Development of Three Molecular Diagnostic Tools for the Identification of the False Codling Moth (Lepidoptera: Tortricidae).

Three molecular protocols using qPCR TaqMan probe, SYBR Green, and loop-mediated isothermal amplification (LAMP) methods were set up for the identification of larvae and adults of an African invasive moth, Thaumatotibia leucotreta (Meyrick, 1913) (Lepidoptera: Tortricidae). The DNA extracts from larval and adult samples of T. leucotreta were perfectly amplified with an average Ct value of 19.47 ± 2.63. All assays were demonstrated to be inclusive for T. leucotreta and exclusive for the nontarget species tested; the absence of false positives for nontarget species showed a 100% of diagnostic specificity and diagnostic sensitivity for all assays. With the SYBR Green protocol, the Cq values were only considered for values less than 22 (cutoff value) to prevent false-positive results caused by the late amplification of nonspecific amplicons. The limit of detection (LoD) for the qPCR probe protocol was equal to 0.02 pg/µl while a value equal to 0.128 pg/µl for the qPCR SYBR Green assay and LAMP method were established, respectively. The intrarun variabilities of reproducibility and repeatability in all the assays evaluated as CV%, ranged between 0.21 and 6.14, and between 0.33 and 9.52, respectively; the LAMP values were slightly higher than other assays, indicating a very low interrun variability. In order for an operator to choose the most desirable method, several parameters were considered and discussed. For future development of these assays, it is possible to hypothesize the setup of a diagnostic kit including all the three methods combined, to empower the test reliability and robustness.

Evolutionary adaptations in four hippoboscid fly species belonging to three different subfamilies.

Lipoptena cervi (Linnaeus, 1758), Lipoptena fortisetosa Maa, 1965, Hippobosca equina Linnaeus, 1758, and Pseudolynchia canariensis (Macquart, 1840) (Diptera: Hippoboscidae) are haematophagous ectoparasites that infest different mammal and bird species and occasionally attack humans. They are known for the health implications they have as vectors of pathogens to humans and animals, and for the injuries they inflict on their host's skin. This study focused on the morphological structures evolved by parasites in terms of their biology and the different environment types that they inhabit. To this aim, we examined four hippoboscid species, as well as their hosts' fur (ungulate and horse), and feather (pigeon) through light and Scanning Electron Microscopy (SEM) observations in order to highlight the main morphological features that evolved differently in these flies and to explain the effect of hosts' fur/feather microhabitats on the morphological specializations observed in the investigated ectoparasites. The studied species showed main convergent characters in mouthparts while remarkable differences have been detected on the antennal sensillar pattern as well as on the leg acropod that displayed divergent characters evolved in relation to the host.

Comparative morphology of the deer ked Lipoptena fortisetosa first recorded from Italy.

Hippoboscidae flies parasitize various animal species. Knowledge about these insects remains sparse, although they are known to cause stress and damage to their hosts, and can also accidentally infest humans, causing different sanitary risks. Research conducted in Tuscany assessing the biology and distribution of Lipoptena cervi (Linnaeus, 1758) (Diptera: Hippoboscidae), the most common ectoparasite of ungulates in Italy, revealed the presence of Lipoptena fortisetosa Maa, 1965 in Italy for the first time. This study includes a morphological comparative description of L. cervi and L. fortisetosa, emphasizing the peculiar differences between the two species to facilitate their accurate identification. The most pertinent morphological differences between the two species are highlighted, such as the external features of the antennae, distribution of bristles, and different features in the external genitalia. In both species, scanning electron microscopy of mouthparts revealed strong adaptive convergence in the feeding apparatus. Modified palps and a very thin proboscis are described in relation to feeding behaviour.

Curcumin induces apoptosis in breast cancer cell lines and delays the growth of mammary tumors in neu transgenic mice.

Breast cancer is a leading cancer in women and despite the benefits of the current therapies a significant number of patients with this tumor is at risk of relapse. Some of the alterations taking place in breast cancer cells are currently exploited by molecularly targeted drugs. Different drugs have been developed which target a single molecule but, given that the tumor originates from the dysregulation of many genes, there is the need to find new drugs that have more than one molecular target. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (CUR), a polyphenolic compound found in the spice turmeric, is a pleiotropic molecule able to interact with a variety of molecular targets and has antitumor, anti-inflammatory, antioxidant, immunomodulatory and antimicrobial activities. Here we demonstrate that CUR inhibits the growth of breast cancer cell lines in a dose dependent manner, with IC50 values in the micromolar range, and induces an increase in the percentage of cells in sub-G0 phase, representing the apoptotic cell population. The activation of apoptosis was confirmed by PARP-1 cleavage and by the increased ratio between the pro-apoptotic Bax and the anti-apoptotic Bcl-2 protein. In addition, in CUR-treated cells the activity of ERK1/ERK2 MAP kinases was down-regulated. The cytotoxic effects of CUR were observed in breast cancer cells expressing either high or low levels of ErbB2/neu. The in vivo antitumor activity of CUR was tested in BALB-neuT mice transgenic for the neu oncogene, which develop atypical hyperplasia of the mammary gland at 6 weeks of age and invasive carcinoma at 16 weeks of age. CUR, administered to mice both early and in an advanced stage of mammary carcinogenesis, induced a significant prolongation of tumor-free survival and a reduction of tumor multiplicity. In addition, CUR administration was safe, since no modification of hematological and clinical chemistry parameters could be observed in BALB-neuT and BALB/c mice treated with this compound for several weeks. These findings support further studies on the therapeutic potential of CUR in combination with standard therapies in breast cancer patients.

