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Objetivos: Identificación de biomarcadores que relacionan la osteoporosis con enfermedades pulmonares ocupacionales y ambientales. Material y métodos: Mediante bases de datos de terminología médica unificada se obtuvieron enfermedades relacionadas con enfermedades pulmonares que, junto con la osteoporosis, fueron analizadas en DisGeNET para obtener los genes asociados a cada enfermedad y formar una red de interacción proteína-proteína (PPI) mediante el uso de STRING dentro de Cytoscape. A través de la aplicación de diferentes algoritmos de centralidad utilizando CythoHubba en Cytoscape, se seleccionaron las 5 proteínas de la red con el mayor grado de centralidad. Resultados: 9 enfermedades fueron incluidas en el grupo de enfermedades pulmonares. Se obtuvieron 2.698 genes asociados a enfermedades pulmonares y a osteoporosis. Los genes vinculados con osteoporosis y con al menos dos de las enfermedades pulmonares incluidas dieron lugar a una red PPI con 152 nodos y 1.378 ejes. Las proteínas con mayor grado de centralidad de la red fueron AKT1, ALB, IL6, TP53 y VEGFA. Conclusiones: Existe una elevada relación entre la osteoporosis y las enfermedades pulmonares ambientales estudiadas, a través de genes con una implicación dual. Nosotros proponemos cinco genes importantes que vinculan estas enfermedades y que podrían constituir una base coherente para investigaciones más profundas en este campo.

Objetivos: Identificación de potenciales biomarcadores implicados en procesos de calcificación vascular para avanzar en el diagnóstico y tratamiento de esta patología en sus estadíos subclínicos. Métodos: Se trata de un trabajo experimental en el que se incluyeron 5 pacientes con diabetes mellitus tipo 2 (DM2) con enfermedad arterial periférica e isquemia crítica. Se realizó una extracción proteica e identificación del proteoma mediante cromatografía líquida y espectrometría de masas (LC-MS/MS) de secciones de arteria femoral calcificada. Las proteínas identificadas fueron analizadas a través de gene ontology y comparadas con otras proteínas específicas de patologías vasculares relacionadas mediante la base de datos DisGeNET. Mediante el programa informático Cytoscape se analizó la red de funciones biológicas de las proteínas seleccionadas para su clasificación en base a la patología en la que están implicadas. Resultados: Se identificaron 530 proteínas en las muestras analizadas con funciones mayoritariamente de unión a calcio y catalítica. 37 de ellas fueron comunes en otras patologías vasculares relacionadas. La exploración de las redes biológicas de las 37 proteínas identificadas, dio lugar a la identificación de 2 potenciales marcadores específicos de calcificación vascular en procesos ateroscleróticos, como la proteína mitocondrial de choque térmico de 10 kDa, y la subunidad flavoproteica de la succinato deshidrogenasa. Conclusiones: Existe una importante expresión de proteínas implicadas en procesos de mineralización ósea en tejido vascular calcificado, sugiriendo la existencia de mecanismos moleculares comunes entre la regulación ósea y vascular. El uso de herramientas bioinformáticas sugiere la implicación de la proteína de choque térmico de 10 kDa mitocondrial y la subunidad flavoproteica de la succinato deshidrogenasa como posibles biomarcadores de calcificación vascular en pacientes con DM2, aunque son necesarios estudios adicionales que confirmen esta hipótesis.

Objectives: Identify potential biomarkers involved in vascular calcification processes to improve DM2 diagnosis and treatment in its subclinical stages. Methods: This experimental study included 5 patients suffering diabetes mellitus type 2 (DM2) with peripheral arterial disease and critical ischemia. Protein extraction and identification of the proteome were carried out using liquid chromatography and mass spectrometry (LC-MS/MS) of calcified femoral artery sections. The identified proteins were analyzed through gene ontology and compared with other specific proteins of related vascular pathologies through the DisGeNET database. Cytoscape software analyzed the network of biological functions of the proteins selected for classification based on the disease in which they are involved. Results: 530 proteins were identified in the analyzed samples with functions mainly of calcium binding and catalytic. 37 of them were common in other related vascular pathologies. The exploration of the biological networks of the 37 proteins identified, led to the identification of 2 potential specific markers of vascular calcification in atherosclerotic processes, such as 10-kDa thermal shock mitochondrial protein, and the flavoprotein subunit of succinate dehydrogenase. Conclusions: There is significant expression of proteins involved in processes of bone mineralization in calcified vascular tissue, suggesting the existence of common molecular mechanisms between bone regulation and vascular. The use of bioinformatics tools suggests the involvement of the mitochondrial 10 kDa heat shock protein and the subunit of the succinate dehydrogenase as potential biomarkers of vascular calcification in patients with DM2, although additional studies are needed to confirm this hypothesis. [ABSTRACT FROM AUTHOR]

