Minimal late radiation toxicity and transient early toxicity following postoperative definitive intent conformal radiation therapy (20 × 2.5 Gy) for canine apocrine gland anal sac adenocarcinoma.

Postoperative radiation therapy (RT) may be beneficial for dogs with anal sac apocrine gland adenocarcinoma (ASAC). Clinically significant late toxicities have been reported in up to 65% of dogs with perianal tumors following non-conformal definitive RT, particularly when fractions of 3 Gy or higher are prescribed. The primary objective of this prospective, descriptive study was to evaluate tolerability of a novel 3D conformal RT (3DCRT) protocol in a group of dogs. Dogs with ASAC were prospectively enrolled if clients elected RT following surgery. The planning target volume was prescribed 50 Gy in 2.5 Gy fractions using 6 MV photons and administered over 26 days. Early and late radiation toxicities were graded according to standardized criteria. Thirteen dogs were initially enrolled but 1 was excluded due to a high risk of anesthesia-related mortality. Seven dogs presented with early stage disease. Median follow up time was 771 days (91-2223). Transient grade 3 dermatitis and anusitis developed in all dogs, with resolution within 4 weeks. Two dogs developed transient grade 2 late colitis. Locoregional failure in the irradiated field was documented in one dog at 738 days. All-cause median survival time was 771 days (95% confidence interval: 510 â†’ 2223 days). Findings indicated that this fractionation may be safely administered to the canine anus and pelvic canal using 3DCRT, although acute toxicity should be anticipated. Further prospective studies are needed in order to confirm long-term tolerability and efficacy.

Protocols for endothelial cell isolation from mouse tissues: brain, choroid, lung, and muscle.

Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).

Protocols for endothelial cell isolation from mouse tissues: kidney, spleen, and testis.

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, critical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kidney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic- and fluorescence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Protocols for endothelial cell isolation from mouse tissues: small intestine, colon, heart, and liver.

Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).

Regional and organ-level responses to local lung irradiation in sheep.

Lung is a dose-limiting organ in radiotherapy. This may limit tumour control when effort is made in planning to limit the likelihood of radiation-induced lung injury (RILI). Understanding the factors that dictate susceptibility to radiation-induced pulmonary fibrosis will aid in the prevention and management of RILI, and may lead to more effective personalized radiotherapy treatment. As the interaction of regional and organ-level responses may shape the chronic consequences of RILI, we sought to characterise both aspects of the response in an ovine model. A defined volume of left pulmonary parenchyma was prescribed 5 fractions of 6 Gy within 14 days while the contralateral lung dose was constrained. Radiographic changes via computed tomography (CT) were documented to define differences in radio-exposed lung relative to non-exposed lung at d21, d63 and d171 (n = 2), and at d21, d147 and d227 (n = 2). Gross and histologic lung changes were evaluated in samples derived at necropsy examination to define the chronic pulmonary response to radiation. Irradiated lung demonstrated reduced radio-density and increased homogeneity as evidenced from texture based radiomic feature analysis, relative to the control lung. At necropsy, the radiation field was readily defined by pallor on the pleural surface, which was also evident on the cut surface of fixed lung specimens. The degree and homogeneity of pallor reflected the sparse presence of erythrocytes in alveolar septal capillaries of radiation-exposed lung. These changes contrasted with dilated and congested microvasculature in the contralateral control lung. Referencing data to measurements made in control lung volumes of sheep experiencing acute RILI indicated that interstitial collagen continues to deposit in the radio-exposed lung field. Overall lung vascularity increased during the chronic response, as evidenced by increased expression of endothelial cell marker (CD31); however, vascularity was consistently decreased in irradiated lung and was negatively correlated with lung collagen. Other organ-level responses included increased expression of alpha smooth muscle actin (ASMA), increased numbers of proliferating cells (Ki67 positive), and cells expressing the dendritic cell-lysosomal associated membrane protein (DC-LAMP) antigen. The chronic response to RILI in this model is effected at both the whole organ and local lung level. Whilst the long-term consequences of exposure to radiation involved the continued deposition of collagen in the radiation field, organ-level responses also included increased vascularization and increased expression of ASMA, Ki67 and DC-LAMP. Interrupting the interplay between these aspects may influence susceptibility to pulmonary fibrosis after radiotherapy. We advocate for the importance of large animal model systems in pursuing these opportunities to target local, organ-level and systemic mechanisms in parallel within the same subject over time.

Single-Cell Transcriptome Atlas of Murine Endothelial Cells.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

Single-Cell RNA Sequencing Reveals Renal Endothelium Heterogeneity and Metabolic Adaptation to Water Deprivation.

BACKGROUND: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. METHODS: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. RESULTS: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and-surprisingly-oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. CONCLUSIONS: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.

Vascular ultrasonographic findings in canine patients with clinically diagnosed phlebitis.

