1 (Bio-Rad Laboratories). Relative changes of mRNA expression were analyzed with the 2–△△Ct method, with 18S RNA serving as an internal reference. These standardized data were used to calculate fold changes in gene expression. All real-time PCR amplifications were performed in

triplicate. ELISA assay was performed on serum samples taken 21 days post-therapy to determine levels of IL-6 and TGF-β protein in the circulation. Briefly, 96-well microtiter Selleckchem BEZ235 plates (MultiSciences, Hang zhou, China, Catalog No. EK2812; EK2062) were coated with serum from tumor-bearing mouse for 2 hours at 37°C. For TGF-β, serum was acidified with 1 N HCl and then neutralized with 1 N NaOH. Biotinylated secondary antibody was then added to the plates for 1 hour at 37°C. Finally, streptavidin conjugated to HRP was added for 45 minutes at 37°C. Color development was achieved using tetramethylbenzidine (TMB) (MultiSciences, Hang zhou, China) solution for 10 to 15 minutes and then stopped. Optical density was measured at 450 nm. The concentration of IL-6 and TGF-β was calculated by comparison to the standard curve. Comparisons between groups were analyzed by means of one-way analysis of variance. A value of PLX4032 solubility dmso P < .05 was designated as statistical significance. The synergistic antitumor effect of rapamycin and sunitinib on tumor growth was evaluated. Subcutaneous

implantation of 4T1 breast cancer Amino acid cells resulted in large tumors in the untreated group, and the mean tumor volume was 1157.02 ± 138.59 mm3 21 days after implantation. There was limited tumor growth in mice treated with sunitinib alone. Rapamycin monotherapy also significantly reduced the tumor growth. The combination treatment induced a robust delay in

tumor growth, with the tumor volume only 357.81 ± 64.14 mm3 (Figure 1, A and B). As expected, the combination group had the lowest tumor weight ( Figure 1C). In addition, the combinational strategy reduced splenomegaly in 4T1 breast cancer models ( Figure 1D). Together, these data suggested that this combinational strategy was effective to retard tumor progression in animal breast tumor models. To determine the effect of combinational therapy on the tumor vessel density in tumor microenvironment, immunostaining against CD31 was performed. Compared with other groups, tumors in the vehicle group had the most vasculature, with large and tortuous morphology. The combinational strategy could robustly reduce the blood vessel density in the tumor microenvironment (Figure 2, A and B). Though rapamycin or sunitinib monotherapy could also inhibit the microvessle density, both were weaker than the combination treatment ( Figure 2, A and B). Myeloid-derived suppressor cells (MDSCs) have been shown contributing to tumor progression through immunosuppression and proangiogenesis. The quantity of MDSCs in the spleen was assessed with flow cytometry.

We suggest that pharmacologically active components of PNV modify the functional expression of AQP4 and GFAP in a distinct manner in the different cerebellum compartments examined based on the molecular, cellular, neuroanatomical and neurochemical characteristics

of each at a given period of post-natal life development. Aquaporin-4 belongs to a family of integral channel proteins that promote the transmembrane diffusion of water through the cell membrane and which is particularly concentrated in the endfeet of astrocytes. AQP4 is also concentrated in astrocyte membrane contacting synaptic sites where promotes potassium siphoning and normal neuronal signal transduction. By removing K+ excess from the extracellular peri-synaptic sites, AQP4 acts as a buffer thus avoiding excytotoxic activity of neurons. Astrocytes

are part of the glio-neural-vascular unit and hence function as intermediaries between neurons and endothelial Y-27632 cells at the BBB. Picomolar changes in the content of ions inside and/or outside astrocytes are enough to induce important changes in the neuronal activity. On the other Metabolism inhibitor hand, such changes lead astrocytes to release neurotransmitters which also affect neuronal activity. We suggest that the upregulation of AQP4 is probably an intrinsic protective mechanism triggered to mediate transcellular water movement out of cerebellum in order to counteract perivascular edema and swelling of astrocyte endfeet caused by P. nigriventer venom. The simultaneous reinforcement of astrocyte cytoskeleton promoted by upregulation of GFAP would be in line with protective mechanism to restore BBB functionality impaired by PNV. Moreover, since PNV causes excytotoxic signals

