Passion fruit by-product was obtained from an industry of fruit pulp located in the city of Jundiai, São Paulo State, Brazil. The peels of passion fruit were dried in oven under air flow at 60 °C until constant weight. The dry peels were reduced to fine

powder in a Bimby processor (model TM 31, Vorwerk®, Wuppertal, Germany). In order to make the mixture of the fiber powder into the reconstituted milk easier, the particle size was standardized to less than 42 μm, measured through sieves (Granutest, São Paulo, Brazil). The passion fruit peel powder (PFPP) was stored in clapped glass bottles Cobimetinib and kept under refrigeration at 4 °C until use. Skimmed milk Molico® and whole milk Ninho® powders (Nestlé, Araçatuba, Brazil) were both reconstituted to 12 g 100 mL−1 of distilled water and each one was divided into two milk samples. In order to define the highest amount of the passion fruit peel powder that caused the minimum volume of whey separation by the end of fermentation, previous fermentation tests were made with the two types of milk in graduated 50 mL Falcon tubes with addition of the powder varying from 0.5 to 1.0 g 100 mL−1 of milk. As result, 0.7 g

of PFPP in 100 mL of milk was added into the two types of milk. Samples without the PFPP were used as control. All milk bases were heat treated at 85 °C for 15 min under agitation in a water bath and then divided into sterile Schott® flasks (500 mL), cooled in an ice bath, and stored at 4 °C for 24 h. We used Pifithrin�� in this study a freeze-dried starter yoghurt culture (CY340. DSM, Moorebank, NSW, Australia) – composed of Streptococcus thermophilus (St) and Lactobacillus delbrueckii subsp.

bulgaricus (Lb) – and four probiotics, namely two strains of Lactobacillus acidophilus (L10. DSM, and NCFM. Danisco, Madison, WI, USA) and two strains of Bifidobacterium animalis subsp. lactis (Bl04 and HN019. Danisco). The lyophilized cultures were diluted in sterilized milk and divided into aliquots into Eppendorf® flasks and frozen at −20 °C. selleck chemicals Each inoculum was prepared by thawing the cultures and diluting them into 50 mL of sterilized skim or whole milk, according to the milk base to be fermented. Each Schott® flask containing 500 mL of reconstituted milk was inoculated with 1 mL of yoghurt starter co-culture with an average count of 8.2 Log CFU mL−1 of St and 5.4 Log CFU mL−1 of Lb and 1 mL of probiotic culture with counts around 6.4 Log CFU mL−1 (P > 0.05). Eight different PFPP-enriched yoghurts were prepared using the four probiotic strains in the two different milk bases, plus eight controls without passion fruit peel powder.

In contrast, the porcine rectal mucosa is not as thick and the relatively narrow lumen leads to better maneuverability of the duodenoscope. Therefore, simulated papillae can be easily find more created in the circumference of the rectal wall in the ex vivo rectum model. In the current study, we established that 13 or more simulated papillae could be created in all models. This allows 1 model to be used by multiple trainees and by using various generator settings. The endoscopist’s tactile sensation during

ES in the native porcine papilla is different from that in humans because of the small orifice without protrusion or papillary roof as well as the thin mucosa. In the current model, the endoscopists (T.I. and R.T.) experienced a similar tactile and visual sensation when cutting the simulated papilla. However, maneuverability of the duodenoscope was quite different in the in vivo and ex vivo stomach models and the ex vivo rectum model; that is, ES was easier to perform in the ex vivo rectum model

than the stomach model because of the stability of the duodenoscope. Our results suggest that the rectum model is suitable for ES training in beginners and the stomach model for the experienced. The same features of the ex vivo rectum model allowed both ES and EP to be performed. To the best of our knowledge, this is first description of a simple and useful EP training model. In terms of the cost per mucosal bleb of the in vivo model, (16 mucosal blebs per US$2000 live pig), MucoUp, which includes 20 mL in a vial, is $100, and in each bleb, approximately 2 mL MucoUp is used, suggesting

