Genome-wide SNP discovery and population structure analysis in pepper (Capsicum annuum) using genotyping by sequencing.

BACKGROUND: Knowledge on population structure and genetic diversity in vegetable crops is essential for association mapping studies and genomic selection. Genotyping by sequencing (GBS) represents an innovative method for large scale SNP detection and genotyping of genetic resources. Herein we used the GBS approach for the genome-wide identification of SNPs in a collection of Capsicum spp. accessions and for the assessment of the level of genetic diversity in a subset of 222 cultivated pepper (Capsicum annum) genotypes. RESULTS: GBS analysis generated a total of 7,568,894 master tags, of which 43.4% uniquely aligned to the reference genome CM334. A total of 108,591 SNP markers were identified, of which 105,184 were in C. annuum accessions. In order to explore the genetic diversity of C. annuum and to select a minimal core set representing most of the total genetic variation with minimum redundancy, a subset of 222 C. annuum accessions were analysed using 32,950 high quality SNPs. Based on Bayesian and Hierarchical clustering it was possible to divide the collection into three clusters. Cluster I had the majority of varieties and landraces mainly from Southern and Northern Italy, and from Eastern Europe, whereas clusters II and III comprised accessions of different geographical origins. Considering the genome-wide genetic variation among the accessions included in cluster I, a second round of Bayesian (K = 3) and Hierarchical (K = 2) clustering was performed. These analysis showed that genotypes were grouped not only based on geographical origin, but also on fruit-related features. CONCLUSIONS: GBS data has proven useful to assess the genetic diversity in a collection of C. annuum accessions. The high number of SNP markers, uniformly distributed on the 12 chromosomes, allowed the accessions to be distinguished according to geographical origin and fruit-related features. SNP markers and information on population structure developed in this study will undoubtedly support genome-wide association mapping studies and marker-assisted selection programs.

Sequence variation of block III segment identifies three distinct lineages within Eggplant mottled dwarf virus isolates from Italy, Spain and Greece.

Partial polymerase (L) gene sequences of 919 nts, including the conserved segments pre-motif A and motif A of block III, of 20 Eggplant mottled dwarf virus (EMDV) isolates were generated, and trimmed sequences of 889 nts, based on the length of available sequences of other isolates, were used to determine phylogenetic relationships. Phylogenetic reconstructions revealed two divergent lineages, designated as genetic group A (Italian isolates) and group B, with the latter further divided into subgroups BI (Greek isolates) and BII (Spanish isolates). No evidence of recombination signals among sequences was detected, whereas analysis of the nonsynonymous/synonymous ratio indicated strong purifying selection, with codons under negative selection uniformly distributed along the sequences. An RT-PCR-RFLP method able to discriminate EMDV isolates of the two main genetic groups was proposed.

First Report of Cucumber mosaic virus Subgroup IA Isolate Infecting Yucca aloifolia in Italy.

