KOH-mounts were performed with 20% KOH and were microscopically c

KOH-mounts were performed with 20% KOH and were microscopically checked for fungal elements after at least 20 min of exposure. Fungal cultures were prepared on Sabouraud glucose agars (bioMérieux, Marcy-l’Etoile, France) containing antibiotics (chloramphenicol 0.05 g l−1 selleck kinase inhibitor and gentamycin 0.01 g l−1) with and without addition of cycloheximide (0.4 g l−1; AppliChem, Darmstadt, Germany). The cultures were

incubated at 26 °C for at least 3 weeks before assessed as negative. Positive cultures were identified by established criteria based on morphology and physiology.12 If necessary, subcultures were prepared on special media according to the specific requirements. In particular, the urease test, cultures on potato-dextrose agar and the hair perforation test were used

if a strain could not be identified on morphological basis. The sample shares allocated to genetic analysis were submitted to DNA-extraction using a QiAmp DNA Mini Kit (Qiagen, Hilden, Germany). A PCR was then performed using a HotStarTaq Plus Master Mix Kit (Qiagen) according to the manufacturer’s instructions. The following primers were used that amplify a 280 base pair fragment containing a GT-microsatellite repeat specific for strains of the closely selleck chemical related T. rubrum/Trichophyton violaceum-clade6: forward 5′TGG TCT GGC CTT GAC TGA CC3′, reversed 5′GTA AGG ATG GCT AGT TAG GGG G3′. The fungal DNA was amplified by 30 cycles in a thermocycler (Thermomixer Comfort; Eppendorf, Hamburg, Germany): 30 s at 95 °C denaturation, 30 s mafosfamide at 60 °C annealing and 45 s at 42 °C extension. The amplification product was then checked for bands with 280 bp by gel electrophoresis in a 2% agarose gel in comparison with a negative control and a positive control with DNA extracted from Trichophyton mentagrophytes (Fig. 1). For a total of 464 samples of scales, a corresponding detection of T. rubrum by PCR and culture was seen in 75

cases. A positive T. rubrum PCR, but negative T. rubrum culture, was found in 40 scale samples, whereas a positive T. rubrum culture, but negative T. rubrum PCR, was found in 13 cases. Trichophyton rubrum culture and T. rubrum PCR were both negative in 336 cases. In Fig. 2, these results are shown in rounded percentage of all scale samples. In 66 samples of scales, other fungi than T. rubrum were detected by cultures; all of these samples had a negative PCR for T. rubrum. Twenty-one scale samples were positive for fungal elements in the KOH-mounts only and were negative in cultures and T. rubrum PCR. For a total of 230 nail samples, a corresponding detection of T. rubrum by PCR and culture was seen in 40 cases. A positive T. rubrum PCR, but negative T. rubrum culture, was found in 47 nail samples, whereas a positive T. rubrum culture, but negative T. rubrum PCR, was found in 8 samples. In Fig. 3, these results are shown in rounded percentage of all nail samples. In 25 samples of nails, other fungi than T.


However, RO4929097 further studies are needed before recommending the use of these drugs safely in clinical situations. “
“There is scarcity of data regarding significance of candiduria in patients with haematologic malignancies and its association with invasive candidiasis. To that end, we retrospectively evaluated all hospitalised, non-intensive care unit patients with haematologic malignancies and candiduria during a 10-year period (2001–2011). To decrease the possibility of bladder colonisation and sample contamination, we excluded all patients with candiduria who had urinary catheters and those with concomitant bacteriuria. Twenty-four such patients (21 females) were identified,

with median age at diagnosis 62 years

(range, 20–82 years). Acute leukaemia was the most common underlying disease (54%); 62% of these cases were not in remission. Twenty-nine percent of the patients had diabetes mellitus and 25% were neutropenic. The most common isolated Candida species was Candida glabrata (37%), followed by C. albicans (29%). Only 8% of them had urinary tract infection symptoms. However, 88% received systemic antifungals. Candidemia and crude mortality rates at 4 weeks were low (4% and 12% respectively). Isolated candiduria in patients with haematologic malignancies selleck chemicals llc has risk factors similar to those in other hospitalised patients, and it does not seem to be a strong predictor of subsequent invasive candidiasis. “
“Two Candida albicans isolates were collected from a HIV-positive patient with recurrent oropharyngeal candidosis (OPC). One isolate was taken during the first episode of oral candidosis [fluconazole susceptible (FLU-S), minimal inhibitory concentration (MIC) = 0.25 mg l−1] and the second after the patient developed refractory OPC and resistance to fluconazole (FLU-R, MIC = 64 mg l−1). Both isolates were clonally identical. Different in vitro studies were carried out to assess putative virulence factors of both isolates. Gene expressions of efflux pumps and CSH1 were determined as well as adherence to human epithelial cells, determination of proteinase secretion and biofilm