that 10 blebs can be learn more made by using a single vial. Thus, the real price of an in vivo bleb is approximately $135 (total $2160/16 blebs). On Histone demethylase the other hand, in terms of the cost per ex vivo model, the esophagus-stomach-duodenum is almost the same as stomach alone ($20) and the cost of ex vivo porcine rectum is $10. Therefore, the real price of each ex vivo bleb is approximately $11 (total $180/16 blebs). Furthermore, the live animal model is costly and requires housing, and the various preparations, anesthesia, and space are time-consuming and cumbersome and poorly simulates the human papilla. Onaya et al18 revealed that blebs were maintained at least for more than 30 minutes after injection. Although it is unknown whether mucosal blebs can be created in a frozen ex vivo model and then transported to a facility, it seems possible that skilled technicians can create them just before hands-on training as has been done for training in ESD. There are several limitations to this pilot study. There was no control group, and the training effects were not measured. Moreover, in the in vivo model, perforation and hemorrhage may occur regardless of the correct direction of the incision. In contrast, the ex vivo model lacks realism because hemorrhage does not occur, and there is no respiratory variation, which is often encountered during ES and EP in humans.

Surveillance for pneumonia or other infection was conducted daily until death or 72 h after the patient had left the ICU. Patients with clinically suspected pneumonia were investigated with a chest X-ray, white blood cell count, blood culture and non-bronchoscopic bronchial lavage. Clinical pneumonia was defined by the presence of new and persistent infiltrates on chest X-ray, considered likely to be associated with pulmonary infection, and at least two of the following three criteria: temperature of ≥38 °C, white blood cell count ≤4 × 109 or ≥12 × 109/l or the presence of purulent tracheal secretions. The microbial cause of the pneumonia was determined

by the isolation of at least one pathogenic microorganism in a blood culture or at least one pathogenic microorganism in the culture of the non-bronchscopic lavage with the bacterial growth ≥105 colony forming Epacadostat supplier units (CFU)/ml. Community-acquired pneumonia was defined as pneumonia developing within 48 h of admission to any

hospital and HCAP as pneumonia developing more than 48 h after admission to any hospital. The diagnosis of pneumonia was confirmed by an independent physician, not otherwise involved in the daily conduct SRT1720 of the study. Blood, 5–8 ml (for adults) or 2–5 ml (for children), was inoculated into BACTEC plus aerobic bottles (Becton Dickinson, Sparks, MD, USA). These bottles contain a resin to adsorb antimicrobials. The bottles were incubated at 37 °C in the BACTEC 9050 automated analyser for 5 days and subcultured when the machine indicated a positive signal. Patients were pre-oxygenated prior to the non-bronchoscopic bronchial lavage.12 They were already sedated by the tetanus therapy. Secretions in the trachea and tracheostomy were removed by sterile suction. A standard 50 cm, 14-gauge tracheal aspiration catheter (Argyle Sherwood Medical, enough London, UK) was attached to a 20 ml syringe filled with 20 ml of sterile saline (10 ml for children). The distal end was lubricated with sterile gel, introduced via the tracheostomy tube and advanced until

significant resistance was encountered. The saline was instilled over 10–15 s, withdrawn 1–2 cm and then immediately re-aspirated and the catheter was removed. Generally 5–10 ml of fluid was recovered. No further aspiration was attempted during removal of the catheter to avoid contamination with tracheal secretions. Samples were processed in the laboratory within 1 h of collection. A Gram stain and Ziehl–Neelsen stain was prepared from the lavage fluid, which was then mixed with an equal volume of freshly prepared dithiothreitol (Sputasol; Oxoid, Basingstoke, UK). The mixture was left at room temperature for 10 min during which time it was shaken vigorously on three occasions. Three serial tenfold dilutions were made by transferring 1 ml of the mixture to 9 ml of maximum recovery diluent (Oxoid, Basingstoke, UK).

However, this behavior is less evident for films plasticized with glycerol. At the same aw, the equilibrium moisture content is higher for amaranth flour films in the presence of glycerol ( Fig. 5a), compared with films containing sorbitol ( Fig. 5b). Therefore, the glycerol-plasticized flour films are able to retain more water at equilibrium, compared with the sorbitol-plasticized samples. In the other words, films prepared

