Cytoplasmic Labeling Research Articles

Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light.

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as "CABL". Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dienophile. 6-(2-Pyridyl)dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation produce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k2 of 106 M-1 s-1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin, or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore were demonstrated. CABL is light-dose dependent, and two-photon excitation promotes CABL at the suborganelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous protein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small molecule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of "ordinary" tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.

Carboxypeptidase A3 expression in canine mast cell tumors and tissue-resident mast cells.

Mast cell tumors (MCTs) are one of the most common cutaneous malignancies in dogs. Previous studies have reported expression of mast cell–specific proteases chymase and tryptase in canine cutaneous MCTs and in connective tissue and mucosal mast cells. In humans and rodents, mast cells express an additional specific protease, carboxypeptidase A3 (CPA3). In this article, we describe CPA3 immunoreactivity in connective tissue, visceral, mucosal, and neoplastic mast cells in dogs. Positive immunolabeling for CPA3 was observed in nonneoplastic mast cells in 20/20 formalin-fixed paraffin-embedded normal tissues (skin, liver, spleen, intestine), and in 63/63 MCTs irrespective of their histological grade. CPA3 protein expression was comparable to that of c-kit in both the nonneoplastic and neoplastic mast cells. Three distinct labeling patterns (membranous, diffuse, and focal cytoplasmic) were observed for CPA3 in MCTs. The focal cytoplasmic labeling pattern was associated with high-grade MCTs staged with the Kiupel 2-tier grading criteria. We propose CPA3 as a novel immunohistochemical marker for canine mast cells in health and disease.

Metastatic Digital Chondrogenic Melanocytic Tumour in a Dog

A 9-year-old intact male Brazilian Mastiff dog with a 1-year history of progressive severe swelling in all four limbs and significant locomotory difficulty was euthanized due to unresponsiveness to medical management. Macroscopically, the distal phalanx of the 5th digit of the left hindlimb was replaced by a cutaneous, non-ulcerated, 3.0cm diameter, multilobulated, black, firm nodule. The cortical bones of the appendicular skeleton were thickened and partially effaced by infiltrative coalescing nodules (0.1-1.0cm diameter). The lungs, heart, pleura, mesentery, adrenal glands and kidneys were infiltrated by similar nodules (0.5-3.5cm diameter). Histological evaluation of the masses revealed a non-encapsulated malignant neoplasm composed of spindloid to polygonal cells that contained variable amounts of intracytoplasmic melanin and were arranged in haphazardly interlacing streams and bundles supported by scant fibrovascular stroma with myxoid and chondroid matrix formation. Neoplastic cells had intense cytoplasmic labeling for S100 and 10% had moderate cytoplasmic immunoreactivity for Melan-A. To the authors' knowledge, this is the first description of a canine metastatic digital chondrogenic melanocytic tumour with strongly suggestive chondroid differentiation of neoplastic melanocytes.

Spontaneous Disseminated Histiocytic Sarcoma in a Bengal Tiger (Panthera tigris tigris)

A 16-year-old, female, captive Bengal tiger (Panthera tigris tigris) had a history of a recurrent subcutaneous mass. After two attempts at surgical removal, euthanasia was elected during the third surgical attempt due to the degree of neoplastic infiltration. At necropsy, a large subcutaneous mass infiltrated the dermis, subcutis and abdominal wall with metastasis to the lungs, spleen and adrenal glands. Microscopically, the neoplasm formed nodules comprising sheets of round cells with large irregular nuclei. Toluidine blue and Giemsa stains were negative. An immunohistochemistry panel revealed membranous and cytoplasmic labelling with ionized calcium-binding adaptor molecule 1 (IBA1), cytoplasmic labelling with vimentin but no labelling with multiple myeloma oncogene 1 (MUM1), CD20 or pancytokeratin markers. Based on the gross distribution and histological and immunohistochemistry features, a diagnosis of disseminated histiocytic sarcoma was made. To the best of our knowledge, this is the first record of disseminated histiocytic sarcoma in a captive Bengal tiger.

ING3 and ING4 immunoexpression and their relation to the development of benign odontogenic lesions.

