Adaptation to chronic hypoxia (CH) enhances myocardial tolerance to ischemia/reperfusion injury and is closely associated with stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), a central transcription factor in hypoxic response. Given the central role of mitochondria in cardiac pathophysiology, we investigated the contribution of HIF-1α to cellular mechanisms underlying CH-induced cardioprotection, with a focus on proteomic remodeling, antioxidant defense, and regulation of the mitochondrial permeability transition pore (mPTP). Adult male wild-type and heterozygous Hif1a knockout mice were exposed to intermittent CH (7000 m, 8 h/day, 4 weeks) or kept under normoxia. Isolated perfused hearts treated with cyclosporine A, an inhibitor of mPTP opening, were subjected to global ischemia/reperfusion insult for infarct size determination. Quantitative label-free proteomics was conducted to assess HIF-1α-dependent changes. We evaluated oxidative stress, measured levels of proteins associated with antioxidant defense and mPTP regulation. In parallel, a proof-of-concept study was performed in transfected AC16 cardiomyocytes exposed to H2O2-induced oxidative stress. CH induced HIF-1α-dependent cardioprotection by limiting infarct size through regulation of mPTP opening. Moreover, CH attenuated oxidative stress and promoted the protective translocation of hexokinase-2 to mitochondria in an HIF-1α-dependent manner. Consistently, HIF-1α overexpression enhanced cardiomyocyte survival under oxidative stress, whereas HIF-1α inhibition by acriflavine reduced cell viability. These findings identify HIF-1α as a key mediator of CH-induced adaptive response and cardioprotection during I/R injury in the mouse heart, acting through mPTP regulation.

Although Dupuytren’s disease (DD) and relapsed Clubfoot (RC) are clinically distinct conditions, both exhibit fibrotic tissue remodeling and contracture. This exploratory study investigated whether DD and RC share molecular features associated with fibroproliferative contracture. Pathological tissues from DD nodules and contracted tissues from RC together with their respective control tissues (n = 6/group), were analyzed using label-free quantitative proteomics. The analysis identified 12 significantly upregulated proteins shared between both pathological conditions relative to their controls (|log2FC| ≥ 1, p ≤ 0.05). These proteins included structural, signaling and tensile stress ECM proteins. Functional enrichment and network analyses revealed partially overlapping dysregulation of pathways associated with ECM organization and degradation, ECM–receptor interaction, matricellular signaling and mechanobiological processes. In DD samples (n = 10), immunohistochemistry confirmed increased expression of fibrosis-associated proteins (α-SMA, TGF-β1, TGFBI, COL III, COL VI, and COL XII) (at least p < 0.01). Despite these similarities, differences in individual protein abundance and collagen crosslinking were observed between tissues. The findings suggest that DD and RC may share aspects of fibrotic ECM-remodeling despite differences in age, localization, and disease origin. These findings provide initial insights into shared ECM-remodeling processes, although their interpretation should consider the relatively small sample size and biological heterogeneity of the analyzed tissues.

Dupuytren’s disease (DD) is a common fibrotic disorder of the hand, characterized by progressive thickening and contracture of the palmar and digital fascia. Surgical excision remains the primary treatment; however, there are currently no therapies to prevent disease progression or recurrence. This study aims to develop a 3D in vitro model to test novel antifibrotic therapies. The model is based on decellularized pathological DD tissue seeded with patient-derived fibroblasts, capturing the role of both cellular and extracellular matrix components in disease progression. Fibrotic DD tissues were obtained from surgical excisions, sectioned, and decellularized. In parallel, primary fibroblasts were isolated from patient samples. The decellularized extracellular matrices (dECMs) were characterized with respect to biochemical composition, collagen structure, and mechanical properties. Fibroblasts were seeded onto the dECMs and cultured stepwise to initially promote proliferation, followed by differentiation into myofibroblasts. Secretomes of cells cultivated on the established 3D model were compared to those from conventional 2D cultivations. To evaluate the model´s relevance and effectiveness we tested the antifibrotic drug minoxidil. The dECMs retained the pathological architecture and mechanical properties of native DD tissue, although individual ECM components were reduced after decellularization. Fibroblasts successfully adhered, proliferated, and repopulated the scaffold. The relevance of the 3D model was demonstrated by the presence of myofibroblasts with disease–relevant secretome. The responsiveness to the drug minoxidil was significantly more complex in the 3D model than in conventional 2D cultures. We demonstrated that dECM seeded with DD fibroblasts represents a relevant 3D in vitro model of Dupuytren’s disease. The model enables antifibrotic drug screening, as demonstrated by the testing of minoxidil. Our model provides a reproducible platform also suitable for the investigation of cells and ECM contributions to palmar fascial fibrosis.