with glycerol are more hygroscopic than films prepared with sorbitol, even at high temperatures. This observation confirms the higher affinity of glycerol for water, which generates a more pronounced plasticizing effect. Chaudhary, Adhikari, and Kasapi (2011) listed several reasons for this behavior, such as the lower molecular weight of glycerol (92.09 g mol−1) compared with sorbitol see more (182 g mol−1) and the better interaction learn more of sorbitol with starch macromolecules. Furthermore, glycerol is highly hydrophilic and a strong humectant; at 25 °C and 50% RH, its hygroscopicity is 25 g H2O/100 g, while the hygroscopicity of sorbitol is 1 g H2O/100 g ( Takahashi, Yamada, & Machida, 1984). Because sorbitol crystallizes at room temperature

and high RH, the edible films plasticized with this compound are less hygroscopic than those plasticized with glycerol ( Talja, Helén, Roos, & Jouppila, 2007). Table 3 shows that glycerol increases the value of the monolayer water content (mo) and the value of constant C, related to the water–substrate interaction energy, at all the studied temperatures. This result suggests that the hydrophilic groups of the starch and protein present in the amaranth flour are

less available for interaction with water molecules Vasopressin Receptor in the presence of sorbitol; and that stronger water association might occur in the presence of glycerol. In other words, sorbitol is more compatible with the polymers existing in the flour, thereby strongly interacting with these macromolecules. Moreover, the mo values found in this study agree with values reported for soy protein isolate/poly(vinyl alcohol)/glycerol blend, methylcellulose/glycerol, cassava starch/sorbitol, and pea protein/sorbitol films ( Kowalczyk & Baraniak, 2011; Mali, Sakanaka, Yamashita, & Grossmann, 2005; Müller, Yamashita, & Borges-Laurindo, 2008; Su et al., 2010; Vargas, Albors, Chiralt, & González-Martínez, 2011). The k values obtained for the films plasticized with glycerol or sorbitol are <1. These values do not appear to be affected by the temperature or plasticizer type. The desirability function (G) was formulated from the models calculated for the tensile strength (TS), elongation at break (E), and solubility (S) of the flour films plasticized with glycerol (equations (6), (7) and (12)) and sorbitol (equations (9), (10) and (13)).

Also, protein ubiquitination in www.selleckchem.com/products/pf-562271.html synapses of rat brains was also studied using this approach [ 28]. Advantages and challenges are also discussed in recent reviews [ 24 and 29]. There are some limitations to this approach in that there is some ambiguity in assigning gly-gly modifications on lysine residues to ubiquitination, as for instance NEDD8 modification also leads to the same tag present on lysine side chains after proteolytic trypsin digestion. To overcome this, other tags on the basis of the detection of LRGG-lysine have been used in MS experiments (Figure 2). However, this approach is not feasible for the detection of protein

modifications with other ubiquitin-like proteins, such as SUMOylation. Recent attempts to overcome this without the need to introduce SUMO C-terminal mutations were reported in which the application of aspartic acid cleavage, caspase, elastase and trypsin digestion protocols were used to generate SUMO tags on lysine residues that can facilitate see more detection of modifications by SUMO1 and SUMO2/3 [30 and 31]. Such approaches permit the survey of a wider range of ubiquitin and ubiquitin-like modification profiles on proteomes under normal physiological and pathological conditions in the future. Advances in the sensitivity and throughput

of mass spectrometry (MS) based discovery capabilities have continued to spur experiments that are focused on characterising the expression of conjugating (E1/E2/E3s) and deconjugating enzymes (DUBs), but also their interactors and/or substrates. For instance, whole Galeterone cell proteome studies can now provide insight into the turnover and levels of several thousands of cellular proteins in one single experiment [32••, 33 and 34••]. Of particular note is a study reporting on a reference proteomes of 11 cell lines illustrating differences

in the steady state level of a number of proteins [32••]. This is the first time that comprehensive information on the abundance of components of the ubiquitin system is available in different cell types. Interestingly, the abundance of ubiquitin-specific enzymes appears to vary to a great extent as demonstrated for a selection of E3 ligases and DUBs (Figure 3). This information can help to better understand their biological function when combined with functional assays, cell type specificity and regulation. Also, direct co-immunoprecipitation of either E3 ligase components or DUBs directly has given better clues about the enzyme’s function through the discovery of interactors and/or substrates [35, 36 and 37]. However, these approaches have their limitations in terms of the identification of cognate substrates as often direct enzyme-substrate affinities are low.

