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Microglia are heterogenous cells characterized by distinct populations each contributing to specific biological processes in the nervous system, including neuroprotection. To elucidate the impact of sex-specific microglia heterogenicity to the susceptibility of neuronal stress, we video-recorded with time-lapse microscopy the changes in shape and motility occurring in primary cells derived from mice of both sexes in response to pro-inflammatory or neurotoxic stimulations. With this morpho-functional analysis, we documented distinct microglia subpopulations eliciting sex-specific responses to stimulation: male microglia tended to have a more pro-inflammatory phenotype, while female microglia showed increased sensitivity to conduritol-B-epoxide (CBE), a small molecule inhibitor of glucocerebrosidase, the enzyme encoded by the GBA1 gene, mutations of which are the major risk factor for Parkinson’s Disease (PD). Interestingly, glucocerebrosidase inhibition particularly impaired the ability of female microglia to enhance the Nrf2-dependent detoxification pathway in neurons, attenuating the sex differences observed in this neuroprotective function. This finding is consistent with the clinical impact of GBA1 mutations, in which the 1.5–2-fold reduced risk of developing idiopathic PD observed in female individuals is lost in the GBA1 carrier population, thus suggesting a sex-specific role for microglia in the etiopathogenesis of PD-GBA1.

Background Nonsteroidal anti-inflammatory drugs (NSAIDs) have been proposed as chemopreventive agents for many tumours; however, the mechanism responsible for their anti-neoplastic activity remains elusive and the side effects due to cyclooxygenase (COX) inhibition prevent this clinical application. Methods Molecular biology, in silico, cellular and in vivo tools, including innovative in vivo imaging and classical biochemical assays, were applied to identify and characterise the COX-independent anti-cancer mechanism of NSAIDs. Results Here, we show that tumour-protective functions of NSAIDs and exisulind (a sulindac metabolite lacking anti-inflammatory activity) occur through a COX-independent mechanism. We demonstrate these NSAIDs counteract carcinogen-induced proliferation by inhibiting the sirtuin 1 (SIRT1) deacetylase activity, augmenting acetylation and activity of the tumour suppressor p53 and increasing the expression of the antiproliferative gene p21. These properties are shared by all NSAIDs except for ketoprofen lacking anti-cancer properties. The clinical interest of the mechanism identified is underlined by our finding that p53 is activated in mastectomy patients undergoing intraoperative ketorolac, a treatment associated with decreased relapse risk and increased survival. Conclusion Our study, for the first-time, links NSAID chemopreventive activity with direct SIRT1 inhibition and activation of the p53/p21 anti-oncogenic pathway, suggesting a novel strategy for the design of tumour-protective drugs.

The progressive decrease of mean SCC in dairy herds worldwide is affecting SCC accuracy as a subclinical mastitis marker. This evidence supports studies aiming to apply differential cell count (DSCC) as a tool to identify mastitis. Two of the major obstacles to apply DSCC were the unavailability of high-throughput milk analysers and the cost of these analyses. Recently availability of high-throughput milk analysers, able to perform a partial DSCC on milk, allowed designing a study aiming to identify subclinical mastitis in individual milk samples. This paper reports the result of this first Italian study performed under field conditions. The study considered 4386 milk test records from four dairy herds with different size, management and milking management. DSCC data were analysed by ROC procedure. This procedure allows identifying the threshold giving the highest accuracy and the highest combined value for sensitivity and specificity, among all the possible thresholds. Among the different ways used to classify milk samples, the analysis applied to days in milk (three classes) showed the highest mean values for sensitivity plus specificity, and the value for accuracy was very close to the highest one observed. At the time of submission, this is the first paper available on peer-reviewed scientific journals reporting the evaluation of DSCC as a marker for subclinical mastitis on individual milk samples collected during routine milk test. The results will help the improvement of mastitis diagnosis and will help dairy farmers to increase the levels of herd management and efficiency.Highlights At the time of submission, this is the first paper available on peer-reviewed scientific journals on the evaluation of DSCC as a marker for subclinical mastitis on individual milk samples. The analysis of data showed as DSCC has not consistent performances, confirming the presence of confounding factors such as parity and days in milk. The thresholds calculated on samples classified by days in milk (three classes) showed to have the overall best test performances with an accuracy of 82.3%.