Investigating prenatal hypoxia is difficult in mammals, as there are confounding factors stemming from maternal adaptations and compensatory mechanisms. We have thus established an avian model of hypoxic incubation (starting after 2 days of normoxia, 15% O2, normobaric, until the time of sampling at embryonic day 8) to study embryonic reactions to low oxygen concentration. Our previous studies have shown increased vascularization, oedema, and ventricular wall thinning preceding the lethality at mid-gestation. Analysis of the cardiac proteome after 6 days of hypoxic incubation showed strong upregulation of enzymes involved in anaerobic glycolysis as well as an increase in apoptosis-related proteins, cell adhesion proteins, and secretory activity.

Fibrotic changes in pediatric clubfoot provide an opportunity to improve corrective therapy and prevent relapses with targeted drugs. This study defines the parameters of clubfoot fibrosis and presents a unique analysis of a simple pseudo-3D in vitro model for disease-specific high-throughput drug screening experiments. The model combines clubfoot-derived fibroblasts with a biomimetic cultivation environment induced by the water-soluble polymers Ficoll and Polyvinylpyrrolidone, utilizing the principle of macromolecular crowding. We achieved higher conversion of soluble collagen into insoluble collagen, accelerated formation of the extracellular matrix layer and upregulated fibrosis-related genes in the mixed Ficoll environment. To test the model, we evaluated the effect of a potential antifibrotic drug, minoxidil, emphasizing collagen content and cross-linking. While the model amplified overall collagen deposition, minoxidil effectively blocked the expression of lysyl hydroxylases, which are responsible for the increased occurrence of specific collagen cross-linking in various fibrotic tissues. This limited the formation of collagen cross-link in both the model and control environments. Our findings provide a tool for expanding preclinical research for clubfoot and similar fibroproliferative conditions.

Valvular heart disease leads to ventricular pressure and/or volume overload. Pressure overload leads to fibrosis, which might regress with its resolution, but the limits and details of this reverse remodeling are not known. To gain more insight into the extent and nature of cardiac fibrosis in valve disease, we analyzed needle biopsies taken from the interventricular septum of patients undergoing surgery for valve replacement focusing on the expression and distribution of major extracellular matrix protein involved in this process. Proteomic analysis performed using mass spectrometry revealed an excellent correlation between the expression of collagen type I and III, but there was little correlation with the immunohistochemical staining performed on sister sections, which included antibodies against collagen I, III, fibronectin, sarcomeric actin, and histochemistry for wheat germ agglutinin. Surprisingly, the immunofluorescence intensity did not correlate significantly with the gold standard for fibrosis quantification, which was performed using Picrosirius Red (PSR) staining, unless multiplexed on the same tissue section. There was also little correlation between the immunohistochemical markers and pressure gradient severity. It appears that at least in humans, the immunohistochemical pattern of fibrosis is not clearly correlated with standard Picrosirius Red staining on sister sections or quantitative proteomic data, possibly due to tissue heterogeneity at microscale, comorbidities, or other patient-specific factors. For precise correlation of different types of staining, multiplexing on the same section is the best approach.

Talipes equinovarus congenitus (clubfoot) is frequently defined as a stiff, contracted deformity, but few studies have described the tissue from the point of view of the extracellular matrix, and none have quantified its mechanical properties. Several researchers have observed that clubfoot exhibits signs of fibrosis in the medial side of the deformity that are absent in the lateral side. Our study aims to quantify the differences between the medial and lateral side tissue obtained from relapsed clubfoot during surgery in terms of the morphological and mechanical properties of the tissue. Combining methods of optical and atomic force microscopy, our study revealed that the medial side has a higher Young’s modulus, contains more collagen and less adipose tissue and that the collagen fibers propagate at a higher frequency of the crimp pattern after surgical dissection of the tissue. Our study offers a multi-correlative approach that thoroughly investigates the relapsed clubfoot tissue.