All patients received an introductory letter and the questionnaire, leaving open the possibility to refuse participation. Ethics approval was obtained from the University of Sydney and Area Health

Service Ethics VE-822 concentration Committees linked to the participating cancer centres. All 18 items of the TiOS consist of a proposition in the third person singular, with a 5-point Likert answering scale (‘strongly disagree’ (1) to ‘strongly agree’ (5)). Three items are negatively phrased. Mean trust (range 1–5) is calculated by averaging the responses. Socio-demographics (gender, age, marital status, education level, ethnicity, mother tongue and religion) and disease characteristics (time since diagnosis, cancer site

and treatments undergone, number of previous consultations with the present oncologist) were assessed. Satisfaction with the oncologist was assessed with the 5-item Patient Satisfaction Questionnaire (PSQ) [15]. An additional item asked whether patients would recommend their oncologist to their friends. Physical and mental Health Related Quality of Life (HRQOL) were measured with the 12-item short form health survey (SF-12) [16]. Finally, one item asked patients how much trust they had in the Australian health care system. For missing values, we used expectation maximization [17]. Using confirmatory factor analysis (CFA), we tested our 4-dimensional model first, then a uni-dimensional representation of

trust. PI3K inhibitor A good model fit would be indicated by non-significant χ2, and Root Mean Square Error of Approximation (RMSEA) < .06 [18]. As in the Dutch sample, we expected uni-dimensionality, but also a reasonably good fit of our 4-dimensional model. We calculated internal consistencies (Cronbach's α), inter-item correlations and item-scale correlations for the TiOS. Construct validity was assessed by calculating Spearman's correlations between trust (TiOS) and its known correlates: satisfaction, trust in health care, and number of previous consultations with the oncologist. We expected that high trust levels would be strongly associated with high satisfaction, and moderately with strong trust in health care and a larger Thymidylate synthase number of previous consultations [2], [3], [19] and [20]. Exploratory, we assessed correlations between trust and patients’ HRQOL, socio-demographics and disease characteristics. No hypotheses were specified with regard to exploratory analyses. Analyses were performed using SPSS 16 [21], and Lisrel 8.5 [22]. In total, 177 questionnaires were returned (response rate 70%, range 56–84% for the different locations). Data from two participants were excluded because of more than 25% missing data. Socio-demographic characteristics of the sample are shown in Table 1. All items, including their psychometric properties, are displayed in Table 2.

, 2006) reveals that across the world’s tropics, the coastal population is expected to grow by 45% to 1.95 billion people by 2050, while the number of people occupying the inland tropics will grow by 71% to 2.26 billion. However, the total area of inland tropical land is four times that of coastal regions, so tropical population density in 2050 is projected to be 57 km−2 inland and 199 km−2 on coasts. Coastal communities will generate increased local environmental stresses, although improved management may keep some or all of this

increase unrealized. Table 1 presents three averaged projections of the physico-chemical http://www.selleckchem.com/products/dabrafenib-gsk2118436.html state of tropical coastal environments in 2050, using three alternative http://www.selleckchem.com/products/PD-0332991.html scenarios developed by the international community associated with the IPCC to describe different policy approaches to GHG emissions. The business-as-usual (BAU) scenario uses RCP8.5 (Vuuren et al., 2011) which approximates the earlier SRES A1FI scenario (Rogelj et al., 2012), and involves high levels of fossil fuel use and minimal efforts to reduce GHG emissions. It is

the future to which we are currently moving. By 2050, under this scenario, global temperatures will approximate 1.7 °C warmer relative to the year 2000, rising towards 4.0 °C warmer in 2100 (Fig. 3 in Rogelj et al., 2012). The MODERATE scenario, RCP4.5 (similar to SRES B1), involves strenuous efforts to rapidly reduce emissions such that atmospheric concentration of CO2 is stabilized at around 450 ppm by 2100. In 2050, average global temperature under RCP4.5 will approximate 1.2 °C warmer than 2000. In the STRONG scenario, RCP3-PD, human emissions of CO2 fall to very low levels within one or two decades with the outcome that average global temperature approximates 0.8 °C warmer than 2000 in 2050 and begins to decline by 2100. Tropical sea surface temperatures (SST) are approximated from average global air temperature assuming a small time lag due to the relatively higher thermal inertia of sea water. Higher ocean temperatures lead to thermal expansion which combines with increased melting

of land ice to raise sea levels. Box 1.  Modeling effects of climate change on next fishery production in Raja Ampat The Raja Ampat archipelago is a representative coral reef system, currently rich and productive. We simulated a loss of coral biomass, incrementally reducing the biomass of coral from 100% of its current (2008) value, to 0%. Throughout these simulations, current fishing effort was maintained. The model of Ainsworth et al. (2008) includes mediation effects that simulate non-trophic dependencies in the ecosystem such as the protection from predators offered by coral to fish. For this study, we have added an additional effect to represent space-limited growth of benthic algae: as coral biomass declines, benthic algal productivity increases.