The sample comprised of 20 odontogenic keratocysts (OKC), 20 ameloblastomas (AM), and 15 adenomatoid odontogenic tumors (AOT) specimens. Nuclear and cytoplasmic immunolabeling of ING3 and ING4 were semi-quantitatively evaluated in epithelial cells of the odontogenic lesions, according to the percentage of immunolabelled cells in each case. Descriptive and statistics analysis were computed, and the p-value was set at 0.05. No statistically significant differences were found in cytoplasmic and nuclear ING3 immunolabeling among the studied lesions. In contrast, AOTs presented higher cytoplasmic and nuclear ING4 labeling compared to AMs (cytoplasmic p-value = 0.01; nuclear p-value < 0.001) and OKCs (nuclear p-value = 0.007). ING3 and ING4 protein downregulation may play an important role in the initiation and progression of more aggressive odontogenic lesions, such as AMs and OKCs.

Pathological and immunohistochemical aspects of acute megakaryoblastic leukaemia in a cat - Short communication.

An adult, mixed-breed, feline leukaemia virus (FeLV-) positive female cat was presented with mucosal jaundice and a history of anorexia and constipation for three days. Physical examination revealed splenomegaly, cachexia, and dehydration. Humane euthanasia was conducted, followed by postmortem examination. Grossly, the cat was icteric, and presented hepatomegaly with multifocal white spots and splenomegaly. Histologically, the bone marrow was nearly completely replaced by a proliferation of megakaryocytes and megakaryoblasts, and there was a proliferation of fibrous connective tissue. Similar neoplastic proliferation was observed infiltrating the liver, lymph nodes, spleen, kidney, skeletal muscle, and lungs. Immunohistochemistry was performed for von Willebrand Factor (VWF), CD79α, CD3, feline immunodeficiency virus, FeLV, and CD61. Marked cytoplasmic labelling was observed in the neoplastic cells for FeLV, VWF and CD61, corroborating the diagnosis of acute megakaryoblastic leukaemia.

Prognostic Significance of JMJD3 Expression in Pleural Mesotheliomas

Pleural mesothelioma is a disease associated with asbestos exposure and patients often have poor prognosis. Biomarkers that can stratify tumours more efficiently are much sought after to enable more personalized treatment options and predict prognosis. Jumonji domain-containing protein D3 (JMJD3) has variable expression in a range of tumours. However, there has been much discordance in the immunohistochemical labelling of JMJD3 between cancers at different sites and ambiguity exists regarding its functional significance. Recent evidence suggests that although nuclear expression of JMJD3 has a demethylase role in most cancers, there are also demethylase-independent actions of JMJD3 that need to be explored including its cytoplasmic expression. We analysed JMJD3 labelling in 99 pleural mesothelioma tissues and correlated nuclear and cytoplasmic expression with survival outcomes. We found that low nuclear and high cytoplasmic expression were associated with poor survival outcomes in our cohort (p = 0.014 and p = 0.041, respectively). Additionally, we found that low nuclear expression of JMJD3 was frequent in the sarcomatoid subtype (p &lt; 0.001). Finally, we showed that cytoplasmic labelling is an independent prognostic marker of poor survival. Our cohort only contained a small number of tumours with high cytoplasmic expression of JMJD3, and a larger cohort study may provide clearer stratification.

Visualization of c-Mpl Expression in Ph-Negative Myeloproliferative Diseases and So-Called Essential Thrombocythemia with Ringed Sideroblasts by Confocal Laser Scanning Microscopy.