Yucca aloifolia L. (Spanish bayonet), family Asparagaceae, is the type species of the genus Yucca. It is native to Mexico and the West Indies and is appreciated worldwide as an ornamental plant. In 2013, during a survey for viruses in ornamental plants in the Campania region of southern Italy, symptoms consisting of bright chlorotic spots and ring spots 1 to 3 mm in diameter with some necrotic streaks were observed on leaves of two plants of Y. aloifolia growing in a nursery located in the Pignataro Maggiore municipality, Caserta Province. Cucumber mosaic virus (CMV) infection was suspected because the symptoms resembled those caused by CMV in Yucca flaccida (1). A range of herbal plant indicators was inoculated with sap extracts of symptomatic Y. aloifolia plants and developed symptoms indicative of CMV. Furthermore, 30 nm isometric virus particles were observed in the same Y. aloifolia sap extracts by transmission electron microscopy. The identity of the virus was confirmed by positive reaction in ELISA tests with CMV polyclonal antisera (Bioreba) conducted on sap extracts of symptomatic Y. aloifolia plants and systemically infected symptomatic hosts (i.e., Nicotiana tabacum, N. glutinosa, Cucumber sativus cv. Marketer, Solanum lycopersicum cv. San Marzano). The presence of CMV in the two naturally infected Y. aloifolia and other mechanically inoculated plants was further verified by reverse transcription (RT)-PCR. Total RNAs were extracted with the E.Z.N.A. Plant RNA Kit (Omega Bio-Tek), according to the manufacturer's instructions. RT-PCR was carried out with the ImProm-II Reverse Transcription System first-strand synthesis reaction (Promega) using the primer pair CMV1 and CMV2 (2). These primers amplify part of the CP gene and part of the 3'-noncoding region of CMV RNA3 and were designed to produce amplicons of different sizes to distinguish CMV isolates belonging to subgroups I or II (3). RT-PCR products were obtained from both naturally infected Y. aloifolia and mechanically inoculated plants as well as from PAE1 isolate of CMV (2), used as positive control, but not from healthy plants. Based on the length of the amplicons obtained (487 bp), the CMV isolate from Y. aloifolia (named YAL) belonged to subgroup I (3). The amplified RT-PCR products were purified with QIAquick PCR Purification Kit (Qiagen), cloned in the pGEMT vector (Promega), and three independent clones were sequenced at MWG (Ebersberg, Germany). Sequences obtained from the two CMV-infected Y. aloifolia plants were identical. This sequence was deposited at GenBank (Accession No. HG965199). Multiple alignments of the YAL sequence with sequences of other CMV isolates using MEGA5 software revealed highest percentage of identity (98.9%) with the isolates Z (AB369269) and SO (AF103992) from Korea and Japan, respectively. Moreover, the YAL isolate was identified as belonging to subgroup IA, based on the presence of only one HpaII restriction site in the 487-bp sequence, as previously proposed (2). Although CMV seems to not be a major threat currently for the production of Y. aloifolia, because the farming of this plant is performed using vegetative propagation, particular attention should be given to the presence of the virus in donor mother plants in order to avoid the dispersion of infected plants that could serve as sources for aphid transmission to other susceptible plant species. To our knowledge, this is the first report of CMV infection of Y. aloifolia in the world. References: (1) I. Bouwen et al. Neth. J. Plant Pathol. 84:175, 1978. (2) G. Parrella and D. Sorrentino. J. Phytopathol. 157:762, 2009. (3) Z. Singh et al. Plant Dis. 79:713, 1995.

Dieback and Wilting Caused by Tomato spotted wilt virus in Arctotis × hybrida in Italy.