Nano- and microsized extracellular vesicles (EVs) are naturally occurring cargo-bearing packages of regulatory macromolecules, and recent studies are increasingly showing that EVs are responsible for physiological intercellular communication. Nanoparticles encapsulating anti-tumor theranostics represent an attractive "exosome-interfering" strategy for cancer therapy. Methods: Herein, by labeling plasma-derived EVs with indocyanine green (ICG) and following their biodistribution by in vivo and ex vivo imaging, we demonstrate the existence of nanoparticles with a highly selective cancer tropism in the blood of colorectal cancer (CRC) patients but not in that of healthy volunteers. Results: In CRC patient-derived xenograft (PDX) mouse models, we show that transplanted EVs recognize tumors from the cognate nanoparticle-generating individual, suggesting the theranostic potential of autologous EVs encapsulating tumor-interfering molecules. In large canine breeds bearing spontaneous malignant skin and breast tumors, the same autologous EV transplantation protocol shows comparable safety and efficacy profiles. Conclusions: Our data show the existence of an untapped resource of intercellular communication present in the blood of cancer patients, which represents an efficient and highly biocompatible way to deliver molecules directly to the tumor with great precision. The novel EV-interfering approach proposed by our study may become a new research direction in the complex interplay of modern personalized cancer therapy.

The Parkinson's disease (PD) evolves over an extended period of time with the onset occurring long before clinical signs begin to manifest. Characterization of the molecular events underlying the PD onset is instrumental for the development of diagnostic markers and preventive treatments, progress in this field is hindered by technical limitations. We applied an imaging approach to demonstrate the activation of Nrf2 transcription factor as a hallmark of neurodegeneration in neurotoxin-driven models of PD. In dopaminergic SK-N-BE neuroblastoma cells, Nrf2 activation was detected in cells committed to die as proven by time lapse microscopy; in the substantia nigra pars compacta area of the mouse brain, the Nrf2 activation preceded dopaminergic neurodegeneration as demonstrated by in vivo and ex vivo optical imaging, a finding confirmed by co-localization experiments carried out by immunohistochemistry. Collectively, our results identify the Nrf2 signaling as an early marker of neurodegeneration, anticipating dopaminergic neurodegeneration and motor deficits.

Extracellular vesicles (EVs), are naturally occurring cargo delivery tools with the potential to be used as drug vehicles of single agents or combination therapies. We previously demonstrated that human lung cancer cell-derived EVs could be used for the systemic delivery of oncolytic virus (OVs) and chemotherapy drugs such as paclitaxel (PTX), leading to enhanced anti-tumor effects in nude mice. In the current work, we evaluated the biodistribution of EVs by using bioluminescence and fluorescence imaging technologies, thus proving the ability of these EVs-formulations to specifically target the neoplasia, while leaving other body tissues unaffected. Moreover, in vivo imaging of NFκB activation in an immunocompetent reporter mouse model allowed to demonstrate the selective ability of EVs to induce tumor-associated inflammatory reactions, which are characterized by immunogenic cell death and CD3+/CD4+/CD8+ T-cell infiltration. While EVs have the potential to induce a systemic immune reaction by pro-inflammatory cytokines, our study provides compelling evidences of a localized inflammatory effect in the peritumoral area. Collectively, our findings strongly support the systemic administration of EVs formulations with OVs alone or in combination with chemotherapy agents as a novel strategy aimed at treating primary and metastatic cancers.

