Oculo-auriculo-vertebral spectrum in Klinefelter syndrome.

We report a boy with classical 47,XXY Klinefelter syndrome (KS) and oculo-auriculo-vertebral spectrum (OAV). Two patients with KS and OAV were reported previously. Also, the combination of bilateral aplasia of the mandibular ramus and condyle and KS has been documented. The present observation supports the view that the cause of hemifacial microsomia appears heterogeneous and that OAV may be part of the spectrum of craniofacial anomalies associated with KS.     

Diversity of European habitat types is correlated with geography more than climate and human pressure

Habitat richness, that is, the diversity of ecosystem types, is a complex, spatially explicit aspect of biodiversity, which is affected by bioclimatic, geographic, and anthropogenic variables. The distribution of habitat types is a key component for understanding broad‐scale biodiversity and for developing conservation strategies. We used data on the distribution of European Union (EU) habitats to answer the following questions: (i) how do bioclimatic, geographic, and anthropogenic variables affect habitat richness? (ii) Which of those factors is the most important? (iii) How do interactions among these variables influence habitat richness and which combinations produce the strongest interactions? The distribution maps of 222 terrestrial habitat types as defined by the Natura 2000 network were used to calculate habitat richness for the 10 km × 10 km EU grid map. We then investigated how environmental variables affect habitat richness, using generalized linear models, generalized additive models, and boosted regression trees. The main factors associated with habitat richness were geographic variables, with negative relationships observed for both latitude and longitude, and a positive relationship for terrain ruggedness. Bioclimatic variables played a secondary role, with habitat richness increasing slightly with annual mean temperature and overall annual precipitation. We also found an interaction between anthropogenic variables, with the combination of increased landscape fragmentation and increased population density strongly decreasing habitat richness. This is the first attempt to disentangle spatial patterns of habitat richness at the continental scale, as a key tool for protecting biodiversity. The number of European habitats is related to geography more than climate and human pressure, reflecting a major component of biogeographical patterns similar to the drivers observed at the species level. The interaction between anthropogenic variables highlights the need for coordinated, continental‐scale management plans for biodiversity conservation.     

Seed to seedling transitions in successional habitats across a tropical landscape

Recognition that tree recruitment depends on the balance between seed arrival and seedling survival has led to a surge of interest in seed‐dispersal limitation and seedling‐establishment limitation in primary forests. Virtually unaddressed are comparisons of this balance in mature and early successional habitats. We assessed seed rain and seedling recruitment dynamics of tree species in primary forest, secondary forest and pasture released from grazing in a tropical agricultural landscape. Seed to seedling ratios (seed effectiveness; Φ_i) for 43 species in southern Mexico determined differences in the extent to which seeds produced seedlings by habitat, life history, and dispersal mode. Reproductive potential as estimated by the transition from seed rain to seedling recruitment, differed by habitats, and varied dramatically by life history and dispersal mode. Expected recruit densities (E_it) were higher for animal‐dispersed than wind‐dispersed species, and for non‐pioneer than pioneer species. Non‐pioneers and animal‐dispersed species had higher expected relative recruit abundance (ε_it) in primary forest (median of 4 seeds recruit^?1) whereas in secondary forest wind‐dispersed pioneers had the highest expected relative recruit abundance (median of 16 seeds per recruit). In pastures, wind‐dispersed pioneer species were most successful with many more seeds per recruit (median of 291) than both forest habitats. Seeds per recruit (Φ_i) appeared to decrease with increase in seed mass for 43 species for which data were available (r = –0.55, P < 0.001). This was associated with a negative correlation of Φ_i with seed size in primary forest (r = –0.50, P = 0.08 for 13 species); Φ_i was not correlated with seed size in secondary forest (n = 16) or pasture (n = 14). Metrics of seeds per recruit, expected recruit density and expected relative recruit abundance dramatically illustrate differences in barriers to recruitment in successional habitats.     

