Tiny insects, big troubles: a review of BOLD's COI database for Thysanoptera (Insecta).

DNA Barcoding is an important tool for disciplines such as taxonomy, phylogenetics and phylogeography, with Barcode of Life Data System (BOLD) being the largest database of partial cytochrome c oxidase subunit I (COI) sequences. We provide the first extensive revision of the information available in this database for the insect order Thysanoptera, to assess: how many COI sequences are available; how representative these sequences are for the order; and the current potential of BOLD as a reference library for specimen identification and species delimitation. The COI database at BOLD currently represents only about 5% of the over 6400 valid thrips species, with a heavy bias towards a few species of economic importance. Clear Barcode gaps were observed for 24 out of 33 genera evaluated, but many outliers were also observed. We suggest that the COI sequences available in BOLD as a reference would not allow for accurate identifications in about 30% of Thysanoptera species in this database, which rises to 40% of taxa within Thripidae, the most sampled family within the order. Thus, we call for caution and a critical evaluation in using BOLD as a reference library for thrips Barcodes, and future efforts should focus on improving the data quality of this database.

Distribution patterns of Triatominae (Hemiptera: Reduviidae) in the Americas: an analysis based on networks and endemicity.

Triatominae, commonly known as kissing bugs, are a group of approximately 150 species of hematophagous reduviids, some of which are vectors of Trypanosoma cruzi, the etiological agent of the Chagas disease. Distributional patterns of triatomines have been studied based on macroecological and historical biogeographic approaches, but the definition of distributional patterns and areas of endemism are yet to be defined based on objective criteria. We used two methods to identify biogeographic units in the Triatominae: the endemicity analysis based on an optimality criterion (NDM/VNDM software) and a network approach aimed to simplify and highlight the underlying structure in species distributions (Infomap Bioregions). Information on species distributions was obtained from a data paper, comprising 21 815 records for 135 triatomine species occurring in the Americas. The resulting areas of each method were clustered using a meta consensus criterion based on dissimilarities and interpreted as recurrent areas. The NDM areas show a nested structure, presenting greater restrictions to the inclusion of species in a given area, requiring broad sympatry. In contrast, bioregions emphasize spatial patterns with better-delimited areas and species occurrences do not need to be highly congruent. When areas were clustered based on their species composition two clear patterns arose from both methods: (i) areas within the southern Amazon and southeast South America, especially in the Chacoan subregion, formed a cluster, and (ii) areas north of the Amazon, Pacific, Mesoamerica, Mexican transition zone and Nearctic formed another cluster. Moreover, within each of these two clusters, there was a latitudinal gradient of the areas in the Americas showing spatial similarity between the areas found in both methods. Results of both methods show well-bound areas separating the triatomine fauna in the Brazilian subregions, resulting in the recognition of areas corresponding to the biomes Chaco, Pampa, Cerrado, and Caatinga, and, to a lesser extent, the Atlantic Forest.

Morphological identification of Glyptothrips species (Thysanoptera: Phlaeothripidae).

Glyptothrips is a New World genus with 14 species of fungivorous Phlaeothripidae found in leaf litter. Identification of species in this genus is difficult due to limited collections of specimens, outdated descriptions, and a lack of keys and images. Here we start addressing these limitations by providing illustrations for each of these species, together with a table of diagnostic characters, and a key to nine species recorded in South America.

A milestone for Pentatomoidea: Grazia emet al./em 2008-What do we know and where can we go?