Data on the prevalence of major contagious pathogens in bulk tank milk (BTM) in Italy are generally not available. The availability of Real-Time PCR procedures (qPCR) to perform BTM analysis by represents an important step to define herd health status. Therefore, a cross-sectional epidemiological study was designed to assess the prevalence of contagious pathogens and Prototheca spp in BTM samples. The study was performed on 581 herds from four districts in the west Lombardy region of Italy. Additionally, the relationship between pathogens in BTM and SCC or milk yield; the presence of an association between four risk factors (district, herd size, average milk yield and SCC) with pathogens in BTM were assessed. The overall data showed that S. aureus was recovered in 42% of the herds, Str. agalactiae in 10%, Prototheca spp in 11% and M. bovis in 1.5% of the herds. The GLM model applied showed a significant influence of BTM results, district, herd size and their interactions on SCC and on milk yield variance. Particularly, S. aureus or Str. agalactiae have a significant effect on milk yield variability and, in a lesser extent, on SCC. The very high prevalence of contagious pathogens significantly affects milk characteristics and yield, thus affecting economic sustainability of the herds, and suggests the need to implement control programmes to decrease the prevalence of contagious pathogens, This will also allow to decrease the use of antimicrobials and to improve cow welfare.HighlightsFirst study on a large sample of Italian dairy herds on the prevalence of contagious pathogens in bulk tank milk samples. The prevalence value observed exceeded 50%.First study estimating the prevalence of M. bovis in bulk tank milk in a large sample of Italian dairy herds, and the prevalence observed was 1.5%.Prevalence of contagious pathogens has a significant influence on milk yield and SCC.Bulk tank milk SCC confirmed to have a low accuracy to identify infected herds. First study on a large sample of Italian dairy herds on the prevalence of contagious pathogens in bulk tank milk samples. The prevalence value observed exceeded 50%. First study estimating the prevalence of M. bovis in bulk tank milk in a large sample of Italian dairy herds, and the prevalence observed was 1.5%. Prevalence of contagious pathogens has a significant influence on milk yield and SCC. Bulk tank milk SCC confirmed to have a low accuracy to identify infected herds.

Oncolytic viruses (OV) are engineered to infect, replicate in and kill cancer cells. Currently, the OV therapeutic approach is mainly restricted to neoplasia amenable to direct local administration of viral particles, while the possibility of a systemic delivery of cancer-tropic viruses would extend the OV application to the treatment of metastatic neoplasia. Herein, we applied in vivo/ex vivo imaging to demonstrate that cancer tropism is achieved when OV are encapsulated inside extracellular vesicles (EV) administered intravenously (i.v.), but not when injected intraperitoneally (i.p.). Moreover, we show that the therapeutic procedure adopted does not alter the immunomodulatory properties of the viruses.

Sex has a role in the incidence and outcome of neuro- logical illnesses, also influencing the response to treatments. Neuroinflammation is involved in the onset and progression of several neurological dis- eases, and the fact that estrogens have anti-inflam- matory activity suggests that these hormones may be a determinant in the sex-dependent manifestation of brain pathologies. We describe significant differ- ences in the transcriptome of adult male and female microglia, possibly originating from perinatal expo- sure to sex steroids. Microglia isolated from adult brains maintain the sex-specific features when put in culture or transplanted in the brain of the opposite sex. Female microglia are neuroprotective because they restrict the damage caused by acute focal cere- bral ischemia. This study therefore provides insight into a distinct perspective on the mechanisms under- scoring a sexual bias in the susceptibility to brain diseases.

Noninvasive in vivo imaging offers a novel approach to preclinical studies opening the possibility of investigating biological events in the spatiotemporal dimension (eg, in any district of the body in time). Toxicological analysis may benefit from this novel approach through precise identification of the time and the target organs of toxicity manifestations, and assessment of the reversibility of toxic insults. The current limitation for routine application of this technology is the lack of appropriate surrogate markers for imaging toxicological events. Here, we demonstrate that in vivo imaging of a proliferation marker is capable of measuring the reduction of cell proliferation due to genotoxic/apoptotic agents, γ rays or antineoplastic drugs, or the increased proliferation associated with the inflammatory and regenerative reactions occurring after a toxic insult. A number of tools are currently available for imaging proliferation in preclinical and clinical settings, however our data provide a novel way to translate the evidence of toxic effects obtained in preclinical animal studies, by the direct, noninvasive measure of dividing cells in humans.