Abscopal effect of boron neutron capture therapy (BNCT): proof of principle in an experimental model of colon cancer

The aim of the present study was to evaluate, for the first time, the abscopal effect of boron neutron capture therapy (BNCT). Twenty-six BDIX rats were inoculated subcutaneously with 1 × 10⁶ DHD/K12/TRb syngeneic colon cancer cells in the right hind flank. Three weeks post-inoculation, the right leg of 12 rats bearing the tumor nodule was treated with BPA-BNCT (BPA-Boronophenylalanine) at the RA-3 nuclear reactor located in Buenos Aires, Argentina, at an absorbed dose of 7.5 Gy to skin as the dose-limiting tissue. The remaining group of 14 tumor-bearing rats were left untreated and used as control. Two weeks post-BNCT, 1 × 10⁶ DHD/K12/TRb cells were injected subcutaneously in the contralateral left hind flank of each of the 26 BDIX rats. Tumor volume in both legs was measured weekly for 7 weeks to determine response to BNCT in the right leg and to assess a potential influence of BNCT in the right leg on tumor development in the left leg. Within the BNCT group, a statistically significant reduction was observed in contralateral left tumor volume in animals whose right leg tumor responded to BNCT (post-treatment/pre-treatment tumor volume <1) versus animals who failed to respond (post/pre ≥1), i.e., 13 ± 15 vs 271 ± 128 mm³. In addition, a statistically significant reduction in contralateral left leg tumor volume was observed in BNCT-responsive animals (post/pre <1) vs untreated animals, i.e., 13 ± 15 vs 254 ± 251 mm³. The present study performed in a simple animal model provides proof of principle that the positive response of a tumor to BNCT is capable of inducing an abscopal effect.     

Boron neutron capture therapy (BNCT) translational studies in the hamster cheek pouch model of oral cancer at the new “B2” configuration of the RA-6 nuclear reactor

Boron neutron capture therapy (BNCT) is based on selective accumulation of B-10 carriers in tumor followed by neutron irradiation. We demonstrated, in 2001, the therapeutic effect of BNCT mediated by BPA (boronophenylalanine) in the hamster cheek pouch model of oral cancer, at the RA-6 nuclear reactor. Between 2007 and 2011, the RA-6 was upgraded, leading to an improvement in the performance of the BNCT beam (B2 configuration). Our aim was to evaluate BPA-BNCT radiotoxicity and tumor control in the hamster cheek pouch model of oral cancer at the new “B2” configuration. We also evaluated, for the first time in the oral cancer model, the radioprotective effect of histamine against mucositis in precancerous tissue as the dose-limiting tissue. Cancerized pouches were exposed to: BPA-BNCT; BPA-BNCT + histamine; BO: Beam only; BO + histamine; CONTROL: cancerized, no-treatment. BNCT induced severe mucositis, with an incidence that was slightly higher than in “B1” experiments (86 vs 67%, respectively). BO induced low/moderate mucositis. Histamine slightly reduced the incidence of severe mucositis induced by BPA-BNCT (75 vs 86%) and prevented mucositis altogether in BO animals. Tumor overall response was significantly higher in BNCT (94–96%) than in control (16%) and BO groups (9–38%), and did not differ significantly from the “B1” results (91%). Histamine did not compromise BNCT therapeutic efficacy. BNCT radiotoxicity and therapeutic effect at the B1 and B2 configurations of RA-6 were consistent. Histamine slightly reduced mucositis in precancerous tissue even in this overly aggressive oral cancer model, without compromising tumor control.     

Sympatric snow leopards and Tibetan wolves: coexistence of large carnivores with human-driven potential competition