Background: Typical bone proteins, such as sclerostin and periostin, have been associated with cardiovascular disease (CVD). Simultaneously, several risk scores have been developed to predict CVD in the general population. Therefore, we aimed to evaluate the association of these bone proteins related to CVD, with the main vascular risk scales: Framingham Risk Score (FRS), REGICOR and SCORE2-Diabetes, in patients with type 2 diabetes. We focus in particular on the SCORE2-Diabetes algorithm, which predicts 10-year CVD risk and is specific to the study population., Methods: This was a cross-sectional study including 104 patients with type 2 diabetes (62 ± 6 years, 60% males). Clinical data, biochemical measurements, and serum bioactive sclerostin and periostin levels were collected, and different risk scales were calculated. The association between bioactive sclerostin or periostin with the risk scales was analyzed., Results: A positive correlation was observed between circulating levels of bioactive sclerostin (p < 0.001) and periostin (p < 0.001) with SCORE2-Diabetes values. However, no correlation was found with FRS or REGICOR scales. Both serum bioactive sclerostin and periostin levels were significantly elevated in patients at high-very high risk of CVD (score ≥ 10%) than in the low-moderate risk group (score < 10%) (p < 0.001 for both). Moreover, analyzing these proteins to identify patients with type 2 diabetes at high-very high vascular risk using ROC curves, we observed significant AUC values for bioactive sclerostin (AUC = 0.696; p = 0.001), periostin (AUC = 0.749; p < 0.001), and the model combining both (AUC = 0.795; p < 0.001). For diagnosing high-very high vascular risk, serum bioactive sclerostin levels > 131 pmol/L showed 51.6% sensitivity and 78.6% specificity. Similarly, serum periostin levels > 1144 pmol/L had 64.5% sensitivity and 76.2% specificity., Conclusions: Sclerostin and periostin are associated with vascular risk in the SCORE2-Diabetes algorithm, opening a new line of investigation to identify novel biomarkers of cardiovascular risk in the type 2 diabetes population., (© 2024. The Author(s).)

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&#95;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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Martínez-Heredia, Muñoz-Torres, Sanabria-de la Torre, Jiménez-Ortas, Andújar-Vera, González-Cejudo, Contreras-Bolívar, González-Salvatierra, Gómez-Vida, García-Fontana and García-Fontana.)

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, IL1β (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., (© 2023. The Author(s).)

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.

Diet is a modifiable factor in bone and muscle health. The Mediterranean diet (MedDiet) is rich in nutrients and contains key bioactive components with probable protective effects on muscle and bone deterioration. Osteoporosis (OP) and sarcopenia are diseases that increase frailty and susceptibility to fracture, morbidity and mortality. Therefore, it is necessary to combat them in the population. In this regard, MedDiet adherence has proven to be beneficial to bone mineral density (BMD), muscle mass, physical function, OP and sarcopenia. Hence, this diet is proposed as a therapeutic tool that could slow the onset of osteoporosis and sarcopenia. However, there is doubt about the interaction between the MedDiet, strength and fracture risk. Perhaps the amount of EVOO (extra virgin olive oil), fruits, vegetables and fish rich in anti-inflammatory and antioxidant nutrients ingested has an influence, though the results remain controversial.

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., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

The pandemic caused by the SARS-CoV-2 virus has triggered great interest in the search for the pathophysiological mechanisms of COVID-19 and its associated hyperinflammatory state. The presence of prognostic factors such as diabetes, cardiovascular disease, hypertension, obesity, and age influence the expression of the disease's clinical severity. Other elements, such as 25-hydroxyvitamin D (25(OH)D3) concentrations, are currently being studied. Various studies, mostly observational, have sought to demonstrate whether there is truly a relationship between 25(OH)D3 levels and the acquisition and/or severity of the disease. The objective of this study was to carry out a review of the current data that associate vitamin D status with the acquisition, evolution, and/or severity of infection by the SARS-CoV-2 virus and to assess whether prevention through vitamin D supplementation can prevent infection and/or improve the evolution once acquired. Vitamin D system has an immunomodulatory function and plays a significant role in various bacterial and viral infections. The immune function of vitamin D is explained in part by the presence of its receptor (VDR) and its activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) in immune cells. The vitamin D, VDR, and Retinoid X Receptor complex allows the transcription of genes with antimicrobial activities, such as cathelicidins and defensins. COVID-19 characteristically presents a marked hyperimmune state, with the release of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β. Thus, there are biological factors linking vitamin D to the cytokine storm, which can herald some of the most severe consequences of COVID-19, such as acute respiratory distress syndrome. Hypovitaminosis D is widespread worldwide, so the prevention of COVID-19 through vitamin D supplementation is being considered as a possible therapeutic strategy easy to implement. However, more-quality studies and well-designed randomized clinical trials are needed to address this relevant question.

The life expectancy of the global population has increased. Aging is a natural physiological process that poses major challenges in an increasingly long-lived and frail population. Several molecular mechanisms are involved in aging. Likewise, the gut microbiota, which is influenced by environmental factors such as diet, plays a crucial role in the modulation of these mechanisms. The Mediterranean diet, as well as the components present in it, offer some proof of this. Achieving healthy aging should be focused on the promotion of healthy lifestyle habits that reduce the development of pathologies that are associated with aging, in order to increase the quality of life of the aging population. In this review we analyze the influence of the Mediterranean diet on the molecular pathways and the microbiota associated with more favorable aging patterns, as well as its possible role as an anti-aging treatment.

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.