formation activity. Virulence was studied using a disseminated mouse model. All mice challenged with the FLU-S isolate survived the experiment when Ergoloid FLU was given. However, when FLU was absent, the mortality of the FLU-S isolate was higher than that of the FLU-R isolate with no mice surviving the experiment. In vitro studies showed pronounced growth rates of the FLU-S isolate and a more intense biofilm-building activity compared with the FLU-R isolate. The FLU-R isolate highly up-regulated MDR1 and CSH1. This isolate also adhered stronger to the epithelial cell line. The results showed that FLU-S and FLU-R isolates exhibit different virulence factors, which enable the survival of both isolates in adapted environments.

2b) IgM increased significantly only in the D-LL + Lc (N) group

2b). IgM increased significantly only in the D-LL + Lc (N) group compared to LL (P < 0·05) and LL + Lc (0), but only on day 28 (P < 0·05) (Fig. 2c). The levels of IgA in serum showed no significant differences among the different groups assayed (Fig. 2a). In order to study whether the specific humoral immune response

induced by the different immunization strategies used in this work increased resistance in mice against a pneumococcal infection, the animals were challenged intranasally with serotypes 3 and 14 of the pathogen. Analysis of the infection was carried out evaluating colonization in lung and pathogen passage into blood on day 2 after challenge (Table 2). All the treatments prevented colonization CP-690550 clinical trial in lung by both S. pneumoniae

serotypes and also prevented dissemination into the BGB324 datasheet blood of serotype 14. In contrast, when animals were infected with serotype 3, only administration of the recombinant bacterium, live (LL) and dead (D-LL), associated with the oral administration of the probiotic strain Lc, prevented dissemination of the pathogen into the bloodstream. Administration of D-LL + Lc (N) did not prevent colonization of the lung by serotype 3. These results demonstrate that immunization with LL + Lc (O) and D-LL + Lc (O) would be the most effective treatment for the prevention against pneumococcal infection of young mice with S. pneumoniae. The effect of different

treatments on the vaccine-induced immune response is important in the selection of a vaccination strategy adequate against a specific pathogen. We assessed the levels of IgG1 and IgG2a anti-PppA post-vaccination (day 42) in order to analyse the Th1/Th2 balance in both BAL and serum. Th1 cells secrete IFN-γ, associated with switching to IgG2a, while Th2 cells secrete mainly IL-4, which promotes switching to IgG1. The results obtained are shown in Table 3 and correspond to the IgG1/IgG2a ratio for each group on day 42 (2 weeks after the third immunization). Administration of LL induced a mixed-type Th1/Th2 response in BAL. The live vaccine associated with the oral administration of Lc [LL + Lc MYO10 (O)] and the inactivated vaccine (D-LL) induced a significant increase in the IgG1/IgG2a ratio, indicating preferential activation of Th2 cells. In contrast, immunization with D-LL + Lc (N) and D-LL + Lc (O) showed a significant decrease in the IgG1/IgG2a ratio compared to the other groups. This would indicate that the probiotic would induce a shift towards the type Th1 response. Similar results were found in serum, although the LL + Lc (O) group did not show significant differences with LL. The type of immune response induced in the respiratory mucosa is decisive in the protection of the host against pathogens that enter the organism through the airways.