Constantly elevated Rad6 expression in primary and metastatic melanomas suggests that Rad6 may play an active role during all phases of melanoma pathogenesis: initiation, maintenance and progression to metastatic disease. It remains to be determined, however, whether the melanoma transformation-inducing

properties of Rad6 are solely PTC124 clinical trial transmitted through β-catenin or through the function of Rad6 as a postreplication DNA repair protein. The postreplication repair pathway enables completion of DNA replication blocked by damaging DNA lesions via error-free and error-prone bypass mechanisms [18], and the ubiquitin conjugating activity of Rad6 is critical to this process [47]. Since cells are challenged by environmental or endogenous processes that induce DNA damage, we posit that the activation of Rad6 postreplication repair pathway in the early phase of melanoma development may be necessary for ensuring completion of stalled DNA replication and hence cell survival. Because postreplication repair is often error prone or mutagenic, it is tempting FDA approved Drug Library to speculate that Rad6 may participate in melanocyte transformation by directly contributing to genomic alterations underlying melanoma pathogenesis. In summary, our data suggest that Rad6 may serve as an early marker for melanoma development. The first detectable increase

in Rad6 expression is correlated with melanocyte transformation, and is further augmented in malignant melanoma, there by implicating Rad6 as a novel anti-melanoma therapeutic target. The authors thank Dr. Michael Tainsky for programmatic support of this project. This work was supported by U.S. Army Medical Research Acquisition W81XWH07-1-0562, NIH R21CA178117-01 (MPS), and startup funds Cyclic nucleotide phosphodiesterase from Wayne State University (KR). “
“The efficacy of drug therapy is partly related to the ability of the therapeutic agent to reach its target. The delivery of chemotherapeutics

to tumors was shown to be influenced by the tumor blood supply, the drug transport through the vascular wall, and the drug diffusion/convection through the interstitial space [1] and [2]. Various methods have been tested to improve drug distribution, including isolated organ perfusion, drug physiochemical property changes, and tumor vessel modulation [3], [4] and [5]. Photodynamic therapy was initially designed to destroy tumor cells and the tumor vasculature. It consists of the administration of a photosensitizer that, after activation by nonthermal light, produces a variety of changes at the cellular level in the treated area [6]. Recently, low-dose photodynamic therapy (L-PDT) was shown to enhance the extravasation of macromolecular compounds into tumors [7] and [8]. For example, vascular L-PDT of sarcoma metastasis in a murine model resulted in a significant and selective enhancement of liposomal doxorubicin (Liporubicin; Regulon Inc, Athens, Greece) in tumors.

, 2004). The Akt family of kinases, i.e., Akt1, Akt2, and Akt3, plays arolein processes that are well known as hallmarks of cancer, such as sustained angiogenesis, unlimited replicative potential, and tissue invasion and metastasis (Hanahan and Weinberg, 2011). Moreover, Akt activation mediates

the expression of N-cadherin and metalloproteinases and plays aroleintum or invasion and metastasis by inducing EMT (Park et al., 2001, Higuchi et al., 2001, Grille et al., 2003 and Wallerand et al., 2010). Recently, Steelman et al. (2011) demonstrated that the activation of AKT-1 increased the resistance of MCF-7 cells to radiation. Additionally, Toker and Yoeli-Lerner (2006) showed that Akt1 might have a dual role in tumorigenesis, not only promoting it by suppressing apoptosis but also inhibiting it by suppressing invasion and metastasis. The specific role of AKT in terms of cell motility and invasion seems Doxorubicin in vivo to depend on the cell type and the pathways that are activated. Many of the enzymes that either mediate the