In winter 2012, some potted plants of African daisy (Arctotis × hybrida L., family Asteraceae) cv. Hannah, propagated by rooted stem cuttings and cultivated for commercial purposes in a greenhouse located at Albenga (Liguria region, Italy), were noticed for a rapid dieback, generalized reddening, following by an irreversible wilting. Around 130 plants on a total of 3,000 cultivated plants showed symptoms (4 to 5%). One gram of fresh leaves, each collected from three different symptomatic plants, was ground in 4 ml of cold (∼5°C) sodium phosphate 0.03 M buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of carborundum (600 mesh). The inoculum was rubbed on healthy indicator herbaceous plants and inoculated plants were maintained in an insect-proof greenhouse with natural illumination and temperatures of 24/18°C day/night. Healthy and buffer inoculated plants were also included in the test and used as negative control in the subsequent serological and molecular analysis. Sap-inoculated plants showed the following symptoms after 1 to 3 weeks: necrotic local lesions in Chenopodium amaranticolor and C. quinoa, yellowing and stunting following by systemic necrosis and death of the plants in tomato (Solanum lycopersicum cv. San Marzano), necrotic local lesions following by systemic necrotic patterns and leaf deformation in tobacco (Nicotiana tabacum cv. Xanthi nc.) and N. glutinosa, necrotic local lesions in petunia (Petunia × hybrida cv. Pink Beauty). No symptoms were recorded on buffer inoculated plants. Leaf samples from both symptomatic hosts and the three original symptomatic African daisy plants were tested by double-antibody sandwich-ELISA with polyclonal antisera against Cucumber mosaic virus (CMV) and tospoviruses (Tospovirus broad-spectrum, Serogroups I, II, and III, Bioreba AG, Switzerland). Positive reaction was obtained with Tospo-groups antibodies, but not with the CMV ones. Total RNA was extracted from infected leaves of African daisy with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to reverse transcription (RT)-PCR by using the tospovirus universal primers BR60/BR65 that amplify part of the nucleocapsid protein gene (1). Target amplicons of 454 bp were produced for all samples tested. The PCR products were cloned and sequenced on both strands (one clone per amplicon cloned). The resulting sequences were 100% identical, so a single sequence was deposited in GenBank (HF913777). The sequence showed highest homology (99%) with the Tomato spotted wilt virus (TSWV) tomato isolate NJ-JN from South Korea (HM581936). The identity of the virus infecting African daisy was further confirmed by sequencing amplicons obtained by RT-PCR using primers partially covering the movement protein gene of TSWV (2). The sequence obtained (HF913776) showed the highest homology (99%) with three TSWV isolates: a tomato isolate from Spain (AY744493), a pepper isolate from South Korea (AB663306), and again the tomato NJ-JN isolate from South Korea (HM581936). To our knowledge, this is the first natural report of TSWV infecting African daisy plants. Moreover, since this ornamental is often cultivated with other flowering plants, it can act as reservoir for the virus that can infect other ornamentals and crops, considering that TSWV has a very broad host range (3). This result also represents the first finding of TSWV in the genus Arctotis, family Asteraceae, the greater botanical family of TSWV hosts (3). References: (1) M. Eiras et al. Fitopatol. Bras. 26:170, 2001. (2) M. M. Finetti et al. J. Plant Pathol. 84:145, 2002. (3) G. Parrella et al. J. Plant Pathol. 85:227. 2003.

Araujia sericifera New Host of Alfalfa mosaic virus in Italy.

Araujia sericifera Brot. (Fam. Apocynaceae) is an evergreen climbing plant native of South America, originally introduced in Europe as an ornamental. In spring 2012, virus-like symptoms including bright yellow mosaic of calico-type and leaf distortion were observed in three A. sericifera plants growing in an abandoned field located in Pomigliano d'Arco (Campania region, Italy). Leaves from the three plants were collected and examined using commercial antisera (Bioreba AG, Reinach, Switzerland) by double antibody sandwich (DAS)-ELISA against Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), and by indirect plate trapped antigen (PTA)-ELISA against potyviruses (Potygroup test). Only AMV was detected serologically in the three A. sericifera samples. The virus was mechanically transmitted from the ELISA-positive samples to four plants each of Chenopodium quinoa, C. amaranticolor, tobacco (Nicotiana tabacum cv. Xanthi nc), cowpea (Vigna unguiculata, cv. Black eyes), basil (Ocimum basilicum, cv. Gigante), and tomato (Solanum lycopersicum cv. San Marzano), using chilled 0.03 M sodium phosphate buffer, containing 0.2% sodium diethyldithiocarbamate, 75 mg/ml of active charcoal, and traces of Carborundum (600 mesh). Inoculated plants were kept in an insect-proof greenhouse with natural illumination and temperatures of 24 and 18°C day/night. Under these conditions, plants showed the following symptoms after 1 to 3 weeks, consistent with symptoms caused by AMV (1): chlorotic local lesions following by mosaic in C. quinoa and C. amaranticolor, reddish local lesions following by mosaic in cowpea, necrotic local lesions followed by systemic necrosis in tomato, bright yellow mosaic (calico type) in basil, and mosaic and strong deformation of the apical leaves in tobacco. The presence of AMV in ELISA-positive A. sericifera and host plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers for the coat protein gene (CP) previously used for the molecular characterization of AMV isolates (2). An Italian isolate of AMV from Lavandula stoechas (GenBank Accession No. FN667967) and RNA extracted from a healthy A. sericifera plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (∼750 bp) was obtained from each of the infected plants assayed, and that derived from A. sericifera isolate Ars2 was purified (QIAqick PCR Purification Kit, Qiagen), cloned in pGEMT easy vector (Promega, Fitchburg, WI) and sequenced (HF570950). Sequence analysis of the CP gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 98% (99% amino acid identity) with the AMV isolate Tef-1 (FR854391), an isolate belonging to subgroup I (3). To our knowledge, this is the first report of AMV infecting A. sericifera in Italy. Since A. sericifera is considered an invasive plant, in continuous expansion to new areas in Italy and in other European countries, particular attention should be paid to the possibility that this species may play a role in the epidemiology of aphid-transmitted viruses such as AMV and CMV, representing a threat to susceptible crops growing nearby. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Plant Dis. 96:249, 2012.