Finally, after incubation with sera, the L1210 cells were stained

Finally, after incubation with sera, the L1210 cells were stained with hematoxylin and eosin (H&E) and visualized by light microscopy. This examination find more revealed that after 4 h incubation, cells treated with cytotoxic sera had the morphology of oncotic necrotic cells

with cellular swelling, membrane disruption, and karyolysis (Fig. 5D). No chromatin condensation or apoptotic body formation, hallmarks of apoptosis, were detected in the stained cell nuclei after incubation with the cytotoxic sera. Due to the antitumor potential of the detected anti-NeuGcGM3 antibodies, we evaluated their presence in cancer patients. We compared 53 NSCLC patients with gender- and age-matched healthy donors. Analysis of antibody levels in the sera from these patients by ELISA revealed statistically significant lower anti-NeuGcGM3 responses in NSCLC patients less than 60 years of age than in healthy donors (Fig. 6A). We detected low levels of anti-NeuGcGM3 antibodies only in six patients, two of which also reacted with NeuAcGM3 ganglioside (Supporting Information Fig. 7). These six NSCLC patients were not able to recognize the L1210 tumor cell line (data not

shown). When we measured the total IgM and IgG concentration in the sera of the cancer patients, although the levels of total IgM and IgG antibodies did not change with age (data not shown), there was a significantly lower total IgM level in cancer patients’ sera when compared Selleckchem JNK inhibitor with that of healthy donors. In contrast, the total levels of IgG in the NSCLC patients were similar to the levels observed for healthy donors (Fig. 6B). Natural antibodies have been considered to be important in the primary defense against invading pathogens [22], the clearance of damaged structures, dying cells and oxidized epitopes [23], and the modulation

of cell functions [24]. But also, naturally occurring antibodies could play a role in the protection against neoplastic transformation [25-29]. In this study, we describe the presence of antibodies against NeuGcGM3 ganglioside, circulating in the sera of of healthy adult individuals. NeuGcGM3 ganglioside is not only overexpressed on tumor cell membranes, but are also important for tumor development due to its suppressive effect on immune system function [2]. Sixty-five healthy donors’ sera out of 100 tested bound to NeuGcGM3 by ELISA, and did not recognize the acetylated form of this ganglioside. This result is in concordance with a previous result about reactivity against different N-glycolylated compounds of 16 healthy donors, reported by Padler-Karavani et al. [30]. Previous reports have shown the existence of a naturally occurring immunity against glycolipidic antigens, specifically gangliosides. Some of these reactivities have been associated with the induction of pathological alterations, as is the case for the antibodies against ganglioside complexes, such as GD1a and GD1b, or GM1 and GD1a in Guillian–Barre syndrome [31].

4) We compared the performance of gene sets with their constitue

4). We compared the performance of gene sets with their constituent genes in profiles from high versus low HAI responders to influenza vaccination. We found that the top-scoring gene sets in TIV responders were more strongly correlated with the high antibody response phenotype than any constituent Ivacaftor purchase gene in either gene set (Supporting Information Fig. 5A). Moreover,

although both complement and antibody genes were present in gene sets enriching in responders, the antibody genes were among those most upregulated (Supporting Information Fig. 5A and B). Thus a gene set based analytic approach identifies signatures of proliferation and immunoglobulin genes that are strongly correlated Idasanutlin with high antibody response. We next sought

to determine if enrichment of the immunoglobulin and/or proliferation gene sets could be used as a predictor of vaccine response, using high or low HAI titers as an outcome. To do this, we selected the most differentially enriched gene set from each of the two clusters, and fitted them into logistic regression models. Both models closely fit the data and yielded an AUC of ∼0.9 (Fig. 3A and B), suggesting that each independent gene set could provide a strongly predictive model of vaccine response. To integrate both biological processes into a single model, we applied Bayes’ rule, and found that the integrated model achieved an AUC of 0.94 (Fig. 3C). To compare our integrated gene set based model with the single-gene level model previously described for this dataset [16], we tested our model in a validation dataset comprised of PBMC samples Montelukast Sodium from an independent trial of TIV vaccination. We found that our predictive model yielded an accuracy of 88% in the test set, comparable

to the performance of the single-gene level predictor [16]. This indicates that gene set based analysis of expression profiles provide accurate predictors of response to vaccination. An advantage of a gene set enrichment analysis is that it can capture subtle changes in gene expression distributed across transcriptional networks. We therefore compared the degree of differential expression of genes in the predictive gene sets (proliferation and immunoglobulin gene sets) with that of the genes selected in the single-gene level predictor originally applied to this dataset (Fig. 4). Predictive genes selected in the study by Nakaya et al. [16] were all highly differentially expressed in day seven PBMC expression profiles from responders compared to nonresponders, as expected (mean fold change 3.36). In contrast, the gene sets identified in our analysis included many genes that were much less differentially expressed (mean fold change of proliferation cluster 2.13; mean fold change of immunoglobulin cluster 2.53) (Fig. 4).