c-Mpl acts as a primary regulator of megakaryocytopoiesis and platelet production and is essential for the maintenance of normal hematopoiesis in humans. An impaired posttranslational processing of Mpl with underglycosylation has been proposed as a molecular defect of polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF; Molinterno and Spivak, Blood 1999; 94: 2555).To visualize the subcellular localization of megakaryocyte c-Mpl protein in myeloproliferative diseases and myelodysplastic/myeloproliferative disorders we performed an imaging study by confocal laser scanning microscopy. Bone marrow trephines from normal bone marrow donors (n=14), patients with essential thrombocythemia (ET, n=15), PV (n=18), CIMF (n=15), myelodysplastic syndromes (MDS) including refractory anemia with ringed sideroblasts (RS; n=12) and so-called essential thrombocythemia with RS (ET/RS; n=67) were examined. Immunohistochemistry was performed using a rabbit polyclonal antibody recognizing glycosylated and unglycosylated c-Mpl and semiquantitatively evaluated by scoring of plasmalemmal vs. cytoplasmic labeling (score 1–3).Our results provide evidence that normal megakaryocytes show a pronounced cell surface expression of c-Mpl. In PV and CIMF, c-Mpl suface expression was significantly reduced (p<0.001), while cytoplasmic expression was often strongly accentuated. In ET, heterogeneous patterns of membraneous labeling were observed ranging from normal to decreased. In MDS, plasmalemmal immunoreacativity was generally down-regulated. In ET/RS c-Mpl imaging revealed a heterogeneous positivity with different degrees of plasmalemmal vs. cytoplasmic immunoreactivity in keeping either with CIMF, true ET or MDS.The role of c-Mpl as a biomarker of Ph-negative MPDs is controversely discussed. A weak megakaryocyte c-Mpl staining which may complement morphologic distinction has been observed in PV (Tefferi et al., Blood 2000; 96: 771). Our findings suggest that the strikingly reduced cell surface c-Mpl expression despite equivalent or increased mRNA levels (Bock et al., J Pathol 2004; 203: 609) may result in impaired receptor domains for TPO in PV and CIMF and is a characteristic epigenetic abnormality of theses entities. Upon consideration of all our clinico-pathological data, so-called ET/RS is now fracturing in clearly defined haematological diseases which could be categorized as CIMF/RS (38/67), true ET/RS (10/67) or different MDS categories (n=19). The heterogeneous patterns of c-Mpl expression further contribute to define positive criteria for the discrimination of the distinct entities within in the ET/RS group.

Validation of Immunohistochemistry for Canine Proteins Involved in Thyroid Iodine Uptake and Their Expression in Canine Follicular Cell Thyroid Carcinomas (FTCs) and FTC-Derived Organoids.

Thyrotropin receptor (TSHR), sodium iodide symporter (NIS), pendrin, and thyroid peroxidase (TPO) are essential for the uptake of iodine by follicular thyroid cells. The aim of this study was to establish immunohistochemistry (IHC) protocols for TSHR, NIS, pendrin, and TPO in canine tissues and characterize their expression in organoids derived from canine follicular cell thyroid carcinoma (FTC) and in the respective primary tumors. This constitutes a fundamental step to establish organoids as a model to study the uptake of iodine in canine FTC. Commercially available antibodies directed against human proteins were selected. Antibody specificity was confirmed by western blot using lysates of the HTori-3 human thyroid cell line and healthy canine thyroid gland. IHC was validated using HTori-3 cells and a set of canine normal tissues including healthy thyroid gland. The expression of TSHR, NIS, pendrin, and TPO was evaluated in 3 organoid lines derived from FTC and respective primary tumors. All 4 antibodies produced specific bands by western blot and cytoplasmic labeling in follicular cells by IHC in both human HTori-3 cells and canine thyroid gland. NIS also showed basolateral membrane immunolabeling in follicular cells. All 4 proteins were highly expressed in organoids derived from FTC. The expression was similar or higher compared to the primary tumors. The results of this study characterize organoids derived from canine FTC as a suitable in vitro model to investigate iodine uptake, opening new research possibilities in the field of canine thyroid cancer therapy.

Efficient photodynamic inactivation of Leishmania parasites mediated by lipophilic water-soluble Zn(II) porphyrin ZnTnHex-2-PyP4+

BackgroundPhotodynamic inactivation (PDI) is emerging as a promising alternative for cutaneous leishmaniasis (CL). The chemotherapy currently used presents adverse effects and cases of drug resistance have been reported. ZnTnHex-2-PyP4+ is a porphyrin with a high potential as a photosensitizer (PS) for PDI, due to its photophysical properties, structural stability, and cationic/amphiphilic character that can enhance interaction with cells. This study aimed to investigate the photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites. MethodsZnTnHex-2-PyP4+ stability was evaluated using accelerated solvolysis conditions. The photodynamic action on promastigotes was assessed by (i) viability assays, (ii) mitochondrial membrane potential evaluation, and (iii) morphological analysis. The PS-promastigote interaction was studied. PDI on amastigotes and the cytotoxicity on macrophages were also analyzed. ResultsZnTnHex-2-PyP4+, under submicromolar concentration, led to immediate inactivation of more than 95% of promastigotes. PDI promoted intense mitochondrial depolarization, loss of the fusiform shape, and plasma membrane wrinkling in promastigotes. Fluorescence microscopy revealed a punctate PS labeling in the parasite cytoplasm. PDI also led to reductions of ca. 64% in the number of amastigotes/macrophage and 70% in the infection index after a single treatment session. No noteworthy toxicity was observed on mammalian cells. ConclusionsZnTnHex-2-PyP4+ is stable against demetallation and more efficient as PS than the ethyl analogue ZnTE-2-PyP4+, indicating readiness for evaluation in in vivo studies as an alternative approach to CL. General significanceThis report highlighted promising photodynamic effects mediated by ZnTnHex-2-PyP4+ on Leishmania parasites, opening up perspectives for applications in CL pre-clinical assays and PDI of other microorganisms.