Identificación de especies cárnicas en productos cárnicos cocidos: utilización de sds-page como método de screening / Meat species identification in cooked meat products: using sds-page as screening method

It is necessary to have methodologies for the detection of meat species used in meat products in order to establish possible adulterations. Twenty cooked meat products produced in pilot plants or commercially available products were analyzed in order to evaluate the SDS-PAGE electrophoresis methodology as a screening method to identify the meat species used. The results found by electrophoresis were compared with an immunochemical method (ELISA kits for detection ofpork, beef and poultry). SDS-PAGE methodology allowed the detection of beef, pork, chicken and /or turkey proteins in most samples. When some of these species were present in low proportion this methodology was not able to detect them. SDS-PAGE method has the advantage that allows the detection of proteins of different meat species in only one electrophoresis run while with the ELISA method it is necessary to analyze the same sample with different species identification kits to confirm the presence of the species used.

Es necesario contar con metodologías que permitan la detección de las especies cárnicas utilizadas en la elaboración de productos cárnicos a los fines de establecer posibles adulteraciones. Con la finalidad de evaluar SDS-PAGE como método de screening para identificar la/las especies cárnicas utilizadas" en el presente trabajo" se analizaron veinte productos cárnicos crudos o cocidos elaborados en plantas piloto o comerciales. Los resultados hallados por electroforesis se compararon con un método inmunoquímico (ELISA). SDS-PAGE permitió la detección de proteínas de carne vacuna" porcina" de pollo y/o de pavo en las mayoría de las muestras analizadas. Sólo en algunas muestras que contenían alguna de las especies cárnicas en baja proporción" esta metodología no permitió su detección. SDS-PAGE tiene como ventaja poder detectar en una sola corrida la presencia de proteínas de diferentes especies cárnicas" mientras que con ELISA es necesario analizar una misma muestra con los diferentes kits de especies cárnicas para confirmar la presencia de las especies utilizadas.

Macrophages and liposomes in inflammatory disease: friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.

Vertebral Body Stenting System for the Treatment of Osteoporotic Vertebral Compression Fracture: Follow-up at 12 Months in 20 Cases.

We describe our preliminary experience with the vertebral body stenting system (VBS) for the treatment of osteoporotic vertebral fracture or traumatic vertebral fracture showing our clinical results at 12 months follow-up. Twenty patients (16 women, four men, mean age 71 years): four with traumatic vertebral fracture (Magerl A1 fractures) and 16 with osteoporotic vertebral compression fracture (VCFs) resistant to conservative therapy, were treated by vertebral body stenting system (VBS) as follows: two at level T11, four at T12, one at L1, two at L2, five at L3 and six at L4. All patients were studied by MR (protocol: sagittal T1W, T2W and T2 STIR) and MDCT with MPR reconstructions. All procedures were performed under local anesthesia with fluoroscopy guidance and a bipeduncular approach. VBS, a new system of implantation of endovertebral stent used as an alternative to conventional vertebroplasty (VP), was implanted in all patients to restore the loss of height in the fractured vertebral body. A clinical and x-ray follow-up was performed at six and 12 months evaluating the result by VAS and ODS scale. New vertebral fractures at a distant level were observed in two cases and treated by VP. VBS was successful and led to an excellent outcome in all patients with clinical improvement stable at six months and one year follow-up. The height in the fractured vertebral body was increased in 12 of the 20 VCFs by an average of 1.5 mm. No vascular, extraforaminal or epidural leakage or other adverse events were observed. In the clinical 12 months follow-up we recorded a reduction of four scores in the VAS evaluation and a 40% reduction in the ODS score compared with the pre-treatment values. Endovertebral stents were stable at 12 months at x-ray control in 19/20 patients. No new vertebral fracture located in adjacent vertebrae were observed at 12 month follow-up. By using a stent, the VBS system reduces the collapsed vertebral body and offers good height restoration. The mechanical scaffold of the stent restores the height and at the same time offers a cavity for injection of highly viscous PMMA bone cement without increasing the rate of new vertebral fracture post-VP. A long-term follow-up is recommended.