A significant association was reported between TB and rs1800896 G

A significant association was reported between TB and rs1800896 G-allele. IL10 GCC and ACC haplotypes distribution showed a significant difference between patients with TB and controls. No statistically significant association was detected between rs1800871, rs1800629, rs1800750, rs361525 polymorphisms, functional TNF-α/IL-10 genotypes and TB. Leprosy.  Leprosy is a mycobacterial disease, caused by Mycobacterium leprae that initially affects the peripheral nervous EX 527 solubility dmso system and patients displaying contrasting clinical, immunological and pathological manifestations. Many factors and metabolic pathways including TLR/LIR-7, VDR, TNF-α and TGF-β have been reported to play role in disease. Goulart and Goulart [35]

reviewed the complex molecular interactions in affected individuals PD0325901 price influenced by the pathogenetic background. A significant association between the TNF rs1800629 A-allele and multibacillary leprosy has been reported from India [36]. In Brazil, this allele was associated with resistance against multibacillary leprosy [37, 38]. A significant association of TNF rs1800629 was found in borderline tuberculoid leprosy patients with the magnitude of in vivo delayed type hypersensitivity skin test reactivity to cutaneously injected M. leprae antigens. It has been reported that signalling deficient mutations in certain Toll-like receptors (TLR2; act upstream of TNF) can be strongly correlated with lepromatous leprosy. TNF rs1800629 regulatory polymorphism

plays an important role in patients with leprosy in a Brazilian population [39], and in patients with leprosy, higher frequency of TNF rs1800629, GG genotype, and a decreased frequency of GA/AA genotypes were reported as compared to the control group. The GG genotype was particularly higher in patients with tuberculoid (TT) and borderline (BB) leprosy. A lower frequency Interleukin-3 receptor of GCC/GCC haplotype of IL-10 in patients with lepromatous leprosy (LL)

than in controls was also reported. TNF-alpha polymorphism rs361525 and rs1800629, and its association with the outcome of different clinical forms of leprosy have been reported by Vanderborght et al. [39]. TNF polymorphism rs361525 and rs1800629 have shown differences in the frequency of the haplotypes along the ethnic groups, but no statistical differences were observed in haplotype frequencies between patients with multibacillary (MB) and paucibacillary (PB). A lower bacteriological index (BI) among the TNF polymorphism rs1800629 carriers was reported, while higher BI in rs361525 carriers [40]. Recurrent acute otitis media.  Acute otitis media (AOM) is caused by bacterial infection in children. Genetic variations in immunoresponse genes are reported to influence susceptibility to infectious diseases [41], and increased expression of TNF-α, IL-1β, IL-6 and IL-10 was observed during experimental otitis media in animals. Polymorphism in immune response genes such as IL10, IL6 and IL4 has been associated with altered cytokine expression levels [42].

The palliative approach to patients with ESKD includes managing a

The palliative approach to patients with ESKD includes managing all aspects of the physical, emotional and spiritual dimensions of the illness and care of the family. That breadth perfectly accords with modern medical beliefs in the interrelatedness

of body, mind and spirit in the experience of illness for all human beings Health PKC412 cost professionals dealing with patients with ESKD need to acquire skills in these areas. Given that no one health professional can provide all treatment, support and assistance needed a critical ethos of the palliative approach is the multidisciplinary team (MDT). Continuing collaboration between renal medicine and palliative medicine is essential. Given that there is currently, and will for the foreseeable future be, a shortage of Palliative Care health professionals the onus should be on all disciplines, including Nephrology, to acquire and nurture basic skills

in the palliative approach to patients, including skills in discussions around the possible withholding of and withdrawal from dialysis, symptom management, psychosocial support and the appropriate care of the dying patient. The cultural and religious beliefs of patients may inform or determine their view on medical decision-making including in relation to the withholding or withdrawing of dialysis and the care of the dying. It is therefore important that clinicians explore these beliefs with patients Lapatinib ic50 and their families. In modern societies patients may or may not have a religious faith but all patients have spirituality. Most religions believe that