Mutations in PTEN-induced kinase 1 (PINK1) result in a recessive familial form of Parkinson’s disease (PD). PINK1 loss is associated with mitochondrial Ca2+ mishandling, mitochondrial dysfunction, as well as increased neuronal vulnerability. Here we demonstrate that PINK1 directly interacts with and phosphorylates LETM1 at Thr192 in vitro. Phosphorylated LETM1 or the phospho-mimetic LETM1-T192E increase calcium release in artificial liposomes and facilitates calcium transport in intact mitochondria. Expression of LETM1-T192E but not LETM1-wild type (WT) rescues mitochondrial calcium mishandling in PINK1-deficient neurons. Expression of both LETM1-WT and LETM1-T192E protects neurons against MPP+–MPTP-induced neuronal death in PINK1 WT neurons, whereas only LETM1-T192E protects neurons under conditions of PINK1 loss. Our findings delineate a mechanism by which PINK1 regulates mitochondrial Ca2+ level through LETM1 and suggest a model by which PINK1 loss leads to deficient phosphorylation of LETM1 and impaired mitochondrial Ca2+ transport.., Mutations in the mitochondrial kinase PINK1 result in familial Parkinson’s disease. Here the authors show that LETM1, a mitochondrial inner membrane protein, is a substrate of PINK1 that regulates Ca2+ handling in mitochondria in response to mitochondrial toxins.

To counteract oxidative stress cells developed several mechanisms, including the transcription factor Nuclear Factor E2-related factor 2 (Nrf2). The aim of the study was to evaluate the activation of Nrf2 in transgenic mice fed saturated or polyunsaturated fatty acids and the anti-inflammatory effect of estrogens on organism. Forty-eight ARE CRE OMO reporter mice were divided into 3 groups, consisting of 16 animals, based on presence/absence of estrogens (ovariectomized or sham female, OVX - SH; male, MA). Each group was further split in 4 subgroups of 4 animals each and fed different diets (7.5% lard, 7.5% tuna oil, 20.0 % lard and 20.0% tuna oil). Two times a week animals were anaesthetized and injected i.p. with 100µL luciferin 15 min before the imaging session. Using the Living Image Software, photon emission was mapped for selected body areas. On day 70, animals were sacrificed after a challenge with Sodium Arsenite. Specific organs were dissected and immediately subjected to ex vivo imaging session. MIXED and GLM procedures of SAS software were used for statistical analysis. Dietary treatments did not affect body weight and feed intake as well as Nrf2 expression in both pre- and post-challenge phases, with the exception of the abdominal region (P=0.031 pre-challenge); in this area, during the pre-challenge phase, OVX showed lower Nrf2 activation (P, International Journal of Health, Animal Science and Food Safety, V. 4, N. 1s (2017)

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.

Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress.

Most of the ageing-associated pathologies are coupled with a strong inflammatory component that accelerates the progress of the physiopathological functional decline related to ageing. The currently available pharmacological tools for the control of neuroinflammation present several side effects that restrict their application, particularly in chronic disorders. The discovery of the potential anti-inflammatory action exerted by endogenous oestrogens, as well as the finding that activation of oestrogen receptor α results in a significant decrease of inflammation at the cellular level and in models of inflammatory diseases, prompted us to embark in a series of studies aimed at the generation of reporter systems, allowing us to (i) understand the anti-inflammatory action of oestrogens at molecular level; (ii) evaluate the extent to which the action of this steroid hormone was relevant in models of pathologies characterised by a strong inflammatory component; and (iii) investigate the efficacy of novel, synthetic oestrogens endowed with anti-inflammatory activity. Accordingly, we conceived the NFκB-luc2 reporter mouse, a model characterised by dual reporter genes for fluorescence and bioluminescence imaging under the control of a synthetic DNA able to bind the transcription factor nuclear factor kappa B, the master regulator of the expression of most of the cytokines responsible for the initial phase of acute inflammation. Here, we summarise the philosophy that has driven our research in the past years, as well as some of the results obtained so far.

Although 17β-estradiol (E2) anti-inflammatory activity has been well described, very little is known about the effects of this hormone on the resolution phase of the inflammatory process. Here, we identified a previously unreported ERα-mediated effect of E2 on the inflammatory machinery. The study showed that the activation of the intracellular estrogen receptor shortens the LPS-induced pro-inflammatory phase and, by influencing the intrinsic and extrinsic programs, triggers the resolution of inflammation in RAW 264.7 cells. Through the regulation of the SOCS3 and STAT3 signaling pathways, E2 facilitates the progression of the inflammatory process toward the IL10-dependent “acquired deactivation” phenotype, which is responsible for tissue remodeling and the restoration of homeostatic conditions. The present study may provide an explanation for increased susceptibility to chronic inflammatory diseases in women after menopause and it suggests novel anti-inflammatory treatments for such disorders.

