Key Odorants Forming Aroma of Polish Mead: Influence of the Raw Material and Manufacturing Processes.

Mead was analyzed by using the concept of molecular sensory science for the identification of key odorants. A total of 29 odor-active compounds were identified in mead by using gas chromatography olfactometry (GCO). Flavor dilution (FD) factors of identified compounds ranged from 1 to 16,384, compounds with FD factors ≥32 were quantitated by using stable isotopically substituted odorants as internal standards or external standard method, and odor activity values (OAVs) were calculated. Fifteen compounds showed OAVs ≥1: aldehydes (2-phenylacetaldehyde, 3-(methylsulfanyl)propanal), 4-hydroxy-3-methoxybenzaldehyde), esters (ethyl 3-methylbutanoate, ethyl propanoate, ethyl octanoate), alcohols (2-phenylethan-1-ol, 3- and 2-methylbutan-1-ol, 3-(methylsulyfanyl)propan-1-ol), furanons (4-hydroxy-2,5-dimethylfuran-3(2H)-one, 3-hydroxy-4,5-dimethylfuran-2(5H)-one), acids (3- and 2-methylbutanoic acid, acetic acid), 1,1-diethoxyethane, and 4-methylphenol. 2-Phenylacetaldehyde (OAV, 3100) was suggested as the compound with the biggest influence on the aroma of mead, followed by 4-hydroxy-2,5-dimethylfuran-3(2H)-one (OAV, 1900), 3-(methylsulfanyl)propanal (OAV, 890), and 2-phenylethan-1-ol (OAV, 680). Quantitative olfactory profile analysis revealed strong honey, malty, and alcoholic impressions. Omission experiments revealed that 3-(methylsulfanyl)propanal, 2-phenylethan-1-ol, 4-hydroxy-2,5-dimethylfuran-3(2H)-one, ethyl propanoate, ethyl 3-methylbutanoate, 2-phenylacetaldehyde, 3- and 2-methylbutanoic acid, 3-hydroxy-4,5-dimethylfuran-2(5H)-one, and 4-hydroxy-3-methoxybenzaldehyde were the key odorants in the mead. Determining concentrations of key odorants in important production steps showed that the fermentation and maturation stages had the strongest effect on the formation of mead aroma.

Molecular Background of the Lychee Aroma of Vitis vinifera L. 'Muscaris'.

Muscaris is a modern white grape variety with good fungal resistance and a pleasant aroma, the molecular background of which was unknown. A comparative aroma extract dilution analysis applied to Muscaris grapes and grapes of the father variety Muskateller revealed little differences and resulted in 39 and 35 odorants, respectively. Sixteen odorants exceeded their odor threshold concentrations. Odor reconstitution and omission experiments showed that the distinct lychee note in the aroma of the Muscaris grapes was generated by the combination of (2S,4R)-rose oxide and geraniol. This finding will guide further molecular research on the transfer of the lychee note into wine and may also be helpful for the targeted breeding of new grape varieties.

Molecular Rearrangement of Four Typical Grape Free Terpenes in the Wine Environment.

In order to analyze the molecular rearrangement of terpenes in wine during aging, the changes in linalool, α-terpineol, nerol, and geraniol in model wine were investigated in the dark at low temperature for 90 days. Headspace-gas chromatograph-mass spectrometer/olfactometry was used for qualitative and relative quantitation of terpenes. Quantum mechanical calculation was used to analyze the Gibbs free energy. The results showed that nerol was converted into d-limonene, terpinolene, linalool, and α-terpineol. Geraniol was converted into ß-ocimene, terpinolene, and linalool. Linalool was converted into terpinolene. The conversion rate of nerol to terpinolene was the highest with 5.94%. α-Terpineol was not converted spontaneously into other terpenes due to its lowest Gibbs free energy, indicating that the cyclization and isomerization could occur spontaneously through an exotherm reaction. However, the dehydroxylation of linalool, nerol, and geraniol required an energy source.

Changes in the Major Odorants of Grape Juice during Manufacturing of Dornfelder Red Wine.