withdrawal from or withholding treatment, including dialysis, find more is acceptable when this is in the patient’s best interests. A core competency of Nephrology should be the capacity to diagnose dying. Failure to do this or procrastination in this recognition may result in neither the clinicians nor the family being prepared for the possibility of death. That unpreparedness may have a significant impact on the bereavement of the family. Withdrawal of dialysis is ethically and legally valid; once the dying phase has been recognized and acknowledged it is important that invasive tests are ceased so as not to add to or prolong suffering. An increasing issue is the need to deprogramme AICD; this specific issue should be discussed with the patient and his/her cardiologist. It is important at this time to be specific that deprogramming AICD does not constitute euthanasia or physician-assisted suicide, that deprogramming AICD will not cause death and that the process of deprogramming is not painful or make the process of death more painful. The time to death after withdrawal varies considerably, averaging 10 days for most patients but 3 weeks or even longer for those with residual renal function.

The ISDR interacts with PKR and regulates replication of HCV in v

The ISDR interacts with PKR and regulates replication of HCV in vitro (28).

Mutations in the ISDR affect the interaction with PKR and may inhibit viral replication. In the case of the IRRDR, the molecular mechanism underlying the possible involvement of this region in IFN responsiveness of the virus Selleckchem Rucaparib is still unknown. The significant difference among IRRDR sequence patterns may suggest genetic flexibility of this region. Thus, changes in the IRRDR might be capable of modulating intracellular antiviral activity, or maybe the genetic flexibility of this region is accompanied by compensatory changes elsewhere in the viral genome and these compensatory changes affect overall viral fitness and responses to IFN therapy (29–31) When we investigated the impact of various sequences patterns at positions 70 and 91 of the core protein, we observed that single point mutation at position 70 (Gln70 CX-5461 ic50 vs non-Gln70) was the only factor that significantly

influenced treatment responses. This result is consistent with recent reports, including a recent multi-center study in Japan that identified Gln70 as a predictive factor for poor responses to PEG-IFN/RBV treatment (14, 13, 30). The core region of HCV interacts with several host factors and modulates expression of numerous genes, including down-regulating IFN-induced antiviral genes, thus inhibiting the antiviral action of IFN (32, 33). Therefore, it would also be interesting to investigate the impact of polymorphism, both at position 70 and of NS5A, on HCV pathogenesis and IFN sensitivity. Multivariate logistic regression analysis of all available data, including those of NS5A and core polymorphisms in this study and the data on NS3 polymorphism in the same patient cohort published elsewhere (16), identified IRRDR ≥ 4 and group A of NS3 as independent viral factors that are significantly associated with a SVR, and IRRDR ≤ 3,

and Gln70 of the core protein as independent factors significantly associated with a null response (Table 5). No combinations of these criteria produced a more significant correlation with virological responses to PEG-IFN/RBV therapy (data not shown). In conclusion, the present results demonstrate that sequence heterogeneity of NS5A, why especially in IRRDR and ISDR, and a single-point mutation at position 70 of the core protein of HCV-1b are significantly correlated with virological responses to PEG-IFN/RBV therapy. Also, the results emphasize the possible functional importance of NS5A and core protein in regulating viral responsiveness to PEG-IFN/RBV. This study was supported in part by Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare, Japan, and a Science and Technology Research Partnership for Sustainable Development grant from the Japan Science and Technology Agency and Japan International Cooperation Agency.

Other fungi previously found in the oral cavity of immunocompromi

Other fungi previously found in the oral cavity of immunocompromised patients include Penicillium, Geotrichum, Aspergillus, Scopulariopsis,

Hemispora, and Hormodendrum [89, 111, 112], although the representation of species seems to correlate with the geographic area of sampling [113]. Recently, Mukherjee et al. used pyrosequencing to characterize the oral microbiota of 12 HIV-infected patients and 12 healthy subjects [114]. The core oral bacterial microbiota comprised 14 genera, of which 13 were common between patients and healthy AZD6738 solubility dmso subjects. In contrast, the core oral mycobiota differed more between HIV-infected and -uninfected individuals, with Candida being the predominant species in immunocompromised patients (98 versus 58% in healthy subjects). Among HIV-infected patients, Candida, Epicoccum, and Alternaria were the most common genera, while in uninfected participants, the most abundant fungi were Candida, Pichia, and Fusarium. Increase in Candida colonization, particularly that of C. albicans, was associated with a concomitant decrease in the abundance of Pichia — a resident oral fungus representing the 33% of healthy oral mycobiota, Palbociclib molecular weight suggesting an antagonistic relationship. Indeed,