Abnormal N-Glycosylation of Human Lens Epithelial Cells in Type-2 Diabetes May Contribute to Cataract Progression.

PurposeIn order to better understand cataract development, we analyzed the glycosylation profile of human lens epithelial cells (HLECs) from anterior lens capsules of type 2 diabetes mellitus (T2DM) and non-diabetic (ND) patients undergoing routine cataract surgery.SettingResearch Department of the Asociación para Evitar la Ceguera, Hospital “Dr. Luis Sánchez Bulnes”, Mexico.DesignExperimental study.MethodsEvaluation of anterior lens capsules from T2DM and ND patients undergoing phacoemulsification and free from other ocular diseases.ResultsHematoxylin-eosin staining revealed HLECs alterations in T2DM samples. From lectins with different sugar specificities used, concanavalin A showed significant differences, labeling homogeneously both in the cytoplasm and in cell membranes in ND capsules, while in T2DM capsules, in addition to membrane and cytoplasm labeling, there were perinuclear vesicles with high concanavalin A labeling. Two-dimensional gel electrophoresis showed that T2DM patients have a ~65-kDa spot with an isoelectric point of 5.5 with a higher density compared to ND capsules, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed 62% homology with type-1 cytokeratin. Immunohistochemistry using anti-pan cytokeratin antibody revealed co-localization with concanavalin A, and a lectin blot revealed with concanavalin A showed a band of ~65 kDa, a molecular weight that corresponds to human type 1 cytokeratin.ConclusionThese results suggest that over-expression of N-glycosidically linked human type 1 cytokeratin may induce capsule disruption and affect selective permeability, allowing the entry of different molecules to the lens that facilitate cataract progression.

An ultrastructural connectomic analysis of a higher-order thalamocortical circuit in the mouse.

Many studies exist of thalamocortical synapses in primary sensory cortex, but much less in known about higher-order thalamocortical projections to higher-order cortical areas. We begin to address this gap using genetic labeling combined with large volume serial electron microscopy (i.e., "connectomics") to study the projection from the thalamic posterior medial nucleus to the secondary somatosensory cortex in a mouse. We injected into this thalamic nucleus a cocktail combining a cre-expressing virus and one expressing cre-dependent ascorbate peroxidase that provides an electron dense cytoplasmic label. This "intersectional" viral approach specifically labeled thalamocortical axons and synapses, free of retrograde labeling, in all layers of cortex. Labeled thalamocortical synapses represented 14% of all synapses in the cortical volume, consistent with previous estimates of first-order thalamocortical inputs. We found that labeled thalamocortical terminals, relative to unlabeled ones: were larger, were more likely to contain a mitochondrion, more frequently targeted spiny dendrites and avoided aspiny dendrites, and often innervated larger spines with spine apparatuses, among other differences. Furthermore, labeled terminals were more prevalent in layers 2/3 and synaptic differences between labeled and unlabeled terminals were greatest in layers 2/3. The laminar differences reported here contrast with reports of first-order thalamocortical connections in primary sensory cortices where, for example, labeled terminals were larger in layer 4 than layers 2/3 (Viaene et al., 2011a). These data offer the first glimpse of higher-order thalamocortical synaptic ultrastructure and point to the need for more analyses, as such connectivity likely represents a majority of thalamocortical circuitry.