Pentatomoidea is the third largest superfamily in Heteroptera. The internal systematics and classification of this superfamily have an intricate history. The paper by Grazia et al. (2008) is a milestone to the phylogenetic hypothesis of Pentatomoidea. Subsequent papers explored the limited conclusions and unanswered questions left by Grazia et al. (2008). We proposed to look at the body of knowledge produced since Grazia et al. (2008) and to compile the molecular data for Pentatomoidea deposited in Genbank to achieve three aims: (i) to evaluate the advances on the phylogenetic relationships of the Pentatomoidea; (ii) to produce a phylogenetic hypothesis based on molecular data deposited in Genbank; and (iii) to highlight the shortcomings and strengths of the available data. We retrieved sequences of four molecular markers (COI, 16S, 18S, and 28S) for 167 terminal taxa, including 149 pentatomoids. A concatenated matrix was analyzed under maximum likelihood (ML) and parsimony (MP). Both methods supported the monophyly of Pentatomoidea, and poorly resolved internal relationships among the families. Acanthosomatidae, Dinidordae, Pentatomidae, Scutelleridae, Thaumastellidae, and Urostylididae were monophyletic (under ML and MP), and also Plataspidae and Thyreocoridae (under ML). Tessaratomidae and Cydnidae were non-monophyletic under both methods. Our results were compared to the phylogenetic hypotheses proposed for Pentatomoidea. The analysis of the data available on the GenBank allowed us to affirm that many problems mentioned previously remain unsolved, even though the sampling of terminals has increased. In summary, the efforts in the last two decades to better understand the relationships within the Pentatomoidea have been insufficient to propose robust advances in phylogenetic hypothesis for the group. We discuss topics we understand are paramount to upcoming developments:1) better taxon sample; 2) collection management; 3) increased markers; and 4) morphology and anatomical ontology.

Efficacy of myo-inositol in the treatment of cutaneous disorders in young women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common endocrine cause of hirsutism, acne and pattern alopecia, often characterised by ovulation disorders (usually manifested as oligo- or amenorrhea). In addition, 30-40% of women with PCOS have impaired glucose tolerance, and a defect in the insulin signalling pathway seems to be implicated in the pathogenesis of insulin resistance. For this reason, insulin-lowering medications represent novel approach in women with PCOS. The aim of this study was to evaluate the effects of myo-inositol (MYO), an isoform of inositol, belonging to the vitamin B complex, in the treatment of cutaneous disorders like hirsutism and acne. METHODS: Fifty patients with PCOS were enrolled in the study. BMI, LH, FSH, insulin, HOMA index, androstenedione, testosterone, free testosterone, hirsutism and acne were evaluated at the baseline and after receiving MYO therapy for 6 months. RESULTS: After 3 months of MYO administration, plasma LH, testosterone, free testosterone, insulin and HOMA index resulted significantly reduced; no significant changes were observed in plasma FSH and androstenedione levels. Both hirsutism and acne decreased after 6 months of therapy. DISCUSSION: MYO administration is a simple and safe treatment that ameliorates the metabolic profile of patients with PCOS, reducing hirsutism and acne.

Soy extract phytoestrogens with high dose of isoflavones for menopausal symptoms.

AIM: The aim of the present study was to assess the efficacy and safety of a standardized compound based on an extract of soy phytoestrogens, with high doses of isoflavones in the management of menopausal hot flushes. METHODS: A total of 180 women aged 40-65 years with a minimum of five moderate-to-severe hot flushes in the last 7 days at baseline and absence of menstruation for at least 6 months participated in a 12-week prospective, randomized, double-blind, placebo-controlled multicenter trial. After a 2-week run-in period, women received one tablet a day of 80 mg isoflavones (corresponding to 60 mg of genistein) or a matching placebo. RESULTS: The mean daily number of moderate-to-severe hot flushes decreased in both study groups, but the reduction was greater in the isoflavones arm at 6 (36.2%) and 12 weeks (41.2%) than in the placebo arm (24.0% at 6 weeks, 29.3% at 12 weeks), with a difference of 1.1 (95% CI [-2.0 to -0.06]) (P = 0.038) at 6 weeks and 1.1 (95% CI [-2.05 to -0.15]) (P = 0.023) at 12 weeks. Similar findings were obtained for hot flushes of any intensity. The Kupperman index decreased in both study groups. Relief of hot flushes was greater when time to menopause was >or=12 months and in cases of BMI >or=27 kg/m(2). CONCLUSION: In daily practice conditions, high doses of isoflavones, particularly genistein, can be used for the management of hot flushes in postmenopausal women not treated with hormone replacement therapy due to their superior efficacy to placebo and very good safety profile.