Aim: Patients with type 2 diabetes (T2DM) have more risk of bone fractures. However, areal bone mineral density (aBMD) by conventional dual-energy x-ray absorptiometry (DXA) is not useful for identifying this risk. This study aims to evaluate 3D-DXA parameters determining the cortical and trabecular compartments in patients with T2DM compared to non-diabetic subjects and to identify their determinants., Materials and Methods: Case-control study in 111 T2DM patients (65.4 ± 7.6 years old) and 134 non-diabetic controls (64.7 ± 8.6-year-old). DXA, 3D-DXA modelling via 3D-Shaper software and trabecular bone score (TBS) were used to obtain aBMD, cortical and trabecular parameters, and lumbar spine microarchitecture, respectively. In addition, biochemical markers as 25-hydroxyvitamin d, type I procollagen N-terminal propeptide (P1NP), C-terminal telopeptide of type I collagen (CTX), and glycated haemoglobin (HbA1c) were analysed., Results: Mean-adjusted values showed higher aBMD (5.4%-7.7%, ES: 0.33-0.53) and 3D-DXA parameters (4.1%-10.3%, ES: 0.42-0.68) in the T2DM group compared with the control group. However, TBS was lower in the T2DM group compared to the control group (-14.7%, ES: 1.18). In addition, sex (β = 0.272 to 0.316) and body mass index (BMI) (β = 0.236 to 0.455) were the most consistent and positive predictors of aBMD (p ≤ 0.01). BMI and P1NP were negative predictors of TBS (β = -0.530 and -0.254, respectively, p ≤ 0.01), while CTX was a positive one (β = 0.226, p=0.02). Finally, BMI was consistently the strongest positive predictor of 3D-DXA parameters (β = 0.240 to 0.442, p<0.05)., Conclusion: Patients with T2DM present higher bone mass measured both by conventional DXA and 3D-DXA, suggesting that 3D-DXA technology is not capable of identifying alterations in bone structure in this population. Moreover, BMI was the most consistent determinant in all bone outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ubago-Guisado, Moratalla-Aranda, González-Salvatierra, Gil-Cosano, García-Fontana, García-Fontana, Gracia-Marco and Muñoz-Torres.)

Sclerostin is most recognized for its role in controlling bone formation; however, it is also expressed in the heart, aorta, coronary, and peripheral arteries. Human studies have associated high circulating sclerostin levels with the presence of different cardiovascular diseases (CVD), surrogate CVD markers, and a high risk of cardiovascular events in some populations. However, this is still a matter of scientific debate, as the results have been very heterogeneous among studies. In the present review, the association between serum sclerostin levels and CVD and/or cardiovascular mortality was analyzed. For this purpose, a scoping review was performed in which articles measuring serum sclerostin levels and cardiovascular risk in patients were selected. Eleven articles answered the research question; of these articles, 8/11 evaluated the association between sclerostin and CVD, of which 4/8 found a positive association, 2/8 found a negative association, and 2/8 found no association between variables. Five (5/11) of the articles included in the study evaluated cardiovascular mortality, of which 3/5 found a positive association, 1/5 found a negative association, and 1/5 found no association between variables. In conclusion, we did not find sufficient results to be able to demonstrate an association between elevated sclerostin levels and the development of CVD and/or cardiovascular mortality in the general population due to heterogeneity in the results. However, there seems to be a tendency to consider increased sclerostin levels as a risk factor for both the development of cardiovascular events and cardiovascular mortality in specific populations. Further studies in this field will help to solve some of the inconsistencies found during this scoping review and allow for the future use of sclerostin measurement as a strategy in the prevention and diagnosis of CVD and/or cardiovascular mortality.

Recent scientific evidence has shown an increased risk of fractures in patients with obesity, especially in those with a higher visceral adipose tissue content. This contradicts the old paradigm that obese patients were more protected than those with normal weight. Specifically, in older subjects in whom there is a redistribution of fat from subcutaneous adipose tissue to visceral adipose tissue and an infiltration of other tissues such as muscle with the consequent sarcopenia, obesity can accentuate the changes characteristic of this age group that predisposes to a greater risk of falls and fractures. Other factors that determine a greater risk in older subjects with obesity are chronic proinflammatory status, altered adipokine secretion, vitamin D deficiency, insulin resistance and reduced mobility. On the other hand, diagnostic tests may be influenced by obesity and its comorbidities as well as by body composition, and risk scales may underestimate the risk of fractures in these patients. Weight loss with physical activity programs and cessation of high-fat diets may reduce the risk. Finally, more research is needed on the efficacy of anti-osteoporotic treatments in obese patients.

Lifestyle changes are causing an exponential increase in the prevalence of obesity and metabolic syndrome (MetS) worldwide. The most frequent complications of these are the development of diabetes (T2D) and cardiovascular disease (CVD). Accurate tools are needed to classify the cardiovascular risk (CVR) in the MetS population. In recent years, numerous biomarkers of bone metabolism have been associated with CVR. The aim of this study was to determine the levels of undercarboxylated osteocalcin (ucOC) in a cohort of patients with MetS and to analyse its association with MetS parameters and CVR as well as with T2D prevalence. A longitudinal study was conducted in which a MetS population was followed for one year. Weight change, adherence to the Mediterranean diet (MedDiet), ucOC levels, MetS parameters and CVR were analysed and CVR was calculated using different scores. Our results showed a decrease of CVR associated with a better adherence to the MetDiet resulting in higher HDL-C and ucOC levels though the improvement of MetS risk factors. This bone protein appeared as a potential biomarker to classify CVR in the MetS population, especially for MetS patients without prevalent T2D. Furthermore, ucOC serum levels could be good predictors of T2D prevalence.

