Corrigendum: Random cellulose acetate nanofibers: a breakthrough for cultivated meat production.

[This corrects the article DOI: 10.3389/fnut.2023.1297926.].

Femoral Neck Thickness Index as an Indicator of Proximal Femur Bone Modeling.

The alteration in the shape of the femoral neck is an important radiographic sign for scoring canine hip dysplasia (CHD). Previous studies have reported that the femoral neck thickness (FNT) is greater in dogs with hip joint dysplasia, becoming progressively thicker with disease severity. The main objective of this work was to describe a femoral neck thickness index (FNTi) to quantify FNT and to study its association with the degree of CHD using the Fédération Cynologique Internationale (FCI) scheme. A total of 53 dogs (106 hips) were randomly selected for this study. Two examiners performed FNTi estimation to study intra- and inter-examiner reliability and agreement. The paired t-test, the Bland-Altman plots, and the intraclass correlation coefficient showed excellent agreement and reliability between the measurements of the two examiners and the examiners' sessions. All joints were scored in five categories by an experienced examiner according to FCI criteria. The results from examiner 1 were compared between FCI categories. Hips that were assigned an FCI grade of A (n = 19), B (n = 23), C (n = 24), D (n = 24), and E (n = 16) had a mean ± standard deviation FNTi of 0.809 ± 0.024, 0.835 ± 0.044, 0.868 ± 0.022, 0.903 ± 0.033, and 0.923 ± 0.068, respectively (ANOVA, p < 0.05). Therefore, these results show that FNTi is a parameter capable of evaluating proximal femur bone modeling and that it has the potential to enrich conventional CHD scoring criteria if incorporated into a computer-aided diagnosis capable of detecting CHD.

Artificial Intelligence in Veterinary Imaging: An Overview.

Artificial intelligence and machine learning have been increasingly used in the medical imaging field in the past few years. The evaluation of medical images is very subjective and complex, and therefore the application of artificial intelligence and deep learning methods to automatize the analysis process would be very beneficial. A lot of researchers have been applying these methods to image analysis diagnosis, developing software capable of assisting veterinary doctors or radiologists in their daily practice. This article details the main methodologies used to develop software applications on machine learning and how veterinarians with an interest in this field can benefit from such methodologies. The main goal of this study is to offer veterinary professionals a simple guide to enable them to understand the basics of artificial intelligence and machine learning and the concepts such as deep learning, convolutional neural networks, transfer learning, and the performance evaluation method. The language is adapted for medical technicians, and the work already published in this field is reviewed for application in the imaging diagnosis of different animal body systems: musculoskeletal, thoracic, nervous, and abdominal.

Femoral parallelism: evaluation and impact of variation on canine hip dysplasia assessment.

Adequate radiographic positioning on the X-ray table is paramount for canine hip dysplasia (HD) screening. The aims of this study were to evaluate femoral parallelism on normal ventrodorsal hip extended (VDHE) view and the effect of femoral angulation (FA) on Norberg Angle (NA) and Hip Congruency Index (HCI). The femoral parallelism was evaluated comparing the alignment of the long femoral axis with the long body axis in normal VDHE views and the effect of FA on NA and HCI on repeated VDHE views with different levels of FA. The femoral long axis in normal VDHE views showed a ranged of FA from -4.85° to 5.85°, mean ± standard deviation (SD) of -0.06 ± 2.41°, 95% CI [-4.88, 4.76°]. In the paired views, the mean ± SD femur adduction of 3.69 ± 1.96° led to a statistically significant decrease NA, and HCI, and femur abduction of 2.89 ± 2.12 led to a statistically significant increase in NA and HCI (p < 0.05). The FA differences were also significantly correlated with both NA differences (r = 0.83) and HCI differences (r = 0.44) (p < 0.001). This work describes a methodology that allows evaluation of femoral parallelism in VDHE views and the results suggest that femur abduction yielded more desirable NA and HCI values and adduction impaired NA and HCI values. The positive linear association of FA with NA and HCI allows the use of regression equations to create corrections, to reduce the influence of poor femoral parallelism in the HD scoring.