The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders.

The dopamine transporter is a plasma membrane protein that controls the spatial and temporal domains of dopamine neurotransmission through the accumulation of extracellular dopamine. The dopamine transporter may play a role in numerous dopamine-linked neuropsychiatric disorders. We review the cloning and organization of the human dopamine transporter gene, polymorphisms in its coding and noncoding sequence, and emerging data on its transcriptional regulation.

Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism.

The importance of the nuclear receptor nurr1 for the appropriate development of mesencephalic dopamine-synthesizing neurons has been clearly demonstrated through the targeted disruption of the nurr1 gene. The persistence of nurr1 expression in adult tissue suggests a possible role for this transcription factor in the maintenance, as well as development, of the dopaminergic phenotype. To address this issue, we analyzed the effects of nurr1 on the transcriptional expression of the human dopamine transporter gene (hDAT), one of the most specific phenotypic markers for dopaminergic neurons. Nurr1 enhanced the transcriptional activity of hDAT gene constructs transiently transfected into a newly described cell line (SN4741) that expresses a dopaminergic phenotype, whereas other members of the NGFI-B subfamily of nuclear receptors had lesser or no effects. Nurr1 activation of hDAT was not dependent upon heterodimerization with the retinoid X receptor. Unexpectedly, functional analysis of a series of gene constructs revealed that a region of the hDAT 5'-flanking sequence devoid of NGFI-B response element (NBRE)-like sites mediated nurr1 activation. Additional experiments using a nurr1 mutant construct suggest that nurr1 activates hDAT transcription via a novel NBRE-independent mechanism.

The Internet and patient education--resources and their reliability: focus on a select urologic topic.

OBJECTIVES: The information revolution triggered by the rapid growth of the Internet has allowed healthcare providers and patients to access a rapidly expanding volume of information. To address the quality of this information, a survey of the data on a single urology-related topic available on the Internet was performed. METHODS: The search on the World Wide Web (Web) was performed using the search engine HotBot and search directory Yahoo. The Web pages were assessed according to their relevancy to the topic chosen. Relevance rates were derived from the number of relevant sites divided by the total number of sites found. Relevant sites were subsequently ranked for quality on the basis of their accuracy, comprehensiveness, and objectivity. HotBot was then subsequently divided by domain, with each assessed separately. Yahoo was analyzed in its entirety. The resources were then compared for relevance and quality of information. RESULTS: When using the keyword "Viagra," HotBot responded with 15,109 hits. Yahoo presented 51 hits under the category, "Health: Pharmacy: Drugs and Medications: Specific Drugs and Medications: Viagra (Sildenafil)." The relevance rate for the first 50 hits in the search engine HotBot was 0.08. The relevance rates for the edu and org domains found by HotBot were 0.22 and 0.24, respectively; those for com and net were both 0.10. The relevance rate for the search directory Yahoo was 0.20. For relevant sites, the quality of the information presented was significantly higher in the Yahoo and in the HotBot domains hosted by nonprofit organizations when compared with HotBot in general and with its commercially oriented domains. HotBot overall was found to contain seven excellent sites, of which only three were found within Yahoo. CONCLUSIONS: Although the medical information available on the Web has proliferated at a remarkable rate, the number of Web sites providing complete, nonbiased information continues to represent only a small portion of the total. We have shown that the search directory Yahoo reduced the number of irrelevant sites significantly, but at the same time, some very valuable information available in HotBot was missing. At present, it may be useful to conduct searches within Yahoo followed by a review of both the edu and org HotBot domains.

Characterization of the 5'-flanking region of the human dopamine transporter gene.

The dopamine transporter (DAT) plays a major role in modulating dopamine (DA) neurotransmission by controlling the levels of this neurotransmitter in the extracellular space. We have isolated 8.3 kb of the 5'-flanking regulatory region of the human DAT (hDAT) gene and identified numerous potential elements involved in transcriptional control of the DAT. A series of hDAT-luciferase reporter constructs encompassing increasing amounts of 5'-flanking sequence was utilized in transient transfection assays assessing basal activity and response to selected stimuli. Our results suggest that the proximal hDAT 5'-flanking region displays a strong, nonselective promoter activity that is silenced through regulatory elements present in the distal portion of the 5'-flanking sequence. Although potential cyclic AMP responsive elements (CRE) were identified on the sequence, hDAT constructs were unresponsive to cyclic AMP induction. The transcription factor nurr1 increases the transcriptional activity of several larger hDAT constructs, consistent with the presence of several putative NGFI-B response elements (NBRE). The cloning and functional analysis of an extensive portion of the 5'-flanking regulatory region of the hDAT gene provides further insights into the factors involved in the regulation of this gene.