Peripheral vein phlebitis (inflammation) is a relatively frequent complication in dogs, however, published information on the ultrasonographic characteristics is currently lacking. This prospective, observational study describes the ultrasound (US) characteristics of normal canine cephalic veins, and veins with clinical phlebitis. Correlations among US findings and between US findings versus time that the intravenous catheter was in place were investigated. Safety of the US procedure was evaluated. Fifty patients were prospectively recruited for the study and 18 met the final inclusion criteria. Each patient underwent daily US examinations and was assessed for multiple criteria (vascular wall appearance, compressibility, spontaneity of flow, color fill, and presence/absence of filling defects, flow contour, direction, non-pulsatility). Characteristics of normal canine cephalic veins were as follows: smooth and thin wall, complete compressibility, no flow disturbances, no filling defects, smooth flow contours, and unidirectional, non-pulsatile flow with no turbulence. Characteristics of cephalic veins with clinical phlebitis were as follows: wall thickening (83%), decreased compressibility (55%), filling defects consistent with intraluminal thrombus (55%), vessel wall hyperechogenicity (44%), and abnormal color Doppler flow (39%). Significant correlations were found between Doppler filling defects and compressibility, Doppler filling defects and presumed thrombosis, and compressibility and presumed thrombosis (P = .001, P = .001, P = .000, respectively). No correlation was found between the US findings and time the intravenous catheter was in place. Findings indicated that duplex and compressibility US are feasible and safe methods for characterizing and monitoring cephalic veins in dogs with clinical phlebitis.

Radiation therapy for intracranial tumours in cats with neurological signs.

OBJECTIVES: The aim of this study was to evaluate the outcome of cats with intracranial tumours presenting with neurological signs treated with radiation therapy. METHODS: This study comprised a retrospective multicentre case series. Medical records of a total of 22 cats with intracranial space-occupying lesions, presenting with neurological signs and/or epileptic seizures and treated with external beam radiation therapy, were reviewed. In the treated cats, patient-, tumour- and treatment-related variables were investigated, including age, sex, tumour location, tumour volume, total radiation dose, equivalent dose in 2 Gy fractions (EQD2), corticosteroid dose, overall treatment time and institution for influence on local tumour control and survival. RESULTS: Based on advanced imaging characteristics, the 22 treated cats presented with meningioma (n = 11), pituitary tumour (n = 8), choroid plexus tumour (n = 2) or glioma (n = 1). Allocated to the neuraxis, 11 lesions were extra-axial, three were intra-axial and eight were located in the pituitary region. At diagnosis, 21 cats exhibited altered neurological status. One cat presented with epileptic seizures and another cat had both seizures and altered neurological status. The mean total physical dose of radiation was 41.63 Gy (± 4.33), range 24-45 Gy. In all but one cat (95.5%), neurological signs improved after radiation therapy. The median progression-free survival was 510 days (95% confidence interval [CI]: 51-969). The proportion free of progression at 1 year was 55.7% (95% CI: 33-78). Fourteen cats died (only in five cases was death related to the intracranial tumour) and eight cats were still alive or lost to follow-up. The median overall survival time was 515 days (95% CI: 66-964). None of the tested variables influenced outcome. CONCLUSIONS AND RELEVANCE: Radiation therapy seems to represent a viable treatment option in cats with intracranial tumours, relieving neurological signs and improving local tumour control. Radiation therapy may be considered for cats with tumours in complicated/inoperable localisations or for cases with a high peri- and postoperative risk.

Nebulisation of synthetic lamellar lipids mitigates radiation-induced lung injury in a large animal model.

Methods to protect against radiation-induced lung injury (RILI) will facilitate the development of more effective radio-therapeutic protocols for lung cancer and may provide the means to protect the wider population in the event of a deliberate or accidental nuclear or radiological event. We hypothesised that supplementing lipid membranes through nebulization of synthetic lamellar lipids would mitigate RILI. Following pre-treatment with either nebulised lamellar lipids or saline, anaesthetised sheep were prescribed fractionated radiotherapy (30 Gray (Gy) total dose in five 6 Gy fractions at 3-4 days intervals) to a defined unilateral lung volume. Gross pathology in radio-exposed lung 37 days after the first radiation treatment was consistent between treatment groups and consisted of deep red congestion evident on the pleural surface and firmness on palpation. Consistent histopathological features in radio-exposed lung were subpleural, periarteriolar and peribronchial intra-alveolar oedema, alveolar fibrosis, interstitial pneumonia and type II pneumocyte hyperplasia. The synthetic lamellar lipids abrogated radiation-induced alveolar fibrosis and reduced alpha-smooth muscle actin (ASMA) expression in radio-exposed lung compared to saline treated sheep. Administration of synthetic lamellar lipids was also associated with an increased number of cells expressing dendritic cell-lysosomal associated membrane protein throughout the lung.