Application of the aroma extract dilution analysis (AEDA) on a distillate prepared from freshly squeezed juice of Dornfelder grapes revealed (3Z)-hex-3-enal and trans-4,5-epoxy-(2E)-dec-2-enal with the highest flavor dilution (FD) factors. In contrast, in the final Dornfelder wine prepared thereof, the highest FD factors were found for 2-phenylethyl acetate, 2-phenylethan-1-ol, and (E)-ß-damascenone. However, for example, among others, (3Z)-hex-3-enal no longer appeared as an important odorant. To monitor the olfactory changes occurring in single processing steps from Dornfelder grapes to the final wine, selected odorants in grape juice, must, and young as well as aged wine from the same batch of Dornfelder grapes were quantitated. In particular, (3Z)-hex-3-enal and hexanal decreased considerably during mashing, while, as to be expected, the concentrations of yeast metabolites, e.g., odor-active alcohols and esters, drastically increased during fermentation. To reveal the influence of barrel aging, the odorants of the same Dornfelder wine aged in either barrique barrels or steel tanks were compared.

Influence of Varying Fermentation Parameters of the Yeast Strain Cyberlindnera saturnus on the Concentrations of Selected Flavor Components in Non-Alcoholic Beer Focusing on (E)-ß-Damascenone.

The diversification of beer flavor is becoming increasingly popular, especially in the field of non-alcoholic beers, where sales are growing steadily. While flavor substances of traditional beers can largely be traced back to defined secondary metabolites, the production of non-alcoholic beers with non-Saccharomyces yeasts generates novel fruity flavors, some of which cannot yet be assigned to specific flavor substances. In a recently published study, besides pear, cool mint sweets, and banana-like flavor, distinctive red berry and apple flavors were perceived in a non-alcoholic beer fermented with the yeast strain Cyberlindnera saturnus TUM 247, whose secondary metabolites were to be elucidated in this study. The trials were carried out using response surface methodology to examine the fermentation properties of the yeast strain and to optimize the beer with maximum fruitiness but minimal off-flavors and ethanol content. It turned out that a low pitching rate, a moderate fermentation temperature, and an original gravity of 10.5 °P gave the optimal parameters. Qualitative analysis of the secondary metabolites, in addition to standard analysis for traditional beers, was first performed using headspace-gas chromatography with olfactometry. (E)-ß-damascenone emerged as the decisive substance for the red berry and apple flavor and so this substance was then quantitated. Although (E)-ß-damascenone is a well-known secondary metabolite in beer and this substance is associated with apple or cooked apple- and berry-like flavors, it has not yet been reported as a main flavor component in non-alcoholic beers.

Phase I/Ib Study of the Efficacy and Safety of Buparlisib and Ibrutinib Therapy in MCL, FL, and DLBCL with Serial Cell-Free DNA Monitoring.

PURPOSE: Activation of Bruton tyrosine kinase (BTK) and phosphatidylinositol-3-kinase (PI3K) represent parallel, synergistic pathways in lymphoma pathogenesis. As predominant PI3Kδ inhibition is a possible mechanism of tumor escape, we proposed a clinical trial of dual BTK and pan-PI3K inhibition. PATIENTS AND METHODS: We conducted a single-center phase I/Ib trial combining a BTK inhibitor (ibrutinib) and a pan-PI3K inhibitor (buparlisib) in 37 patients with relapsed/refractory (R/R) B-cell lymphoma. Buparlisib and ibrutinib were administered orally, once daily in 28-day cycles until progression or unacceptable toxicity. The clinical trial is registered with clinicaltrials.gov, NCT02756247. RESULTS: Patients with mantle cell lymphoma (MCL) receiving the combination had a 94% overall response rate (ORR) and 33-month median progression-free survival; ORR of 31% and 20% were observed in patients with diffuse large B-cell lymphoma and follicular lymphoma, respectively. The maximum tolerated dose was ibrutinib 560 mg plus buparlisib 100 mg and the recommended phase II dose was ibrutinib 560 mg plus buparlisib 80 mg. The most common grade 3 adverse events were rash/pruritis/dermatitis (19%), diarrhea (11%), hyperglycemia (11%), and hypertension (11%). All grade mood disturbances ranging from anxiety, depression, to agitation were observed in 22% of patients. Results from serial monitoring of cell-free DNA samples corresponded to radiographic resolution of disease and tracked the emergence of mutations known to promote BTK inhibitor resistance. CONCLUSIONS: BTK and pan-PI3K inhibition in mantle cell lymphoma demonstrates a promising efficacy signal. Addition of BCL2 inhibitors to a BTK and pan-PI3K combination remain suitable for further development in mantle cell lymphoma.