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.

Hypophosphatasia (HPP) a rare disease caused by mutations in the ALPL gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ALPL results in high clinical heterogeneity, which makes it difficult to establish a specific HPP symptomatology. In the present study, three variants of ALPL gene with uncertain significance and no previously described (p.Del Glu23&#95;Lys24, p.Pro292Leu and p.His379Asn) were identified in heterozygosis in patients diagnosed with HPP. These variants were characterized at phenotypic, functional and structural levels. All genetic variants showed significantly lower in vitro ALP activity than the wild-type (WT) genotype ( p -value <0.001). Structurally, p.His379Asn variant resulted in the loss of two Zn 2+ binding sites in the protein dimer which may greatly affect ALP activity. In summary, we identified three novel ALPL gene mutations associated with adult HPP. The correct identification and characterization of new variants and the subsequent study of their phenotype will allow the establishment of genotype-phenotype relationships that facilitate the management of the disease as well as making it possible to individualize treatment for each specific patient. This would allow the therapeutic approach to HPP to be personalized according to the unique genetic characteristics and clinical manifestations of each patient., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sanabria-de la Torre, Martínez-Heredia, González-Salvatierra, Andújar-Vera, Iglesias-Baena, Villa-Suárez, Contreras-Bolívar, Corbacho-Soto, Martínez-Navajas, Real, García-Fontana, Muñoz-Torres and García-Fontana.)

Bone fragility is a common complication in subjects with type 2 diabetes mellitus (T2DM). However, traditional techniques for the evaluation of bone fragility, such as dual-energy X-ray absorptiometry (DXA), do not perform well in this population. Moreover, the Fracture Risk Assessment Tool (FRAX) usually underestimates fracture risk in T2DM. Importantly, novel technologies for the assessment of one microarchitecture in patients with T2DM, such as the trabecular bone score (TBS), high-resolution peripheral quantitative computed tomography (HR-pQCT), and microindentation, are emerging. Furthermore, different serum and urine bone biomarkers may also be useful for the evaluation of bone quality in T2DM. Hence, in this article, we summarize the limitations of conventional tools for the evaluation of bone fragility and review the current evidence on novel approaches for the assessment of quality and bone microstructure alterations in patients with T2DM.

The identification of common targets in Alzheimer's disease (AD) and cardiovascular disease (CVD) in recent years makes the study of the CVD/AD axis a research topic of great interest. Besides aging, other links between CVD and AD have been described, suggesting the existence of common molecular mechanisms. Our study aimed to identify common targets in the CVD/AD axis. For this purpose, genomic data from calcified and healthy femoral artery samples were used to identify differentially expressed genes (DEGs), which were used to generate a protein-protein interaction network, where a module related to AD was identified. This module was enriched with the functionally closest proteins and analyzed using different centrality algorithms to determine the main targets in the CVD/AD axis. Validation was performed by proteomic and data mining analyses. The proteins identified with an important role in both pathologies were apolipoprotein E and haptoglobin as DEGs, with a fold change about +2 and -2, in calcified femoral artery vs healthy artery, respectively, and clusterin and alpha-2-macroglobulin as close interactors that matched in our proteomic analysis. However, further studies are needed to elucidate the specific role of these proteins, and to evaluate its function as biomarkers or therapeutic targets.

Recent evidence has revealed anti-inflammatory properties of vitamin D as well as extra-skeletal activity. In this context, vitamin D seems to be involved in infections, autoimmune diseases, cardiometabolic diseases, and cancer development. In recent years, the relationship between vitamin D and insulin resistance has been a topic of growing interest. Low 25-hydroxyvitamin D (25(OH)D) levels appear to be associated with most of the insulin resistance disorders described to date. In fact, vitamin D deficiency may be one of the factors accelerating the development of insulin resistance. Vitamin D deficiency is a common problem in the population and may be associated with the pathogenesis of diseases related to insulin resistance, such as obesity, diabetes, metabolic syndrome (MS) and polycystic ovary syndrome (PCOS). An important question is the identification of 25(OH)D levels capable of generating an effect on insulin resistance, glucose metabolism and to decrease the risk of developing insulin resistance related disorders. The benefits of 25(OH)D supplementation/repletion on bone health are well known, and although there is a biological plausibility linking the status of vitamin D and insulin resistance supported by basic and clinical research findings, well-designed randomized clinical trials as well as basic research are necessary to know the molecular pathways involved in this association.

