Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo. IMPORTANCE: Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

A Rare Case of Recurrent Cystitis in a Primary Care Setting.

Uncomplicated cystitis is common in women and typically presents with symptoms such as increased urinary frequency, dysuria, suprapubic pain, and urgency. Escherichia coli is the most frequently identified pathogen in these cases. Colovesical fistulas constitute an uncommon etiology of recurrent urinary tract infections, and they are even rarer in women due to the protective barrier provided by the uterus. Faecaluria and pneumaturia are the pathognomonic symptoms of these types of fistulas that help differentiate them from recurrent cystitis. While the gold standard imaging is the abdominopelvic CT scan, in some instances, MRI may be necessary to identify fistulous tracts. This case report describes a scenario of recurrent urinary tract infection caused by a colovesical fistula, in a woman with a history of diverticular disease. In contrast to uncomplicated recurrent cystitis, the treatment of the fistula is surgical. The aim of this article is to raise awareness of this potential and rare cause of recurrent urinary tract infection encountered in a primary healthcare setting, in order to prevent the prescription of multiple cycles of ineffective antibiotic therapy in these patients and the consequent development of antimicrobial resistance, a global public health issue. Our intention is to alert general practitioners about the diagnosis of a rare cause of recurrent cystitis, the treatment of which is surgical and warrants referral to secondary care.

Characterization of Functional Coatings on Cork Stoppers with Laser-Induced Breakdown Spectroscopy Imaging.

Evaluating the efficiency of surface treatments is a problem of paramount importance for the cork stopper industry. Generically, these treatments create coatings that aim to enhance the impermeability and lubrification of cork stoppers. Yet, current methods of surface analysis are typically time-consuming, destructive, have poor representativity or rely on indirect approaches. In this work, the use of a laser-induced breakdown spectroscopy (LIBS) imaging solution is explored for evaluating the presence of coating along the cylindrical surface and in depth. To test it, several cork stoppers with different shaped areas of untreated surface were analyzed by LIBS, making a rectangular grid of spots with multiple shots per spot, to try to identify the correspondent shape. Results show that this technique can detect the untreated area along with other features, such as leakage and holes, allowing for a high success rate of identification and for its performance at different depths, paving the way for future industry-grade quality control solutions with more complex surface analysis.

Toxoplasma gondii harbors a hypoxia-responsive coproporphyrinogen dehydrogenase-like protein.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that TgCPDH potentially functions as an alternative enzyme to perform the same reaction as CPOX under low oxygen conditions. In this study, we demonstrated that TgCPDH exhibits coproporphyrinogen dehydrogenase activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wildtype and heme-deficient parasites did not alter their intracellular growth under both ambient and low oxygen conditions. This research marks the first report of a coproporphyrinogen dehydrogenase-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings suggest that it does not directly affect intracellular infection or the pathogenesis of Toxoplasma parasites.

Cardiac Corpuscles: A "New" Morphofunctional Entity? / Corpúsculos Cardíacos: ¿Una "Nueva" Entidad Morfofuncional?

SUMMARY: The existence of "transitional muscular structures" between subendocardial branches (Purkinje fibers) and ventricular working muscle fibers (WF) was first described by the German anatomist, Kurt Goerttler, in 1964. He designated them as "subendocardial nucleus organs." He supposed such fibers functioned as mechanoreceptors, controlling of the intensity of contraction of the ventricular musculature. Brazilian anatomist Ferraz de Carvalho described similar structures in 1993. A thorough literature search failed to identify any other research articles confirming or denying their existence. The objective of this work was to find such structures in subendocardial ventricular walls in human hearts. We collected fifteen formalin-preserved hearts from the Anatomy Department of São Paulo University and sectioned the apical portions on the right and left ventricles according to method used by Goerttler. We utilized conventional histology (light microscopy- LM), scanning electron microscopy (SEM), and a new preservation method called micro- plastination (MP). At the anterior wall of the right ventricle in the subendocardial region between the interventricular septum and moderator band, we found several bundles of fusiform and helicoidal fibers of similar histology to the WF. The bundles measured between 400 and 1150 µm in length and were separated from adjacent muscular fibers by thin collagen fiber, thus acting as a "pseudo capsule." Some structures seemed to be linked to PF and were appeared to be lymphatic and blood vessels and nerves. We called those structures "cardiac corpuscles" (CC). The observation of the previously "unknown" CC in this initial study confirmed the previous descriptions and its discovery may contribute to new perspectives in the study of cardiac muscle structure and function.