Variable effects of cyclophosphamide in rodent models of experimental allergic encephalomyelitis.

In this study, we have evaluated the effects of cyclophosphamide on the development of experimental allergic encephalomyelitis (EAE) in four EAE rodent models: monophasic EAE in Lewis rats, protracted relapsing (PR)-EAE in DA rats, myelin oligodendrocyte protein (MOG)-induced EAE in C57Bl/6 mice and proteolipid protein (PLP)-induced EAE in Swiss/Jackson Laboratory (SJL) mice. Cyclophosphamide, administered either prophylactically or therapeutically, suppressed most strongly the clinical symptoms of PR-EAE in DA rats. Treated rats in this group also exhibited the lowest degree of inflammatory infiltration of the spinal cord, as well as the lowest levels of nuclear factor kappa B, interleukin-12 and interferon-gamma. Cyclophosphamide prophylactically, but not therapeutically, also delayed significantly the onset of EAE in Lewis rats. In contrast, regardless of the treatment regimen used, was unable to influence the clinical course of EAE in either MOG-induced EAE in C57Bl/6 mice or PLP-induced EAE in SJL mice. This heterogeneous pharmacological response to cyclophosphamide suggests that significant immunopathogenic differences exist among these EAE rodent models that must be considered when designing preclinical studies. In addition, the effectiveness of cyclophosphamide in dark Agouti (DA) rats with PR-EAE suggests that this may be a particularly useful model for studying novel therapeutic approaches for refractory and rapidly worsening multiple sclerosis in human patients.

Combined Percutaneous and Endovascular Treatment of Symptomatic Aneurysmal Bone Cyst of the Spine: Clinical Six Months. Follow-up of Six Cases.

We describe the usefulness of endovascular and direct percutaneous treatment as a therapy option for aneurysmal bone cysts (ABCs) of the spine. From January 2007 to December 2008, we treated six consecutive patients with symptomatic ABCs resistant to continuous medical management or with acute clinical onset of paraparesis at cervical, thoracic and lumbar spine level. Two patients were treated after emergency laminectomy. All patients were studied with an MRI protocol and multidetector CT with MPR reconstructions followed by angiographic control before treatment. The procedure was performed under general anaesthesia for all patients. Under CT or fluoroscopy guidance, percutaneous treatment was performed either by direct injection of Glubran(®) diluted at 30% with Lipiodol(®) only, or combined with endovascular treatment by Onyx® injection. Clinical and X-ray follow-up was performed at three and six months. Combined endovascular and percutaneous treatment for ABCs was successful and led to an excellent outcome in five out of six patients with clinical improvement. There were no periprocedural or subsequent clinical complications and the glue resulted in successful selective permanent occlusion with intralesional penetration. Direct sclerotherapy resulted in immediate thrombosis of the malformation with no progression of symptoms. Complete healing was observed in five out of six aggressive lesions. No major complications were noted. At six month follow-up the symptoms had completely resolved and X-ray control showed a partial or total sclerotic reaction of the lesion with stable clinical results (no partial or clinical abnormalities). One patient had a recurrence of the ABC with spinal cord cervical clinical symptomatology. Combined endovascular and percutaneous treatment or direct percutaneous sclerotherapy with glue alone are important, safe, effective therapy options for symptomatic aneurysmal bone cyst. Results are stable and confirmed by clinical and X-ray follow-up six months after treatment.