Osteoglycin (OGN) could be a biomarker of mild kidney function impairment in type 2 diabetes (T2D). Our study aimed to determine the association between serum OGN and impaired kidney function risk in T2D patients and to analyze its potential role as an estimator of kidney disturbances in this population. This cross-sectional study included 147 T2D patients (65 ± 8 years, 58.5% males), and 75 healthy controls (63 ± 10 years, 36% males). Circulating OGN levels were determined by ELISA. Linear regression modeling was performed to determine the variables influencing circulating OGN, and an ROC curve was plotted to assess the usefulness of OGN as an estimator of diabetic kidney disease risk. Circulating OGN was significantly increased in T2D patients compared to controls (18.41 (14.45-23.27) ng/mL vs. 8.74 (7.03-12.35) ng/mL; p < 0.001). We found a progressive increase in serum OGN according to the severity of kidney impairment in T2D patients (normal kidney function: 16.14 (12.13-20.48) ng/mL; mildly impaired kidney function: 19.15 (15.78-25.90) ng/mL; moderate impaired kidney function: 21.80 (15.06-29.22) ng/mL; p = 0.006). Circulating OGN was an independent estimator of mildly impaired kidney function risk in T2D patients. We suggest that serum OGN could act as an albuminuria-independent biomarker of incipient kidney dysfunction in T2D patients.

Hypophosphatasia (HPP) is a rare genetic disease characterized by a decrease in the activity of tissue non-specific alkaline phosphatase (TNSALP). TNSALP is encoded by the ALPL gene, which is abundantly expressed in the skeleton, liver, kidney, and developing teeth. HPP exhibits high clinical variability largely due to the high allelic heterogeneity of the ALPL gene. HPP is characterized by multisystemic complications, although the most common clinical manifestations are those that occur in the skeleton, muscles, and teeth. These complications are mainly due to the accumulation of inorganic pyrophosphate (PPi) and pyridoxal-5'-phosphate (PLP). It has been observed that the prevalence of mild forms of the disease is more than 40 times the prevalence of severe forms. Patients with HPP present at least one mutation in the ALPL gene. However, it is known that there are other causes that lead to decreased alkaline phosphatase (ALP) levels without mutations in the ALPL gene. Although the phenotype can be correlated with the genotype in HPP, the prediction of the phenotype from the genotype cannot be made with complete certainty. The availability of a specific enzyme replacement therapy for HPP undoubtedly represents an advance in therapeutic strategy, especially in severe forms of the disease in pediatric patients.

Currently, there are not many in-depth studies focusing on the protein analysis of antioxidants involved in the calcification of the femoral artery. In this context, this study aimed to increase the knowledge of the molecular redox mechanisms involved in this process. Samples from calcified femoral artery sections of seven patients diagnosed with type 2 diabetes (T2D) and critical ischemia were analyzed. The isolated proteins were identified using liquid chromatography and mass-mass spectrometry and were used to generate a protein-protein interaction (PPI) network. Subsequently, highly interconnected regions within the PPI network were identified to obtain a representative module linked to oxidative stress. The proteins of this module with a higher degree of centrality (hubs) were selected to validate them by datamining, transcriptomic and proteomic assays. The analysis of modules of the femoral PPI network showed a module with mainly antioxidant function in which superoxide dismutase 2 (SOD2) was reported as the most important hub. SOD2 was validated at transcriptomic and proteomic level and confirmed by datamining. These results indicate that SOD activity is highly linked to the atherosclerotic process. We suggest that SOD2 could be a potential therapeutic target to prevent the calcification of the femoral artery. The maintenance of optimal SOD2 levels and its cofactors could be used as a preventive strategy for vascular calcification and the related cardiovascular complications in T2D patients., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Desiccation-tolerant plants are able to survive for extended periods of time in the absence of water. The molecular understanding of the mechanisms used by these plants to resist droughts can be of great value for improving drought tolerance in crops. This understanding is especially relevant in an environment that tends to increase the number and intensity of droughts. The combination of certain microorganisms with drought-sensitive plants can improve their tolerance to water scarcity. One of these bacteria is Microbacterium sp. 3J1, an actinobacteria able to protect pepper plants from drought. In this study, we supplemented drought-tolerant and drought-sensitive plant rhizospheres with Microbacterium sp. 3J1 and analyzed their proteomes under drought to investigate the plant-microbe interaction. We also compare this root proteome with the proteome found in desiccation-tolerant plants. In addition, we studied the proteome of Microbacterium sp. 3J1 subjected to drought to analyze its contribution to the plant-microbe interaction. We describe those mechanisms shared by desiccation-tolerant plants and sensitive plants protected by microorganisms focusing on protection against oxidative stress, and production of compatible solutes, plant hormones, and other more specific proteins., (Copyright © 2020 García-Fontana, Vilchez and Manzanera.)

Undercarboxylated osteocalcin (ucOC) could be a biomarker of glucose disturbances and cardiovascular risk. Our study aimed to determine the association between serum levels of ucOC and cardiovascular risk in metabolic syndrome (MetS) patients and to analyse its potential role as estimator of type 2 diabetes (T2D) risk in this population. This cross-sectional study included 235 patients with MetS, 53.2% women, aged 55-75 years. Circulating ucOC levels were measured by ELISA. Cardiovascular risk was determined as Z-score of the diagnostic criteria for MetS (CV-ZS). Linear regression model was performed to analyse the association between circulating ucOC and CV-ZS. A receiver operating curve (ROC) was performed to analyse the usefulness of ucOC as T2D risk estimator. Patients above the CV-ZS median showed significant lower ucOC levels. We found an inverse association between ucOC levels and CV-ZS in MetS patients without T2D. Patients with ucOC levels below the 25 th percentile showed worse cardiometabolic profile and higher cardiovascular and T2D risk. The area under the curve performed better when ucOC levels were included along with the classic T2D risk factors. The measurement of circulating ucOC could be a useful tool to identify increased cardiovascular and T2D risk in MetS patients without T2D.

