Stability of housekeeping genes in inflamed joints of spontaneous and collagen-induced arthritis in DBA/1 mice.

BACKGROUND: DBA/1 mice arthritis models have contributed to our understanding of human rheumatoid arthritis (RA) and spondyloarthritis (SpA) pathogenesis, as well as the exploration of therapeutic targets for treatment. Quantitative polymerase chain reaction (qPCR) is an indispensable tool in molecular research, which requires reference gene validation to obtain consistent and reliable results. OBJECTIVE: To determine the stability of candidate reference genes for qPCR in the joint of collagen-induced arthritis (CIA) and spontaneous arthritis (SpAD) DBA/1 mice. METHODS: The expression of eleven commonly used reference genes (ACTB, B2M, EF1a, GAPDH, HMBS, HPRT, PPIB, RPL13A, SDHA, TBP, and YWHAZ) was assessed by qPCR and the data were compared using delta-Ct methods and the geNorm, NormFinder, and RefFinder software packages. Genes identified as stable in each model were used for the quantification of inflammatory cytokines RESULTS: The gene stabilities differed between the two arthritis models in the DBA/1 mice. EF1a and RPL13A were the best reference genes for SpAD, while RPL13A and TBP were the best for the CIA. These genes allowed the data normalization for the quantification of the inflammatory cytokines in both models; these results showed an increase in the expression of IL-1B, IL-12B, IL-17A, and IL-6 in the inflamed joints. The use of different primer sequences for the same reference gene resulted in different relative quantification values. CONCLUSION: This study demonstrates that commonly used reference genes may not be suitable for arthritic tissues from DBA/1 mice, and strengthening the principle that meticulous validation of reference genes is essential before each experiment to obtain valid and reproducible qPCR data for analysis or interpretation.

Positive transcriptional response on inflammation and joint remodelling influenced by physical exercise in proteoglycan-induced arthritis: An animal study.

AIMS: To assess the effect of physical exercise (PE) on the histological and transcriptional characteristics of proteoglycan-induced arthritis (PGIA) in BALB/c mice. METHODS: Following PGIA, mice were subjected to treadmill PE for ten weeks. The tarsal joints were used for histological and genetic analysis through microarray technology. The genes differentially expressed by PE in the arthritic mice were obtained from the microarray experiments. Bioinformatic analysis in the DAVID, STRING, and Cytoscape bioinformatic resources allowed the association of these genes in biological processes and signalling pathways. RESULTS: Arthritic mice improved their physical fitness by 42.5% after PE intervention; it induced the differential expression of 2,554 genes. The bioinformatic analysis showed that the downregulated genes (n = 1,371) were significantly associated with cellular processes that mediate the inflammation, including Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), Notch, and cytokine receptor interaction signalling pathways. Moreover, the protein interaction network showed that the downregulated inflammatory mediators interleukin (IL) 4, IL5, IL2 receptor alpha (IL2rα), IL2 receptor beta (IL2rß), chemokine ligand (CXCL) 9, and CXCL12 were interacting in several pathways associated with the pathogenesis of arthritis. The upregulated genes (n = 1,183) were associated with processes involved in the remodelling of the extracellular matrix and bone mineralization, as well as with the processes of aerobic metabolism. At the histological level, PE attenuated joint inflammatory infiltrate and cartilage erosion. CONCLUSION: Physical exercise influences parameters intimately linked to inflammatory arthropathies. Research on the effect of PE on the pathogenesis process of arthritis is still necessary for animal and human models.Cite this article: Bone Joint Res. 2020;9(1):36-48.

Improving glucose and xylose assimilation in Azotobacter vinelandii by adaptive laboratory evolution.

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8 g L-1) and glucose (2 g L-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.

Oral health and orofacial function in patients with rheumatoid arthritis.