in rats, AQP4 intense upregulation around neurons of the cerebellar cortex may be a reactive response of astrocytes against a probable increase in glutamate and K+ ( Prado et al., 1996; Mafra et al., 1999; Reis et al., 2000; Vieira et al., 2003) resulting from neuronal activation by PNV ( Cruz-Höfling et al., 2007) and changes in the electric activity of neurons ( Ferrari et al., 2010). Taken together, the findings allow us to speculate that the upregulation of AQP4 in response to PNV may represent the involvement of this protein in neural signal transduction, particularly Pyruvate dehydrogenase in neurotransmitter and K+ siphoning and edema resolving thus with impact on the physiology of BBB impairment caused by PNV. The authors thank Instituto Butantan (São Paulo, SP, Brazil) for donation of venom, Ms. Stephanie Souto Maior for technical assistance and Mr. Miguel Silva for excellent animal care. The authors are indebted to Professor L. Sodek for revising the language. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (Fapesp # 2008/55748-1) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, # 302206/2008-6 and 481316/2008-6). L.M.S. was supported by a MSc studentship from CNPq and M.A.C.H.

However, the high effective concentrations (IC50 > 75 μM) of NSC23766 limit its use as a therapeutic agent [36]. Other known Rac inhibitors also have IC50s of 10 to 50 μM [44] and [45]; including the recently published Rac inhibitors

AZA1, ZINC69391, and IA-116 [46] and [47]. At concentrations ranging from 5 to 20 μM, AZA1 acted as a dual inhibitor for Rac and Cdc42, and blocked prostate cancer cell proliferation, cell migration, and reduced Cyclin D1, and PAK and Akt activities [46]. Another compound ZINC69361, which inhibited Rac activity with an IC50 of 61 μM and reduced lung metastasis, was used as a lead to derive IA-116, which was selective for Rac and inhibited the FRAX597 ic50 interaction between Rac and the Rac GEF p-Rex1; albeit, at μM effective concentrations [47]. Recent studies have also shown the utility of the NSC23766 derivative

AZA197, which was identified as a selective inhibitor for the closely related Rac homolog Cdc42. AZA197, at 1 to 10 μM, inhibited the Cdc42 GEF Dbs activity, PAK and ERK activities, and reduced Cyclin D levels, colon cancer cell proliferation, AC220 and cancer progression in a mouse model [48]. The potency of this inhibitor is similar to that of ML141 (CID2950007), another Cdc42 selective inhibitor with an IC50 ~ 3 to 5 μM [49], that was shown to inhibit melanoma cell migration [50]. These data demonstrate the utility of developing chemical probes to target both Rac and Cdc42 in malignant cancer. To improve the efficacy of NSC23766 and its derivatives, we developed a panel of related compounds [51], and identified EHop-016 as a Rac inhibitor that is 100 times more potent than NSC23766, and binds to the effector domain of Rac1 with a tighter interaction [52]. To our knowledge, EHop-016 is one of the most potent Rac inhibitors that has been published, and is an effective

tool for probing Rac function in cell and mouse models; as has been shown by us and others, in studies using breast cancer cell lines, leukemia, melanoma, and T lymphocytes [50], [52], [53] and [54]. We reported that EHop-016 inhibits the Rac activity of metastatic cancer cells with an IC50 of 1 μM by blocking the specific interaction of Rac with the Rac GEF and oncogene Vav. EHop-016 Adenosine also inhibits the activity of the Rac downstream effector PAK, lamellipodia extension, and cell migration in metastatic cancer cells at concentrations less than 10 μM, while concentrations ≥ 10 μM inhibits the activity of the close Rac homolog Cdc42, and cell viability [52] and [53]. The aim of this study was to test the In Vivo effects of EHop-016 in cancer progression. We used metastatic cancer cell lines and a mouse model of experimental metastasis to demonstrate the efficacy of EHop-016 at reducing mammary fat pad tumor growth, metastasis, and angiogenesis.