Studying and Analyzing Humane Endpoints in the Fructose-Fed and Streptozotocin-Injected Rat Model of Diabetes.

This work aimed to define a humane endpoint scoring system able to objectively identify signs of animal suffering in a rat model of type 2 diabetes. Sprague-Dawley male rats were divided into control and induced group. The induced animals drink a 10% fructose solution for 14 days. Then, received an administration of streptozotocin (40 mg/kg). Animals' body weight, water and food consumption were recorded weekly. To evaluate animal welfare, a score sheet with 14 parameters was employed. Blood glucose levels were measured at three time points. After seven weeks of initiating the protocol, the rats were euthanized. The induced animals showed weight loss, polyuria, polyphagia, and polydipsia. According to our humane endpoints table, changes in animal welfare became noticeable after the STZ administration. None of the animals hit the critical score limit (four). Data showed that the most effective parameters to assess welfare in this type 2 diabetes rat induction model were dehydration, grooming, posture, abdominal visualization, and stool appearance. The glycemia was significantly higher in the induced group when compared to the controls (p < 0.01). Induced animals' murinometric and nutritional parameters were significantly lower than the controls (p < 0.01). Our findings suggest that in this rat model of type 2 diabetes with STZ-induced following fructose consumption, our list of humane endpoints is suitable for monitoring the animals' welfare.

Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212 nm vs. 284 ± 130 nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Random cellulose acetate nanofibers: a breakthrough for cultivated meat production.

Overcoming the challenge of creating thick, tissue-resembling muscle constructs is paramount in the field of cultivated meat production. This study investigates the remarkable potential of random cellulose acetate nanofibers (CAN) as a transformative scaffold for muscle tissue engineering (MTE), specifically in the context of cultivated meat applications. Through a comparative analysis between random and aligned CAN, utilizing C2C12 and H9c2 myoblasts, we unveil the unparalleled capabilities of random CAN in facilitating muscle differentiation, independent of differentiation media, by exploiting the YAP/TAZ-related mechanotransduction pathway. In addition, we have successfully developed a novel process for stacking cell-loaded CAN sheets, enabling the production of a three-dimensional meat product. C2C12 and H9c2 loaded CAN sheets were stacked (up to four layers) to form a ~300-400 µm thick tissue 2 cm in length, organized in a mesh of uniaxial aligned cells. To further demonstrate the effectiveness of this methodology for cultivated meat purposes, we have generated thick and viable constructs using chicken muscle satellite cells (cSCs) and random CAN. This groundbreaking discovery offers a cost-effective and biomimetic solution for cultivating and differentiating muscle cells, forging a crucial link between tissue engineering and the pursuit of sustainable and affordable cultivated meat production.

Active learning for data efficient semantic segmentation of canine bones in radiographs.

X-ray bone semantic segmentation is one crucial task in medical imaging. Due to deep learning's emergence, it was possible to build high-precision models. However, these models require a large quantity of annotated data. Furthermore, semantic segmentation requires pixel-wise labeling, thus being a highly time-consuming task. In the case of hip joints, there is still a need for increased anatomic knowledge due to the intrinsic nature of the femur and acetabulum. Active learning aims to maximize the model's performance with the least possible amount of data. In this work, we propose and compare the use of different queries, including uncertainty and diversity-based queries. Our results show that the proposed methods permit state-of-the-art performance using only 81.02% of the data, with O ( 1 ) time complexity.

Acetabular Coverage Area Occupied by the Femoral Head as an Indicator of Hip Congruency.