The aim of the study was to describe the oral health and orofacial function of Mexican patients with rheumatoid arthritis (RA) and their association with clinical and radiological aspects of the disease. Patients with RA received a complete odontological exam, which also included a clinical and radiographic assessment of the temporomandibular joint (TMJ). The rheumatologic assessment included detailed profiling of the disease and serological and radiographic parameters. The study included 62 RA patients; the median (min-max) age was 51 (18-72) years old and 8.5 (1-39) years of disease duration. The 63.6% of the patients had DAS28 ≥ 3.2, and a median (min-max) of Sharp/van der Heijde score (SvdHS) of 41 (0-214). 98.3% of the patients presented caries, which were severe in 53.3% of the cases. The 73.8% of the patients were missing teeth due to caries, with a median (min-max) of 4 (0-32) teeth missing per patient. Oral hygiene was classified as bad in 49.1% of patients and only 15.3% of them had a healthy periodontium. The TMJ function was abnormal in 98.4% of the patients and 62.9% of them presented moderate or severe TMJ disorder (TMD). The radiographic damage of the TMJ correlated positively with the SvdHS. No correlations were found between disease activity or structural progression and orofacial variables, including periodontitis. There are severe oral and orofacial health problems in RA patients despite having medical attention for their disease. Multidisciplinary management remains an area of opportunity for both the medical specialists and the health system in our country.

Temporomandibular and Odontological Abnormalities in Patients with Rheumatoid Arthritis. / Alteraciones temporomandibulares y odontológicas en pacientes con artritis reumatoide.

OBJECTIVE: To characterize the orofacial abnormalities in patients with rheumatoid arthritis (RA) and compare them with those in a reference population. METHODS: The study included 30 RA patients and 30 consecutive patients in an odontology clinic in whom RA was ruled out. Patients underwent a clinical dental examination which included: 1) clinical and radiographic abnormalities of the temporomandibular joint; 2) biomechanical craniocervical analysis; 3) state of dentition and treatment needs; 4) periodontal status; 5) oral hygiene status; and 6) facial pain, which was compared among study groups. In addition, the association between the variables studied was determined through correlation tests. RESULTS: Patients with RA showed a higher prevalence of temporomandibular abnormalities, both clinical (100.0% vs. 60.0%, P<.001) and radiographic, including erosions (50.0% vs. 16.0%, P=.010), compared with individuals in the control group. Likewise, patients with RA had a greater number of missing teeth (6.9±5.7 vs. 3.0±2.0, P=.001), more caries (13.4±5.4 vs. 4.9±6.5, P=.001), periodontitis (1.3±0.9 vs. 0.8±0.8, P=.015), poorer oral hygiene (43.3% vs. 13.3%, P=.005) and greater facial pain (66.7% vs. 20.0%, P <.001). The cephalometric analysis of Rocabado showed differences in the craniocervical angle and hyoid triangle between RA and controls. Significant correlations were obtained between oral and temporomandibular abnormalities. CONCLUSIONS: Patients with RA showed a greater orofacial deterioration, which reflects the importance of multidisciplinary care, including periodic dental examination.

Exercise Exacerbates the Transcriptional Profile of Hypoxia, Oxidative Stress and Inflammation in Rats with Adjuvant-Induced Arthritis.

Physical exercise (PE) is recommended for Rheumatoid Arthritis (RA), but the molecular and biological mechanisms that impact the inflammatory process and joint destruction in RA remain unknown. The objective of this study was to evaluate the effect of PE on the histological and transcriptional changes in the joints of adjuvant-induced arthritis (AIA) rat model. AIA rats were subjected to PE on a treadmill for eight weeks. The joints were subjected to histological and microarray analysis. The differentially expressed genes (DEGs) by PE in the arthritic rats were obtained from the microarray. The bioinformatic analysis allowed the association of these genes in biological processes and signaling pathways. PE induced the differential expression of 719 genes. The DEGs were significantly associated with pathogenic mechanisms in RA, including HIF-1, VEGF, PI3-Akt, and Jak-STAT signaling pathways, as well as response to oxidative stress and inflammatory response. At a histological level, PE exacerbated joint inflammatory infiltrate and tissue destruction. The PE exacerbated the stressed joint environment aggravating the inflammatory process, the hypoxia, and the oxidative stress, conditions described as detrimental in the RA joints. Research on the effect of PE on the pathogenesis process of RA is still necessary for animal models and human.

Glucose limitation and glucose uptake rate determines metabolite production and sporulation in high cell density continuous cultures of Bacillus amyloliquefaciens 83.