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of beta-thalassemia.

The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent beta-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

Extralesional detection and load of human papillomavirus DNA: a possible marker of preclinical tumor spread in cervical cancer.

OBJECTIVE: Because the interaction between viral DNA products and cellular regulatory mechanisms is the first step leading to cancerous transformation, the detection of its presence in histologically negative lymph nodes may represent a very early biological step in cancer spread. The quantitative estimate may represent and an indirect sign of active cellular replication. MATERIALS AND METHODS: Cervical and lymph nodes tissues of 13 cases of invasive cervical cancer were analyzed for human papillomavirus (HPV)-DNA presence and viral load by HPV typing and quantification by real-time polymerase chain reaction. RESULTS: HPV-DNA was demonstrated in all tissue samples (primary tumor, positive lymph nodes, negative lymph nodes) with the most prevalence of HPV 16 (61.5%) and single-type infection (69.3%), whereas viral load (mean quantity of DNA copies) is statistically different in negative versus positive lymph nodes (p =.005). CONCLUSIONS: Concordance of viral type and lymph nodes viral load may represent as a useful tool in identifying early metastatic risk of tumor spread.

Holopothrips diversity-a Neotropical genus of gall-inducing insects (Thysanoptera, Phlaeothripidae).

The genus Holopothrips represents the main Neotropical group of thrips associated with plant galls, and several of the 36 currently described species are known to induce or invade galls of other organisms. The existence of several Holopothrips specimens in collections that do not belong to any of the already described species, allied to the absence of basic biological information for several species, such as host plants and habit, shows that the current knowledge on the genus is severely lacking. Here we start addressing these problems, by describing 24 new species: H. acrioris, H. atlanticus, H. bicolor, H. brevicapitatum, H. cardosoi, H. curiosus, H. flavisetis, H. graziae, H. inconspicuus, H. infestans, H. irregularis, H. johanseni, H. kaminskii, H. longihamus, H. longisetus, H. magnus, H. maiae, H. nigrisetis, H. nigrum, H. punctatus, H. reticulatus, H. singularis, H. spermathecus, H. varicolor. This study also includes information on galls for several species; an updated and illustrated key to species; and comments on the morphological diversity of the group. With that, we hope to lay the taxonomic and morphological bases for future studies in this group, focusing on its diversity, ecology and phylogenetic relationships.

Different approaches for noninvasive prenatal diagnosis of genetic diseases based on PNA-mediated enriched PCR.

The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

Revision of the South American genus Xiphiola Bolívar, 1896 (Orthoptera: Acrididae: Ommatolampidinae: Abracrini).

The Neotropical genus Xiphiola Bolívar, 1896 is revised. It includes two valid species: Xiphiola borellii Giglio-Tos, 1900 and Xiphiola cyanoptera (Gerstaecker, 1889). The genus and the species are redescribed and diagnostic characters are provided along with illustrations of each species. A lectotype and paralectoype for X. cyanoptera are here designated. We also include new morphological characters from male and female genitalia, and external characters that have not been employed until now. We present the distribution map based on material examined, as well as a key to species.

Metabolism of tumour-derived urokinase receptor and receptor fragments in cancer patients and xenografted mice.