The Value of Calretinin in the Diagnosis of Hirschsprung&amp;apos;s Disease in Dakar

<i>Introduction</i>: Hirschsprung's disease is a congenital anomaly characterised by a rarefaction or absence of ganglion cells associated with schwannian hyperplasia or hypertrophy involving the colon or a segment of the colon. Its diagnosis is based on clinical, radiological and histological evidence. Haematoxylin-eosin examination is used in cases of suspected Hirschsprung's disease. However, in some situations it is insufficient to confirm or refute the diagnosis. Immunohistochemical and histo-enzymatic techniques can be used to demonstrate aganglionosis. The aim of our study is to evaluate the interest of calretinin in the diagnosis of Hirschsprung's disease in Dakar. <i>Materials and methods</i>: This was a retrospective and descriptive study from 1 January 2018 to 31 August 2019. It was conducted at the pathology laboratory of Idrissa Pouye General Hospital where 51 paraffin blocks from suspected Hirschsprung's Disease cases were included. All blocks were immunohistochemically studied with anti-calretinin antibody. The diagnosis of Hirschsprung's disease on standard histological examination was based on the absence of lymph node cells in the submucosal and myenteric plexuses. Calretinin immunoreactivity was shown by nuclear and cytoplasmic labelling of ganglion cells and nerve nets. In Hirschsprung's disease, there is an absence of labelling of the nerve plexuses. <i>Results</i>: The majority of patients (73.5%) were aged 2 years or older with a mean age of 3.4 years. The sex ratio was 2. Biopsies constituted 56.86% of the specimens and surgical specimens 43.14%. Concordance between haematoxylin-eosin examination and calretinin immunohistochemistry was observed in 47 cases (92.15%) and discordance in 4 cases (7.15%). The sensitivity of calretinin was 93.75% and the specificity 89.47%. The kappa index was 0.92. The recto-sigmoid form was the most frequent topographic form observed in 83.3% of patients. <i>Conclusion</i>: Morphological examination with haematoxylin-eosin remains a good diagnostic method for Hirschsprung's disease. Calretinin immunohistochemistry is necessary in equivocal cases, in neonates and infants and in case of superficial biopsies.

Recurrent giant phyllodes tumour in a 17-year-old female: a rare case report

Phyllodes tumours (PTs) are rare fibroepithelial tumours of the breast with incidence accounting for <1% in the general population. Availability of reports on PTs with size of 31 cm or more in diameter in the literature are extremely rare. Herein, the case of a 17-year-old female patient with a giant recurrent right breast PT is reported. Histologically, the tumour showed proliferation of stromal tumour cells consisting of spindle cells with uniform nuclear chromatin, inconspicuous nucleoli, abundant eosinophilic cytoplasm and proliferation labelling index for Ki67 of <10%. PTs require meticulous surgical excision due to the fact that they have a high rate of recurrence and status of surgical margins must be included in the pathology report. This is because recurrent cases of PTs tend to grow faster than the primary ones with a high chance of transforming to malignancy.

Fluorescent Copolymers for Bacterial Bioimaging and Viability Detection.

Novel fluorescent labels with high photostability and high biocompatibility are required for microbiological imaging and detection. Here, we present a green fluorescent polymer chain (GFPC), designed to be nontoxic and water-soluble, for multicolor bioimaging and real-time bacterial viability determination. The copolymer is synthesized using a straightforward one-pot reversible addition-fragmentation chain-transfer (RAFT) polymerization technique. We show that GFPC does not influence bacterial growth and is stable for several hours in a complex growth medium and in the presence of bacteria. GFPC allows the labeling of the bacterial cytoplasm for multicolor bacterial bioimaging applications. It can be used in combination with propidium iodide (PI) to develop a rapid and reliable protocol to distinguish and quantify, in real time, by flow cytometry, live and dead bacteria.

Neuropathologic description of CHCHD10 mutated amyotrophic lateral sclerosis.

ObjectiveTo present the postmortem neuropathologic report of a patient with a CHCHD10 mutation exhibiting an amyotrophic lateral sclerosis (ALS) clinical phenotype.MethodsA 54-year-old man without significant medical history or family history presented with arm weakness, slowly progressed over 19 years to meet the El Escorial criteria for clinically probable ALS with bulbar and respiratory involvement, and was found to have a CHCHD10 p.R15L mutation. Postmortem neuropathologic examination took place including immunohistochemical staining with CHCHD10, and double immunofluorescence combining CHCHD10 with TDP43 and neurofilament was performed and the results were compared with normal controls and sporadic ALS cases.ResultsPostmortem examination of the CHCHD10 mutation carrier showed severe loss of hypoglossal and anterior horn motor neurons, mild corticospinal tract degeneration, and a relative lack of TDP43 immunopathology. CHCHD10 immunohistochemistry for the 3 controls and the 5 sporadic ALS cases showed strong neuronal cytoplasmic and axonal labeling, with the CHCHD10 mutation carrier also having numerous CHCHD10 aggregates within their anterior horns. These aggregates may be related to the CHCHD10 aggregates recently described to cause mitochondrial degeneration and disease in a tissue-selective toxic gain-of-function fashion in a CHCHD10 knock-in mouse model. The CHCHD10 aggregates did not colocalize with TDP43 and were predominantly extracellular on double immunofluorescence labeling with neurofilament.ConclusionsThe neuropathology of CHCHD10 mutated ALS includes predominantly lower motor neuron degeneration, absent TDP43 immunopathology, and aggregates of predominantly extracellular CHCHD10, which do not contain TDP43.