Accurate radiographic screening evaluation is essential in the genetic control of canine HD, however, the qualitative assessment of hip congruency introduces some subjectivity, leading to excessive variability in scoring. The main objective of this work was to validate a method-Hip Congruency Index (HCI)-capable of objectively measuring the relationship between the acetabulum and the femoral head and associating it with the level of congruency proposed by the Fédération Cynologique Internationale (FCI), with the aim of incorporating it into a computer vision model that classifies HD autonomously. A total of 200 dogs (400 hips) were randomly selected for the study. All radiographs were scored in five categories by an experienced examiner according to FCI criteria. Two examiners performed HCI measurements on 25 hip radiographs to study intra- and inter-examiner reliability and agreement. Additionally, each examiner measured HCI on their half of the study sample (100 dogs), and the results were compared between FCI categories. The paired t-test and the intraclass correlation coefficient (ICC) showed no evidence of a systematic bias, and there was excellent reliability between the measurements of the two examiners and examiners' sessions. Hips that were assigned an FCI grade of A (n = 120), B (n = 157), C (n = 68), D (n = 38) and E (n = 17) had a mean HCI of 0.739 ± 0.044, 0.666 ± 0.052, 0.605 ± 0.055, 0.494 ± 0.070 and 0.374 ± 0.122, respectively (ANOVA, p < 0.01). Therefore, these results show that HCI is a parameter capable of estimating hip congruency and has the potential to enrich conventional HD scoring criteria if incorporated into an artificial intelligence algorithm competent in diagnosing HD.

Early hip laxity screening and later canine hip dysplasia development.

Background and Aim: Passive hip laxity (PHL) is considered the primary risk factor for canine hip dysplasia (HD) and is estimated, in stress hip radiographs, using the distraction index (DI). The study aimed to associate the early PHL using the hip Distractor of University of Trás-os-Montes and Alto Douro (DisUTAD) and the late HD grades. Materials and Methods: A total of 41 dogs (82 hips) were submitted to a follow-up study. First, between 4 and 12 months of age, dogs were radiographed using the DisUTAD hip distractor and were determined the DI for each hip joint. Then, after 12 months of age, dogs were reevaluated for HD using the conventional hip ventrodorsal projection and hips were evaluated for HD using the Fédération Cynologique Internationale (FCI) scoring system. Results: Hips of dogs' in the second examination with FCI grades of A (n=28), B (n=11), C (n=22), and D and E (n=21) had an early DI of 0.32±0.1, 0.38±0.08, 0.50±0.12, and 0.64±0.11, respectively. Statistical analysis using the general linear model univariate, with the DI as dependent variable and the FCI grades, side and sex as fixed factors, and the post hoc Bonferroni correction test showed significant differences among FCI grades (p<0.05). Conclusion: These results show the association between early DI and the late FCI HD grades and the DisUTAD is recommended for the early canine HD diagnosis.

Transcriptomic characterization of the molecular mechanisms induced by RGMa during skeletal muscle nuclei accretion and hypertrophy.

BACKGROUND: The repulsive guidance molecule a (RGMa) is a GPI-anchor axon guidance molecule first found to play important roles during neuronal development. RGMa expression patterns and signaling pathways via Neogenin and/or as BMP coreceptors indicated that this axon guidance molecule could also be working in other processes and diseases, including during myogenesis. Previous works from our research group have consistently shown that RGMa is expressed in skeletal muscle cells and that its overexpression induces both nuclei accretion and hypertrophy in muscle cell lineages. However, the cellular components and molecular mechanisms induced by RGMa during the differentiation of skeletal muscle cells are poorly understood. In this work, the global transcription expression profile of RGMa-treated C2C12 myoblasts during the differentiation stage, obtained by RNA-seq, were reported. RESULTS: RGMa treatment could modulate the expression pattern of 2,195 transcripts in C2C12 skeletal muscle, with 943 upregulated and 1,252 downregulated. Among them, RGMa interfered with the expression of several RNA types, including categories related to the regulation of RNA splicing and degradation. The data also suggested that nuclei accretion induced by RGMa could be due to their capacity to induce the expression of transcripts related to 'adherens junsctions' and 'extracellular-cell adhesion', while RGMa effects on muscle hypertrophy might be due to (i) the activation of the mTOR-Akt independent axis and (ii) the regulation of the expression of transcripts related to atrophy. Finally, RGMa induced the expression of transcripts that encode skeletal muscle structural proteins, especially from sarcolemma and also those associated with striated muscle cell differentiation. CONCLUSIONS: These results provide comprehensive knowledge of skeletal muscle transcript changes and pathways in response to RGMa.