Quality management systems like ISO, EFQM and TQM evaluate structures and processes but do not assess the related outcome. They were first used in industry and transferred Raf activity to healthcare systems thereafter. The necessity that an individual organization has to define its own quality goals, as well as the processes to achieve them, could be considered as a weakness. Moreover, those programs are addressing entire hospitals rather than specific diseases or functional units. Pure industrial process optimization programs

are addressing processes without considering best practices from other organizations. After defining their own quality goals, the processes to achieve them have to be developed by the organization itself. Finally, process consulting is helpful in order to solve individual problems, and best practice transfer is the basis of this type of optimization. Most consulting projects selleckchem are very long lasting, however, and put a high burden of the organization regarding human resources. According to our experience, all above-mentioned programs are addressing relevant parts of clinical process optimization in stroke

care. None of them provides a holistic solution, however. Reviewing the literature, Donabedian [15] has defined three different qualities in medical care describing the basis for optimization in stroke care. The structural quality is covered by guideline adherence. In this context it is important that the guidelines are defined by the medical societies and based on clinical and scientific evidence. Lonafarnib ic50 However, the guidelines have to be implemented into clinical processes resulting in a positive impact on process quality. By combining both efforts, the quality of care is expected to

increase but this effect has to be monitored in order the proof outcome quality. In order to address these three qualities, a methodology for process optimization in stroke care has to include all the relevant clinical guidelines and to reflect the organizational structure which is defined by specific guidelines. Moreover, such a methodology has to have the capability to support optimization of clinical processes addressed by management consulting tools. Additionally, transfer of best practices will be helpful in achieving this goal. Our focus should be on support processes as well, which contributes in improving the process quality, e.g. providing optimized imaging infrastructure. An essential part is also to measure quality parameters thus addressing structural, procedural and outcome performance indicators. Keeping all these requirements in mind, so called “process maturity models” seem to best meet our needs. They are generally accepted in software industry or aeronautics.

,

2011). Here, we show that primary monocytes loaded with NGF using Bioporter can secrete NGF in a time-dependent manner over 24 h. This is also true for endogenous cytokines indicating that protein secretion is active rather than a result of proteolytic degradation, however, further investigation is required. On the other hand, whether or not monocyte cell death does indeed occur, the more important point is that NGF is released from our cells. Other studies have reported that Aβ1–42 significantly elevates the release of inflammatory cytokines in monocytes (Fiala et al., 1998). Differences in our findings may be due to culturing variations, a longer incubation period and higher doses of Aβ. Our future studies will involve administrating IWR 1 Bioporter-NGF-loaded primary monocytes and observing whether these cells can deliver therapeutically relevant levels of NGF this website as well as help reduce β-amyloid deposition and cholinergic neurodegeneration. The present study illustrates that primary rat monocytes can be efficiently loaded with NGF using lentivirus vectors or Bioporter. It further shows that NGF secreted from these cells is

bioactive and that Bioporter does not disrupt monocyte functional properties. These findings provide insights into the use of peripheral monocytes as brain delivery vehicles for NGF and this approach may have implications in the future for the treatment of AD and other neurodegenerative diseases. This study was supported by the Austrian Science Funds (P24541-B24). L.A.H. was supported in part by a U.S. Student Fulbright HAS1 Research grant, sponsored by the Austrian-American Education Commission. We thank Ursula Kirzenberger-Winkler and Kathrin Schanda for their excellent technical assistance. We thank Dr. Martin Offterdinger for his help with the confocal microscopy. We also

thank Celine Ullrich and Daniela Ehrlich for preparing organotypic brain slices and Veronika Rauch for help with lentiviral transductions. “
“The publisher regrets that the above mentioned article was published with an incorrect copyright statement and would like to apologize for any inconvenience caused. The correct copyright statement is given below as: 2012 Elsevier B.V. All rights reserved. “
“The human pentraxin proteins, serum amyloid P component (SAP) (Pepys et al., 1997) and C‐reactive protein (CRP) (Pepys and Hirschfield, 2003), are normal circulating plasma proteins which are important in routine clinical diagnosis. They are also targets for novel therapies currently being developed for major diseases (Pepys et al., 2002, Pepys et al., 2006, Kolstoe et al., 2009, Bodin et al., 2010 and Gillmore et al., 2010). However some of their putative roles in health and disease are controversial.