The snow leopard Panthera uncia coexists with the wolf Canis lupus throughout most of its distribution range. We analysed the food habits of snow leopards and wolves in their sympatric range in the Karakoram mountains of Pakistan. A total of 131 genotyped scats (N?=?74, snow leopard; N?=?57, Tibetan wolf) were collected during the cold periods (i.e. winter and spring) of 2011 and 2012 in the Hushey valley. Large mammals, i.e. livestock and ibex, accounted for 84.8 and 83.1% of the diet (relative frequency) of the snow leopard and the wolf, respectively. Domestic prey was the staple of the diet of both snow leopards (66.6%) and wolves (75.1%). Ibex Capra ibex, the only wild ungulate in our study area, contributed 18.2 and 16.9% of relative frequencies in the diets of the snow leopard and the wolf, respectively. In winter, the snow leopard heavily relied on domestic sheep (43.3%) for food, whereas the wolf preyed mainly on domestic goats (43.4%). Differently from other study areas, both snow leopards and wolves showed no apparent prey preference (Jacobs index: snow leopard min. ??0.098, max. 0.102; Tibetan wolf min. ??0.120, max. 0.03). In human depauperate areas, with livestock and only a few wild prey, should competitive interactions arise, two main scenarios could be expected, with either predator as a winner. In both cases, the best solution could primarily impinge on habitat restoration, so that a balance could be found between these predators, who have already coexisted for thousands of years.     

A robust screen for novel antibiotics: specific knockout of the initiator of bacterial DNA replication

We have developed a novel type of a positive screen for the discovery of antibacterial compounds that target the Escherichia coli replication initiator protein DnaA. DnaA is an essential replication protein, conserved in (almost) all bacteria--including all human pathogens--and no existing antibiotics target the main components of the DNA replication machinery. This makes DnaA an attractive target and compounds discovered by this screen will constitute a new group of antibiotics. The conditional mutant, dnaA219, has a cold sensitive phenotype due to overreplication. In the screen, a DnaA inhibitor will reduce DnaA overactivity and thus restore growth at the nonpermissive temperature. This positive type of selection utilizes the rare phenomenon of lethal overactivity. In addition, the mutant strain has been made independent of DnaA activity by introduction of an alternative initiation pathway that allows growth under conditions of complete knockdown of DnaA. The resulting dnaA219rnhA strain is the basis of a robust, cell-based assay amenable to high-throughput screening. The screening assay has been validated against (1) a library of microbial fermentation extracts and (2) a known intracellular DnaA inhibitor.     

Structure and evolution of the mitochondrial control region of the pollen beetle Meligethes thalassophilus (Coleoptera: Nitidulidae).

The organization of the mitochondrial DNA (mtDNA) control region (CR) of the pollen beetle Meligethes thalassophilus is described. This mtDNA CR represents the longest sequenced for beetles so far, since the entire nucleotide sequence ranges from approximately 5000 to approximately 5500 bp. The CR of M. thalassophilus is organized in three distinct domains: a conserved domain near the tRNAIle gene, a variable domain flanking the 12S rRNA gene, and a relatively large central tandem array made up of a variable number of approximately 170 bp repeats that is responsible for the intraspecific length variation observed. Like other CRs found in insects, the M. thalassophilus CR contains two long homopolymeric runs that may be involved in mtDNA replication. Furthermore, conserved stem-and-loop structures in the repetitive domain were identified and their possible role in generating length variation is examined. Intraspecific comparison of the tandem repeat elements of M. thalassophilus suggests mechanisms of concerted evolution leading to homogenization of the repetitive region. The utility of such an array of tandem repeats as a genetic marker for assessing population-level variability and evolutionary relationships among populations is discussed. Finally, the technical difficulties found in isolating the mtDNA CR in beetles are remarked upon.     

Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4⁺ and CD8⁺ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4⁺ and CD8⁺ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8⁺ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4⁺ and CD8⁺ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations.Human papillomavirus (HPV) infection represents the most important risk factor for the development of cervical cancer. Although more than 100 distinct HPV genotypes have been described, and at least 20 are associated with cervical cancer, HPV type 16 (HPV-16) is by far the most frequently detected in cervical neoplasia regardless of the geographical origin of the patients (4). In the last few years significant advances have been made in the development of candidate prophylactic vaccine against cervical cancer and HPV-related infections. In several large prospective randomized studies, virus-like particles consisting of the HPV-16 and HPV-18 major capsid protein L1 (L1-VLPs) have shown promise in protecting young healthy females against persistent infection with HPV-16 and HPV-18 and their associated cervical intraepithelial neoplasia (reviewed in reference 12). These data strongly suggest that the implementation of large-scale L1-VLP-based prophylactic vaccinations have the potential to dramatically reduce worldwide cervical cancer rates in the years to come.Unfortunately, because HPV infection is endemic in humans and there is a long latency from HPV infection to the development of invasive cervical cancer in women, even if prophylactic L1-based vaccinations are implemented on a worldwide scale today it would take decades to perceive any significant benefit. Consistent with this view, an estimated 5 million cervical cancer deaths will occur in the next 20 years due to existing HPV infections (4, 12). Thus, the current development of therapeutic vaccines for protection against persistent HPV infections, cervical cancer, and its precursor lesions remains an area of great interest.Although the interactions between the host immune system and HPV-infected cells are still not completely understood, several lines of evidence suggest that protection against HPV-related infections by L1-VLP-based vaccines is likely conferred by the generation of high levels of neutralizing antibodies (12, 38). Nevertheless, a potential crucial role of L1-specific T-cell responses and the involvement of T cells in mediating the production of neutralizing antibodies and antiviral effect in infected hosts has been previously hypothesized (8, 24). This point may be particularly noteworthy in patients harboring HPV-infected cervical lesions because several studies have demonstrated the critical importance of both cytotoxic (CD8⁺) and helper (CD4⁺) T cells in achieving clinical responses (1, 5, 16-18, 20, 23). However, limited information is currently available to evaluate whether cell-mediated immune responses to L1-VLP may have any significant therapeutic effect in cervical cancer patients harboring HPV-16 positive tumors. Furthermore, to our knowledge, no direct comparison of the therapeutic efficacy of L1 and E7-specific immune responses against naturally HPV-16-infected cervical cancer have been yet reported in human patients.In the present study we have analyzed and quantified by highly sensitive real-time PCR (RT-PCR) the RNA levels of E6, E7, and L1 in flash-frozen biopsy specimens obtained from HPV-16-infected cervical carcinomas and in short- and long-term primary cultures of HPV-16-positive cervical tumors. In addition, we have studied the kinetics of expression of these genes and proteins during the establishment of HPV-16-positive primary tumors in vitro. Finally, using completely autologous systems of naturally infected HPV-16-positive human tumors, we have carefully studied the phenotype and function of L1-specific CD4⁺ and CD8⁺ T-lymphocyte responses generated by VLP-loaded dendritic cells (DCs) and compared their therapeutic potential to those elicited by DC loaded with the E7 oncoprotein.     

Neonatal handling induces alteration in progesterone secretion after sexual behavior but not in angiotensin II receptor density in the medial amygdala: implications for reproductive success

Neonatal handling affects the hypothalamus-pituitary-gonadal axis in female rats. Indeed, postnatal handling induces anovulatory estrous cycles and decreases sexual receptiveness. On the other hand, Angiotensin II (Ang II) infused into the medial amygdala (MeA) reduces sexual behavior in male and female rats. Considering this, and that gonadal steroid secretion after copulatory behavior is important for reproductive success, the purpose of the present study was to investigate whether the reduction in sexual receptiveness in neonatally handled female rats is mediated by changes in Ang II receptor density in MeA. Moreover, gonadal steroid secretion after sexual behavior was analyzed. Two groups of female Wistar rats were studied: nonhandled (pups were left undisturbed) and handled (pups were handled for 1 min once a day during the first 10 days of life). Once they were 80-85 days old in the evening of the proestrus day, sexual receptiveness was recorded and after that the animals were killed by decapitation. Trunk blood samples were collected, and plasma estradiol and progesterone were measured by radioimmunoassay. The brains were removed for Ang II receptor autoradiography in MeA. The decreased lordosis quotient in the neonatally handled group was confirmed in the present study. Neonatal handling also reduced the progesterone concentration in the plasma, but did not change the estradiol and the density of Ang II receptors in MeA. The reduced progesterone could be due to the decreased lordosis frequency of handled females. However, this decreased sexual receptiveness is not mediated by changes in Ang II receptors in MeA.