Bacillus amyloliquefaciens spores have been used as the principal ingredient of biocontrol products. However, during the process of spore production, wild-type strains produce poly-γ-glutamic acid (γ-PGA), an undesirable byproduct that increases broth viscosity and hinders recovery and drying. This work examined the influence of specific glucose uptake rates (qGluc) in glucose-controlled overflow metabolism. Diverse scenarios, from glucose limitation to glucose sufficiency, were evaluated in continuous cultures to control qGluc. Cell yields of glucose were higher at low qGluc, while the opposing trend was found for γ-PGA and other overflow metabolic byproducts yields. However, γ-PGA production was still detectable in cultures with the highest glucose limitation (D = 0.06 h-1), even though high sporulation incidence was observed in these cultures. Indeed, in such conditions, nonsporulating vegetative cells seem to maintain glucose overflow metabolism, allowing limited γ-PGA production. These findings can be used to establish fed-batch culture strategies for high cell density Bacillus amyloliquefaciens cultures where γ-PGA production (and apparent viscosity) is significantly reduced. This is the first time that the dependence of qGluc on growth, sporulation and carbon overflow metabolism of a spore and biofilm producer, Bacillus amyloliquefaciens strain, has been reported.

Removal of Tetracycline Pollutants by Adsorption and Magnetic Separation Using Reduced Graphene Oxide Decorated with α-Fe2O3 Nanoparticles.

Nanocomposites of reduced graphene oxide (RGO) with ferromagnetic α-Fe2O3 nanoparticles have been prepared in-situ by thermal treatment. The structure and morphology of the hybrid material were studied by X-ray photoelectron spectroscopy, Raman, X-ray diffraction, and transmission electron microscopy. The results show a hybrid material highly modified with α-Fe2O3 nanoparticles distributed on the graphene surface. The adsorption kinetics show the presence of α-Fe2O3 nanoparticles on the RGO surface, and the amount of remaining functional groups dominated by ionization and dispersion. The adsorption kinetics of this adsorbent was characterized and found to fit the pseudo-second-order model. The α-Fe2O3 nanoparticles on RGO modify the electrostatic interaction of RGO layers and tetracycline, and adsorption properties decreased in the hybrid material. Adsorption isotherms fit with the Langmuir model very well, and the maximum capacity adsorption was 44.23 mg/g for RGO and 18.47 mg/g for the hybrid material. Magnetic characterization of the hybrid material shows ferromagnetic behavior due to the nanosize of α-Fe2O3 with a saturation magnetization, Ms = 7.15 Am²/kg, a remanence Mr = 2.29 Am²/kg, and a coercive field, Hc = 0.02 T.

DNA Damage and Deficiencies in the Mechanisms of Its Repair: Implications in the Pathogenesis of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a perplexing and potentially severe disease, the pathogenesis of which is yet to be understood. SLE is considered to be a multifactorial disease, in which genetic factors, immune dysregulation, and environmental factors, such as ultraviolet radiation, are involved. Recently, the description of novel genes conferring susceptibility to develop SLE even in their own (monogenic lupus) has raised the interest in DNA dynamics since many of these genes are linked to DNA repair. Damage to DNA induces an inflammatory response and eventually triggers an immune response, including those targeting self-antigens. We review the evidence that indicates that patients with SLE present higher levels of DNA damage than normal subjects do and that several proteins involved in the preservation of the genomic stability show polymorphisms, some of which increase the risk for SLE development. Also, the experience from animal models reinforces the connection between DNA damage and defective repair in the development of SLE-like disease including characteristic features such as anti-DNA antibodies and nephritis. Defining the role of DNA damage response in SLE pathogenesis might be strategic in the quest for novel therapies.

Surgical outcomes in Alagille syndrome and PFIC: A single institution's 20-year experience.

