1-weighted MRI signal for marrow edema and cortical erosion has strong interobserver agreement.

Diagnosing and managing spinal injury in patients with ankylosing spondylitis.

BACKGROUND: Individuals with ankylosing spondylitis are at an increased risk of vertebral fractures. These are often unstable, leading to primary and secondary neurological injury and conferring high levels of morbidity and mortality. Fractures in these patients can occur after minimal trauma and are easily missed, with potentially disastrous consequences. OBJECTIVES: To educate health professionals who may be involved in the initial assessment and management of ankylosing spondylitis patients with possible spinal injuries, despite not being spinal specialists. CASE REPORTS: We present three cases from our own hospital, which illustrate the pitfalls associated with traumatic spinal injury in ankylosing spondylitis. Case 1 shows why delayed presentation of spinal injury is common, as well as demonstrating the need for multiple imaging modalities in some patients. Case 2 is an example of primary neurological injury in this patient group, and case 3 highlights the risk of secondary neurological injury, as well as the effect of multiple comorbidities on patient outcomes. CONCLUSIONS: It is important that staff in the Emergency Department have an understanding of the extreme caution that is needed in the management of possible spinal injuries in patients with or suspected of having ankylosing spondylitis.

Review: indications for interventional radiology in the management of patients with spinal cord injuries.

OBJECTIVES: To outline a range of minimally invasive image-guided procedures that benefit spinal cord-injured patients and may expedite clinical care. STUDY DESIGN: Pictorial review. RESULTS/CONCLUSIONS: Image-guided procedures have made a significant impact in medical management in many specialties. These techniques continue to evolve rapidly and afford opportunities to reduce patient morbidity and in-patient length of stay.

Systemic effects of hypophosphatasia characterization of two novel variants in the ALPL gene.

Introduction: Hypophosphatasia (HPP) is an inborn metabolic error caused by mutations in the ALPL gene encoding tissue non-specific alkaline phosphatase (TNSALP) and leading to decreased alkaline phosphatase (ALP) activity. Although the main characteristic of this disease is bone involvement, it presents a great genetic and clinical variability, which makes it a systemic disease. Methods: Patients were recruited based on biochemical assessments. Diagnosis was made by measuring serum ALP and pyridoxal 5-phosphate levels and finally by Sanger sequencing of the ALPL gene from peripheral blood mononuclear cells. Characterization of the new variants was performed by transfection of the variants into HEK293T cells, where ALP activity and cellular localization were measured by flow cytometry. The dominant negative effect was analyzed by co-transfection of each variant with the wild-type gene, measuring ALP activity and analyzing cellular localization by flow cytometry. Results: Two previously undescribed variants were found in the ALPL gene: leucine 6 to serine missense mutation (c.17T>C, L6S) affecting the signal peptide and threonine 167 deletion (c.498_500delCAC, T167del) affecting the vicinity of the active site. These mutations lead mainly to non-pathognomonic symptoms of HPP. Structural prediction and modeling tools indicated the affected residues as critical residues with important roles in protein structure and function. In vitro results demonstrated low TNSALP activity and a dominant negative effect in both mutations. The results of the characterization of these variants suggest that the pleiotropic role of TNSALP could be involved in the systemic effects observed in these patients highlighting digestive and autoimmune disorders associated with TNSALP dysfunction. Conclusions: The two new mutations have been classified as pathogenic. At the clinical level, this study suggests that both mutations not only lead to pathognomonic symptoms of the disease, but may also play a role at the systemic level.

Magnetic resonance imaging of pressure sores in spinal cord injured patients: accuracy in predicting osteomyelitis.

OBJECTIVE: Identify key magnetic resonance imaging (MRI) features that have a significant correlation with osteomyelitis of pressure ulcers in spinal injury patients. DESIGN: Retrospective review study. PARTICIPANTS: Adult patients admitted to the National Spinal Injuries Centre with spinal cord injury (SCI) and signs of pressure ulceration investigated with MRI. METHODS: Analysis of MRI examinations and clinical records collected over a 4-year period. Images were independently assessed by 2 experienced radiologists for osteomyelitis based on assigned predictive indicators including cortical bone erosion, soft tissue edema, deep collections, heterotopic new bone, hip effusion, and abnormal signal change of the marrow. RESULTS: Thirty-seven patients underwent 41 MRI scans. The prevalence of osteomyelitis was highly correlated with cortical bone erosion (r = 0.84) and abnormal bone marrow changes on T1-weighted images (r = 0.82).