, 2010). The data present here suggest that LM-PLA2-I, a PLA2 isolated from L. muta snake venom generates LPC that in turn enhance ganglion cells survival through PKC pathway, probably the PKCδ isoform; and also the JNK is involved. Surprisingly, data from literature have demonstrated the participation of JNK enzyme in apoptosis ( Dhanasekaran and Reddy, 2008 and Brnjic et al., 2010). But, this enzyme is also involved in the trophic effect elicited

by ouabain in retinal ganglion cell survival Apoptosis Compound Library in vitro ( de Rezende Corrêa et al., 2010) and now, elicited by LM-PLA2-I. The local production of LPC in retina may be an important step to stimulate such cells to enhance their survival. Several works have been demonstrated the presence of PLA2 activity in retina (Jacob et al., 1998, Giusto et al., 2000, Masamune et al., 2001, Farooqui and Horrocks, 2006 and Wang and Kolko, 2010), and when retina cells are treated with PLA2 inhibitors ABT-737 price the viability of them is diminished (Forlenza et al., 2007 and Suburo and Cei de Job, 1987). In neurodegenerative diseases, the activity of PLA2 plays a central role in the development on the pathophysiological

processes (Farooqui and Horrocks, 2006). Exogenous LPC or the one formed by LM-PLA2-I enzymatic activity may act as a trophic molecule modulating retinal survival, thus protecting cells from death and this phenomena is related to the concentration many of LPC formed; where low concentrations increase cells survival and high ones damaged them. We thank to FAPERJ, CNPq and

CAPES for financial support. And also, the authors would like to thank Alexandre José Fernandes, Bernardino Matheus dos Santos and Alecsandro de Jesus Rezende for technical assistance. “
“Please find attached the correct Fig. 1 as there was some mistake in the published paper. “
“Figure options Download full-size image Download as PowerPoint slide “
“In spite of the wide range of pharmacological classes and drugs that have been used as analgesics for decades, there is a continuing search for new alternatives, both because low efficacy or safety of many of them (Melnikova, 2010 and Woodcock, 2009). Among the new alternatives that have been evaluated, products obtained from animals, plants and microorganisms are promising. Many of them exhibit a plethora of biological activities, including inhibition of nociceptive behaviour in experimental models of pain. Although the honeybee (Apis mellifera) sting induces local pain and oedema ( Vetter and Visscher, 1998), the A. mellifera venom (AMV) has traditionally been used to treat inflammatory diseases and to relieve pain ( Lee et al., 2005 and Son et al., 2007). Various components of AMV have been identified, but there is not a consensus about their concentration.

[50]). The major risk factors for hepatic cancer include chronic infection with hepatitis B and C (accounting for 54% and 31% of cases worldwide respectively), the consumption of food grains contaminated with mycotoxins (produced by fungi during storage in tropical or sub-tropical

climatic www.selleckchem.com/products/AZD8055.html countries) and last, but not the least, heavy alcohol consumption [1], [2] and [3]. Hepatocarcinogenesis involves initial genotoxic insult (initiation), clonal expansion from premalignant to malignant lesions (promotion) and finally tumor progression by means of further clonal expansion [4]. To date, surgery remains the best choice of treatment that could prolong HCC patients’ survival. However, poor prognosis at times after surgery along with side effects of various chemotherapeutic drugs are also being seen as causes of relapse [5]. In addition

to surgery, chemoprevention is another key approach to control HCC, where one or more nontoxic, naturally occurring or synthetic agents are administrated to prevent, improve or reverse the occurrence of disease substantially. Thus, chemopreventive intervention may serve as a feasible alternative strategy for prevention of liver tumorigenesis. In recent years, considerable efforts have been made to search naturally occurring substances for the intervention NVP-BKM120 in vivo of carcinogenesis [6] and [7]. Nexrutine® (NX), a commercially available herbal extract from Phellodendronamurense, widely used for the treatment of inflammation, gastroenteritis, abdominal pain and diarrhea, has shown to exhibit minimal toxicity to normal tissues [8]. Active components of NX are isoquinoline alkaloids, phenolic compounds and flavone glycosides. A recent study revealed that NX inhibited the proliferation of

L-gulonolactone oxidase prostate and lung cancer cells through the modulation of Akt and CREB-mediated signaling pathways, and that its anti-proliferative effects are comparable to that of berberine, a well-known chemopreventive agent [9], [10] and [11]. Other findings also established NX to be effective against early-stage prostate tumor development as well as tumor progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model [8] and [12]. In addition, recently our group showed that NX inhibited the promotion of skin tumorigenesis in the two-stage mouse skin tumorigenesis model [13]. Although NX has proven to be a potent anti-cancer agent for prostate, skin and lung cancer, no study so far has reported the anti-tumor effects of NX on liver cancer. Therefore, in this study, anti-inflammatory and anti-tumor promoting potential of NX was demonstrated in partially modified Solt-Farber rat liver tumorigenesis model.