The urokinase-type plasminogen activator receptor (uPAR) is involved in cell migration and tissue remodelling, as a receptor for pro-uPA, as a cell adhesion component, and in a soluble form as a chemoattractant.We have analyzed the presence and the molecular forms of uPAR and uPAR-fragments in urine of ovarian cancer patients in comparison with tumour tissue, ascites, and serum. Carcinoma tissue contained high levels of uPAR, but more abundantly the D2D3-fragment. Ascitic fluid contained similar ratio's of suPAR fragments as corresponding tumour tissue, but serum only contained intact suPAR. Interestingly, urine contained predominantly the uPAR-fragments D1 and D2D3, and the pattern of these fragments was different in cancer patients as compared to healthy individuals. To confirm the hypothesis that circulating and urinary suPAR and suPAR-fragments originate from the tumour tissue, the presence of human suPAR (fragments) was analyzed in mice xenografted with human tumours. Indeed, high levels of urinary D1 fragment were found in mice carrying a tumour displaying cleaved uPAR on the cell surface, but little or no D1 was found in the urine from mice carrying a tumour with full-length uPAR. Mouse serum contained only intact suPAR. Our data demonstrate that the enhanced levels of suPAR fragments in the urine of cancer patients is likely to originate from uPAR expressed in the tumour tissue. Considering the biological activities that uPAR fragments display, the level and typing of uPAR fragments in urine might therefore be clinically more relevant than the plain serum uPAR content.

Comparative morphology of selected characters of the Pentatomidae foreleg (Hemiptera, Heteroptera).

Heteropteran legs are very diverse within and among taxa, and such variation is frequently correlated with life habits. Structural modifications are commonly present in the legs of the Pentatomoidea but are poorly studied. Using scanning electron microscopy, the tibia and pretarsal microstructure of 82 species of Pentatomidae (Heteroptera), three species of Scutelleridae, and ten species of Thyreocoridae were described, focusing on the pretarsal structure, the foretibial apparatus, and the foretibial comb. The Pentatomidae, the Scutelleridae, and the Thyreocoridae have uniform pretarsal structures. Variation can be found in the length of the parempodial setae and in the shape of the parempodial projections. The foretibial combs of the Pentatomidae, the Thyreocoridae, and the Scutelleridae are described for the first time, and we have demonstrated that there is low structural variation in the foretibial comb complex of the studied species. The setae organization and distribution on the foretibial apparatus is uniform in the families studied. However, the Asopinae (Pentatomidae) bear a foretibial apparatus that is uniquely organized. The taxonomic and phylogenetic relevance of the pretarsal traits, the foretibial apparatus, and the foretibial comb are discussed.

Isolated tubal torsion in pregnancy.

Adnexal torsion is an uncommon cause of acute abdomen in pregnancy and isolated fallopian tube twisting accounts for a very small number of these cases. These conditions, either in pregnancy or in non-gestational circumstances, are known to be due to both genital and non-genital causes and, in most cases, predisposing factors can be identified. We reviewed the literature and retrieved only 19 cases of isolated fallopian tube torsion in pregnancy treated surgically from 1936 to today, including one recently published case from our experience. The clinical presentation was lower quadrant abdominal pain in all cases. The right side was involved in 90% of the cases. Tenderness was usually present but peritoneal irritation with guarding or rebound was exceptional. Symptoms were nausea and vomiting, scanty vaginal bleeding and dysuria. Signs suggestive of necrosis such as leucocytosis, increased CRP and mild hyperpyrexia were uncommon. Preoperative ultrasound evaluation was performed in eight patients and in all cases an adnexal cyst was detected on the ipsilateral side of the abdominal pain. The case we recently published was carefully investigated preoperatively by Doppler flow ultrasound techniques which allowed for a precise differential diagnosis with total adnexal torsion. This aspect has never been previously considered. The surgical approach showed acute isolated fallopian tube torsion in all the cases and a predisposing factor was identified in 75% of the patients. Foetal and maternal outcome were always excellent. In cases of acute abdomen in pregnancy, with detailed Doppler flow ultrasound evidence of normal ovaries and of a pelvic cyst, an isolated tubal-paratubal cyst torsion should be considered and appropriate ovary-sparing surgical treatment foreseen.

Premature ovarian failure in suspected Whipple's disease.