La existencia de "estructuras musculares de transición" entre los ramos subendocárdicos (fibras de Purkinje) y las fibras musculares ventriculares activas(FMV) fue descrita por primera vez por el anatomista alemán Kurt Goerttler en 1964, quien las denominó "órganos del núcleo subendocárdico". Supuso que tales fibras funcionaban como mecanoreceptores, controlando la intensidad de la contracción de la musculatura ventricular. El anatomista brasileño Ferraz de Carvalho describió estructuras similares en 1993. Una búsqueda bibliográfica exhaustiva no logró identificar ningún otro artículo de investigación que confirmara o negara su existencia. El objetivo de este trabajo fue encontrar dichas estructuras en las paredes ventriculares subendocárdicas de corazones humanos. Recolectamos 15 corazones conservados en formalina del Departamento de Anatomía de la Universidad de São Paulo y seccionamos las porciones apicales de los ventrículos derecho e izquierdo según el método utilizado por Goerttler. Utilizamos histología convencional (microscopía de luz-LM), microscopía electrónica de barrido (SEM) y un nuevo método de conservación llamado microplastinación (MP). En la pared anterior del ventrículo derecho en la región subendocárdica entre el tabique interventricular y la banda moderadora, encontramos varios haces de fibras fusiformes y helicoidales de histología similar a la FMV. Los haces medían entre 400 y 1150 µm de longitud y estaban separados de las fibras musculares adyacentes por una fina fibra de colágeno, actuando así como una "pseudocápsula". Algunas estructuras parecían estar vinculadas a la fibras de purkinje y parecían ser vasos linfáticos, sanguíneos y nerviosos. Llamamos a esas estructuras "corpúsculos cardíacos" (CC). La observación del CC previamente "desconocido" en este estudio inicial confirmó las descripciones anteriores y su descubrimiento puede contribuir a nuevas perspectivas en el estudio de la estructura y función del músculo cardíaco.

Identification of Toxoplasma calcium-dependent protein kinase 3 as a stress-activated elongation factor 2 kinase.

Toxoplasma gondii is an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle, tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites, the elongation phase of protein synthesis is regulated by the Toxoplasma oxygen-sensing protein, PHYb. PHYb acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PHYb, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to the Saccharomyces cerevisiae eEF2 kinase, RCK2. In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions, CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular stress-induced eEF2 kinase.IMPORTANCEAlthough it is an obligate intracellular parasite, Toxoplasma must be able to survive in the extracellular environment. Our previous work indicated that ensuring that elongation continues during protein synthesis is part of this stress response and that this is due to preventing phosphorylation of elongation factor 2. But the identity of the eEF2 kinase has remained unknown in Toxoplasma and other protozoan parasites. Here, we identify CDPK3 as the first protozoan eEF2 kinase and demonstrate that it is part of a stress response initiated when parasites are exposed to extracellular stress. We also demonstrate that CDPK3 engages eEF2 as a result of its relocalization from the plasma membrane to the cytosol.

Transnasal endoscopy-guided percutaneous access to the sphenopalatine ganglion for neurostimulation in the treatment of primary headache: operative technique and feasibility

Abstract Objectives: Cluster headache is considered a trigeminal autonomic cephalalgia and may present with characteristic symptoms of sympathetic/parasympathetic activation on the affected side of the face, such as nasal discharge, tearing, and conjunctival injection. Invasive therapies targeting the sphenopalatine ganglion have been performed in these headache syndromes and can have a medication-sparing effect, especially in refractory, difficult-to-manage cases. The gate control theory of pain suggests that electric pulses delivered to nerve tissues can modulate neuronal activity, thus aiding in management of nociceptive or neuropathic pain, and studies have demonstrated the efficacy and safety of sphenopalatine ganglion neurostimulation. Within this context, we sought to assess the feasibility of a new surgical technique for neurostimulation of the sphenopalatine ganglion in a cadaver dissection model. Methods: The technique was developed through dissection of two cadaver heads. We divided the procedure into two stages: an endonasal endoscopic approach to expose the sphenopalatine ganglion and confirm electrode placement, and a cervicofacial approach to introduce the electrode array and position the internal pulse-generator unit. Computed tomography was performed to confirm implant placement at the end of the procedure. Results: The pulse-generator unit was successfully placed through a retroauricular incision, as is already standard for cochlear implant placement. This should reduce the incidence of perioperative sequelae, especially pain and swelling in the oral region, which are a common complication of previous approaches used for this purpose. Control imaging confirmed proper electrode placement. The device used in this study allows the patient to modulate the intensity of the stimulus, reducing or even obviating the need for drug therapy. Conclusion: The novel technique described herein, based on percutaneous access guided by transmaxillary endoscopy, can provide great precision in electrode array positioning and decreased perioperative morbidity, combining the advantages of endoscopic approaches with those of the retroauricular route. Level of evidence: 3.