BACKGROUND: Alagille Syndrome (AGS) and Progressive Familial Intrahepatic Cholestasis (PFIC) are rare pediatric biliary disorders that lead to progressive liver disease. This study reviews our experience with the surgical management of these disorders over the last 20years. METHODS: We retrospectively reviewed the records of children diagnosed with AGS or PFIC from January 1996 to December 2016. Data collected included demographics, surgical intervention (liver transplant or biliary diversion), and complications. RESULTS: Of 37 patients identified with these disorders, 17 patients (8 AGS,9 PFIC) underwent surgical intervention. Mean postsurgical follow-up was 6.9±4.7years. Liver transplantation was the most common procedure (n=14). Two patients who were initially thought to have biliary atresia underwent hepatoportoenterostomy, but were subsequently shown to have Alagille syndrome. Biliary diversion procedures were performed in 3 patients (external n=1, internal n=2). PFIC patients tended to be older at the time of liver transplant compared to AGS (4.3±3.9years vs. 2.4±1.1years, p=0.25). The AGS patient with external diversion had resolution of symptoms and no complications (follow-up: 12.5years). Both PFIC patients with internal diversion (conduit between gallbladder and transverse colon) had resolution of pruritus and no progression of liver disease (follow-up: 3.8 and 4.5years). CONCLUSIONS: AGS and PFIC are rare biliary disorders in children which result in pruritus and progressive liver failure. Three patients in this series (8%) benefited from biliary diversion for control of pruritus and have not to date required transplantation for progressive liver disease. 38% underwent transplantation owing to pruritus and severe liver dysfunction. LEVEL OF EVIDENCE: 2b.

Hypoxia and its implications in rheumatoid arthritis.

Alterations in tissue oxygen pressure contribute to a number of diseases, including rheumatoid arthritis (RA). Low partial pressure of oxygen, a condition known as hypoxia, is a relevant feature in RA since it is involved in angiogenesis, inflammation, apoptosis, cartilage degradation, energy metabolism, and oxidative damage. Therefore, alterations in hypoxia-related signaling pathways are considered potential mechanisms of disease pathogenesis. The objective of this review is to highlight and update our current knowledge of the role of hypoxia in the pathogenesis of RA. We describe the experimental evidence that RA synovial tissue exists in a hypoxic state, as well as the origin and involvement of synovial hypoxia in different aspects of the pathogenic process.

Oxidative Stress Relevance in the Pathogenesis of the Rheumatoid Arthritis: A Systematic Review.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease whose pathogenic mechanisms remain to be elucidated. The oxidative stress and antioxidants play an important role in the disease process of RA. The study of oxidants and antioxidants biomarkers in RA patients could improve our understanding of disease pathogenesis; likely determining the oxidative stress levels in these patients could prove helpful in assessing disease activity and might also have prognostic implications. To date, the usefulness of oxidative stress biomarkers in RA patients is unclear and the evidence supporting them is heterogeneous. In order to resume and update the information in the status of oxidants and antioxidants and their connection as biomarkers in RA, we performed a systematic literature search in the PubMed database, including clinical trials published in the last five years using the word combination "rheumatoid arthritis oxidative stress". In conclusion, this review supports the fact that the oxidative stress is an active process in RA pathogenesis interrelated to other better known pathogenic elements. However, some controversial results preclude a definite conclusion.

Analysis of respiratory activity and carbon usage of a mutant of Azotobacter vinelandii impaired in poly-ß-hydroxybutyrate synthesis.

In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-ß-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).

Rheumatic Diseases in Chihuahua, México: A COPCORD Survey.

BACKGROUND: Rheumatic diseases (RDs) represent a global problem for health care systems and patients. Community Oriented Program for Control of Rheumatic Diseases (COPCORD) is a low-cost screening tool for detecting musculoskeletal (MSK) pain and RDs. OBJECTIVE: The aim of this study was to examine the pattern of MSK pain and RDs in clinic population in Chihuahua City, Mexico. METHODS: A cross-sectional study was conducted in 7 primary health clinics using the COPCORD methodology in subjects older than 18 years. People with MSK pain not induced by trauma (positive cases) were evaluated by primary care physicians and rheumatologists. RESULTS: The study included 1006 individuals with a mean age of 46.0 (SD, 15.8) years; 751 (74.7%) were women. Musculoskeletal pain in the previous 7 days was reported by 571 individuals (56.75%; 95% confidence interval [CI], 53.8%-60.1%), and 356 cases (35.4%; 95% CI, 32.5%-38.4%) were COPCORD positive. The mean pain intensity in visual analog scale was 6.62 (SD, 2.4). The most common painful joint was the knee (54.7%; 95% CI, 51.1%-59.0%). Two hundred eighty subjects with MSK pain (49.0%) previously sought medical attention, and 375 (65.7%) were under treatment. Functional impairment was reported by 69.8% of the COPCORD-positive subjects. The prevalence of RDs was 21.4% (95% CI, 18.9%-23.8%). The most prevalent disease was osteoarthritis (10.3%; 95% CI, 8.6%-12.4%), followed by regional pain syndromes (5.5%; 95% CI, 4.1%-7.0%), rheumatoid arthritis (1.4%; 95% CI, 0.8%-2.2%), and mechanical low-back pain (1.4%; 95% CI, 0.7%-2.2%). CONCLUSIONS: Musculoskeletal pain is an important problem that affects our community. The data provided in this study will be presented to the local authorities to help in the development of prevention strategies.