INTRODUCTION: Whipple's disease is a relapsing systemic infectious disease probably caused by the Gram-positive bacillus Tropheryma whippelii. The diagnosis can be established based on the characteristic histopathological features found in the affected organ (foamy macrophages with a coarsely granular cytoplasm, which stains with PAS, and by means of polymerase chain reaction (PCR) technology). CASE REPORT: We report a case of a 23-year-old woman affected by suspected Whipple's disease. She presented encephalopathy and neuropathy with inveterate hyperpyrexia and alteration of the hypothalamic-pituitary-ovary axe. She was amenorrheic because of an hypergonadotropic hypogonadism. DISCUSSION: This hypogonadism is possibly due to follicular depletion caused by inveterate hyperpyrexia or T. whipplii localization.

Myo-inositol in patients with polycystic ovary syndrome: a novel method for ovulation induction.

BACKGROUND: Polycystic ovary syndrome (PCOS) is often characterized by chronic oligo- or anovulation (usually manifested as oligo- or amenorrhea), and hyperandrogenism. In addition, 30-40% of PCOS women have impaired glucose tolerance, and a defect in the insulin signaling pathway (inositol-containing phosphoglycan mediators) seems to be implicated in the pathogenesis of insulin resistance. PCOS patients are subfertile as a consequence of such ovulatory disorders and often need drugs, such as clomiphene citrate or follicle-stimulating hormone, for ovulation induction, which increases the risk of multiple pregnancy and ovarian hyperstimulation syndrome. We hypothesized that the administration of an isoform of inositol (myo-inositol), belonging to the vitamin B complex, would improve the insulin-receptor activity, restoring normal ovulatory function. MATERIALS AND METHODS: Twenty-five PCOS women of childbearing age with oligo- or amenorrhea were enrolled in the study. Ovulatory disorder due to PCOS was apparently the only cause of infertility; no tubal defect or deficiency of male semen parameters was found. Myo-inositol combined with folic acid (Inofolic) 2 g twice a day was administered continuously. During an observation period of 6 months, ovulatory activity was monitored with ultrasound scan and hormonal profile, and the numbers of spontaneous menstrual cycles and eventually pregnancies were assessed. RESULTS: Twenty-two out of the 25 (88%) patients restored at least one spontaneous menstrual cycle during treatment, of whom 18 (72%) maintained normal ovulatory activity during the follow-up period. A total of 10 singleton pregnancies (40% of patients) were obtained. Nine clinical pregnancies were assessed with fetal heart beat at ultrasound scan. Two pregnancies evolved in spontaneous abortion. CONCLUSION: Myo-inositol is a simple and safe treatment that is capable of restoring spontaneous ovarian activity and consequently fertility in most patients with PCOS. This therapy did not cause multiple pregnancy.

IFN-gamma produced by human papilloma virus-18 E6-specific CD4+ T cells predicts the clinical outcome after surgery in patients with high-grade cervical lesions.

Cervical neoplastic lesions are associated with infection by high-risk human papilloma viruses (HPVs). HPV-16 and HPV-18 are the most common genotypes. It has been proposed that development of HPV-16-positive cervical lesions is associated with impaired CD4(+) T cell immunity against early Ags. The aim of the study was to evaluate whether this impairment also applies to HPV-18. We investigated the presence and the quality of anti-HPV-18 E6 CD4(+) T cell responses in the blood of 37 consecutive patients with high-grade cervical lesions, 25 normal donors, and 20 cord bloods. The immune infiltrate in the cervical lesions was also evaluated. The characteristics of the responses were correlated to the clinical outcome. We found that one or more HPV-18 E6 peptides, containing naturally processed epitopes, were able to induce a response in 40-50% of the patients, depending on the effector function tested. Importantly, these percentages rose to 80-100% when HPV-18-positive patients were considered. HPV-18 E6-specific CD4(+) T cells produced mixed Th1/Th2 responses and statistical analysis of the cytokines produced revealed that the amount of IFN-gamma released could predict infection persistence and/or disease relapse after surgery. Finally, we found that a higher number of infiltrating CD4(+) and T-bet(+) T cells in the lesions correlated with a favorable clinical outcome. Our results strongly suggest a relevant role for CD4(+) T cells in the control of the HPV-18 compared with HPV-16 infections in patients with high-grade cervical lesions and identify an immunologic parameter potentially useful for patients' stratification.