SAP30 Gene Is a Probable Regulator of Muscle Hypertrophy in Chickens.

Animals with muscle hypertrophy phenotype are targeted by the broiler industry to increase the meat production and the quality of the final product. Studies characterizing the molecular machinery involved with these processes, such as quantitative trait loci studies, have been carried out identifying several candidate genes related to this trait; however, validation studies of these candidate genes in cell culture is scarce. The aim of this study was to evaluate SAP30 as a candidate gene for muscle development and to validate its function in cell culture in vitro. The SAP30 gene was downregulated in C2C12 muscle cell culture using siRNA technology to evaluate its impact on morphometric traits and gene expression by RNA-seq analysis. Modulation of SAP30 expression increased C2C12 myotube area, indicating a role in muscle hypertrophy. RNA-seq analysis identified several upregulated genes annotated in muscle development in treated cells (SAP30-knockdown), corroborating the role of SAP30 gene in muscle development regulation. Here, we provide experimental evidence of the involvement of SAP30 gene as a regulator of muscle cell hypertrophy.

Refinement of Animal Model of Colorectal Carcinogenesis through the Definition of Novel Humane Endpoints.

This study aimed to define appropriate humane endpoints (HEs) for an animal model of colorectal carcinogenesis (CRC). Twenty-nine male Wistar rats were divided into two control groups (CTRL1 and CTRL2) injected with ethylenediamine tetraacetic acid (EDTA)-saline solutions and two induced groups (CRC1 and CRC2) injected with 1,2-dimethylhydrazine (DMH) for seven weeks. A score sheet with 14 biological parameters was used to assess animal welfare. Groups CRC1 and CTRL1 and groups CRC2 and CTRL2 were euthanized 11 and 17 weeks after the first DMH administration, respectively. Five animals from the induced groups died unexpectedly during the protocol (survival rates of 75.0% and 66.7% for groups CRC1 and CRC2, respectively). The final mean body weight (BW) was smaller in the CRC groups when compared with that in the CTRL groups. A uniformity of characteristics preceding the premature animals' death was observed, namely an increase of 10% in mean BW, swollen abdomen, diarrhea, and priapism. The surface abdominal temperature of group CRC2 was significantly higher, when compared with that of group CTRL2. The parameters already described in other cancer models proved to be insufficient. For the CRC model, we considered assessing the abdominal temperature, priapism, and sudden increase in the BW.

RGMa can induce skeletal muscle cell hyperplasia via association with neogenin signalling pathway.

Although originally discovered inducing important biological functions in the nervous system, repulsive guidance molecule a (RGMa) has now been identified as a player in many other processes and diseases, including in myogenesis. RGMa is known to be expressed in skeletal muscle cells, from somites to the adult. Functional in vitro studies have revealed that RGMa overexpression could promote skeletal muscle cell hypertrophy and hyperplasia, as higher efficiency in cell fusion was observed. Here, we extend the potential role of RGMa during C2C12 cell differentiation in vitro. Our results showed that RGMa administrated as a recombinant protein during late stages of C2C12 myogenic differentiation could induce myoblast cell fusion and the downregulation of different myogenic markers, while its administration at early stages induced the expression of myogenic markers with no detectable morphological effects. We also found that RGMa effects on skeletal muscle hyperplasia are performed via neogenin receptor, possibly as part of a complex with other proteins. Additionally, we observed that RGMa-neogenin is not playing a role as an inhibitor of the BMP signalling in skeletal muscle cells. This work contributes to placing RGMa as a component of the mechanisms that determine skeletal cell fusion via neogenin receptor.