Key Odorants of Raw and Cooked Green Kohlrabi (Brassica oleracea var. gongylodes L.).

Volatile compounds of raw and cooked green kohlrabi were investigated using a sensomics approach. A total of 55 odor-active compounds were detected and identified in raw and cooked green kohlrabi using GC-O. Twenty-eight odor-active compounds with high flavor dilution (FD) factors ranging from 64 to 1024 were quantitated, and odor activity values (OAVs) were determined. Eight compounds showed high OAVs in raw and cooked kohlrabi: five sulfur compounds (dimethyl trisulfide, methyl 2-methyl-3-furyl disulfide, and three isothiocyanates (1-isothiocyanato-3-(methylsulfanyl)propane, benzyl isothiocyanate, and 1-isothiocyanato-4-(methylsulfanyl)butane)), two lipid oxidation products (1-octen-3-one and trans-4,5-epoxy-(2E)-dec-2-enal), and 2-isopropyl-3-methoxypyrazine. Among these, the sulfur compounds contributed most to the overall smell of the raw and cooked vegetables. The quantitation analysis indicates that the eight odorants are the backbone compounds for raw and cooked kohlrabi. The OAVs for the backbone compounds and also for minor odorants are clearly higher in raw kohlrabi than in the cooked one. Differences can be explained by the influence of the cooking process.

Investigation of neonatal disorders in New World camelids and factors associated with death during and after hospitalization of affected crias.

OBJECTIVE: To assess signalment, clinical findings, and treatments for New World camelids (NWCs) hospitalized for evaluation and treatment of neonatal disorders and investigate associations between these factors and death during and after hospitalization. ANIMALS: 267 NWCs ≤ 30 days of age. PROCEDURES: Medical records of a veterinary teaching hospital were retrospectively reviewed to identify NWCs admitted for evaluation and treatment of neonatal disorders between 2000 and 2010. Signalment, physical examination data, diagnostic findings, treatments, and outcomes were recorded. Factors were examined for association with death during hospitalization and the overall hazard of death by use of multivariable logistic regression and Cox proportional hazards analysis, respectively. RESULTS: The sample comprised alpacas (n = 255) and llamas (12). Median age at admission was 3 days, and median hospitalization time was 2 days; 208 of the 267 (77.9%) neonatal NWCs survived to hospital discharge. Factors associated with increased odds of death during hospitalization included prematurity or dysmaturity, hypothermia, sepsis, toxic changes in neutrophils, and undergoing surgery. The odds of death during hospitalization also increased as anion gap increased. After discharge, 151 of 176 (85.8%) animals had follow-up information available (median follow-up time, 2,932 days); 126 (83%) were alive and 25 (17%) had died. Prematurity or dysmaturity, congenital defects, sepsis, oxygen administration, and undergoing surgery as a neonate were associated with an increased hazard of death; the hazard of death also increased as serum chloride concentration at the time of hospitalization increased. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested the prognosis for survival during and after hospitalization is good for most NWCs hospitalized because of neonatal disorders.

Molecular background of the undesired odor of polypropylene materials and insights into the sources of key odorants.