Two forms of competitive encounters namely Randori (free fight) and Kata (highly ritualized fight) were studied in 22 professional male judo fighters. The dyadic, symmetrical (in terms of body weight and fighting ability) encounters were videotaped to assess relationships between agonistic behavior and individual variations in plasma levels of testosterone (T), cortisol (C) and interleukins (IL-6 and IL-1 beta) measured before and after the competition. Unremarkably, winners showed longer attack but devoted less time to defensive behaviors when compared to losers. T increased only during Randori but the individual pre- and post-competition T levels recorded in such fights were strongly correlated with the corresponding measures in the Kata for the same individuals. Interestingly, the pre- and post-Randori competition T levels were higher in losers than in winners and T variations positively correlated with the frequencies of attacks and with the duration of defensive postures. The T response shows individual variation and seems to reflect evaluation of the likelihood of winning or losing. Both Randori and Kata induced a marked C increase, although the pre- and post-Randori hormonal titers were higher than those found for the Kata. IL-6 significantly increased between the pre- and the post-Randori competition, but no such changes occurred during the Kata. No correlations were found between individual pre- and post-competition C and IL-6 and IL-1 beta levels in either Randori or Kata. This suggests that C and cytokine release are unrelated to emotional or cognitive perception of the possible outcome of fighting but are a consequence of general motor activity. Martial arts appear to provide good human models to understand: (a) the relationships between conflict, hormones and the immune system and (b) the relationships between mood and physiological responses to competitive aggression. Language: en

The superconducting material YBa2Cu3O7−x was prepared by microwave heating of an oxide mixture (Y2O3 ,B aO, CuO). The tim er equired for the synthesis is reduced to about 3.5 h compared to 1–2 days if conventional heating is used. If during the microwave heating the boat containing the starting powders is surrounded by SiC, the Y2BaCuO5 insulating phase does not appear.

Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover, heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts. In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli.

CdCl2 salt is widely used in exposure oriented studies, while the biological exposure of Cadmium (Cd) occurs mostly through diet. Hence, we designed a in vivo imaging methodology with sexually immature male ERE-Luc reporter mice to test the estrogen-like (EL) effects of Cd as a natural component in wheat and flax bread based diets (containing 17.57 and 49.22 ug/kg Cd concentrations respectively) and CdCl2 per-oral dose of 1 ug/kg/bw/day. Total exposure of ingested and % bioaccumulation of Cd in selected organs were estimated as 547 ng (4.4%), 776 ng (0.3%) and 2131.8 ng (0.1%) corresponding to CdCl2, wheat and flax bread based diet treatments respectively. Cd from CdCl2 bioaccumulated more readily, despite the exposure of Cd is higher with bread based diets. Longitudinal in vivo imaging did not reveal significant changes in luciferase activity. White adipose tissue (WAT) and prostate were identified as novel target organs of Cd. Indeed, the rest of the observed EL effects, endogenous target gene expression and necropsy findings are not consistent to any particular organ or treatment. This implies that, the observed EL effects due to low doses of Cd (either from CdCl2 or dietary form) occur only as subtle changes at the molecular level, but inadequate to cause significant changes at the anatomo-pathological level during the 21 day exposure period. The study demonstrates the sensitivity of the methodology to assess EL effects of food embedded Cd and underlines the limitations of directly extrapolating the results of suspected chemicals in their pure form to dietary exposure scenarios.