As documented above, de novo genetic variation has an important role in risk for an ASD phenotype. From an evolutionary perspective, this is unsurprising because interest in reproductive success B-Raf assay is typically low in individuals with autism, such that genetic variants would be subject to negative selection. If inherited variation were to contribute significantly in ASD risk, it would need to be shielded, at least partly, from this selection [ 90]. Possible mechanisms

include sex-differential expression (i.e., 4:1 male:female), recessive inheritance [ 61], parent-of-origin effects (e.g., maternal 15q11–13 duplications), and gene–gene or gene–environment interactions [ 91]. In addition, variation must be replenished by de novo risk events at a rate matching the selection differential. Indeed CNV

and exome sequencing studies suggest that some 10–15% of ASD subjects carry a de novo risk variant and this value may rise as whole genome sequencing efforts reveal further previously undetected events. De novo events are ‘genetic’, and identifying them can yield deep insight into the biology of ASD, but they are not necessarily heritable variation in the traditional sense [ 92]. They also do not fit in the design of twin studies that estimate Baf-A1 chemical structure heritability from twin recurrence. Assuming strict independence of de novo events in dizygotic twins, de novo mutations play only a minor role in recurrence, but their impact increases as the probability of recurrence of de novo events within the same family increases. This within-family dependence has not been quantified, but the

fact that mutation rate is a function of parental age [ 93] and germline or gonadal mosaicism is evoked fairly often to explain concordant selleck kinase inhibitor mutations in ASD sibs (and other disorders) suggests the dependence is non-negligible. This and other features of twin study designs [ 94] limit their general applicability for accurate estimates of heritability. An important paper by Risch et al. [ 91] suggested that the inheritance patterns of ASD could be due to gene–gene interaction, but not simply to a few genes of major effect, even if they interacted to generate risk. Research in the past decade has begun to uncover numerous genes and loci and the mechanisms that govern their action, but there are hundreds of other ASD risk loci estimated to exist [ 20••, 38•, 80, 81 and 82] that await further genetic and functional characterization. Moreover, there has been rudimentary progress in identifying multiple ‘mutations’ in single individuals [ 95, 96 and 97], suggesting possible multigenic threshold models for ASD. These variants include multiple CNVs [ 95 and 96], smaller sequence-level changes [ 97], variants affecting apparent non-coding regions of the genome [ 20•• and 72•], and combinations of each [ 24 and 72•], all of which are predicted to be etiologic due to both the rarity in populations and the presumed damaging effect on the genes.

For PAR, OGG1-positive nuclei, and OGG1-positive cytoplasm, agreement of the respective pattern with tumor incidences appeared less striking (see Fig. 2). With

a more detailed histopathological examination using a multiple-step section approach with at least 60 slides per lung for tumor evaluation (Kolling et al., 2008), concordance of the data patterns between marker Kinase Inhibitor Library order expression and tumor incidence was even more striking including, in addition, PAR-positive nuclei (data not shown). In the present study, we provided evidence that immunohistochemical detection and quantification of different genotoxicity markers in formalin-fixed, paraffin-embedded rat lung tissue samples is a suitable methodological approach to determine local genotoxicity in situ in the lung after particle exposure, even in a retrospective manner. Up to now, only few methods are available for detecting local genotoxicity in lung tissue after MNP exposure, for example Comet assay and micronucleus assay, both of which involve certain limitations regarding applicability

and informative value. Comet assay, which mostly uses single-cell suspensions from whole lung tissue, is unable to differentiate between cell types and to display localization of DNA damage within the tissue. In addition, Comet assay requires living cells/tissue and is therefore not applicable for retrospective studies. Micronucleus assay, on the other hand, can theoretically be performed on fixed and embedded lung tissue by DNA staining, but induction of micronuclei

requires cell proliferation and is restricted to dividing cell populations. This assay VX-770 concentration thus might underestimate DNA damage when applied to whole lung tissue and its informative value is limited to clastogenic and aneugenic genotoxic effects, without identifying detailed types of DNA damage. Oxidative DNA base lesions also cannot be detected by micronucleus assay. Immunohistochemical in situ detection and quantification Gefitinib price of genotoxic insults in fixed lung tissue thus could be a step forward in the investigation of potential genotoxic modes of action of MNP, even in a retrospective manner, if a meaningful panel of genotoxicity markers with different degrees of informative value is used. In the present study, we therefore analyzed the applicability of this methodological approach and the usefulness and informative value of various well known markers of genotoxic stress. As described in the following, all these markers have specific advantages and disadvantages, but overall, some of them are quite useful in better understanding the processes involved in genotoxicity. Given that particle-induced tumor development involves genotoxic events and based on the tumor data, one would expect clearly enhanced numbers of PAR-positive nuclei in particle treated animals, as PAR indicates an early response to DNA strand-break induction.