Screening the volatiles isolated from a standard polypropylene material consisting of a polypropylene homopolymer, the filler talcum, and a mixture of antioxidants, for odor-active compounds by application of an aroma extract dilution analysis revealed 30 odorants with flavor dilution factors ranging from 1 to 64. Eighteen odor-active compounds were subsequently quantitated by gas chromatography-mass spectrometry using stable isotopically substituted odorants as internal standards, and their odor activity values (OAVs) were calculated as ratios of the concentrations to the odor threshold values. Five odorants showed OAVs ≥1, among which were hex-1-en-3-one (OAV 12), butanoic acid (OAV 3), as well as 4-methylphenol, butan-1-ol, and 2-tert-butylphenol (all OAV 1). A comparative analysis of polypropylene materials with different additives suggested plastic-like, pungent smelling hex-1-en-3-one as an ubiquitous key odorant. Odor-active amounts of alkylphenols, in particular plastic-like, phenolic smelling 2-tert-butylphenol, were additionally formed in the presence of talcum and phenolic antioxidants. Whereas the precursors of the phenols were thus obvious, the origin of hex-1-en-3-one was unknown. Injection molding showed only little influence on odorant concentrations.

Endothelin 1 Is Associated with Heart Failure Hospitalization and Long-Term Mortality in Patients with Heart Failure with Preserved Ejection Fraction and Pulmonary Hypertension.

BACKGROUND: The prevalence of pulmonary hypertension (PH) in heart failure with preserved ejection fraction (HFpEF) is increasing. We aim to study the role of big endothelin 1 (Big ET1), endothelin 1 (ET1), and neprilysin (NE) in HFpEF with PH. METHOD: This was a single center prospective cohort study including 90 HFpEF patients; 30 with no PH, 30 with postcapillary PH, and 30 with combined post- and precapillary PH. After enrollment, pulmonary venous and pulmonary arterial samples of Big ET1, ET1, and NE were collected during right heart catheterization. Subjects were then followed long term for adverse outcomes which included echocardiographic evidence of right ventricular dysfunction, heart failure hospitalization, and all-cause mortality. RESULTS: Patients with HFpEF-PH were found to have increased ET1 in pulmonary veins (endothelin from the wedge; ET1W) compared to controls (2.3 ± 1.4 and 1.6 ± 0.9 pg/mL, respectively). ET1W levels were associated with both PH (OR 2.7, 95% CI 1.5-4.7, p = 0.01) and pulmonary vascular resistance (OR 1.6, 95% CI 1.04-2.3, p = 0.03). No evidence of right ventricular dysfunction was observed after 1 year of follow-up. ET1W (OR 1.8, 95% CI 1.2-2.6, p = 0.01) and ET1 gradient (ET1G; OR 1.4, 95% CI 1.04-2, p = 0.03) were predictive of 1-year hospitalization. ET1G ≥0.2 pg/mL was associated with long-term mortality (log-rank 4.8, p = 0.03). CONCLUSION: In HFpEF patients, ET1W and ET1G are predictive of 1-year heart failure hospitalization, while elevated ET1G levels were found to be associated with long-term mortality in HFpEF. This study highlights the role of ET1 in developing PH in HFpEF patients and also explores the potential of ET1 as a prognostic biomarker.

Divergent clonal evolution of a common precursor to mantle cell lymphoma and classic Hodgkin lymphoma.

Clonal heterogeneity and evolution of mantle cell lymphoma (MCL) remain unclear despite the progress in our understanding of its biology. Here, we report a 71-yr-old male patient with an aggressive MCL and depict the clonal evolution from initial diagnosis of typical MCL to relapsed blastoid MCL. During the course of the disease, the patient was diagnosed with classic Hodgkin lymphoma (CHL) and received a CHL therapeutic regimen. Molecular analysis by next-generation sequencing of both MCL and CHL demonstrated clonally related CHL with characteristic immunophenotype and PDL1/2 gains. Moreover, our data illustrate the clonal heterogeneity and acquisition of additional genetic aberrations including a rare fusion of SEC22B-NOTCH2 in the process of clonal evolution. Evidence obtained from our comprehensive immunophenotypic and genetic studies indicates that MCL and CHL can originate from a common precursor by divergent clonal evolution, which may pose a therapeutic challenge.

