Ultrasonographic and CT-scan findings of splenic active chylous effusion and postlymphangiography enhancement in a cat with chylothorax associated with splenic angiosarcoma.

A 12-year-old domestic shorthair cat was presented with relapsing noncardiogenic chylothorax. CT-lymphangiography of the thorax confirmed bilateral pleural effusion without evidence of an underlying intra-thoracic origin. Abdominal CT-lymphangiography revealed a 2.6 cm diameter splenic mass surrounded by chylous effusion actively collecting during ultrasonographic assessment. Following splenectomy, histopathological analysis revealed that the splenic mass exhibited characteristics indicative of splenic angiosarcoma. This case report highlights the utility of advanced thoracic and abdominal imaging, notably CT-lymphangiography, in the diagnostic evaluation of chylothorax in cats. The identification of a splenic mass encased in chylous effusion should prompt a proactive case management strategy.

Heterotopic Ossification Bone Formation in the Frontal Bones of an African Grey Parrot (Psittacus erithacus).

A 5-year-old, male African grey parrot (Psittacus erithacus) was presented with multiple, slow-growing, firm, bilateral masses around the dorsal orbital rims. Computer tomographic imaging revealed mild, incomplete bridging bone formation on the rostrodorsal aspects of the head. A moderate amount of smooth bone formation was identified at the rostrodorsal aspect to the left orbit, with minimal associated soft tissue swelling. Surgical biopsies were collected from the masses and histopathological analysis of the most rostral right mass showed well-differentiated bone, surrounded by dense fibrous connective tissue. Scattered, well-differentiated osteocytes were present within the bone. No evidence of neoplastic changes or infectious agents were identified. The histopathological changes were consistent with metaplastic bone formation. History obtained from the owner revealed recent head trauma, which likely induced the cranial heterotopic ossification in the African grey parrot.

Salinomycin treatment reduces metastatic tumor burden by hampering cancer cell migration.

BACKGROUND: Tumor spreading is the major threat for cancer patients. The recently published anti-cancer drug salinomycin raised hope for an improved treatment by targeting therapy-refractory cancer stem cells. However, an unambiguous role of salinomycin against cancer cell migration and metastasis formation remains elusive. FINDINGS: We report that salinomycin effectively inhibits cancer cell migration in a variety of cancer types as determined by Boyden chamber assays. Additionally, cells were treated with doxorubicin at a concentration causing a comparable low cytotoxicity, emphasizing the anti-migratory potential of salinomycin. Moreover, single-cell tracking by time-lapse microscopy demonstrated a remarkable effect of salinomycin on breast cancer cell motility. Ultimately, salinomycin treatment significantly reduced the metastatic tumor burden in a syngenic mouse tumor model. CONCLUSIONS: Our findings clearly show that salinomycin can strongly inhibit cancer cell migration independent of the induction of cell death. We furthermore demonstrate for the first time that salinomycin treatment reduces metastasis formation in vivo, strengthening its role as promising anti-cancer therapeutic.

Intracellular cyclophilin A is an important Ca(2+) regulator in platelets and critically involved in arterial thrombus formation.

Platelet adhesion and aggregation play a critical role in primary hemostasis. Uncontrolled platelet activation leads to pathologic thrombus formation and organ failure. The decisive central step for different processes of platelet activation is the increase in cytosolic Ca(2+) activity ([Ca(2+)](i)). Activation-dependent depletion of intracellular Ca(2+) stores triggers Ca(2+) entry from the extracellular space. Stromal interaction molecule 1 (STIM1) has been identified as a Ca(2+) sensor that regulates store-operated Ca(2+) entry through activation of the pore-forming subunit Orai1, the major store-operated Ca(2+) entry channel in platelets. In the present study, we show for the first time that the chaperone protein cyclophilin A (CyPA) acts as a Ca(2+) modulator in platelets. CyPA deficiency strongly blunted activation-induced Ca(2+) mobilization from intracellular stores and Ca(2+) influx from the extracellular compartment and thus impaired platelet activation substantially. Furthermore, the phosphorylation of the Ca(2+) sensor STIM1 was abrogated upon CyPA deficiency, as shown by immunoprecipitation studies. In a mouse model of arterial thrombosis, CyPA-deficient mice were protected against arterial thrombosis, whereas bleeding time was not affected. The results of the present study identified CyPA as an important Ca(2+) regulator in platelets, a critical mechanism for arterial thrombosis.

Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes.