Specimens from 53 pituitary adenomas (PAs), including 17 NFPA, 16 PRL-, 9 ACTH-, 9 GH- and 2 TSH-secreting tumors, underwent cytogenetic analysis by the direct and short-term culture methods. Only 8 tumors (15%) appeared to have an abnormal karyotype. To increase the resolution of cytogenetic analysis, direct preparations from 31 PAs were investigated by interphase FISH with probes specific for chromosomes 5, 8, 12 and X, for which gain in pituitary tumors has been reported. Of these 31 PAs, 17 (54.8%) had an abnormal dosage of one or more of the 4 chromosomes tested. Separate or combined trisomies of chromosomes 5, 8 and 12 were found in 10/10 prolactinomas and in 4/9 NFPA, whereas the combined loss of chromosomes 5 and 8 was observed in 1/6 ACTH- and 1/6 GH-secreting PAs. Present and earlier data on 23 PAs showed that tumors with the highest frequency of abnormal karyotypes revealed by cytogenetics and/or interphase FISH were PRL (78%), followed by NFPA (26%) and GH (18%). Recurrent structural rearrangements affecting chromosomes 1, 3 and 12 were also identified in prolactinomas, which therefore appear to be the only pituitary adenoma sub-type with a defined trend of tumor-specific chromosomal changes. Cytogenetic and FISH analyses of different pituitary tumor sub-types indicate that they may harbour genetically distinct lesions. Int. J. Cancer 86:344–350, 2000. © 2000 Wiley-Liss, Inc.

Cardiac ryanodine receptor (RyR2) mutations are associated with autosomal dominant catecholaminergic polymorphic ventricular tachycardia, suggesting that alterations in Ca 2+ handling underlie this disease. Here we analyze the underlying Ca 2+ release defect that leads to arrhythmia in cardiomyocytes isolated from heterozygous knock-in mice carrying the RyR2 R4496C mutation. RyR2 R4496C−/− littermates (wild type) were used as controls. [Ca 2+ ] i transients were obtained by field stimulation in fluo-3–loaded cardiomyocytes and viewed using confocal microscopy. In our basal recording conditions (2-Hz stimulation rate), [Ca 2+ ] i transients and sarcoplasmic reticulum Ca 2+ load were similar in wild-type and RyR2 R4496C cells. However, paced RyR2 R4496C ventricular myocytes presented abnormal Ca 2+ release during the diastolic period, viewed as Ca 2+ waves, consistent with the occurrence of delayed afterdepolarizations. The occurrence of this abnormal Ca 2+ release was enhanced at faster stimulation rates and by β-adrenergic stimulation, which also induced triggered activity. Spontaneous Ca 2+ sparks were more frequent in RyR2 R4496C myocytes, indicating increased RyR2 R4496C activity. When permeabilized cells were exposed to different cytosolic [Ca 2+ ] i , RyR2 R4496C showed a dramatic increase in Ca 2+ sensitivity. Isoproterenol increased [Ca 2+ ] i transient amplitude and Ca 2+ spark frequency to the same extent in wild-type and RyR2 R4496C cells, indicating that the β-adrenergic sensitivity of RyR2 R4496C cells remained unaltered. This effect was independent of protein expression variations because no difference was found in the total or phosphorylated RyR2 expression levels. In conclusion, the arrhythmogenic potential of the RyR2 R4496C mutation is attributable to the increased Ca 2+ sensitivity of RyR2 R4496C , which induces diastolic Ca 2+ release and lowers the threshold for triggered activity.

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)–linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 μM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below ∼0.5 mM.

Background : Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by adrenergically mediated polymorphic or bidirectional ventricular tachycardia (BVT). Andersen-Tawil syndrome (ATS1), which are mainly caused by KCNJ2 mutations, phenocopies CPVT and may manifest the typical adrenergically mediated BVT. The purpose of this study was assess whether patients (pts) lacking periodic paralysis typical of ATS1 and diagnosed as CPVT because of BVT carry KCNJ2 mutations. Methods and Results : Mutational analysis of KCNJ2 was performed in 23 RyR2 and CASQ2 genotype-negative CPVT pts with normal QT interval. Two novel missense mutations (S220I and T305I) were identified. Mutations were absent in >400 reference alleles. Both of the pts present exercise or isoproterenol induced BVT, baseline ECG with prominent U wave and mild facial abnormalities. In vitro characterization showed that no current is detectable when S220I and T305I mutants clones are expressed; on the contrary co-expression of WT and mutant KCNJ2 to mimic heterozygosis present in patients, caused significant dominant negative effect (see figure ). Confocal laser microscopy revealed normal sarcolemmal localization of the mutant channels and of the heterozygous channels. Conclusions : KCNJ2 mutation with loss of function are present in a minority of pts with clinical diagnosis of CPVT. Given the limited number of CPVT pts with KCNJ2 mutations it is impossible to determine whether their prognosis is identical to that of RyR2 and CASQ2 related CPVT. Screening of KCNJ2 should be considered in CPVT pts without mutations in RyR2 and CASQ2 genes.