Radiographic assessment of humeroulnar congruity in a medium and a large breed of dog.

Elbow joint incongruity is recognized as an important factor in the development, treatment, and prognosis of canine elbow dysplasia. Elbow incongruity has been measured based on radiographic joint space widths, however these values can be affected by the degree of elbow joint flexion. Recent studies have reported radiographic curvature radii as more precise measures of humeroulnar congruity in dogs. The aim of this prospective observational study was to describe radiographic curvature radii measured from flexed and extended elbow radiographs for a sample of dogs representing a medium breed (Portuguese Pointing Dog) and a large breed (Estrela Mountain Dog). The curvature radii from the ulnar trochlear notch and humeral trochlea were measured in 114 mediolateral elbow extended radiographic views (30 Portuguese Pointing Dog and 27 Estrela Mountain Dog), and 84 mediolateral flexed views (22 Portuguese Pointing Dog and 20 Estrela Mountain Dog). The sampled animals' ages ranged from 12 to 84 months (34.6 ± 17.8 months). Good agreement was observed between curvature radii measurements for flexed vs. extended views in both breed groups. Ulnar trochlear notch curvature radii measurements were greater than humeral trochlea curvature radii measurements in both breed groups. Both curvature radii were greater in the large-breed dog group vs. the medium-breed dog group. Both breed groups had ulnar and humeral curves with similar typology. However, the large breed group had greater intermediate differences between the humeroulnar surface curvature radii. Results from this study supported the use of curvature radii as measures of humeroulnar congruity in mediolateral flexed elbow radiographs of medium and large breed dogs.

Synergistic Effect of Carboplatin and Piroxicam on Two Bladder Cancer Cell Lines.

BACKGROUND/AIM: This study aimed to evaluate the in vitro efficacy of carboplatin and piroxicam, both in isolation and combined, against T24 and 5637 human urinary bladder cancer cell lines. MATERIALS AND METHODS: Cell viability, drug interaction, cell morphology, cell proliferation, apoptosis and autophagy were analyzed after 72 h of drug exposure. Statistical analysis was performed and values of p<0.05 were considered statistically significant. RESULTS: Drug exposure in combination led to a significant reduction of cell viability comparatively to single-drug exposure. These combinations resulted in a synergistic interaction in the T24 (combination index for 50% effect (CI50)=0.65) and 5637 (CI50=0.17) cell lines. Notable increase of morphological alterations, a marked decrease of Ki-67 expression, a considerable enhancement of autophagic vacuoles and a minimal effect on apoptosis was observed in both cell lines treated with combined drugs. CONCLUSION: Data showed that in vitro combination of carboplatin and piroxicam produced a more potent antiproliferative effect when compared to single drugs.

FGF2, FGF3 and FGF4 expression pattern during molars odontogenesis in Didelphis albiventris.

Odontogenesis is guided by a complex signaling cascade in which several molecules, including FGF2-4, ensure all dental groups development and specificity. Most of the data on odontogenesis derives from rodents, which does not have all dental groups. Didelphis albiventris is an opossum with the closest dentition to humans, and the main odontogenesis stages occur when the newborns are in the pouch. In this study, D. albiventris postnatals were used to characterize the main stages of their molars development; and also to establish FGF2, FGF3 and FGF4 expression pattern. D. albiventris postnatals were processed for histological and indirect immunoperoxidase analysis of the tooth germs. Our results revealed similar dental structures between D. albiventris and mice. However, FGF2, FGF3 and FGF4 expression patterns were observed in a larger number of dental structures, suggesting broader functions for these molecules in this opossum species. The knowledge of the signaling that determinates odontogenesis in an animal model with complete dentition may contribute to the development of therapies for the replacement of lost teeth in humans. This study may also contribute to the implementation of D. albiventris as model for Developmental Biology studies.