Metabotropic glutamate 7 receptor subtype modulates motor symptoms in rodent models of Parkinson's disease.

Metabotropic glutamate (mGlu) receptors modulate synaptic transmission in the central nervous system and represent promising therapeutic targets for symptomatic treatment of Parkinson's disease (PD). Among the eight mGlu receptor subtypes, mGlu7 receptor is prominently expressed in the basal ganglia, but its role in restoring motor function in animal models of PD is not known. The effects of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), the first selective allosteric activator of mGlu7 receptors, were thus tested in different rodent models of PD. Here, we show that oral (5 mg/kg) or intrastriatal administration (0.1 and 0.5 nmol) of AMN082 reverses haloperidol-induced catalepsy in rats. AMN082 (2.5 and 5 mg/kg) reduces apomorphine-induced rotations in unilateral 6-hydroxydopamine (6-OHDA)-lesioned rats. In a more complex task commonly used to evaluate major akinetic symptoms of PD patients, 5 mg/kg AMN082 reverses the increased reaction time to respond to a cue of bilateral 6-OHDA-lesioned rats. In addition, AMN082 reduces the duration of haloperidol-induced catalepsy in a mGlu7 receptor-dependent manner in wild-type but not mGlu7 receptor knockout mice. Higher doses of AMN082 (10 and 20 mg/kg p.o.) have no effect on the same models of PD. Overall these findings suggest that mGlu7 receptor activation can reverse motor dysfunction associated with reduced dopamine activity. Selective ligands of mGlu7 receptor subtypes may thus be considered as promising compounds for the development of antiparkinsonian therapeutic strategies.

Administration of a monomeric CCL2 variant to EAE mice inhibits inflammatory cell recruitment and protects from demyelination and axonal loss.

Based on gene expression data, we tested the P8A-CCL2 variant of the chemokine CCL2, able to interfere with the chemotactic properties of the parental molecule, in relapsing-remitting (RR)-EAE SJL. Only preventive treatment significantly delayed disease onset in a dose dependent manner. P8A-CCL2 administration, however, decreased demyelination, axonal loss and number of CNS infiltrating T cells and macrophages. Immunological analysis revealed that P8A-CCL2 does not act on Ag-specific T cell proliferation and does not interfere with the differentiation of IFNgamma-releasing effectors T cells. These results suggest that the therapeutic mechanism of P8A-CCL2 may rely on interference with immune cell recruitment.

Beneficial effects of r-h-CLU on disease severity in different animal models of peripheral neuropathies.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

Complete nodal staging in male breast cancer. (Biopsy of axillary and internal mammary sentinel node).

Carcinoma of the male breast is an uncommon phenomenon, accounting for < 1% of all malignancies in men. Searching for a more conservative treatment we introduced in our clinical practice axillary sentinel node biopsy and, if present, sentinel node biopsy of the internal mammary chain. The potential clinical implications of complete nodal staging are far-reaching, and give us a major new opportunity to stratify male patients with breast cancer for appropriate surgery as well as giving valuable prognostic information.

Response to male odours in progestin receptor- and oestrogen receptor-containing cells in female rat brain.