Transnasal endoscopy-guided percutaneous access to the sphenopalatine ganglion for neurostimulation in the treatment of primary headache: operative technique and feasibility.

OBJECTIVES: Cluster headache is considered a trigeminal autonomic cephalalgia and may present with characteristic symptoms of sympathetic/parasympathetic activation on the affected side of the face, such as nasal discharge, tearing, and conjunctival injection. Invasive therapies targeting the sphenopalatine ganglion have been performed in these headache syndromes and can have a medication-sparing effect, especially in refractory, difficult-to-manage cases. The gate control theory of pain suggests that electric pulses delivered to nerve tissues can modulate neuronal activity, thus aiding in management of nociceptive or neuropathic pain, and studies have demonstrated the efficacy and safety of sphenopalatine ganglion neurostimulation. Within this context, we sought to assess the feasibility of a new surgical technique for neurostimulation of the sphenopalatine ganglion in a cadaver dissection model. METHODS: The technique was developed through dissection of two cadaver heads. We divided the procedure into two stages: an endonasal endoscopic approach to expose the sphenopalatine ganglion and confirm electrode placement, and a cervicofacial approach to introduce the electrode array and position the internal pulse-generator unit. Computed tomography was performed to confirm implant placement at the end of the procedure. RESULTS: The pulse-generator unit was successfully placed through a retroauricular incision, as is already standard for cochlear implant placement. This should reduce the incidence of perioperative sequelae, especially pain and swelling in the oral region, which are a common complication of previous approaches used for this purpose. Control imaging confirmed proper electrode placement. The device used in this study allows the patient to modulate the intensity of the stimulus, reducing or even obviating the need for drug therapy. CONCLUSION: The novel technique described herein, based on percutaneous access guided by transmaxillary endoscopy, can provide great precision in electrode array positioning and decreased perioperative morbidity, combining the advantages of endoscopic approaches with those of the retroauricular route.

Cardiac corpuscles: a “new” morphofunctional entity?

he existence of “transitional muscular structures” between subendocardial branches (Purkinje fibers) and ventricular working muscle fibers (WF) was first described by the German anatomist, Kurt Goerttler, in 1964. He designated them as “subendocardial nucleus organs.” He supposed such fibers functioned as mechanoreceptors, controlling of the intensity of contraction of the ventricular musculature. Brazilian anatomist Ferraz de Carvalho described similar structures in 1993. A thorough literature search failed to identify any other research articles confirming or denying their existence. The objective of this work was to find such structures in subendocardial ventricular walls in human hearts. We collected fifteen formalin-preserved hearts from the Anatomy Department of São Paulo University and sectioned the apical portions on the right and left ventricles according to method used by Goerttler. We utilized conventional histology (light microscopy- LM), scanning electron microscopy (SEM), and a new preservation method called micro- plastination (MP). At the anterior wall of the right ventricle in the subendocardial region between the interventricular septum and moderator band, we found several bundles of fusiform and helicoidal fibers of similar histology to the WF. The bundles measured between 400 and 1150 µm in length and were separated from adjacent muscular fibers by thin collagen fiber, thus acting as a “pseudo capsule.” Some structures seemed to be linked to PF and were appeared to be lymphatic and blood vessels and nerves. We called those structures “cardiac corpuscles” (CC). The observation of the previously “unknown” CC in this initial study confirmed the previous descriptions and its discovery may contribute to new perspectives in the study of cardiac muscle structure and function.