They were Shiluan 02-1 (HMW-GS 1Ax1, 1Bx7 + 1By9, 1Dx5 + 1Dy10) and Jinan 17 (1Ax1, 1Bx7 + 1By8, 1Dx4 + 1Dy12) with strong gluten strength, Yannong 24 (1Ax1, 1Bx7 + 1By8, 1Dx5 + 1Dy10) with medium gluten strength, Lumai 21 (1Ax1, 1Bx7 + 1By8, 1Dx5 + 1Dy10)

with weak gluten strength. Shiluan 02-1, Yannong 24, and Lumai 21, were used in the growing season of 2010–2011. The 0–20 cm soil layer contained 83.6 mg kg− 1 of available nitrogen, 18.2 mg kg− 1 of available phosphate and 95.2 mg kg− 1 of available potassium. Wheat cultivars Jinan 17 and Lumai 21 were used in the 2009–2010 growing season when the soil contained available nitrogen-phosphate-potassium at 81.5, 17.6 PD0325901 concentration and 93.6 mg kg− 1, respectively. Two contrasting water regimes (irrigated and rainfed) were used. The irrigated treatment was two irrigations with the total water amount of 1500 m3 ha− 1 over the whole growth period (750 m3 ha− 1 each at jointing and booting stages, respectively), whereas the rainfed treatment had no irrigation. The moisture content in soil after anthesis is shown in Fig. 1. The experiment was a complete randomized block design with three replicates. Plot dimension was 3 m × 3 m. Plants were sown on 12 October 2010 and 15 October 2009, respectively, at a density of 180 seeds m− 2. Normal crop farming practices were implemented to minimize pest, disease and weed incidence.

CH5424802 After full heading, spikes flowering on the same date were labeled with thread. At maturity (14 June 2011 and 15 June 2010, respectively), the labeled heads were sampled and used to determine the GMP particle distributions. GMP and HMW-GS contents were also determined. The content Wilson disease protein of GMP was analyzed as follows: 0.05 g of flour was dispersed into and mixed with 1 mL of SDS and then centrifuged at 15,500 ×g for 15 min using an Allegra X-64R centrifuge (Beckman, San Francisco, CA, USA) and the supernatant was retained. Glutenin macropolymer content was measured using TU-1901

dual-wavelength spectrophotometer (Persee Instruments, Beijing, China). Glutenin macropolymer content was calculated using a set of Kjeldahl protein values. Glutenin macropolymer-gel was isolated by dispersing 1.4 g of defatted flour in 0.05 mol L− 1 SDS (pasteurized, 28 mL) and then centrifuged at 80,000 ×g for 30 min at 20 °C using a Beckman L-60 ultracentrifuge (Beckman, San Francisco, CA, USA) as described [16]. The GMP gel-layer was collected from the top of the supernatant. For Coulter laser particle size analysis, 1 g of GMP-gel was added to 8 mL of 0.05 mol L− 1 SDS solvent. The tube was sealed and placed on a roller-bank for 3 h at room temperature and analyzed with a Coulter Laser LS13320 (Beckman Coulter Instruments, San Francisco, CA, USA). The GMP surface area distribution and volume distribution were measured and calculated from the resulting pattern. Quantification of HMW-GS was performed according to the following method [17].

Six studies [19], [32], [33], [35], [38] and [42] reported on dietary

outcomes; two [33] and [38] had positive effects. Stem Cell Compound Library datasheet Thirty-six intervention features were included in the analysis, of which 11 were associated with a positive rate difference (see Table 2). Refer to the online supplemental data for more information on percent success rate differences (Table 3) and analysis of features within each individual outcome (Tables 4–7). DSME programs are complex interventions with various content and delivery components necessary for the education and skills building required for diabetes self-management. However, limited efforts have been made to investigate which intervention features are associated with a positive outcome, specifically for women of diverse ethnic backgrounds. Studies mainly concentrated on glycemic control (i.e., HbA1c levels) (10 studies) or anthropometric outcomes (11 studies), as opposed to behavioral outcomes such as diet (5 studies) and physical Osimertinib cost activity (5 studies). Since

behavioral outcomes strongly reflect the lifestyle changes needed to achieve the desirable metabolic outcomes [18] and [44], it is imperative to understand how intervention features affect these intermediary outcomes as well. Only five (of 38) intervention features had positive success rate differences for at least three of the outcomes examined in this review: hospital-based intervention setting; group intervention format; situational problem-solving; high intensity (10 or more intervention sessions); and incorporating dietitians as interventionists. Because of their broad influence, we recommend the features

that demonstrate success across multiple outcomes in DSME programming for the populations of interest. Many of these features are also recommended in DSME programming for the general population by the American Diabetes Association (ADA) and the Canadian Diabetes Association (CDA). Specifically, group programming and situational HDAC inhibitor problem-solving are recommended by both national organizations [45] and [45], as these features are shown to be effective in improving HbA1c outcomes [46]. Furthermore, the CDA recommends nutritional counseling of clients with diabetes by a dietitian, either one-on-one or in small group settings, to lower HbA1c levels [45]. A recent study supports this recommendation; it found that visits by a dietitian are associated with lower hospitalization rates and charges in persons of varied cultural backgrounds compared to diabetes classes and one-on-one visits from non-dietitian health professionals [47]. Our analysis suggests that incorporating dietitians has positive success rate differences on anthropometrics, and physical activity, in addition to HbA1c. We are unsure why hospital-based interventions appear more successful across outcomes.