Systematic Generation of Patient-Derived Tumor Models in Pancreatic Cancer.

In highly aggressive malignancies like pancreatic cancer (PC), patient-derived tumor models can serve as disease-relevant models to understand disease-related biology as well as to guide clinical decision-making. In this study, we describe a two-step protocol allowing systematic establishment of patient-derived primary cultures from PC patient tumors. Initial xenotransplantation of surgically resected patient tumors (n = 134) into immunodeficient mice allows for efficient in vivo expansion of vital tumor cells and successful tumor expansion in 38% of patient tumors (51/134). Expansion xenografts closely recapitulate the histoarchitecture of their matching patients' primary tumors. Digestion of xenograft tumors and subsequent in vitro cultivation resulted in the successful generation of semi-adherent PC cultures of pure epithelial cell origin in 43.1% of the cases. The established primary cultures include diverse pathological types of PC: Pancreatic ductal adenocarcinoma (86.3%, 19/22), adenosquamous carcinoma (9.1%, 2/22) and ductal adenocarcinoma with oncocytic IPMN (4.5%, 1/22). We here provide a protocol to establish quality-controlled PC patient-derived primary cell cultures from heterogeneous PC patient tumors. In vitro preclinical models provide the basis for the identification and preclinical assessment of novel therapeutic opportunities targeting pancreatic cancer.

NRG1 Fusions in KRAS Wild-Type Pancreatic Cancer.

We used whole-genome and transcriptome sequencing to identify clinically actionable genomic alterations in young adults with pancreatic ductal adenocarcinoma (PDAC). Molecular characterization of 17 patients with PDAC enrolled in a precision oncology program revealed gene fusions amenable to pharmacologic inhibition by small-molecule tyrosine kinase inhibitors in all patients with KRAS wild-type (KRASWT) tumors (4 of 17). These alterations included recurrent NRG1 rearrangements predicted to drive PDAC development through aberrant ERBB receptor-mediated signaling, and pharmacologic ERBB inhibition resulted in clinical improvement and remission of liver metastases in 2 patients with NRG1-rearranged tumors that had proved resistant to standard treatment. Our findings demonstrate that systematic screening of KRASWT tumors for oncogenic fusion genes will substantially improve the therapeutic prospects for a sizeable fraction of patients with PDAC.Significance: Advanced PDAC is a malignancy with few treatment options that lacks molecular mechanism-based therapies. Our study uncovers recurrent gene rearrangements such as NRG1 fusions as disease-driving events in KRASwt tumors, thereby providing novel insights into oncogenic signaling and new therapeutic options in this entity. Cancer Discov; 8(9); 1087-95. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.

TRPM8 is required for survival and radioresistance of glioblastoma cells.

TRPM8 is a Ca2+-permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca2+ signaling and resulting K+ channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy.

Actomyosin contractility and RhoGTPases affect cell-polarity and directional migration during haptotaxis.

Although much is known about chemotaxis- induced by gradients of soluble chemical cues - the molecular mechanisms involved in haptotaxis (migration induced by substrate-bound protein gradients) are largely unknown. We used micropatterning to produce discontinuous gradients consisting of µm-sized fibronectin-dots arranged at constant lateral but continuously decreasing axial spacing. Parameters like gradient slope, protein concentration and size or shape of the fibronectin dots were modified to determine optimal conditions for directional cell migration in gradient patterns. We demonstrate that fibroblasts predominantly migrate uphill towards a higher fibronectin density in gradients with a dot size of 2 × 2 µm, a 2% and 6% slope, and a low fibronectin concentration of 1 µg ml-1. Increasing dot size to 3.5 × 3.5 µm resulted in stationary cells, whereas rectangular dots (2 × 3 µm) orientated perpendicular to the gradient axis preferentially induce lateral migration. During haptotaxis, the Golgi apparatus reorients to a posterior position between the nucleus and the trailing edge. Using pharmacological inhibitors, we demonstrate that actomyosin contractility and microtubule dynamics are a prerequisite for gradient recognition indicating that asymmetric intracellular forces are necessary to read the axis of adhesive gradients. In the haptotaxis signalling cascade, RhoA and Cdc42, and the atypical protein kinase C zeta (aPKCζ), but not Rac, are located upstream of actomyosin contractility.