This is in contrast to the proposed method of PP-50 mediated trehalose delivery [27]. In the current study, the techniques for the cryopreservation of cells using trehalose and PP-50 developed by Lynch et al. [27] were extended to successfully preserve nucleated human cells. The Human osteosarcoma derived cell line SAOS-2 [16] and [35]

was used as a model for nucleated, adherent human cells. Unless otherwise stated, all reagents were purchased from Sigma–Aldrich (UK). Materials for the PP-50 polymer synthesis were sourced as previously described [25]. Foetal bovine serum (FBS), l-glutamine, and penicillin/streptomycin were purchased Ipilimumab supplier from Invitrogen (UK). Dulbecco’s Phosphate-Buffered Saline (DPBS), 10 × DPBS and trypsin–EDTA were purchased from Life Technologies™ (UK). The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (UK). The SAOS-2 cells were purchased from the European Collection of Cell Cultures. The Annexin V-FITC Apoptosis Detection Kit was purchased from BD Biosciences (UK). The synthesis and characterisation Dabrafenib datasheet of the PP-50 polymer were as previously described by Lynch et al. [25]. SAOS-2

cells were grown in tissue culture flasks containing “growth media”: Dulbecco’s Modified Eagle’s Medium – high glucose (DMEM), supplemented with 10% (v/v) FBS, l-glutamine (2 mM), penicillin (100 IU/ml) and streptomycin (100 μg/ml). At approximately 70% confluency, the cells were subcultured with trypsin (0.05% w/v) and EDTA (0.02% w/v), and were subsequently split at a ratio of 1:6. The cells were maintained Loperamide in a humidified incubator at 37 °C with 5% CO2. The cells were used between passages 4 and 20. Calcein, which is membrane impermeable, was used as a tracer for hydrophilic species delivery into the cells. The viability of the

cells was assessed using propidium iodide (PI) staining. SAOS-2 cells were seeded into 35 mm glass bottom culture dishes (PAA, UK) at 2 × 105 cells/dish, in growth media. After 48 h of incubation in a humidified incubator at 37 °C with 5% CO2, a positive control for PI staining was prepared by fixation with paraformaldehyde solution (4% w/v, in DPBS) for 10 min, followed by washing (×3) with DPBS. For the remaining dishes, the cells were washed twice with DPBS. Afterwards, the cells were incubated for 4 h in serum-free media supplemented with 0.2 M trehalose, 2 mM calcein, and with or without PP-50 (200 μg/ml), at pH 7.05. The cells were washed twice with DPBS, and incubated with growth media containing Hoechst 33342 (2 μg/ml) and PI (2 μg/ml) for 15 min. Following three washes with DPBS, the cells were imaged using a TCS SP5 inverted laser scanning confocal microscope (Leica, Germany). SAOS-2 cells were seeded into 96-well tissue culture plastic plates (Corning, UK) at 5000 cells/well. After 24 h, the cells were washed twice with DPBS at either pH 7.4 or pH 7.05.

The wheat flour used was wheat flour

type 1 (Nita – Moinho Paulista Ltda., Santos, Brazil). The water absorption capacity, stability, mixing tolerance index were 65.3 g/100 g, 10.5 min, 20 BU, respectively, determined through Method 54-21.01 (AACC, 2010); maximum resistance (135 min) and extensibility (135 min) were 900 BU and 128 mm, respectively, determined through Method 54-10.01 (AACC, 2010); and its Falling Number was 547 ± 4 s, determined through Method 56-81.03 (AACC, 2010). Whole chia flour was obtained by milling chia seeds (A. Sturla, Buenos Aires, Argentina) in a laboratory CAL-101 datasheet scale mill (Quadrumat Senior Mill, Brabender GmbH & Co. KG, Duisburg, Germany). The hydrogenated vegetable fat used was Pan Advance S550 (Cargill Agrícola S/A, São Paulo, Brazil). The other ingredients were obtained at the local market: sugar (Guarani, Olímpia, Brazil), baking powder (Kraft Foods, Curitiba, Brazil) and whole milk powder (Itambé, Belo Horizonte, Brazil). The protein, lipid, ash, total fibre, soluble and insoluble fibre contents of the wheat and chia flours were determined by the following AACC methods: 46-13.01, 30-10.01, 08-12.01 and 32-10.01 (AACC, 2010), respectively, and the carbohydrate content calculated by difference. The particle size of the raw materials was determined using AOAC Method 965.22.A (AOAC, 2000) with 8″diameter sieves and 20, 32, 60, 80 and