Ultrasonography as the Gold Standard for In Vivo Volumetric Determination of Chemically-induced Mammary Tumors.

BACKGROUND/AIM: In this study, we evaluated the dimensions and volume of rat mammary tumors and the association of these variables with tumor invasiveness. MATERIALS AND METHODS: Tumors were measured by caliper and ultrasonography. Volume was determined by water displacement and by application of four formulas using tumor length (L), width (W) and depth (D) or tumor weight. RESULTS: Results confirmed the data obtained in our previous work, where we verified that mammary tumors grow as oblate spheroids. CONCLUSION: The determination of mammary tumor volume by applying the formula V=(4/3)×π×(L/2)×(L/2)×(D/2) is the best way to evaluate tumor volume in vivo. Beyond volume evaluation by water displacement, the determination on the basis of tumor weight is the most accurate way to evaluate tumor volume after animal sacrifice or tumor excision. According to our results, it is not possible to predict if a tumor is invasive or non-invasive by its dimensions, volume or weight. Future work in chemically-induced mammary cancer should use ultrasonography and water displacement or tumor weight to determine tumor volume in vivo and after animal sacrifice or tumor excision, respectively.

The oxygen uptake slow component at submaximal intensities in breaststroke swimming.

The present work proposed to study the oxygen uptake slow component (VO2 SC) of breaststroke swimmers at four different intensities of submaximal exercise, via mathematical modeling of a multi-exponential function. The slow component (SC) was also assessed with two different fixed interval methods and the three methods were compared. Twelve male swimmers performed a test comprising four submaximal 300 m bouts at different intensities where all expired gases were collected breath by breath. Multi-exponential modeling showed values above 450 ml·min-1 of the SC in the two last bouts of exercise (those with intensities above the lactate threshold). A significant effect of the method that was used to calculate the VO2 SC was revealed. Higher mean values were observed when using mathematical modeling compared with the fixed interval 3rd min method (F=7.111; p=0.012; η2=0.587); furthermore, differences were detected among the two fixed interval methods. No significant relationship was found between the SC determined by any method and the blood lactate measured at each of the four exercise intensities. In addition, no significant association between the SC and peak oxygen uptake was found. It was concluded that in trained breaststroke swimmers, the presence of the VO2 SC may be observed at intensities above that corresponding to the 3.5 mM-1 threshold. Moreover, mathematical modeling of the oxygen uptake on-kinetics tended to show a higher slow component as compared to fixed interval methods.

Synergistic effect between cisplatin and sunitinib malate on human urinary bladder-cancer cell lines.

The aim of this paper is to analyse sunitinib malate in vitro ability to enhance cisplatin cytotoxicity in T24, 5637, and HT1376 human urinary bladder-cancer cell lines. Cells were treated with cisplatin (3, 6, 13, and 18 µM) and sunitinib malate (1, 2, 4, 6, and 20 µM), either in isolation or combined, over the course of 72 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, acridine orange, and monodansylcadaverine staining and flow cytometry were performed. The combination index (CI) was calculated based on the Chou and Talalay method. In isolation, cisplatin and sunitinib malate statistically (P < 0.05) decrease cell viability in all cell lines in a dose-dependent manner, with the presence of autophagic vacuoles. A cell cycle arrest in early S-phase and in G0/G1-phase was also found after exposure to cisplatin and sunitinib malate, in isolation, respectively. Treatment of urinary bladder-cancer cells with a combination of cisplatin and sunitinib malate showed a synergistic effect (CI < 1). Autophagy and apoptosis studies showed a greater incidence when the combined treatment was put into use. This hints at the possibility of a new combined therapeutic approach. If confirmed in vivo, this conjugation may provide a means of new perspectives in muscle-invasive urinary bladder cancer treatment.