Toxoplasma gondii F-Box Protein L2 Silences Feline-Restricted Genes Necessary for Sexual Commitment.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages requires substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct nuclear sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNA seq data show that TgFBXL2 conditional depletion induces the expression of genes necessary for sexual commitment. We suggest that TgFBXL2 is a latent guardian of sexual stage development in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of sexual development.

Development of an interactive multidisciplinary platform for the bovine neuroanatomy study / Desarrollo de una plataforma interactiva multidisciplinar para el estudio de la neuroanatomía bovina

SUMMARY: The anatomy study is part of the basic cycle of disciplines that composes Veterinary Medicine college curriculum, and its comprehension is essential for other courses subject understanding. However, the current student's profile, the reduced time frame of superior education programs, and the multidisciplinary approach nowadays have made anatomy teaching method outdated and ineffective. Addressing the problem we developed an interactive and multidisciplinary platform based on the blended learning methodology, which could serve as a valuable tool for bovine neuroanatomy comprehension. To produce a new study tool, photos from bovine specimens fixed in formaldehyde, platinated brain pieces sectioned in a metameric order, as well as histological slides of the bovine central nervous system were used. These materials were applied to photos and schemes production, that were correlated with image exams correlation, as well as written content and videotaped classes. The obtained content was compiled into a digital platform, that can serve as an effective additional method to bovine central nervous system study. Furthermore, our results serve as a guide for the development of other blended learning methodologies in veterinary medicine and anatomy teaching. The platform provides a great tool for those who wish to accomplish a better understanding of bovine neuroanatomy and its clinical, surgical and image diagnosis correlations.

El estudio de la anatomía forma parte del ciclo básico de disciplinas que componen el currículo de la facultad de Medicina Veterinaria, y su comprensión es fundamental para el entendimiento de las materias de otros cursos. Sin embargo, el perfil del estudiante actual, la reducción de los tiempos de los programas de educación superior y el enfoque multidisciplinario actual han hecho que el método de enseñanza de la anatomía sea obsoleto e ineficaz. Abordando el problema desarrollamos una plataforma interactiva y multidisciplinar basada en la metodología blended learning, que podría servir como una valiosa herramienta para la comprensión de la neuroanatomía bovina. Para producir una nueva herramienta de estudio, se utilizaron fotografías de especímenes bovinos fijados en formaldehído, piezas de cerebro plastinadas y seccionadas en un orden metamérico, así como láminas histológicas del sistema nervioso central bovino. Estos materiales se utilizaron en la producción de fotos y esquemas, que se correlacionaron con exámenes de imágenes, así como contenido escrito y clases grabadas en video. El contenido obtenido se compiló en una plataforma digital, que puede servir como un método adicional y eficaz para el estudio del sistema nervioso central bovino. Además, nuestros resultados sirven como guía para el desarrollo de otras metodologías de aprendizaje semipresencial en la enseñanza de la medicina veterinaria y la anatomía. La plataforma proporciona una gran herramienta para aquellos que deseen lograr una mejor comprensión de la neuroanatomía bovina y sus correlaciones clínicas, quirúrgicas y de diagnóstico por imágenes.

The impact of properly diagnosed sarcopenia on postoperative outcomes after gastrointestinal surgery: A systematic review and meta-analysis.

BACKGROUND: Sarcopenia is defined as the loss of muscle mass combined with loss of muscle strength, with or without loss of muscle performance. The use of this parameter as a risk factor for complications after surgery is not currently used. This meta-analysis aims to assess the impact of sarcopenia defined by radiologically and clinically criteria and its relationship with complications after gastrointestinal surgeries. MATERIALS AND METHODS: A review of the literature was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (PROSPERO registration number: CRD42019132221). Articles were selected from the PUBMED and EMBASE databases that adequately assessed sarcopenia and its impact on postoperative complications in gastrointestinal surgery patients. Pooled estimates of pre-operative outcome data were calculated using the odds ratio (OR) and 95% confidence interval (CI). Subgroup analysis were performed to assess each type of surgery. RESULTS: The search strategy returned 1323, with 11 studies meeting the inclusion criteria. A total of 4265 patients were analysed. The prevalence of sarcopenia between studies ranged from 6.8% to 35.9%. The meta-analysis showed an OR for complications after surgery of 3.01 (95% CI 2.55-3.55) and an OR of 2.2 (95% CI 1.44-3.36) for hospital readmission (30 days). CONCLUSION: Sarcopenia, when properly diagnosed, is associated with an increase in late postoperative complications, as well as an increase in the number of postoperative hospital readmissions for various types of gastrointestinal surgery. We believe that any preoperative evaluation should include, in a patient at risk, tests for the diagnosis of sarcopenia and appropriate procedures to reduce its impact on the patient's health.