Pseudomonas aeruginosa is an opportunistic human pathogen causing infections in a variety of plant and animal hosts. The gene mcpB, part of the chemosensory gene cluster II, encodes a soluble chemoreceptor whose function remains unknown. Previous studies show that the cheB2 gene, also located in the chemosensory cluster II, is involved in a specific response during infection and it is required for full pathogenicity of P. aeruginosa. To determine whether the McpB (or Aer2) chemoreceptor is involved in virulence processes, we generated a mcpB mutant and tested its phenotype using a virulence-measuring system. This system was developed by our group and is based on different bioassays using organisms living at different soil trophic levels, including microbial, nematode, arthropod, annelid, and plant model systems. The deletion of mcpB resulted in an attenuation of bacterial virulence in different infection models, and wild-type virulence was restored following genetic complementation of the mutant strain. Our study indicates that the McpB chemoreceptor is linked to virulence processes and may constitute the basis for the development of alternative strategies against this pathogen.

Hypophosphatasia (HPP) is a genetic disease caused by one or several mutations in ALPL gene encoding the tissue-nonspecific alkaline phosphatase affecting the mineralization process. Due to its low prevalence and lack of recognition, this metabolic disorder is generally confused with other more frequent bone disorders. An assessment of serum total alkaline phosphatase (ALP) levels was performed in 78,590 subjects. Pyridoxal-5'-phosphate (PLP) concentrations were determined and ALPL gene was sequenced in patients potentially affected by HPP. Functional validation of the novel mutations found was performed using a cell-based assay. Our results showed persistently low serum ALP levels in 0.12% of subjects. Among the studied subjects, 40% presented with HPP-related symptoms. Nine of them (~28%) had a history of fractures, 5 (~16%) subjects showed chondrocalcinosis and 4 (~13%) subjects presented with dental abnormalities. Eleven subjects showed increased PLP concentrations. Seven of them showed ALPL gene mutations (2 of the mutations corresponded to novel genetic variants). In summary, we identified two novel ALPL gene mutations associated with adult HPP. Using this protocol, almost half of the studied patients were diagnosed with HPP. Based on these results, the estimated prevalence of mild HPP in Spain could be up to double than previously reported.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in polluted environments and are included in the priority list of toxic compounds. Previous studies have shown that the fungus Penicillium oxalicum, isolated from a hydrocarbon-polluted pond, has a great capability to transform different PAHs in short periods under submerged fermentation conditions. Although cytochrome p450s (CYPs) seems to be the main responsible enzyme in this process, changes in proteome profile remains poorly understood. The aim of this work was to characterise molecular disturbances in the cytosolic and microsomal sub-proteomes of P. oxalicum by applying two-dimensional (2D) gel electrophoresis and label-free quantitative proteomics during anthracene biodegradation. Our results showed that by using 2D-gels, 10 and 8 differential proteins were over-expressed in the cytosolic and microsomal fractions, respectively. Most of them were related to stress response. Shotgun proteomics allowed the identification of 158 and 174 unique protein species that differentially accumulated during anthracene biotransformation, such as CYPs, epoxide hydrolases and transferases enzymes, belonging to Phase I and Phase II of the metabolism of xenobiotics, contributing to the anthracene biodegradation pathway. These results confirm the biological significance of ascomycetes fungi the rol of CYPs on biodegradation and the need of a deeper knowledge on fungal proteomics for the application of the appropriate microorganisms in biodegradation processes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cardiovascular diseases are a health problem throughout the world, especially in people with diabetes. The identification of cardiovascular disease biomarkers can improve risk stratification. Sclerostin is a modulator of the Wnt/β-catenin signalling pathway in different tissues, and it has recently been linked to vascular biology. The current study aimed to evaluate the relationship between circulating sclerostin levels and cardiovascular and non-cardiovascular mortality in individuals with and without type 2 diabetes. We followed up a cohort of 130 participants (mean age 56.8 years; 48.5% females; 75 with type 2 diabetes; 46 with prevalent cardiovascular disease) in which serum sclerostin levels were measured at the baseline. Time to death (both of cardiovascular and non-cardiovascular causes) was assessed to establish the relationship between sclerostin and mortality. We found that serum sclerostin concentrations were significantly higher in patients with prevalent cardiovascular disease (p<0.001), and independently associated with cardiovascular mortality (p = 0.008), showing sclerostin to be a stronger predictor of mortality than other classical risk factors (area under the curve = 0.849 vs 0.823). The survival analysis showed that an increase of 10 pmol/L in the serum sclerostin level resulted in a 31% increase in cardiovascular mortality. However, no significant association was observed between sclerostin levels and non-cardiovascular mortality (p = 0.346). From these results, we conclude that high sclerostin levels are related to mortality due to cardiovascular causes. The clinical implication of these findings is based on the possible use of serum sclerostin as a new biomarker of cardiovascular mortality risk in order to establish preventive strategies., Competing Interests: The authors have declared that no competing interests exist.