Scaffolds made of degradable polymers, such as collagen, polyesters or polysaccharides, are promising matrices for fabrication of bioartificial vascular grafts or patches. In this study, collagen isolated from porcine skin was processed into a gel, reinforced with collagen particles and with incorporated adipose tissue-derived stem cells (ASCs). The cell-material constructs were then incubated in a DMEM medium with 2% of FS (DMEM_part), with added polyvinylalcohol nanofibers (PVA_part sample), and for ASCs differentiation towards smooth muscle cells (SMCs), the medium was supplemented either with human platelet lysate released from PVA nanofibers (PVA_PL_part) or with TGF-β1 + BMP-4 (TGF + BMP_part). The constructs were further endothelialised with human umbilical vein endothelial cells (ECs). The immunofluorescence staining of alpha-actin and calponin, and von Willebrand factor, was performed. The proteins involved in cell differentiation, the extracellular matrix (ECM) proteins, and ECM remodelling proteins were evaluated by mass spectrometry on day 12 of culture. Mechanical properties of the gels with ASCs were measured via an unconfined compression test on day 5. Gels evinced limited planar shrinkage, but it was higher in endothelialised TGF + BMP_part gel. Both PVA_PL_part samples and TGF + BMP_part samples supported ASC growth and differentiation towards SMCs, but only PVA_PL_part supported homogeneous endothelialisation. Young modulus of elasticity increased in all samples compared to day 0, and PVA_PL_part gel evinced a slightly higher ratio of elastic energy. The results suggest that PVA_PL_part collagen construct has the highest potential to remodel into a functional vascular wall.

Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We compared the expression of hypoxia-related proteins in the tissue of the contracted (medial) side of relapsed clubfoot, and in the tissue of the non-contracted (lateral) side of relapsed clubfoot. Tissue samples from ten patients were analyzed by immunohistochemistry and image analysis, Real-time PCR and Mass Spectrometry to evaluate the differences in protein composition and gene expression. We found a significant increase in the levels of smooth muscle actin, transforming growth factor-beta, hypoxia-inducible factor 1 alpha, lysyl oxidase, lysyl oxidase-like 2, tenascin C, matrix metalloproteinase-2, matrix metalloproteinase-9, fibronectin, collagen types III and VI, hemoglobin subunit alpha and hemoglobin subunit beta, and an overexpression of ACTA2 , FN1 , TGFB1 , HIF1A and MMP2 genes in the contracted medial side tissue of clubfoot. In the affected tissue, we have identified an increase in the level of hypoxia-related proteins, together with an overexpression of corresponding genes. Our results suggest that the hypoxia-associated pathway is potentially a factor contributing to the etiology of clubfoot relapses, as it stimulates both angioproliferation and fibroproliferation, which are considered to be key factors in the progression and development of relapses.

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, β-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10–40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient’s cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes. The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus. Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.

To the Editor: Angiogenesis defines a common process in physiological fetal growth, wound healing, and endometrial hyperplasia within the menstrual cycle. Under these conditions, it is intricately regulated: that is, “turned on” for a limited period of time and then inhibited.1 However, many pathologic states are marked by an upregulated and persistent vessel generation. Angiogenesis is a complex dynamic process that is discontinuous in time and space, and its continuous perpetuation generates a structural and functional variety of the vessel sizes, shapes, connecting patterns, and blood flow.1 Recently, Novotny et al2 published a research article entitled “Increased microvessel and arteriole density in the contracted side of the relapsed clubfoot” aimed at comparing the microvessel density (MVD), arteriole density, and concentration of proangiogenic proteins in the contracted medial side versus the noncontracted lateral side of relapsed clubfeet in tissue samples taken from 10 young patients. They reported a statistically significant increase of MVD and arteriole number for cm (nearly 1300 and 600, respectively) in contracted relapsed lateral side tissues by using a subjective counting all vessels with CD31 endothelial cells without a muscular wall. Arterioles were identified by detecting their internal elastic lamina with the use of orcein and hematoxylin staining. In addition, arterioles were defined as completely surrounded by dense irregular connective tissue. This was accompanied by a significant increase in the levels of vascular endothelial growth factor, vascular endothelial growth factor receptor 2, β-catenin, and active βcatenin.2 In 2002, Hlatky et al3 stated that MVD is a subjective parameter (ie, observer-dependent estimate), and not a valid measure to guide or evaluate the angiogenic status. Furthermore, MVD may not vary in accordance with the tissue or blood levels of any single proangiogenic molecule. Quantitative descriptors of vascular irregularity can be, however, abstracted from the fractal geometry introduced by Benoit Mandelbroit (1924-2010) in 1975 and others.1 In addition, since the 1950s and 1960s, electron microscopy studies have demonstrated evident structural differences among capillaries. First, Novotny et al2 do not mention the functional heterogeneity of endothelial cells lining venous and arterial vessels in the contracted side of the relapsed clubfoot. Second, although clubfoot deformity is one of the most common birth defect, the low number of examined tissues by Novotny et al2 limits the methodological reproducibility and their scientific conclusions. Moreover, the above considerations make it indubitable that the irregular geometry of vasculature1 and its functional heterogeneity4,5 cannot be measured on the basis of MVD estimate alone or in combination with proangiogenic and antiangiogenic mediators amounts. Only introducing an appropriate sample size and quantitative indicators the findings reported in the study by Novotny et al2 can be helpful and reproducible. Aristotle (384-322BC) was the first Greek philosopher and thinker to devise a logical system that can be objectively determined by its “quantity” (ie, the extension of an object) “qualities” (ie, the functional nature of an object) and configuration pattern (ie, “where” is the position in relation to the surrounding environment). Scientific knowledge advances through the introduction of new concepts, and this process is driven by more appropriate methodologies that provide previously unavailable observations. The broad applicability of quantitative methods in a logical Aristotle way makes it possible to explore the range of the morphologic variability of neovascularity that can be observed in nature, thus increasing its importance in preclinical and clinical research. Fabio Grizzi, PhD* Daniel Yiu, MD† Maurizio Chiriva-Internati, PhD‡§ Gianluigi Taverna, MD∥ *Department of Immunology and Inflammation, Humanities Clinical and Research Center, Rozzano, Milan ∥Department of Urology, Humanitas Mater Domini, Castellanza, Varese, Italy †Oxford University Hospitals NHS Foundation Trust, Oxford, UK ‡Department of Gastroenterology Hepatology & Nutrition, Division of Internal Medicine, The University of Texas MD Anderson Cancer Center §Kiromic Biopharma Inc., Houston, TX