Safety and feasibility of stereotactic radiotherapy using computed portal radiography for canine intracranial tumors.

Stereotactic radiotherapy is a highly conformal treatment option for intracranial and extracranial malignancies. Stereotactic radiotherapy utilizes specialized equipment specifically designed to avoid normal tissue while delivering ablative treatments with submillimeter precision and accuracy. Linear accelerator based stereotactic radiotherapy incorporates on-board image guidance utilizing cone beam computed tomography (CT). Many institutions lack the ability to provide image guidance with cone beam CT but delivery of highly conformal treatments with submillimeter precision and accuracy is still feasible. The purpose of this retrospective, pilot study was to describe clinical outcomes for a group of dogs with neurological disease that were treated with an stereotactic radiotherapy technique utilizing intensity modulated radiation therapy, megavoltage computed portal radiography, a bite plate, thermoplastic mold, and mask based positioning system. Twelve dogs with neurological clinical signs were included. The diagnosis of intracranial tumor was made based on advanced imaging (12/12) and confirmed via histopathology (3/12). Twelve courses of stereotactic radiotherapy, utilizing three fractions of 8.0 Gy, were delivered on alternating days. Self-resolving neurological deterioration was observed in two patients during stereotactic radiotherapy. Neurological progression free interval and median survival time were 273 days (range: 16-692 days) and 361 days (range: 25-862 days). Stereotactic radiotherapy using computed portal radiography may be a safe treatment option for dogs with intracranial tumors.

IMAGING DIAGNOSIS-NUCLEAR SCINTIGRAPHIC, RADIOLOGICAL, AND PATHOLOGIC CHARACTERISTICS OF METASTATIC PILOMATRICOMA IN A DOG.

A 6-year-old castrated Goldendoodle dog was presented for left-sided lameness of 3 weeks' duration. Focal, moderate to marked increased 99m Tc-methylene diphosphonate (99m Tc-MDP) uptake was detected in the right caudal lung lobe, caudal angle of the left scapula, and the distal aspect of the left femur with whole body bone phase scintigraphy. Radiographs identified a well-circumscribed, oval-shaped soft tissue opaque mass in the right caudal lung lobe; a suspect oval-shaped osteolytic lesion in the proximal third of the left scapula; and an osteolytic lesion in the distal aspect of the left femur. Metastatic pilomatricoma was confirmed histologically at all three sites.

SYNTHESIS, STRUCTURE AND PHARMACOLOGICAL EVALUATION OF 1-(1H-PYRROL-1-YLMETHYL)-4-AZATRICYCLO[5.2.1.0(2,6)] DEC-8-ENE-3,5-DIONE.

A set of arylpiperazine derivatives with imide fragments, 1-(1H-pyrrol-1-ylmethyl)-10-oxa-4-azatricyclo[5.2.1.0(2,6)-]dec-8-ene-3,5-dione connected by propyl and butyl linkers, were synthesized and tested for the potential anxiolytic and antidepressant activities. Compounds 3a and 3b demonstrated antidepressant activity in the forced swimming tests in mice and were devoid of neurotoxic effects. (chimney test in mice).

Spectroscopic (FT-IR, FT-Raman) investigations and quantum chemical calculations of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-{3-[4-(3-methoxyphenyl)piperazin-1-yl]propyl}-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione.

The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-{3-[4-(3-methoxyphenyl) piperazin-1-yl]propyl}-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione have been investigated experimentally and theoretically using Gaussian09 software package. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. Gauge-including atomic orbital (1)H NMR chemical shifts calculations were carried out and compared with experimental data. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. Molecular Electrostatic Potential was performed by the DFT method and the infrared and Raman intensities have also been reported. First hyperpolarizability is calculated in order to find its role in non-liner optics. The calculated geometrical parameters (SDD) are in agreement with that of similar derivatives. Mulliken's net charges have been calculated and compared with the atomic natural charges.

Vibrational spectroscopic and computational study of 1,7,8,9-Tetrachloro-4-(4-bromo-butyl)-10,10-dimethoxy-4-aza-tricyclo[5.2.1.0(2,6)] dec-8-ene-3,5-dione.

The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of 1,7,8,9-Tetrachloro-4-(4-bromo-butyl)-10,10-dimethoxy-4-aza-tricyclo[5.2.1.0(2,6)] dec-8-ene-3,5-dione (TDAD) have been investigated experimentally and theoretically using Gaussian09 software package. Potential energy distribution of normal modes of vibrations was done using GAR2PED program. Gauge-including atomic orbital (1)H NMR chemical shifts calculations were carried out and compared with experimental data. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. Molecular Electrostatic Potential was performed by the DFT method and infrared intensities and Raman activities are also reported. Mulliken's net charges have been calculated and compared with the atomic natural charges. First hyperpolarizability is calculated in order to find its role in non-liner optics. The calculated geometrical parameters are in agreement with that of similar derivatives.