The DNA molecule is associated with the role of encoding information required to produce RNA which is translated into proteins needed by the cell. This encoding involves information transmission to offspring or to other organisms by horizontal transfer. However, despite the abundance of this molecule in both the cell and the environment, its physiological role seems to be restricted mainly to that of a coding and inheritance molecule. In this paper, we report a new physiological role for the DNA molecule as involved in protection against desiccation, in addition to its well-established main information transfer and other recently reported functions such as bio-film formation in eDNA form. Desiccation-tolerant microorganisms such as Microbacterium sp. 3J1 significantly upregulate genes involved in DNA synthesis to produce DNA as part of their defensive mechanisms to protect protein structures and functions from drying according to RNA-seq analysis. We have observed the intracellular overproduction of DNA in two desiccation-tolerant microorganisms, Microbacterium sp. 3J1 and Arthrobacter siccitolerans 4J27, in response to desiccation signals. In addition, this conclusion can be made from our observations that synthetic DNA protects two proteins from drying and when part of a xeroprotectant preparation, DNA from various organisms including desiccation-sensitive species, does the same. Removal of DNA by nuclease treatment results in absence of this additive protective effect. We validated this role in biochemical and biophysical assays in proteins and occurs in trans even with short, single chains of synthetically produced DNA.

A collection of desiccation-tolerant xeroprotectant-producing microorganisms was screened for their ability to protect plants against drought, and their role as plant growth-promoting rhizobacteria was investigated in two different crops (tomato and pepper). The most commonly described biochemical mechanisms for plant protection against drought by microorganisms including the production of phytohormones, antioxidants and xeroprotectants were analyzed. In particular, the degree of plant protection against drought provided by these microorganisms was characterized. After studying the findings and comparing them with results of the closest taxonomic relatives at the species and strain levels, we propose that trehalose produced by these microorganisms is correlated with their ability to protect plants against drought. This proposal is based on the increased protection of plants against drought by the desiccation-sensitive microorganism Pseudomonas putida KT2440, which expresses the ots AB genes for trehalose biosynthesis in trans .

The genome sequence for Microbacterium sp. strain 3J1, a desiccation-tolerant organism isolated from the Nerium oleander rhizosphere, is reported here. The genome is estimated to be approximately 3.5 Mb in size, with an average G+C content of 67.7% and a predicted number of protein-coding sequences of 3,310., (Copyright © 2015 Manzanera et al.)

Arthrobacter koreensis 5J12A is a desiccation-tolerant organism isolated from the Nerium oleander rhizosphere. Here, we report its genome sequence, which may shed light on its role in plant growth promotion. This is believed to be the first published genome of a desiccation-tolerant plant growth promoter from the genus Arthrobacter., (Copyright © 2015 Manzanera et al.)

The genome sequence for Rhodococcus sp. 4J2A2, a newly described desiccation-tolerant strain that removes aromatic hydrocarbons, is reported here. The genome is estimated to be around 7.5 Mb in size, with an average G+C content of 60.77% and a predicted number of protein-coding sequences of 6,354., (Copyright © 2015 Manzanera et al.)

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules., (© 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Methyltransferases of the CheR family and methylesterases of the CheB family control chemoreceptor methylation, and this dynamic posttranslational modification is necessary for proper chemotaxis of bacteria. Studies with enterobacteria that contain a single CheR or CheB show that, in addition to binding at the methylation site, some chemoreceptors bind CheR or CheB through additional high-affinity sites at distinct pentapeptide sequences in the chemoreceptors. We investigated the recognition of chemoreceptors by CheR proteins in the human pathogen Pseudomonas aeruginosa PAO1. Of the four methyltransferases in PAO1, we detected an interaction only between CheR2 and the chemoreceptor methyl-accepting chemotaxis protein B (McpB), which contains the pentapeptide GWEEF at its carboxyl terminus. Furthermore, CheR2 was also the only paralog that methylated McpB in vitro, and deletion of the pentapeptide sequence abolished both the CheR2-McpB interaction and the methylation of McpB. When clustered according to protein sequence, bacterial CheR proteins form two distinct families-those that bind pentapeptide-containing chemoreceptors and those that do not. These two families are distinguished by an insertion of three amino acids in the β-subdomain of CheR. Deletion of this insertion in CheR2 prevented its interaction with and methylation of McpB. Pentapeptide-containing chemoreceptors are common to many bacteria species; thus, these short, distinct motifs may enable the specific assembly of signaling complexes that mediate different responses.

Isothermal titration calorimetry (ITC) is based on a simple titration of one ligand with another and the small heat changes caused by the molecular interaction are detected. From one ITC experiment the complete set of thermodynamic parameters of binding including association and dissociation constants as well as changes in enthalpy, entropy, and free energy can be derived. Using this technique almost any type of molecular interaction can be analyzed. Both ligands are in solution, and there is no need for their chemical derivatization. There are no limits as to the choice of the analysis buffer, and the analysis temperature can be set between 4 and 80 °C. This technique has been primarily applied to study the interaction between various proteins of Pseudomonas with small molecule ligands. In addition, ITC has been used to study the binding of Pseudomonas proteins to target DNA fragments.