Prevalence of rheumatic diseases in Raramuri people in Chihuahua, Mexico: a community-based study.

This study aimed to determine the prevalence of musculoskeletal (MSK) pain and rheumatic diseases in the Raramuri population (also known as Tarahumaras) who are an indigenous group in the northern state of Chihuahua in Mexico. We used the Community-Oriented Program for Control of Rheumatic Diseases (COPCORD) methodology. An analytical cross-sectional study was conducted including indigenous Raramuri aged ≥18 years from communities settled in Chihuahua City. Subjects with positive MSK pain were evaluated by primary care physicians and rheumatologists. Demographic and occupational factors such as gender and job type associated with rheumatic disease were investigated. A total of 380 indigenous Raramuri (mean age 33.6 ± 13.1 years; 37.9 % male) were interviewed. Seventy-six individuals (20 %) reported MSK pain in the last 7 days. Pain intensity was reported as "severe" and "the most severe" in 30 % of the cases. Fifty-six individuals (14.7 %) reported pain in the past and 86 (22.6 %) had either past or current pain. The prevalence of rheumatic diseases was 10.5 %. Diagnosed diseases were osteoarthritis (6.6 %), low back pain (1.6 %), spondyloarthritis (0.8 %), rheumatoid arthritis (0.5 %), non-specific arthritis (0.5 %), rheumatic regional pain syndromes (0.3 %), and fibromyalgia (0.3 %). Rheumatic disease was associated with the following variables: age (odds ratio (OR) 1.04, 95 % confidence interval (CI) 1.02-1.08; p = 0.006), family history of rheumatic symptoms (OR 6.9; 95 % CI 2.6-18.7; p < 0.001), and Health Assessment Questionnaire-Disability Index (OR 28.9; 95 % CI 2.8-289.7; p < 0.001). A high prevalence of non-traumatic MSK pain suggests the need for a rheumatic disease prevention program in the Raramuri people in Chihuahua, Mexico.

Diffusional and transcriptional mechanisms involved in laccases production by Pleurotus ostreatus CP50.

The independent effects of hydrodynamic stress (assessed as the Energy Dissipation/Circulation Function, EDCF) and dissolved oxygen tension (DOT) on the growth, morphology and laccase production by Pleurotus ostreatus CP50 were studied using a 3(2) factorial design in a 10L reactor. A bell-shape function for fungus growth between 8 and 22% DOT was observed, as well as a significant negative effect on laccase production and the expression of poxc, the gene encoding for the most abundant laccase produced by P. ostreatus CP50. Increasing EDCF from 1 to 21 kW/m(3)s, had a positive effect on fungus growth, whereas no effect on poxc gene expression was observed. However, the increase in EDCF favored the specific laccase production due to the generation of smaller pellets with less diffusional limitations and increased metabolically active biomass. The results show, for the first time, that hydrodynamic effects on growth and laccase production are mainly physical and diffusional, while the influence of the dissolved oxygen is at transcriptional level.

Molecular mechanisms of bone formation in spondyloarthritis.