Extraction of DNA from plastinated tissues.

INTRODUCTION: Plastination allows anatomical samples to be preserved in excellent condition for an indefinite period, free of formalin, and in a format that allows biosafe manipulation by students, academics, and researchers. As with other tissue preservation techniques, it is important to establish the level of conservation of deoxyribonucleic acid (DNA) for use in future applications. The object of the present work was to extract and evaluate DNA from plastinated tissues. METHODS: We used samples of liver from Canis lupus familiaris and skeletal muscle from Rattus norvegicus, Sprague-Dawley strain, extracted from specimens plastinated with silicone at room temperature. The tissue samples were deplastinated by incubation in 5% sodium methoxide dissolved in methanol for 24 or 48 h. The samples were divided into two equal parts and DNA was extracted using two different protocols. After extraction, the DNA was quantified by fluorometry and its integrity was assessed by electrophoresis in a 1% agarose gel. RESULTS AND DISCUSSION: A high yield of DNA was obtained from the deplastinated samples and the DNA was intact. Plastinated tissues have proven to be stable and easily managed. They can also be used for examination under light and electron microscopes. The DNA extraction technique used here allowed us to obtain intact DNA from samples plastinated with silicone at room temperature, without previous fixing. This technique may allow tissue specimens to be preserved for retrospective studies of archived samples of normal and pathological anatomy in the fields of basic, clinical, forensic, and epidemiological sciences. CONCLUSIONS: The extracted DNA was intact and suitable for use in subsequent applications. Obtaining whole DNA from plastinated samples using tissue preservation protocols that preserve the tissue for use in subsequent applications, like real-time PCR, opens up many possibilities, with applications in the basic and clinical sciences, epidemiology, and forensic science.

Development of an ultrathin sheet plastination technique in rat humeral joints with osteoarthritis induced by monosodium iodoacetate for neovascularization study.

Injection with monosodium iodoacetate (MIA) is widely used to produce osteoarthritis (OA). Ultrathin sheet plastination has been used to study the morphology of structures, with strong application in anatomical education and research. Our aim was to carry out, for the first time, ultrathin sheet plastination of rat humeral joints to observe the neovascularization provoked by OA. We injected 0.1 mL of MIA into the left humeral joints of ten Sprague-Dawley rats. The right shoulders of the same rats were used as control. Sixteen weeks after the injection, the animals were euthanized and were given an immediate red epoxy resin injection through the thoracic aorta. The samples were fixed in 10% formalin, prior to the plastination process, without decalcification. Samples were dehydrated with acetone (100%) at - 25 °C, for 10 days. Later, for degreasing, samples were immersed in methylene chloride at room temperature during 1 week. Forced impregnation was performed inside a stove within a vacuum chamber. The plastinated blocks obtained were cut with a slow velocity diamond blade saw. Slices were placed in curing chambers to achieve curing and final tissue transparentation. 230 µm thickness slices were obtained. The slices were analyzed under magnifying glass and microscope, achieving visualization of OA neovascularization. The cartilage affected by OA loses its ability to remain avascular, and blood vessels invade it from the subchondral bone to the calcified and uncalcified cartilage. Ultra-thin sheet plastination is useful to observe articular cartilage neovascularization, caused by OA induced with MIA in humeral rat joint.

Plastination-A scientific method for teaching and research.