A primary driving force during bacterial evolution was the capacity to access compounds necessary for growth and survival. Since the species of the genus Pseudomonas are characterized by metabolic versatility, these bacteria have developed chemotactic behaviors towards a wide range of different compounds. The specificity of a chemotactic response is determined by the chemoreceptor, which is at the beginning of the signaling cascade and to which chemoattractants and chemorepellents bind. The number of chemoreceptor genes of Pseudomonas species is significantly higher than the average number in motile bacteria. Although some of the receptors have been annotated with a function, the cognate signal molecules for the majority of them still need to be identified. Different qualitative and quantitative methods are presented that can be used to study flagellum-mediated taxis.

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14-2.2 μM) than the substrate S-adenosylmethionine (KD of 22-43 μM), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

The paralogous receptors PctA, PctB and PctC of Pseudomonas aeruginosa were reported to mediate chemotaxis to amino acids, intermediates of amino acid metabolism and chlorinated hydrocarbons. We show that the recombinant ligand binding regions (LBRs) of PctA, PctB and PctC bind 17, 5 and 2 l-amino acids respectively. In addition, PctC-LBR recognized GABA but not any other structurally related compound. l-Gln, one of the three amino acids that is not recognized by PctA-LBR, was the most tightly binding ligand to PctB suggesting that PctB has evolved to mediate chemotaxis primarily towards l-Gln. Bacteria were efficiently attracted to l-Gln and GABA, but mutation of pctB and pctC, respectively, abolished chemoattraction. The physiological relevance of taxis towards GABA is proposed to reside in an interaction with plants. LBRs were predicted to adopt double PDC (PhoQ/DcuS/CitA) like structures and site-directed mutagenesis studies showed that ligands bind to the membrane-distal module. Analytical ultracentrifugation studies have shown that PctA-LBR and PctB-LBR are monomeric in the absence and presence of ligands, which is in contrast to the enterobacterial receptors that require sensor domain dimers for ligand recognition., (© 2013 John Wiley & Sons Ltd.)

A number of bacteria can use toxic compounds as carbon sources and have developed complex regulatory networks to protect themselves from the toxic effects of these compounds as well as to benefit from their nutritious properties. As a model system we have studied the responses of Pseudomonas putida strains to toluene. Although this compound is highly toxic, several strains are able to use it for growth. Particular emphasis was given to the responses in the context of taxis, resistance and toluene catabolism. P. putida strains analysed showed chemotactic movements towards toluene. Strain DOT-T1E was characterised by an extreme form of chemotaxis, termed hyperchemotaxis, which is mediated by the McpT chemoreceptor encoded by plasmid pGRT1. Close McpT homologs are found in a number of other plasmids encoding degradation pathways of toxic compounds. The pGRT1 plasmid harbours also the genes for the TtgGHI efflux pump which was identified as the primary determinant for the resistance of strain DOT-T1E towards toluene. Pump expression is controlled by the TtgV repressor in response to a wide range of different mono- and biaromatic compounds. Strain DOT-T1E is able to degrade toluene, benzene and ethylbenzene via the toluene dioxygenase (TOD) pathway. The expression of the pathway operon is controlled by the TodS/T two component system. The sensor kinase TodS recognizes toluene with nanomolar affinity, which in turn triggers an increase in its autophosphorylation and consequently transcriptional activation. Data suggest that transcriptional activation of the TOD pathway occurs at very low toluene concentrations whereas TtgV mediated induction of pump expression sets in as the toluene concentration further increases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The two-component system TmoS/TmoT controls the expression of the toluene-4-monooxygenase pathway in Pseudomonas mendocina RK1 via modulation of P(tmoX) activity. The TmoS/TmoT system belongs to the family of TodS/TodT like proteins. The sensor kinase TmoS is a 108 kDa protein composed of seven different domains. Using isothermal titration calorimetry we show that purified TmoS binds a wide range of aromatic compounds with high affinities. Tightest ligand binding was observed for toluene (K(D)  = 150 nM), which corresponds to the highest affinity measured between an effector and a sensor kinase. Other compounds with affinities in the nanomolar range include benzene, the 3 xylene isomers, styrene, nitrobenzene or p-chlorotoluene. We demonstrate that only part of the ligands that bind to TmoS increase protein autophosphorylation in vitro and consequently pathway expression in vivo. These compounds are referred to as agonists. Other TmoS ligands, termed antagonists, failed to increase TmoS autophosphorylation, which resulted in their incapacity to stimulate gene expression in vivo. We also show that TmoS saturated with different agonists differs in their autokinase activities. The effector screening of gene expression showed that promoter activity of P(tmoX) and P(todX) (controlled by the TodS/TodT system) is mediated by the same set of 22 compounds. The common structural feature of these compounds is the presence of a single aromatic ring. Among these ligands, toluene was the most potent inducer of both promoter activities. Information on the TmoS/TmoT and TodS/TodT system combined with a sequence analysis of family members permits to identify distinct features that define this protein family., (© 2011 The Authors. Microbial Biotechnology © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be monomeric, which rules out a role of the TPR domain in self-association.