Tubulopapillary Carcinoma of the Mammary Gland in a Mare

Background: Mammary gland neoplasia are often observed in bitches and cats, rarely in cows, goats, sheep, sows, and mares. Among the neoplasia that affect mares, breast tumors are rare, as they occur more frequently in older animals. In horses, mammary gland tumors occur more frequently in older animals. In this species there are reports of cases describing carcinomas and adenocarcinomas and several subtypes. Mammary neoplasms in equines tend to be locally aggressive and promote metastases to the regional lymph nodes and other organs, including the lungs, and may cause pleural effusion. The aim objective of the present case report was to describe the clinical-pathological and immunohistochemical findings of a tubulopapillary carcinoma of the mammary gland in mare.Case: Was received a biopsy of a tumor mass of the mammary gland of a 15-year-old Quarter Horse mare. According to the veterinarian, the mare presented an increase in volume with ulceration of the mammary gland three months before the biopsy. Macroscopically, the mass was firm to the cut with white surface measuring 12x12 cm. The fragment was fixed in 10% formalin, routinely processed for histology, paraffin embedded, and stained with hematoxylin and eosin. Microscopically, we observed proliferation of epithelial neoplastic cells arranged in tubules and papillary projections, some with two to three layers of cells. Neoplastic cells had oval, moderate and distinct cytoplasm. The nuclei were rounded with dense chromatin and an apparent nucleolus. There were two figures of mitosis per field of high magnification. Proliferation of neoplastic cells invaded adjacent tissues, and in lymph vessels of the region, neoplastic cells were observed as well as inflammation in adjacent tissue. In addition, the breast fragment was submitted to the immunohistochemistry technique using anti-pancytokeratin and anti-vimentin antibody. In immunohistochemistry for pancytokeratin, there was immunoreactivity with marked cytoplasm in neoplastic epithelial cells. In immunohistochemistry for vimentin, there was cytoplasmic labeling in cells of the supporting tissue (fibroblasts and endothelial cells). After approximately six months of the initial diagnosis, the mare died.Discussion: The diagnosis of tubulopapillary carcinoma of the mammary gland in a mare was based on the clinical-pathological and immunohistochemical findings. In mares, descriptions of breasts neoplasia are uncommon and found in animals aged over 12 years. In a study carried out in slaughterhouses in France, with approximately 40,000 horses slaughtered, only 45 breast neoplasms (0.11%) were observed, most of them consisting of carcinomas. In these equines, the absolute incidence of neoplasia of the reproductive system increases with age and tumors generally appear as deep, multifocal, and ulcerating skin nodules. Thus, important differential diagnoses include chronic mastitis and prepartum breast edema. In mares with neoplasia of the mammary gland, unilateral or bilateral volume increase, pain, warm area, redness and suppurative serous discharge are frequently observed. There are also reports of skin ulceration or necrosis. In the present report, the mare showed increased volume of the mammary gland, with focal hyperthermia, fistulated area and serum-blood secretion. The literature suggests a reserved prognosis for any mare presenting mammary neoplasia. Histopathology and immunohistochemistry were fundamental for the diagnosis of tubulopapillary carcinoma of the mammary gland in the present study. Mammary neoplasiss in mares are rare, but should be considered in the diagnosis of mammary pathologies in this species.

A Safe and Effective Magnetic Labeling Protocol for MRI-Based Tracking of Human Adult Neural Stem Cells.