Sensory cues from male rats, such as odours and vaginal-cervical stimulation (VCS), play a modulatory role in female rat sexual behaviour. For example, exposure to male odours and VCS appears to be at least partially responsible for increases in sexual behaviour following repeated mating of oestradiol-primed female rats. Although there is evidence that VCS influences sexual behaviour via a ligand-independent progestin receptor (PR)-dependent mechanism, the mechanism by which odours influence sexual behaviour is not known. We tested the hypothesis that, similar to VCS, the effects of male odours on sexual behaviour are mediated by progestin receptors. Female rats were injected with the progestin antagonist, RU486, or oil vehicle and were then exposed to male-soiled bedding or clean bedding. Although exposure to male-soiled bedding resulted in higher levels of Fos immunoreactivity in brain areas associated with female sexual behaviour, the progestin antagonist did not reduce this effect. Furthermore, there was minimal coexpression of odour-induced Fos and progestin receptors in brain areas associated with female sexual behaviour. Together, these results suggest that the effects of male odours are not mediated by a PR-dependent mechanism. Therefore, we tested the hypothesis that oestrogen receptor (ER)-containing cells are involved in the effects of olfactory cues. Although there was virtually no coexpression of ERbeta and odour-induced Fos in brain areas associated with female sexual behaviour, exposure to male odours slightly increased the number of cells coexpressing ER(alpha) and odour-induced Fos in the posterodorsal medial amygdala. Although, these results do not support the hypothesis that the effects of odours are mediated by a PR-dependent mechanism, they suggest that integration of male odours and hormonal cues may occur in ER(alpha)-containing cells in the posterodorsal medial amygdala.

A progestin antagonist blocks vaginocervical stimulation-induced fos expression in neurones containing progestin receptors in the rostral medial preoptic area.

Vaginocervical stimulation (VCS) has a variety of effects on the brain, physiology and behaviour. Previous work demonstrated that a progestin antagonist blocked neuronal response to VCS (i.e. Fos expression) in the absence of progesterone in some neurones, and suggested that some of the effects of VCS on the brain are mediated by ligand-independent activation of progestin receptors (PRs). Although it had been reported previously that some of the cells in which VCS induces Fos expression also contain PRs, it had not been determined if a progestin antagonist blocked Fos expression in these particular neurones. The purpose of this experiment was to determine if a progestin antagonist decreases Fos expression specifically in cells that also express PRs in the preoptic area and ventromedial hypothalamus. As has been shown previously, VCS increased Fos-immunoreactive (ir) expression in the particular areas studied. In the rostral medial preoptic area, VCS increased Fos expression in cells that coexpressed PRs, as well as in cells that do not. However, in the caudal medial preoptic area, VCS only increased Fos expression in cells that did not coexpress PRs. Injection of the progestin antagonist, RU 486, decreased Fos expression in the rostral, but not caudal medial preoptic area, and it decreased Fos expression only in cells that coexpressed PR-ir. In contrast to a previous report, in the present study, the progestin antagonist did not inhibit VCS-induced Fos expression in the ventromedial hypothalamic area. The results of this experiment suggest that the progestin antagonist inhibits VCS-induced Fos expression in some neurones by blocking PRs, and they provide further support for the idea that VCS influences neuronal response in some cells by ligand-independent activation of PRs in those cells.

Coexpression of ER beta with ER alpha and progestin receptor proteins in the female rat forebrain: effects of estradiol treatment.

Estrogen and progestin receptors (ER, PgR) play a critical role in the regulation of neuroendocrine functions in females. The neuroanatomical distribution of the recently cloned, ER beta, overlaps with both ER alpha and PgR. To determine whether ER beta is found within ER alpha- or PgR-containing neurons in female rat, we used dual label immunocytochemistry. ER beta-immunoreactivity (ER beta-ir) was primarily detected in the nuclei of cells in the periventricular preoptic area (PvPO), the bed nucleus of the stria terminalis (BNSTpr), the paraventricular nucleus, the supraoptic nucleus, and the medial amygdala (MEApd). Coexpression of ER beta-ir with ER alpha-ir or PgR-ir was observed in the PvPO, BNSTpr, and MEApd in ovariectomized rats. E2 treatment decreased the number of ER beta-ir cells in the PvPO and BNSTpr and the number of ER alpha-ir cells in the MEApd and paraventricular nucleus, and therefore decreased the number of cells coexpressing ER beta-ir and ER alpha-ir in the PvPO, BNSTpr, and MEApd. E2 treatment increased the amount of PgR-ir in cells of the PvPO, BNSTpr, and MEApd, a portion of which also contained ER beta. These results demonstrate that ER beta is expressed in ER alpha- or PgR-containing cells, and they suggest that E can modulate the ratios of these steroid receptors in a brain region-specific manner.