Cardiac Calsequestrin (CASQ2) is a high affinity low capacity calcium binding protein essential in the regulation of intracellular Ca storage and release. Mutations in CASQ2 have been linked to the recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVTr). To elucidate the mechanisms of arrhythmogensis in CPVTr, we generated a knock-in mouse model carrier of the R33Q mutation identified in one of our patients (pts) with a severe CPVT phenotype. Continuous ECG recording (DSI implantable monitors) showed that homozygous (CASQ2 R33Q/R33Q ) mice develop bidirectional and polymorphic VT upon exposure to adrenergic triggers (noise, physical contact). CASQ2 R33Q/R33Q mice have no sign of structural cardiac abnormality (normal heart/body weight ratio, histology no fibrosis nor myofibrillar disarray). Calcium-binding affinity of CASQ2 R33Q/R33Q is identical to that of wild type (WT) CASQ2, its localization evaluated with confocal microscopy is superimposable to that of WT. Western blot analysis shows that CASQ2 R33Q/R33Q content in myocytes is reduced by 40% but Real Time PCR showed that levels of mRNA are not reduced suggesting an abnormal protein turn-over. Evaluation of electrophysiological properties of isolated CASQ2 R33Q/R33Q myocytes revealed that pacing (1–5 Hz) induces delayed afterdepolarizations (DADs; 18/32 cells 56%) and triggered activity (TA; 9/31 29%) addition of adrenalin (200 nM) enhances DADs and TA (DADs 15/16 cells 93%; TA 8/17 cells 47%) and also elicits (25% of myocytes) early after depolarizations (EADs) and EADs-mediated TA. Our data demonstrate that in analogy with our knock-in model of the dominant form of CPVT due to mutations in the cardiac ryanodine receptor (RyR2) the CASQ2 R33Q/R33Q mice 1) do not present signs of cardiomyopathy, 2) develop DADs and TA in vitro and bidirectional and polymorphic VT in vivo. At variance with RyR2 mice, CASQ2 R33Q/R33Q mice 1) present a more malignant phenotype, lower threshold for DADs, TA and VT; 2) EADs concur to arrhythmogenesis 3) overcoming with a knock-in the limitations of previous in vitro and in vivo models overexpressing mutant CASQ2 on the background of the WT protein we demonstrated that a reduction in CASQ2 is implicated in the arrhythmogenesis of recessive CPVT.

YBa2Cu3Ox (Y-123) and Bi2Sr2Ca1Cu2Ox (Bi-2212) films on various substrates have been prepared by Metal-Organic Deposition starting from different metallorganic fluorine-free compounds and using a very simple instrumentation. The processing conditions include a rapid pyrolysis step in air and an annealing step in oxygen for Y-123 and in air for Bi-2212. The films obtained have been characterized by X-ray diffraction (XRD) and the formation of a superconducting phase of Y-123 or Bi-2212 was confirmed measuring the critical temperature (T c) with Ac-susceptibility and resistive measurements. Microstructure and final cationic ratios have been studied by scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS).

Isodicentrics (idic) are structural anomalies of the Y chromosome associated with a 45,X cell line and a broad spectrum of phenotypes. We characterized the rearranged Y chromosomes from three azoospermic males by fluorescence in-situ hybridization (FISH) and PCR. Chromosome study was performed on lymphocytes and testicular biopsy. FISH analysis and PCR established the degree of mosaicism and analysed specific Y regions. Two patients showed a 45,X/46,X,?idic(Y) karyotype with varying degrees of mosaicism. FISH demonstrated the presence of two centromeres and two SRY regions. In the lymphocytes of the third patient, the presence of a small Y-derived marker was also observed. An additional cell line with two idic(Y) was present in the testicular biopsy of the same patient. PCR showed the breakpoint between SY182 (KALY) and SY121 in Yq11.221-q11.222 region in all the cases. For the evaluation of the mosaicism, different tissues must be investigated. The phenotypical sex depends more on the number of copies of the SRY gene rather than on the percentage of 45,X cells, at least in the gonads. The combined use of classical and molecular cytogenetics is necessary for delineating the chromosome regions involved allowing a better genotype-phenotype correlation.