The cationizable nature of 'proton-sponge' transfection agents facilitates pDNA delivery in several steps. Protonated amines account for electrostatic DNA binding and cellular uptake, buffering amines mediate polyplex escape from acidifying intracellular vesicles. As demonstrated with a sequence-defined library of oligo(ethanamino)amides containing selected oligoethanamino acids and histidines, the total protonation capacity as well as the cationization pH profile within the endolysosomal range have critical impact on gene transfer. Building blocks with even numbered amine groups (Gtt, Sph) exhibited higher total endolysosomal buffer capacity than odd number (Stp) analogs. Within the endolysosomal range, Gtt has the highest buffer capacity around pH5, whereas Stp has its maximum around pH7. Histidines increased the total buffer capacity, resulted in a more continuous cationization pH profile and greatly improved transgene expression in vitro and in vivo. Using receptor targeted and polyethylene glycol shielded polyplexes, better endosomal escape and >100-fold enhanced transfection was detected. FROM THE CLINICAL EDITOR: Proton-sponge transfection agents for pDNA delivery are characterized in this study, demonstrating over 100-fold enhanced transection and better endosomal escape by using receptor targeted and polyethylene glycol shielded polyplexes.

Specific immune modulation of experimental colitis drives enteric alpha-synuclein accumulation and triggers age-related Parkinson-like brain pathology.

Background: In some people with Parkinson's disease (PD), α-synuclein (αSyn) accumulation may begin in the enteric nervous system (ENS) decades before development of brain pathology and disease diagnosis. Objective: To determine how different types and severity of intestinal inflammation could trigger αSyn accumulation in the ENS and the subsequent development of αSyn brain pathology. Methods: We assessed the effects of modulating short- and long-term experimental colitis on αSyn accumulation in the gut of αSyn transgenic and wild type mice by immunostaining and gene expression analysis. To determine the long-term effect on the brain, we induced dextran sulfate sodium (DSS) colitis in young αSyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses. Results: A single strong or sustained mild DSS colitis triggered αSyn accumulation in the submucosal plexus of wild type and αSyn transgenic mice, while short-term mild DSS colitis or inflammation induced by lipopolysaccharide did not have such an effect. Genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αSyn. Remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-Serine 129 αSyn in the midbrain (including the substantia nigra), in 21- but not 9-month-old αSyn transgenic mice. This increase in midbrain αSyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons. Conclusions: Our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of PD.

Intranasal proliferative fibro-osseous dysplasia in a domestic longhair cat.

CASE SUMMARY: A 13-year-old female domestic longhair cat was presented for further investigation of chronic sneezing combined with a right-sided nasal discharge. A CT scan of the head revealed a locally invasive, aggressive right nasal mass radiographically consistent with a malignant neoplastic process. Histopathology on rhinoscopically guided biopsies revealed an unusual pathology consistent with fibro-osseous hyperplasia/dysplasia. Surgical treatment via a ventral rhinotomy and curettage was performed, and the diagnosis confirmed by repeat histopathology. The cat's clinical signs significantly improved postoperatively. RELEVANCE AND NOVEL INFORMATION: This case report describes an unusual feline nasal pathology. To our knowledge, there are no previous reports of a non-neoplastic, non-inflammatory expansile feline nasal tumour. Also described are the CT and histological appearance of the mass, and the difficulties encountered obtaining the definitive diagnosis. Information regarding the prognosis following surgical removal of proliferative fibro-osseous lesions in cats is poor, especially from the nasal cavity where clean margins may well be impossible to obtain. In this case, surgical resection improved clinical signs and the cat remains well at 15 months post-procedure.

Pointing to the right side? An ERP study on anaphora resolution in German Sign Language.

Sign languages use the horizontal plane to refer to discourse referents introduced at referential locations. However, the question remains whether the assignment of discourse referents follows a particular default pattern as recently proposed such that two new discourse referents are respectively assigned to the right (ipsilateral) and left (contralateral) side of (right handed) signers. The present event-related potential study on German Sign Language investigates the hypothesis that signers assign distinct and contrastive referential locations to discourse referents even in the absence of overt localization. By using a semantic mismatch-design, we constructed sentence sets where the second sentence was either consistent or inconsistent with the used pronoun. Semantic mismatch conditions evoked an N400, whereas a contralateral index sign engendered a Phonological Mismatch Negativity. The current study provides supporting evidence that signers are sensitive to the mismatch and make use of a default pattern to assign distinct and contrastive referential locations to discourse referents.

Production and Comprehension of Prosodic Markers in Sign Language Imperatives.