Spondyloarthritis comprise a group of inflammatory rheumatic diseases characterized by its association to HLA-B27 and the presence of arthritis and enthesitis. The pathogenesis involves both an inflammatory process and new bone formation, which eventually lead to ankylosis of the spine. To date, the intrinsic mechanisms of the pathogenic process have not been fully elucidated, and our progress is remarkable in the identification of therapeutic targets to achieve the control of the inflammatory process, yet our ability to inhibit the excessive bone formation is still insufficient. The study of new bone formation in spondyloarthritis has been mostly conducted in animal models of the disease and only few experiments have been done using human biopsies. The deregulation and overexpression of molecules involved in the osteogenesis process have been observed in bone cells, mesenchymal cells, and fibroblasts. The signaling associated to the excessive bone formation is congruent with those involved in the physiological processes of bone remodeling. Bone morphogenetic proteins and Wnt pathways have been found deregulated in this disease; however, the cause for uncontrolled stimulation remains unknown. Mechanical stress appears to play an important role in the pathological osteogenesis process; nevertheless, the association of other important factors, such as the presence of HLA-B27 and environmental factors, remains uncertain. The present review summarizes the experimental findings that describe the signaling pathways involved in the new bone formation process in spondyloarthritis in animal models and in human biopsies. The role of mechanical stress as the trigger of these pathways is also reviewed.

Bone lineage proteins in the entheses of the midfoot in patients with spondyloarthritis.

OBJECTIVE: Patients with juvenile-onset spondyloarthritis (SpA) may develop ankylosis of the midfoot resembling the spinal changes seen in patients with ankylosing spondylitis (AS). The study of the histopathology of the feet of patients with tarsitis could help us understand the pathogenesis of bone formation in affected structures in the SpA. The objective of our study was to describe the histopathologic characteristics of the midfoot in patients with tarsitis associated with SpA. METHODS: We obtained synovial sheaths, entheses, and bone samples from 20 patients with SpA with midfoot pain/tenderness and swelling. Tissue samples underwent H&E staining; immunohistochemistry for CD3, CD4, CD8, CD68, and CD20 cell identification; and immunofluorescence for bone lineage proteins, including osteocalcin, osteopontin, parathyroid hormone-related protein, bone sialoprotein, and alkaline phosphatase. RESULTS: Slight edema and hyalinization were found in some tendon sheaths, and few inflammatory cells were detected in the entheses. In bones, we found some changes suggesting osteoproliferation, including endochondral and intramembranous ossification, but no inflammatory cells. In entheses showing bone proliferation, we detected osteocalcin and osteopontin in cells with a fibroblast-mesenchymal phenotype, suggesting the induction of entheseal cells toward an osteoblast phenotype. CONCLUSION: Osteoproliferation and abnormal expression of bone lineage proteins, but no inflammatory infiltration, characterize midfoot involvement in patients with SpA. In this sense, tarsitis (or ankylosing tarsitis) resembles the involvement of the spine in patients with AS. Ossification may be in part explained by the differentiation of mesenchymal entheseal cells toward the osteoblastic lineage.

Expression of alginases and alginate polymerase genes in response to oxygen, and their relationship with the alginate molecular weight in Azotobacter vinelandii.

The transcription of genes involved in alginate polymerization and depolymerization, as well as the alginase activity (extracellular and intracellular) under oxygen-limited and non oxygen-limited conditions in cultures of A. vinelandii, was studied. Two levels of dissolved oxygen tension (DOT) (1% and 5%, oxygen-limited and non-oxygen-limited, respectively) strictly controlled by gas blending, were evaluated in a wild type strain. In cultures at low DOT (1%), in which a high molecular weight alginate (1200 kDa) was synthesized, the transcription levels of alg8 and alg44 (genes encoding alginate polymerase complex), and algX (encoding a protein involved in polymer transport through periplasmic space) were considerably higher as compared to cultures conducted at 5% DOT, under which an alginate with a low MW (42 kDa) was produced. In the case of genes encoding for intracellular and extracellular alginases, the levels of these transcripts were higher at 1% DOT. However, intracellular and extracellular alginase activity were lower (0.017 and 0.01 U/mg protein, respectively) in cultures at 1% DOT, as compared with the activities measured at 5% DOT (0.027 and 0.052 U/mg protein for intracellular and extracellular maximum activity, respectively). The low alginase activity measured in cultures at 1% DOT and the high level of transcription of genes constituting alginate polymerase complex might be mechanisms by which oxygen regulates the production of alginates with a high MW.