Background— Four distinct mutations in the human cardiac calsequestrin gene ( CASQ2 ) have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). The mechanisms leading to the clinical phenotype are still poorly understood because only 1 CASQ2 mutation has been characterized in vitro. Methods and Results— We identified a homozygous 16-bp deletion at position 339 to 354 leading to a frame shift and a stop codon after 5aa (CASQ2 G112+5X ) in a child with stress-induced ventricular tachycardia and cardiac arrest. The same deletion was also identified in association with a novel point mutation (CASQ2 L167H ) in a highly symptomatic CPVT child who is the first CPVT patient carrier of compound heterozygous CASQ2 mutations. We characterized in vitro the properties of CASQ2 mutants: CASQ2 G112+5X did not bind Ca 2+ , whereas CASQ2 L167H had normal calcium-binding properties. When expressed in rat myocytes, both mutants decreased the sarcoplasmic reticulum Ca 2+ -storing capacity and reduced the amplitude of I Ca -induced Ca 2+ transients and of spontaneous Ca 2+ sparks in permeabilized myocytes. Exposure of myocytes to isoproterenol caused the development of delayed afterdepolarizations in CASQ2 G112+5X . Conclusions— CASQ2 L167H and CASQ2 G112+5X alter CASQ2 function in cardiac myocytes, which leads to reduction of active sarcoplasmic reticulum Ca 2+ release and calcium content. In addition, CASQ2 G112+5X displays altered calcium-binding properties and leads to delayed afterdepolarizations. We conclude that the 2 CASQ2 mutations identified in CPVT create distinct abnormalities that lead to abnormal intracellular calcium regulation, thus facilitating the development of tachyarrhythmias.

Myelotoxicity is the well known side effect of most anti-cancer treatments, thus the study of the myelosuppressive effect of a given treatment is tested routinely during the preclinical phase of investigation. Current ethodologies require the use of a very large number of animals to measure the effect of the investigated compound in time and the study is generally carried out by bone marrow dissection and subsequent analysis with a myeloid clonogenic assay. The goal of our study was to apply non invasive imaging technologies to generate a novel, simple and robust assay to test the state of proliferation of bone marrow reducing the number of animals to be sacrificed and increasing the power of the analysis thanks t the possibility to follow in time, in the same animal the effect of the treatment. To this aim, we have utilized a trangenic mouse, the repTOP™mitoIRE reporter mouse recently developed to image proliferative events in vivo (Goeman et al 2012, Oliva et al 2012). This transgenic mouse carries a luciferase reporter gene under the transcriptional control of a minimal human cyclin B2 promoter.Our in vitro and in vivo results show that the luciferase expression and photon emission in the different body regions of the repTOP™mitoIRE is strictly proportional to the proliferative state of the cells in the tissues examined. Our results demonstrate that the extent of bone marrow proliferation can be measured by in vivo imaging photon emission of femur and sternum in each mouse prior the treatment and then monitored during the longitudinal study. The repTOP™mitoIRE mouse model, was successfully applied to the study of the differential immunosuppressive potential of several well known anti-neoplastic treatments demonstrating the validity of the novel test over currently available methodologies: we measured the differential effects on immunosuppression and on regeneration of 5-fluorouracil, docetaxel, bortezomid, temozolomide and radiotherapy in dose-response experiments. The results led us to conclude that the imaging-based test here proposed is reproducible and provides a reliable measure of bone marrow cell proliferation in time and to propose it as a routine test in for the pre-clinical of novel anti-neoplastic compounds. Citation Format: Nicoletta Rizzi, Isabella Manni, Laura Mancini, Chiara Roncoroni, Electra Brunialti, Paolo Oliva, Maria Pia Gentileschi, Stefano Di Giovine, Enrico A. Pesenti, Giulia Piaggio, Adriana Maggi, Paolo Ciana. A reporter mouse to measure drug myelotoxicity in time. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2013-4416