Magnetic resonance imaging (MRI) provides a unique tool for in vivo visualization and tracking of stem cells in the brain. This is of particular importance when assessing safety of experimental cell treatments in the preclinical or clinical setup. Yet, specific imaging requires an efficient and non-perturbing cellular magnetic labeling which precludes adverse effects of the tag, e.g., the impact of iron-oxide-nanoparticles on the critical differentiation and integration processes of the respective stem cell population investigated. In this study we investigated the effects of very small superparamagnetic iron oxide particle (VSOP) labeling on viability, stemness, and neuronal differentiation potential of primary human adult neural stem cells (haNSCs). Cytoplasmic VSOP incorporation massively reduced the transverse relaxation time T2, an important parameter determining MR contrast. Cells retained cytoplasmic label for at least a month, indicating stable incorporation, a necessity for long-term imaging. Using a clinical 3T MRI, 1 × 103 haNSCs were visualized upon injection in a gel phantom, but detection limit was much lower (5 × 104 cells) in layer phantoms and using an imaging protocol feasible in a clinical scenario. Transcriptional analysis and fluorescence immunocytochemistry did not reveal a detrimental impact of VSOP labeling on important parameters of cellular physiology with cellular viability, stemness and neuronal differentiation potential remaining unaffected. This represents a pivotal prerequisite with respect to clinical application of this method.

A facile synthesis of nontoxic luminescent carbon dots for detection of chromium and iron in real water sample and bio‐imaging

AbstractIn this study, we have reported the synthesis of luminescent carbon dots (CDs) from indigenous potato sources by simple heating reactions. The as‐synthesized CDs exhibited an average size of ~ 5.97 nm with a quantum yield (QY) of 6.08%. Furthermore, the CDs possessed high water‐solubility, possibly due to the presence of ─COOH and ─OH groups on their surfaces. The quenching of luminescence of the CDs specifically by Cr6+ and Fe3+ ions was used to detect chromium and iron in the water sample. The minimum limit of detection (LOD) for Cr6+ and Fe3+ ions was found to be 0.012 μM and 0.000549 μM, respectively, in a linear range of 0.5 μM‐100 μM and 0.5 μM‐5 μM for Cr6+ and Fe3+, respectively, which was well below the concentration specified by WHO. We used our sensing system to detect the metal ions in water from the Brahmaputra River as well as in tannery water. In addition, the MTT‐based cell viability experiments showed that the CDs were nontoxic within 200 μg/mL. High quantum yield and the easy uptake of CDs enabled the quick labelling of cytoplasm of the HeLa cells, which can be further attributed to bioimaging applications.

Inhibition of motility by NEO100 through the calpain-1/RhoA pathway.

Glioblastoma (GBM) is the most aggressive type of brain tumor with a high rate of tumor recurrence, and it often develops resistance over time to current standard of care chemotherapy. Its highly invasive nature plays an essential role in tumor progression and recurrence. Glioma stem cells (GSCs) are a subpopulation of glioma cells highly resistant to treatments and are considered responsible for tumor recurrence. Patient-derived populations of GSCs were analyzed by western blot, MTT, and cytoplasmic calcium labeling to determine the cytotoxicity of NEO100. High-performance liquid chromatography was used to evaluate the levels of NEO100 in the cell culture supernatants. The effects of the compound on GSC motility were studied using Boyden chamber migration, 3D spheroid migration and invasion assays, and an mRNA expression PCR array. A RhoA activation assay, western blot, and immunofluorescence techniques were employed to confirm the signaling pathways involved. Intracranial implantation of GSCs in athymic mice was used to evaluate the effects of NEO100 in vivo on tumor progression and overall survival. Here, the authors show how NEO100, a highly purified good manufacturing practices-quality form of perillyl alcohol, is cytotoxic for different subtypes of GSCs, regardless of the mechanisms of DNA repair present. At doses similar to the IC50 (half maximal inhibitory concentration) values, NEO100 induces ER stress and activates apoptotic pathways in all GSC populations tested. At subcytotoxic doses in the micromolar range, NEO100 blocks migration and invasion of GSCs. These results correlate with a decrease in calpain-1 expression and an increase in RhoA activation, leading to enhanced contractility of the GSCs. In addition, NEO100 blocks the activation of the kinases Src, p42/44 MAPK, Akt, and Stat3, all related to cell proliferation and migration. Intranasal administration of NEO100 in mice with GSC-derived intracranial tumors led to a decrease in tumor progression and a 32% increase in overall survival. Immunostaining studies showed that NEO100 induces apoptosis and reduces GSC invasion in vivo. NEO100 could have significant value targeting GSCs and could be used for GBM therapy as either monotherapy or a coadjuvant therapy during temozolomide rest cycles.