In signed and spoken language sentences, imperative mood and the corresponding speech acts such as for instance, command, permission or advice, can be distinguished by morphosyntactic structures, but also solely by prosodic cues, which are the focus of this paper. These cues can express paralinguistic mental states or grammatical meaning, and we show that in American Sign Language (ASL), they also exhibit the function, scope, and alignment of prosodic, linguistic elements of sign languages. The production and comprehension of prosodic facial expressions and temporal patterns therefore can shed light on how cues are grammaticalized in sign languages. They can also be informative about the formal semantic and pragmatic properties of imperative types not only in ASL, but also more broadly. This paper includes three studies: one of production (Study 1) and two of comprehension (Studies 2 and 3). In Study 1, six prosodic cues are analyzed in production: temporal cues of sign and hold duration, and non-manual cues including tilts of the head, head nods, widening of the eyes, and presence of mouthings. Results of Study 1 show that neutral sentences and commands are well distinguished from each other and from other imperative speech acts via these prosodic cues alone; there is more limited differentiation among explanation, permission, and advice. The comprehension of these five speech acts is investigated in Deaf ASL signers in Study 2, and in three additional groups in Study 3: Deaf signers of German Sign Language (DGS), hearing non-signers from the United States, and hearing non-signers from Germany. Results of Studies 2 and 3 show that the ASL group performs significantly better than the other 3 groups and that all groups perform above chance for all meaning types in comprehension. Language-specific knowledge, therefore, has a significant effect on identifying imperatives based on targeted cues. Command has the most cues associated with it and is the most accurately identified imperative type across groups-indicating, we suggest, its special status as the strongest imperative in terms of addressing the speaker's goals. Our findings support the view that the cues are accessible in their content across groups, but that their language-particular combinatorial possibilities and distribution within sentences provide an advantage to ASL signers in comprehension and attest to their prosodic status.

Distribution of FcRn Across Species and Tissues.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I type molecule that binds to, transports, and recycles immunoglobulin G (IgG) and albumin, thereby protecting them from lysosomal degradation. Therefore, besides the knowledge of FcRn affinity, FcRn protein expression is critical in understanding the pharmacokinetic behavior of Fc-containing biotherapeutics such as monoclonal antibodies. The goal of this investigation was to achieve for the first time a comparative assessment of FcRn distribution across a variety of tissues and species. FcRn was mapped in about 20 tissues including placenta from human and the most frequently used species in non-clinical safety testing of monoclonal antibodies (mouse, rat, cynomolgus monkey). In addition, the FcRn expression pattern was characterized in two humanized transgenic mouse lines (Tg32 and Tg276) expressing human FcRn under different promoters, and in the severe combined immunodeficient (SCID) mouse. Consecutive sections were stained with specific markers, namely, anti-CD68 for macrophages and anti-von Willebrand Factor for endothelial cells. Overall, the FcRn expression pattern was comparable across species and tissues with consistent expression of FcRn in endothelial cells and interstitial macrophages, Kupffer cells, alveolar macrophages, enterocytes, and choroid plexus epithelium. The human FcRn transgenic mouse Tg276 showed a different and much more widespread staining pattern of FcRn. In addition, immunodeficiency and lack of IgG in SCID mice had no negative effect on FcRn expression compared with wild-type mice.

Multifunctional polymer-capped mesoporous silica nanoparticles for pH-responsive targeted drug delivery.

A highly stable modular platform, based on the sequential covalent attachment of different functionalities to the surface of core-shell mesoporous silica nanoparticles (MSNs) for targeted drug delivery is presented. A reversible pH-responsive cap system based on covalently attached poly(2-vinylpyridine) (PVP) was developed as drug release mechanism. Our platform offers (i) tuneable interactions and release kinetics with the cargo drug in the mesopores based on chemically orthogonal core-shell design, (ii) an extremely robust and reversible closure and release mechanism based on endosomal acidification of the covalently attached PVP polymer block, (iii) high colloidal stability due to a covalently coupled PEG shell, and (iv) the ability to covalently attach a wide variety of dyes, targeting ligands and other functionalities at the outer periphery of the PEG shell. The functionality of the system was demonstrated in several cell studies, showing pH-triggered release in the endosome, light-triggered endosomal escape with an on-board photosensitizer, and efficient folic acid-based cell targeting.

Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer.

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.

Experience with examination of the spinal cord and peripheral nervous system (PNS) in mice: A brief overview.