100 mesh screens. The cakes were prepared according to the formulation of Borges, Pirozi, Lucia, Pereira, selleck products Moraes and Castro (2006), adding 100 g sugar/100 g flour instead of 86.7/100 g flour. Thus, the basic formulation was the following: flour mixture (wheat flour and whole chia flour) (100 g), sugar (100 g), in natura egg (40 g), baking powder (3.3 g) and whole milk powder (11.2 g). The base formulation adopted in this study is a formulation that is typically used in the production of cakes in Brazil.

The amounts of whole chia flour (WCF) and hydrogenated vegetable fat (HVF) were established according to a 22 central composite rotational design (CCRD) with a total of 11 assays ( Rodrigues & Iemma, 2005). Phosphoglycerate kinase The amount of WCF added ranged between 0 and 30 g/100 g flour mixture and the amount of HVF between 12 and 20 g/100 g flour mixture ( Table 1). Water was added to hydrate the whole milk powder (75 g water/11.2 g whole milk powder), but the moisture contents of the wheat flour, WCF and whole milk powder were taken into consideration in this calculation, decreasing the amount of water added, since they also contributed water. Thus the water added to the formulations ranged between 60.5 and 60.9 g, according to the assay. For cake preparation, a cream was initially made as follows: the sugar, eggs and fat were mixed for 2 min at high speed in a K45SS high speed planetary mixer (Kitchenaid, St. Joseph, USA).

Even when needs were expressed by relatives, they were not considered as a potential client “How can I put it? Even though I was sad, they did not ask why I was feeling that way…” (R25T2). There was a perception of inequality, of inconsistency in services received by relatives where those who were themselves (or a close one) part of the health care system were favored “I told them that my wife used to be a nurse, so maybe it played in www.selleckchem.com/products/sorafenib.html my favor” (S14T2) or “I think it might have helped communication

with the social worker once they knew that she was also a social worker” (S15T1). Communication abilities of health professionals emerged as a key factor to foster respect and confidence toward health professionals “He gave me his hand, and he explained me this and that. I liked it when they introduced themselves” (R2T1) or “They were always available and always smiling all the time, as if we were not disturbing them, you know” (S1T1). Good communication between health professionals was appreciated but was perceived as a challenge in acute care settings “I would repeat in the evening, repeat over the next morning, I would repeat every hour, because I was there on a working shift, you know. You tell yourself ok, at some point,

I have other things to do. Always repeating…” (R4T1). Information-seeking on the part of relatives was perceived as being the norm “I tell you, it’s the same everywhere. Here [in rehabilitation] or in acute care, it’s just the same thing. If you want information, you have to run after it yourself, that’s all” (R23T2). As relatives needed to seek for services, availability Anti-cancer Compound Library and attitudes of health professionals emerged as a determinant factor which was perceived as a facilitator when for example doctors would do systematic daily rounds “…we had a doctor who would

come almost every day. He took time to talk with us… he would come early in the morning or in the afternoon at around 3 pm” (S9T2) or when the physical environment was supportive “Yes, and P-type ATPase of course we would pass by, we would walk around, and they were very close… on the ward, three doors away, and the physiotherapist and occupational therapist were there” (R1T2) or when there was a stability in personnel which was mentioned more frequently in the context of rehabilitation as compared to acute care “So we would walk by, and with time they would recognize us because it was always the same staff. And they would talk to us and ask how we were doing and all that” (R1T2). In contrast, barriers mentioned were high staff turnover “In the first few days, there was a lot of staff turnover, and it was difficult to get new information” (R10T1), scheduling issues such as personnel availability only during day time and weekdays (working hours) “…we did not really speak to the neurologist because he was working days, and we could not be there during the day” (R20T1) or “But you know, treatments were during the day.