Saliva contains possible biomarkers that are associated with dental caries. The present study aimed to analyse differences in the abundance of proteins in the saliva between caries-positive (CP; N = 15) and caries-free (CF; N = 12) males and to compare differences in the abundance of proteins between two saliva sample fractions (supernatant and pellet). We found 14 differently significantly expressed proteins in the CF group when comparing the supernatant fractions of the CP and CF groups, and three proteins in the pellet fractions had significantly higher expression in the CP group. Our results indicate very specific protein compositions of the saliva in relation to dental caries resistance (the saliva of the CP group mainly contained pellet proteins and the saliva of the CF group mainly contained supernatant proteins). This was the first time that the saliva pellet fraction was analysed in relation to the dental caries status. We detected specific calcium-binding proteins that could have decalcified enamel in the saliva pellet of the CP group. We also observed significantly up-regulated immune proteins in the saliva supernatant of the CF group that could play an important role in the caries prevention. The particular protein compositions of the saliva pellet and supernatant in the groups with different susceptibilities to tooth decay is a promising finding for future research.

Background: Clubfoot deformity (pes equinovarus) is one of the most common birth defects, and its etiology is still unknown. Initial clubfoot treatment is based on the Ponseti method throughout most of the world. Despite the effectiveness of this therapy, clubfoot may relapse. Recent studies confirm the theory of active fibrotic remodeling processes in the extracellular matrix of the affected tissue. The aim of this study was to clarify whether relapses in clubfoot therapy are associated with altered angiogenesis and to suggest possible regulatory pathways of this pathologic process. Methods: We compared microvessel density, arteriole density, and concentration of angioproliferative-related proteins found between tissues in the contracted, that is, the medial side (M-side), and noncontracted, that is, the lateral side (L-side) of the relapsed clubfeet. Tissue samples from 10 patients were analyzed. Histopathologic analysis consisted of immunohistochemistry and image analysis. Real-time polymerase chain reaction was used to study mRNA expression. Results: An increase in microvessel and arteriole density was noted in contracted, relapsed clubfoot tissue. This was accompanied by a significant increase in the levels of the vascular endothelial growth factor, vascular endothelial growth factor receptor 2, beta catenin and active beta catenin. Vascular endothelial growth factor, vascular endothelial growth factor receptor 2, and CD31 overexpression was also seen with mRNA analysis. Conclusions: Increased microvessel and arteriole density in the contracted side of the relapsed clubfoot was noted. These processes are mediated by specific proangiogenic proteins that are overexpressed in the contracted tissue. These findings contribute to the etiology and the development of relapses in the treatment of clubfoot.

Aim Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed thein vitroantifibrotic effects of minoxidil on clubfoot-derived cells. Methods Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. Results MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to >= 0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). Conclusions Minoxidil exerts anin vitroinhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.