Pichia has been shown to inhibit growth of pathogenic fungi such as Aspergillus and Candida by inhibiting the ability of these genera to adhere, germinate, and form biofilms in vitro [114]. Oral Candida colonization is a known risk factor for invasive Candidiasis [115]. Similarly, fungal caused periodontal disease is associated with rheumatoid arthritis [116] and atherosclerosis 4-Aminobutyrate aminotransferase [117], suggesting that bacterial and fungal microbiota from the oral cavity may contribute to the development

of certain human diseases. The human respiratory tract represents the major entry point for numerous microorganisms, primarily airborne viruses, bacteria, and fungal spores. Certain characteristics of these microorganisms, such as Aspergillus spp., coupled with the local host immune response, determine whether the microorganisms will be cleared by the immune system or adhere to and colonize the airways, leading to acute or chronic pulmonary disease [118]. The lower respiratory tract (trachea, bronchi, and pulmonary tissue), previously thought to be sterile when healthy, has recently been shown to clearly harbor a low level bacterial microbiota, which changes during disease (reviewed in [119]). Any microbe, be it a bacterium or a fungus, reaching the lower respiratory tract encounters the efficient cleansing action of the ciliated epithelium. Microorganisms are also subsequently removed by coughing, sneezing, and swallowing. However, if the respiratory tract epithelium becomes damaged, as in the case of bronchitis or viral pneumonia, the individual may become susceptible to infection by pathogens descending from the nasopharynx (upper respiratory tract).

Future developments to enable dynamic in vivo imaging would be ad

Future developments to enable dynamic in vivo imaging would be advantageous to allow in vivo quantification of variations in vessel diameters Selleck STI571 down to the arteriolar level while under the influence of in vivo flow conditions and in vivo factors, both local and circulating. In particular, we are currently lacking

micro-CT evidence supporting the arteriolar level as a major contributor to vascular resistance (unpublished) potentially because vascular tone is missing from the ex vivo trees that we have studied. Nevertheless, the current ex vivo methods are effective in quantitatively and statistically evaluating the anatomic variation in branching patterns during development, and in response to genetic and environmental factors. Understanding the factors https://www.selleckchem.com/products/PLX-4032.html regulating the growth and development of the fetoplacental arterial tree is necessary to understand why the tree fails to develop normally in human pregnancy pathologies. Given advances in micro-CT imaging and analysis, together with a growing resource of mouse models, we are poised for rapid progress. We anticipate that new insights into the etiology of fetoplacental arterial development will advance our understanding of vascular development and ultimately lead to improved pregnancy outcomes.

The authors gratefully acknowledge operating grant support from the Heart and Stroke Foundation of Ribociclib manufacturer Ontario (Grants NA5804 and T6297) and the Canadian Institute of Health Research (Grants MOP231389 and MOP93618). MYR was funded by an Ontario Graduate Scholarship and an Oregon Health and Science University Gerlinger Research Award. SLA was supported by the Anne and Max Tanenbaum

Chair in Molecular Medicine at Mount Sinai Hospital. Monique Y. Rennie: Dr. Rennie is a postdoctoral research fellow at the Heart Research Center of Oregon Health and Science University. She uses mouse models to explore fetoplacental vascular alterations in growth restricted fetuses. She has a particular interest in understanding how placental vascular defects alter hemodynamics, and uses chicken embryos to studies the effect of such hemodynamic changes on heart development. Dr. John G. Sled: Dr. Sled is a Senior Scientist at the Hospital for Sick Children and Associate Professor of Medical Biophysics at the University of Toronto. His research program at the Mouse Imaging Centre (http://www.mouseimaging.ca) focuses on the development of novel medical imaging technologies with applications for studying mouse models of disease and for clinical research. An area of particular interest is the patterning of the microcirculation and the role of patterning defects in disease. S. Lee Adamson: Dr. Adamson is a Principal Investigator in the Samuel Lunenfeld Research Institute of Mount Sinai Hospital, and a Professor in Obstetrics and Gynaecology, and Physiology at the University of Toronto.