Over the last four decades, plastination has been one of the best processes of preservation for organic tissue. In this process, water and lipids in biological tissues are replaced by polymers (silicone, epoxy, polyester) which are hardened, resulting in dry, odourless and durable specimens. Nowadays, after more than 40 years of its development, plastination is applied in more than 400 departments of anatomy, pathology, forensic sciences and biology all over the world. The most known polymers used in plastination are silicone (S10), epoxy (E12) and polyester (P40). The key element in plastination is the impregnation stage, and therefore depending on the polymer that is used, the optical quality of specimens differs. The S10 silicone technique is the most common technique used in plastination. Specimens can be used, especially in teaching, as they are easy to handle and display a realistic topography. Plastinated silicone specimens are used for displaying whole bodies, or body parts for exhibition. Transparent tissue sections, with a thickness between 1 and 4 mm, are usually produced by using epoxy (E12) or polyester (P40) polymer. These sections can be used to study both macroscopic and microscopic structures. Compared with the usual methods of dissection or corrosion, plastinated slices have the advantage of not destroying or altering the spatial relationships of structures. Plastination can be used as a teaching and research tool. Besides the teaching and scientific sector, plastination becomes a common resource for exhibitions, as worldwide more and more exhibitions use plastinated specimens.

E12 technique: Conventional epoxy resin sheet plastination.

Epoxy plastination techniques were developed to obtain thin transparent body slices with high anatomical detail. This is facilitated because the plastinated tissue is transparent and the topography of the anatomical structures well preserved. For this reason, thin epoxy slices are currently used for research purposes in both macroscopic and microscopic studies. The protocol for the conventional epoxy technique (E12) follows the main steps of plastination-specimen preparation, dehydration, impregnation and curing/casting. Preparation begins with selection of the specimen, followed by freezing and slicing. Either fresh or fixed (embalmed) tissue is suitable for epoxy plastination, while slice thickness is kept between 1.5 and 3 mm. Impregnation mixture is made of epoxy E12 resin plus E1 hardener (100 ppw; 28 ppw). This mixture is reactive and temperature sensitive, and for this reason, total impregnation time under vacuum at room laboratory temperature should not last for more than 20-24 hr. Casting of impregnated slices is done in either flat chambers or by the so-called sandwich method in either fresh mixture or the one used for impregnation. Curing is completed at 40°C to allow a complete polymerization of the epoxy-mixture. After curing, slices can be photographed, scanned or used for anatomical study under screen negatoscope, magnification glass or fluorescent microscope. Based on epoxy sheet plastination, many anatomical papers have recent observations of and/or clarification of anatomical concepts in different areas of medical expertice.

P40 polyester sheet plastination technique for brain and body slices: The vertical and horizontal flat chamber methods.

The P40 technique produces high-quality brain and body slices and is the user-friendliest of the polyester techniques. The P40 polyester technique follows the same classical steps for plastination. That is, preparation of the specimen, fixation (optional), dehydration by freeze substitution, forced impregnation and curing. Two methods used to prepare two different types of specimens, that is, brain slices and body slices are described. Each method has its own characteristics depending on the specimen type used. Brain slices were used to illustrate the vertical small chamber method while the body slices were used to illustrate the horizontal large chamber method. The brain slices obtained using P40 are of very good quality presenting good contrast between grey and white matter. The body slices are also of very good quality. The physical appearance of these slices makes them an exceptional instrument for diagnostic imaging and anatomical correlation. Body slices prepared with P40 retain the natural colour of the tissue and preserve the anatomical relationships.

The International Society for Plastination.

The International Society for Plastination (ISP) was conceived as a means of defining plastination as an area of professional activity and serving as an agency for disseminating information relative to the art and science of plastination. In this paper, we present the purpose of ISP, the Journal of Plastination, the Plastination Index and the different international meetings organised by ISP.

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

E12 sheet plastination: Techniques and applications.

Plastination is an anatomical technique that consists of replacing the liquids and fat of specimens by reactive polymers through forced impregnation in a vacuum. These are then polymerized to achieve the final result. E12 sheet plastination involves epoxy resin impregnation of thin (2-4 mm) and ultra-thin (<2 mm) tissue sheets, producing dry, transparent, odorless, non-toxic and long-lasting sheets. E12 sheet plastination techniques were reviewed using MEDLINE, EMBASE and SciELO databases, and manual searches. After searching, 616 records were found using the online and manual searches (MEDLINE, n: 207; EMBASE, n: 346; SciELO, n: 44; Manual search: 23). Finally, 96 records were included in this review (after duplicates and articles unrelated to the subject were excluded). The aim of this work was to review the E12 sheet plastination technique, searching for articles concerning views of it, identifying the different variants implemented by researchers since its creation by Gunther von Hagens, and to identify its applications from teaching and research in anatomy to morphological sciences. Clin. Anat. 31:742-756, 2018. © 2017 Wiley Periodicals, Inc.