The representative areas for examination of the mouse peripheral nervous system are the spinal cord, containing central components of the peripheral nervous system that needs to be examined at least at cervical and lumbar level, the sciatic and the tibial nerve. Skeletal muscle samples should include the soleus muscle and the quadriceps femoris or long digital extensor, as well as the medial gastrocnemius. Examination can be extended to the thoracic spinal cord, lumbar dorsal root ganglia and spinal nerve roots, as well as the plantar nerve, and other areas of interest. Perfusion fixation is considered optimal for the nervous system; however, immersion fixation allows producing microscopic sections of excellent quality as well. Paraffin-embedded, hematoxylin and eosin-stained sections can be made from all areas, save for small nerves such as the tibial or plantar nerve, which are examined with advantage in hard plastic sections. It is possible to produce hard plastic sections also of the vertebral column, including the spinal cord, dorsal root ganglia and nerve roots. For special investigations, mice can be fixed in toto, decalcified, embedded and sectioned to reveal the areas of interest. In the mouse peripheral nerves, myelination progresses until the adult age. In aging peripheral nerves there is axonal atrophy, degeneration, nerve fiber loss, increase of collagen and sporadic demyelination, especially radiculoneuropathy. The dorsal root ganglia of untreated control animals show frequent cytoplasmic vacuolation. Axonal degeneration is distally, primary demyelination proximally accentuated. Mouse is not very sensitive to peripheral neurotoxicity: to induce toxic peripheral neuropathy mostly parenteral administration and/or newborn animals are used. Naturally occurring infection affecting the spinal cord and peripheral nerves is Theiler's encephalomyelitis virus inducing acute poliomyelitis or chronic demyelination. Any experimental results are to be assessed taking into account spontaneous, age-related, background changes.

Identification of metabolites, clinical chemistry markers and transcripts associated with hepatotoxicity.

Early and accurate pre-clinical and clinical biomarkers of hepatotoxicity facilitate the drug development process and the safety monitoring in clinical studies. We selected eight known model compounds to be administered to male Wistar rats to identify biomarkers of drug induced liver injury (DILI) using transcriptomics, metabolite profiling (metabolomics) and conventional endpoints. We specifically explored early biomarkers in serum and liver tissue associated with histopathologically evident acute hepatotoxicity. A tailored data analysis strategy was implemented to better differentiate animals with no treatment-related findings in the liver from animals showing evident hepatotoxicity as assessed by histopathological analysis. From the large number of assessed parameters, our data analysis strategy allowed us to identify five metabolites in serum and five in liver tissue, 58 transcripts in liver tissue and seven clinical chemistry markers in serum that were significantly associated with acute hepatotoxicity. The identified markers comprised metabolites such as taurocholic acid and putrescine (measured as sum parameter together with agmatine), classical clinical chemistry markers like AST (aspartate aminotransferase), ALT (alanine aminotransferase), and bilirubin, as well as gene transcripts like Igfbp1 (insulin-like growth factor-binding protein 1) and Egr1 (early growth response protein 1). The response pattern of the identified biomarkers was concordant across all types of parameters and sample matrices. Our results suggest that a combination of several of these biomarkers could significantly improve the robustness and accuracy of an early diagnosis of hepatotoxicity.

Sequential Salinomycin Treatment Results in Resistance Formation through Clonal Selection of Epithelial-Like Tumor Cells.

Acquiring therapy resistance is one of the major obstacles in the treatment of patients with cancer. The discovery of the cancer stem cell (CSC)-specific drug salinomycin raised hope for improved treatment options by targeting therapy-refractory CSCs and mesenchymal cancer cells. However, the occurrence of an acquired salinomycin resistance in tumor cells remains elusive. To study the formation of salinomycin resistance, mesenchymal breast cancer cells were sequentially treated with salinomycin in an in vitro cell culture assay, and the resulting differences in gene expression and salinomycin susceptibility were analyzed. We demonstrated that long-term salinomycin treatment of mesenchymal cancer cells resulted in salinomycin-resistant cells with elevated levels of epithelial markers, such as E-cadherin and miR-200c, a decreased migratory capability, and a higher susceptibility to the classic chemotherapeutic drug doxorubicin. The formation of salinomycin resistance through the acquisition of epithelial traits was further validated by inducing mesenchymal-epithelial transition through an overexpression of miR-200c. The transition from a mesenchymal to a more epithelial-like phenotype of salinomycin-treated tumor cells was moreover confirmed in vivo, using syngeneic and, for the first time, transgenic mouse tumor models. These results suggest that the acquisition of salinomycin resistance through the clonal selection of epithelial-like cancer cells could become exploited for improved cancer therapies by antagonizing the tumor-progressive effects of epithelial-mesenchymal transition.

Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes.