Gonadotropin-releasing hormone antagonist and metformin for treatment of polycystic ovary syndrome patients undergoing in vitro fertilization-embryo transfer.

AIM: The combination of gonadotropin-releasing hormone (GnRH) antagonist and gonadotropin represents a valid alternative to the classical protocol with GnRH agonist for ovulation induction in patients with polycystic ovary syndrome (PCOS). The use of metformin is of benefit to women with PCOS. The aim of the present study was to compare the stimulation characteristics and in vitro fertilization (IVF)-embryo transfer (ET) outcomes of the standard short GnRH antagonist protocol for ovarian stimulation with or without metformin. MATERIALS AND METHODS: We recruited 40 PCOS patients. The population studied was divided into two groups (A and B). Group A was pretreated for 2 months with metformin 1.5 g/day (Glucophage(R); Merck Pharm), and then stimulated with recombinant follicle-stimulating hormone (rFSH) 150 UI/day (Gonal F(R) 75 UI; Serono). GnRH antagonist, cetrorelix acetate 0.25 mg/day (Cetrotide(R); Serono), was started when the leading follicle reached 14 mm diameter on ultrasound scan. Group B was treated only with rFSH 150 UI/day and GnRH antagonist 0.25 mg/day when the leading follicle was >or=14 mm in diameter. RESULTS: In group A we found a statistically significant (p < 0.05) decrease in the number of ampoules of rFSH (A vs. B: 18+/-6 vs. 24+/-8) and estradiol levels (A vs. B: 2400+/-600 vs. 3370+/-900 pg/ml) (all values mean+/-standard deviation). Group A had significantly fewer cancelled cycles (A vs. B: 1 vs. 3; p < 0.05). The incidence of ovarian hyperstimulation syndrome was 5% in group A and 15% in group B (p < 0.05). In patients treated with metformin, the total number of follicles on the day of human chorionic gonadotropin treatment (23+/-1.2 vs. 33+/-2.6) was decreased with no change in the number of follicles >or=14 mm in diameter (A vs. B: 18+/-1.2 vs. 19+/-1.7). However, the mean number of mature oocytes (A vs. B: 8.4+/-1.5 vs. 5.0+/-1.5) was increased with metformin treatment (p < 0.05). No difference was found in the number of cleaved embryos (A vs. B: 2.5+/-0.5 vs. 2.2+/-0.3). CONCLUSIONS: The use of metformin with GnRH antagonist improves the outcome of ovarian stimulation in IVF-ET cycles in PCOS patients.

Psychological assessment of the effects of treatment with phytoestrogens on postmenopausal women: a randomized, double-blind, crossover, placebo-controlled study.

OBJECTIVE: To investigate the effects of soy isoflavones on mood and cognitive function in postmenopausal women. DESIGN: Randomized, double-blind, cross-over, placebo-controlled trial. SETTING: University Hospital, Milan, Italy; A.G.UN.CO. Obstetrics and Gynaecology Centre, Rome, Italy. PATIENT(S): Seventy-eight postmenopausal women. INTERVENTION(S): We administered 60 mg/day isoflavones or placebo for 6 months. After a washout period of 1 month, the patients who had been treated with phytoestrogens received placebo, and those who previously received placebo were administered phytoestrogens (for 6 months). MAIN OUTCOME MEASURE(S): Cognitive performance and mood were assessed by a battery of tests at the end of each treatment period. At the end of the study, the patients were also asked whether they preferred the first or second treatment. RESULT(S): The 17 scores on cognitive performance test and the 6 for mood assessments 6 showed an advantage for the treatment with phytoestrogens. Similarly, of the 8 visual analogue scales used to indicate mood, 7 improved significantly after the treatment with phytoestrogens. Moreover, 49 patients preferred phytoestrogens, 9 placebo, and 18 had no preference. The preference was not related to the order of treatment. CONCLUSION(S): These results suggest that isoflavones may have positive effects on postmenopausal women improving cognitive performance and mood.