Spectroscopic (FT-IR, FT-Raman), first order hyperpolarizability, NBO analysis, HOMO and LUMO analysis of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-[3-(4-phenylpiperazin-1-yl)propyl]-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione by density functional methods.

The optimized molecular structure, vibrational frequencies, corresponding vibrational assignments of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-[3-(4-phenylpiperazin-1-yl)propyl]-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione (TDPPAD) have been investigated experimentally and theoretically using Gaussian09 software package. Gauge-including atomic orbital (1)H NMR chemical shifts calculations were carried out and compared with experimental data. The HOMO and LUMO analysis is used to determine the charge transfer within the molecule. The stability of the molecule arising from hyper-conjugative interaction and charge delocalization has been analyzed using NBO analysis. Molecular Electrostatic Potential was performed by the DFT method and the infrared and Raman intensities have also been reported. Mulliken's net charges have been calculated and compared with the atomic natural charges. Fist hyperpolarizability is calculated in order to find its role in non-liner optics. The calculated geometrical parameters (SDD) are in agreement with that of similar derivatives.

Synthesis and pharmacological activity of 1,8,11,11-tetramethyl-4-azatricyclo[5.2.2.0(2,6)]undec-8-ene-3,5-dione derivatives. [corrected].

The synthesis and pharmacological activity of N-substituted derivatives of 1,8,11,11-tetramethyl-4-azatricyclo[5.2.2.0(2,6)]undec-8-ene-3,5-dione (1) are described. The molecular structure of starting compound (1) was confirmed by elemental analysis, 13C NMR and X-ray crystallography. The structures of derivatives were confirmed by 1H NMR and mass spectra. The compounds were investigated for antibacterial activity, including Gram-positive cocci, Gram-negative rods, and antifungal activity. Studied compounds were evaluated also for their cytotoxicity and anti-HIV-1 activity in MT-4 cells.

17-Hy-droxy-1,8-dimethyl-17-aza-penta-cyclo-[6.6.5.0(2,7).0(9,14).0(15,19)]nona-deca-2,4,6,9(14),10,12-hexa-ene-16,18-dione.

In the title compound, C20H17NO3 (alternative name: N-hy-droxy-9,10-dimethyl-9,10-ethano-anthracene-11,12-dicarboximide), the rigid ethano-anthracene-dicarboximide moiety has a roof-shaped geometry, the inter-planar angle between the two terminal phenyl rings being 124.9 (6)°. In the crystal, mol-ecules are linked via O-H⋯O hydrogen bonds, forming chains along [010]. C-H⋯O and C-H⋯π inter-actions link adjacent chains, leading to the formation of a three-dimensional structure.

Ultrastructure of granular osmiophilic material deposits (GOM) in arterioles of CADASIL patients.

Granular osmiophilic material (GOM) is a pathognomonic feature of CADASIL that may be a consequence of pathological processes triggered by Notch3 mutations. Since knowledge of the effects of CADASIL-associated GOM deposits is important to understand the molecular pathogenesis of this disorder, we performed a thorough ultrastructural analysis of GOM morphology in the skin and muscle arterioles in CADASIL patients. Electron microscopy revealed numerous GOM deposits with different morphology including size, shape and osmiophilic density. Osmiophilic granular material of high density was frequently observed in part of GOM deposits located near vascular smooth muscle cells (VSMC) while a part localized distally from the cell body was less dense and loose. On the basis of our observations we postulate that GOM can be formed on the surface of VSMC in the arteriolar wall and penetrate from these cells into the basement membrane and/or extracellular matrix. The dispersion of granules, which form GOM deposits, may be one of the factors triggering the thickening and changes in the basement membrane and/or extracellular matrix.

Synthesis and evaluation of in vitro biological activity of 4-substituted arylpiperazine derivatives of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione.

A series of twenty arylpiperazine derivatives of 1,7,8,9-tetrachloro-10,10-dimethoxy-4-azatricyclo[5.2.1.0(2,6)]dec-8-ene-3,5-dione have been prepared. These derivatives were tested in vitro with the aim of identifying novel lead compounds active against emergent and re-emergent human and cattle infectious diseases (AIDS, hepatitis B and C, tuberculosis, bovine viral diarrhea). In particular, these compounds were evaluated in vitro against representatives of different virus classes, such as a HIV-1 (Retrovirus), a HBV (Hepadnavirus) and the single-stranded RNA(+) viruses Yellow fever virus (YFV) and Bovine viral diarrhea virus (BVDV), both belonging to the Flaviridae. Compounds 2c, 2g and 3d showed a modest activity against CVB-2. The molecular structures of the starting imide 1 and one of propyl-piperazine derivatives, 3b, have been determined by an X-ray crystallography study.