Neuropeptide Y inhibits estrous behavior and stimulates feeding via separate receptors in Syrian hamsters.

Central injections of neuropeptide Y (NPY) increase food intake in Syrian hamsters; however, the effect of NPY on sexual behavior in hamsters is not known nor are the receptor subtypes involved in feeding and sexual behaviors. We demonstrate that NPY inhibits lordosis duration in a dose-related fashion after lateral ventricular injection in ovariectomized, steroid-primed Syrian hamsters. Under the same conditions, we compared the effect of two receptor-differentiating agonists derived from peptide YY (PYY), PYY-(3-36) and [Leu(31),Pro(34)]PYY, on lordosis duration and food intake. PYY-(3-36) produced a 91% reduction in lordosis duration at 0.24 nmol. [Leu(31),Pro(34)]PYY was less potent, producing a reduction in lordosis duration (66%) only at 2.4 nmol. These results suggest NPY effects on estrous behavior are principally mediated by Y2 receptors. PYY-(3-36) and [Leu(31),Pro(34)]PYY stimulated comparable dose-related increases in total food intake (2 h), suggesting Y5 receptors are involved in feeding. The significance of different NPY receptor subtypes controlling estrous and feeding behavior is highlighted by results on expression of Fos immunoreactivity (Fos-IR) elicited by either PYY-(3-36) or [Leu(31),Pro(34)]PYY at a dose of each that differentiated between the two behaviors. Some differences were seen in the distribution of Fos-IR produced by the two peptides. Overall, however, the patterns of expression were similar. Our behavioral and anatomic results suggest that NPY-containing pathways controlling estrous and feeding behavior innervate similar nuclei, with the divergence in pathways controlling the separate behaviors characterized by linkage to different NPY receptor subtypes.

Maternal behavior stimulates c-fos activity within estrogen receptor alpha-containing neurons in lactating rats.

Estradiol and other hormones are thought to be critical for the onset, but not maintenance, of maternal behavior in rats. Maternal behavior is instead maintained postpartum by tactile stimulation that dams receive during interactions with pups, and many neural sites implicated in the control of maternal behavior show elevated c-fos activity in response to this stimulation. Many of these sites also contain neurons that express the alpha subtype of the estrogen receptor (ERalpha). Because of possible interactions between tactile stimulation from pups, c-fos, and ERalpha in the lactating rat brain, we determined if populations of cells that show increased c-fos activity after maternal behavior in lactating rats also contain ERalpha. Dams were separated from their pups for 48 h beginning on day 5 postpartum. On day 7 postpartum, experimental dams were reunited with pups and mother-litter interactions were observed for 60 min. Control dams received no pup stimulation. Subjects were sacrificed 60 min later and brain sections were double immunolabeled for the Fos and ERalpha proteins. As expected, the number of ERalpha-immunoreactive (ERalpha-ir) neurons did not differ between the two groups in the eight areas analyzed (lateral region of the lateral septum, posterodorsal medial amygdala, dorsal and ventral medial preoptic area, dorsal and ventral bed nucleus of the stria terminalis, lateral habenula, and ventrolateral caudal periaqueductal gray). Consistent with previous reports, maternal dams had 2- to 7-fold more Fos-immunoreactive (Fos-ir) neurons in these sites compared with nonstimulated controls. Maternal dams had significantly more Fos-ir neurons that also contained ERalpha-ir in all sites, with the greatest increases in the ventral medial preoptic area, lateral habenula, and ventral bed nucleus of the stria terminalis. Between approximately 25 and 45% of the Fos-ir cells in the sites examined also expressed ERalpha. Thus, a substantial number of neurons that are genomically activated during maternal behavior contain ERalpha, raising the possibility that the postpartum display of maternal behavior can be influenced by ERalpha activity.