Akt signal, such as MDM2 (Zhou et al., 2001), or regulate Akt activity, such as the tumor suppressor PTEN (Li et al., 1997), are frequently mutated in human tumors. As such, Akt activity is up-regulated, thus increasing tumor cell growth and survival. In several mammalian systems, activated Akt1 correlates with cell migration and invasion. While constitutively active Akt1 can enhance the ability of some cells to invade (Steelman et al., 2011, Kim et al., 2001 and Arboleda et al., 2003), Akt1 can also have the this website opposite effect

in normal or less invasive cells (Arboleda et al., 2003). Moreover, the increased activation of Akt1 correlates with increased proliferation and anchorage-independent growth. However, the effects of activated Akt1 on cell migration and invasiveness depend on the type of cells and tissues in which its action is being studied (Steelman et al., 2011, Kim et al., 2001, Arboleda et al., 2003, Enomoto et al., 2005, Irie et al., 2005 and Yoeli-Lerner et al., 2005). Yoeli-Lerner et al. (2005) and Toker and Yoeli-Lerner (2006) revealed that the expression of activated selleck inhibitor Akt1 potently blocks the migration and invasion of three distinct breast cancer cell lines through Matrigel in vitro. In fibroblasts, Akt signaling enhances the activation of various small GTPases, leading to remodeling of the actin cytoskeleton and enhancing cell motility ( Enomoto et al., 2005). Similarly, the expression of activated Akt in fibrosarcoma or pancreatic cancer cells increases their ability to invade through Matrigel ( Park et al., 2001 and Kim et al., 2001). Liu et al. (2006) demonstrated that cells expressing activated Akt1 show increased proliferation and resistance to apoptosis. Additionally, the invasiveness and motility of the cells were substantially decreased by the down-regulation of Rho activity.

Allele and genotype frequencies were calculated using SPSS v.20 (IBM Corp., Armonk, NY). Allele and genotype frequencies were calculated

for a) the studied population as a whole, b) the R-DEB patient group, and c) 13 selected families in which, apart from the patient, both parents could be genotyped. We improved the detection efficiency of the c.2470insG mutation by being able to screen 96 samples in 2 h with 100% sensitivity and specificity. Forty-five samples of R-DEB patients and their relatives that had been genotyped previously by DNA sequencing and included representatives of all genotype options (−/−,−/G, and G/G) (6) were genotyped identically by our real-time allelic discrimination method (Figure 1). The 89 subjects who participated in this study come from 32 unrelated Mexican families in which at least one ERK inhibitors member suffered from R-DEB. There were 39 (40%) R-DEB patients (36 children, two fathers and one mother) and 50 healthy individuals (24 mothers, 18 fathers, and eight siblings). In the studied population of 89 subjects, the G allele was contributed only by heterozygous genotypes. HKI-272 in vitro The frequency of the G allele was 3.4%. The observed genotype frequencies were 93.3% for the homozygous wild-type (−/−) and 6.7% for the heterozygous genotype

(−/G). The homozygous mutant genotype (G/G) was not found (Table 1). Among subjects suffering from R-DEB (n = 39), the mutant G allele frequency was 3.8%; a 0.4% increase as compared to the studied population (n = 89). The genotype frequencies among patients suffering R-DEB were 92.3% for the −/− and 7.7% for −/G genotypes, respectively ( Table 1). The genotype distribution in the 13 selected families was as follows: the homozygous tuclazepam wild-type (−/−) was found in 13 healthy mothers, 11 healthy fathers and 16 offspring, of which three were healthy but 13 were affected. The heterozygous genotype (−/G) corresponded to two healthy fathers and four offspring, of which three were affected and one was healthy. The nucleotide sequencing method is considered a

gold standard for mutation detection (11). However, its low sensitivity may lead to inadequate analysis and false negatives in samples with a low DNA concentration (13). Furthermore, sequencing is more laborious, time-consuming, and expensive as compared to real-time PCR genotyping (12). We succeeded in developing a real-time PCR genotyping assay that detects the c.2470insG mutation with a 100% concordance as compared to the standard method of nucleotide sequencing. Advantages of the real-time PCR genotyping assay are that a) less input DNA input is required (20 ng), b) high-throughput option facilitates the parallel analysis of a large number of patient samples (96), c) results are obtained rapidly (2 h), d) analysis is simplified as genotypes can be read from allelic discrimination plots (Figure 1), and e) low cost per test ($3 US). The allele frequency of the c.2470insG mutation in our R-DEB population in the current study (3.