Nine sequence-defined, polycationic oligomers were synthesized containing motifs of three consecutive tyrosines (Y3) as stabilizing components for pDNA and siRNA polyplex assembly. For pDNA, a combination of terminal oligotyrosines and cysteines was necessary and sufficient for stable polyplex formation. Stable siRNA binding required a combination of terminal cysteines and oligotyrosines, as well as a central hydrophobic modification (oligotyrosines or fatty acids). The phenolic group within the aromatic amino acids of Y3 containing oligomers further increased the endosomal buffer capacity. As a result, the new class of oligotyrosine containing oligomers was efficient in pDNA and siRNA transfection, in most cases superior to a previously established cysteine-containing, dioleic acid modified oligomer without the Y3 motif. Additionally, increased serum stability of the new oligomers with terminal Y3 motifs was demonstrated by gel shift and fluorescence correlations spectroscopy (FCS). In vivo stability and biodistribution was monitored by intravenous administration of chemically stabilized Cy7 siRNA either as free form, or complexed with the nine Y3 containing oligomers or control oligomers. Oligomer 332, with the overall most beneficial in vitro and in vivo characteristics, was applied in RAN siRNA polyplexes for intratumoral treatment of neuroblastoma-bearing mice. This resulted in significantly reduced tumor growth compared to animal treated with control siRNA polyplexes.

Lexical prediction via forward models: N400 evidence from German Sign Language.

Models of language processing in the human brain often emphasize the prediction of upcoming input-for example in order to explain the rapidity of language understanding. However, the precise mechanisms of prediction are still poorly understood. Forward models, which draw upon the language production system to set up expectations during comprehension, provide a promising approach in this regard. Here, we present an event-related potential (ERP) study on German Sign Language (DGS) which tested the hypotheses of a forward model perspective on prediction. Sign languages involve relatively long transition phases between one sign and the next, which should be anticipated as part of a forward model-based prediction even though they are semantically empty. Native speakers of DGS watched videos of naturally signed DGS sentences which either ended with an expected or a (semantically) unexpected sign. Unexpected signs engendered a biphasic N400-late positivity pattern. Crucially, N400 onset preceded critical sign onset and was thus clearly elicited by properties of the transition phase. The comprehension system thereby clearly anticipated modality-specific information about the realization of the predicted semantic item. These results provide strong converging support for the application of forward models in language comprehension.

A polyphosphoester conjugate of melphalan as antitumoral agent.

The low molecular weight of many chemotherapeutics causes their untargeted distribution in the body and fast renal clearance, which leads to a loss of therapeutic activity and to unspecific toxic side effects. Therefore, there is a growing interest in conjugating anticancer drugs to water soluble polymers and thus, take advantage of the 'enhanced permeability and retention' (EPR) effect in tumors. In this study, water soluble polyphosphoesters were used as polymer carriers of melphalan hydrochloride (hydrochloride of p-bis(2-chloroethyl)amino-L-phenylalanine), which is a multifunctional alkylating agent. Melphalan was chemically immobilized by covalent bonding to poly(oxyethylene H-phosphonate) under Atherton-Todd reaction conditions. Novel polymer-melphalan complexes with ionic and hydrogen bonds were designed as controls, basing on two other biodegradable polyphosphoesters: poly(hydroxyoxyethylene phosphate) and poly(methyloxyethylene phosphate). The structure of the formed products was elucidated by (1)H, (13)C, (31)P NMR and FT-IR spectroscopy. The cytotoxic effect of the melphalan formulations was evaluated on different tumor cell lines. The novel polymer formulations showed a concentration dependent antitumoral activity, comparable to the effect of unmodified melphalan. The polymer-melphalan conjugate was also evaluated in vivo in the human hepatocellular carcinoma HuH7 xenograft mouse model. It improved the therapeutic efficacy of pure melphalan without causing side effects.

Analysis of adult neurogenesis: evidence for a prominent "non-neurogenic" DCX-protein pool in rodent brain.

Here, we have developed a highly sensitive immunoassay for Dcx to characterize expression in brain and cerebrospinal fluid (CSF) of rodents. We demonstrate that Dcx is widely expressed during development in various brain regions and as well can be detected in cerebrospinal fluid of rats (up to 30 days postnatal). While Dcx protein level decline in adulthood and were detectable in neurogenic regions of the adult rodent brain, similar levels were also detectable in brain regions expected to bear no neurogenesis including the cerebral cortex and CA1/CA3 enriched hippocampus. We monitored DCX protein levels after paradigms to increase or severely decrease adult hippocampal neurogenesis, namely physical activity and cranial radiation, respectively. In both paradigms, Dcx protein- and mRNA-levels clearly reflected changes in neurogenesis in the hippocampus. However, basal Dcx-levels are unaffected in non-neurogenic regions (e.g. CA1/CA3 enriched hippocampus, cortex). These data suggest that there is a substantial "non-neurogenic" pool of Dcx- protein, whose regulation can be uncoupled from adult neurogenesis suggesting caution for the interpretation of such studies.