Subcellular stretch-induced cytoskeletal response of single fibroblasts within 3D designer scaffolds.

In vivo, cells are exposed to mechanical forces in many different ways. These forces can strongly influence cell functions or may even lead to diseases. Through their sensing machinery, cells are able to perceive the physical information of the extracellular matrix and translate it into biochemical signals resulting in cellular responses. Here, by virtue of two-component polymer scaffolds made via direct laser writing, we precisely control the cell matrix adhesions regarding their spatial arrangement and size. This leads to highly controlled and uniform cell morphologies, thereby allowing for averaging over the results obtained from several different individual cells, enabling quantitative analysis. We transiently deform these elastic structures by a micromanipulator, which exerts controlled stretching forces on primary fibroblasts grown in these scaffolds on a subcellular level. We find stretch-induced remodeling of both actin cytoskeleton and cell matrix adhesions. The responses to static and periodic stretching are significantly different. The amount of paxillin and phosphorylated focal adhesion kinase increases in cell matrix adhesions at the manipulated pillar after static stretching whereas it decreases after periodic stretching.

Reconstitution of the flavor signature of Dornfelder red wine on the basis of the natural concentrations of its key aroma and taste compounds.

By application of aroma extract dilution analysis (AEDA) on the volatile fraction isolated from a Dornfelder red wine, 31 odor-active compounds were identified by means of HRGC-MS and comparison with reference compounds. A total of 27 odorants, judged with high FD factors by means of AEDA, was quantitated by means of stable isotope dilution assays, and acetaldehyde was determined enzymatically. In addition, 36 taste-active compounds were analyzed by means of HPLC-UV, HPLC-MS/MS, and ion chromatography. The quantitative data obtained for the identified aroma and taste compounds enabled for the first time the reconstruction of the overall flavor of the red wine. Sensory evaluation of both the aroma and taste profiles of the authentic red wine and the recombinate revealed that Dornfelder red wine was closely mimicked. Moreover, it was demonstrated that the high molecular weight fraction of red wine is essential for its astringent taste impression. By comparison of the overall odor of the aroma recombinate in ethanol with that of the total flavor recombinate containing all tastants, it was shown for the first time that the nonvolatile tastants had a strong influence on the intensity of certain aroma qualities.

The Treatment for Adolescents With Depression Study (TADS): outcomes over 1 year of naturalistic follow-up.

OBJECTIVE: The Treatment for Adolescents With Depression Study (TADS) evaluates the effectiveness of fluoxetine, cognitive-behavioral therapy (CBT), and their combination in adolescents with major depressive disorder. The authors report effectiveness outcomes across a 1-year naturalistic follow-up period. METHOD: The randomized, controlled trial was conducted in 13 academic and community sites in the United States. Stages I, II, and III consisted of 12, 6, and 18 weeks of acute, consolidation, and continuation treatment, respectively. Following discontinuation of TADS treatments at the end of stage III, stage IV consisted of 1 year of naturalistic follow-up. The participants were 327 subjects between the ages of 12 and 17 with a primary DSM-IV diagnosis of major depressive disorder. No TADS treatment was provided during the follow-up period; treatment was available in the community. The primary dependent measures, rated by an independent evaluator blind to treatment status, were the total score on the Children's Depression Rating Scale-Revised and the rate of response, defined as a rating of much or very much improved on the Clinical Global Impressions improvement measure. RESULTS: Sixty-six percent of the eligible subjects participated in at least one stage IV assessment. The benefits seen at the end of active treatment (week 36) persisted during follow-up on all measures of depression and suicidality. CONCLUSIONS: In contrast to earlier reports on short-term treatments, in which worsening after treatment is the rule, the longer treatment in the TADS was associated with persistent benefits over 1 year of naturalistic follow-up.