The PD-0332991 cell line latter may serve to “fine tune” their actions in vivo (Schwartz-Albiez, 2012). These results also indicate that this IgG class-dependent cross-reactivity can be reduced by the introduction of PEGs, and this is considered important for the accurate detection of analytes in particular in an artificial array system as the SGA. In order to determine the contribution of non-target binding we assayed P1-, PEG23-, and PEG60-P1 modified beads with fetal calf serum-derived and presumably heterophilic antibodies. The results (Fig. 6B) demonstrate that no substantial binding to all three types of beads was observed for IgM (MFI of around

20) whereas some binding (MFI of around 500) was detected for IgG for the regular P1-beads. This observation is in concordance to the previous experiments (Figs. 5B

and 6A). These IgG signals were reduced for the PEG60-P1 beads to about 100 MFI and for the PEG23-P1 beads to 15 MFI. In summary, unspecific binding was observed almost exclusively when IgGs, but not IgMs were applied as glycan-binding antibodies or as secondary detection antibodies. These data seem to be consistent with existing evidence regarding anti-glycan antibodies. It is known that naturally occurring anti-glycan antibodies are predominantly of IgM class and are produced by CD5 positive B1 cells expressing a distinct pattern of surface markers, but not conventional B cells (Viau and Zouali, 2005, Vollmers and Brandlein, 2009, Griffin et al., 2011 and Bovin, 2013). Despite their polyreactive nature anti-glycan IgMs appear to be highly specific in terms of affinity distinctions. Specific recognition of certain glycan structure strongly GSK1120212 in vitro depends on its natural molecular context (Bovin, 2013). Pentameric IgMs have ten Fab regions and therefore possess a theoretical valency of 10. Multivalent recognition is very important for glycan–protein interaction, providing stable and affine binding to multiple oligosaccharide structures. On the contrary, IgGs are only divalent, their interactions with glycans

may be weaker that is why this antibody class is typically not ascribed to recognize glycans in nature. Due to the same reasons IgGs may be more predisposed to unspecific binding than IgMs upon profiling with glycoarrays. To further exploit the possibility to reduce anti-glycan Parvulin antibody cross-reactivity by using heterobifunctional PEGs, we linked PEG23 and PEG60 to the bead surface, coupled Pk trisaccharide to these beads, and compared the binding of monoclonal human anti-P1 IgM either to P1-coupled beads or to Pk-coupled beads (without or with heterofunctional PEGs) as a function of the antibody dilution (Fig. 7). The results showed that the binding of the anti-P1 IgM antibodies, regardless of the dilution, to Pk-beads was several-fold lower than to P1-beads, indicating that indeed anti-P1 antibodies bind to Pk trisaccharide with much lower affinity than P1 trisaccharide.

Persisting challenges remain with regard to the time spent to formulate and write the feedbacks and to the implementation of the technology. According to the therapists’ evaluation in the CWP and http://www.selleckchem.com/products/XL184.html the T2DM studies, the average time to

write a tailored feedback was about 10–15 min, with substantially more time used on the initial feedbacks given. The therapist reported that using information and text segments (for example mindfulness exercises) from earlier feedbacks made the feedback process more time efficient [8] and [22]. It may be possible to develop a coding system for the different kinds of feedbacks the therapist wants to give and to let the therapist select suitable, more or less standardized feedback messages from pre stored examples. Modifications should then be made to adjust the feedback to each patient’s special needs. To utilize the technology resources even more, it could be possible to use the diary

input to automate the feedback from a registered databank. This databank could be automatically extended with new feedbacks given for specific situations, and a “self-learning” data system could be developed taking the results of each feedback into account. The patients reported that personalized feedback was important. It is therefore essential to find a balance between automating the process and making it more effective while taking into account the relevance of giving selleck inhibitor personalized feedback to the patients. These new developments result in a new type of intervention, requiring a new round of studies on efficacy and feasibility. Automation of the feedback is one way of making the intervention more time efficient. Another timesaving action could be to give weekly feedbacks instead of daily ones. In the diabetes project the feedbacks were given daily for 4 weeks and weekly for 8 weeks. Although the patients preferred the daily feedbacks they became used to the weekly feedbacks and continued to fill in the daily diaries as before. This indicates that the web-based intervention could be used to maintain adherence to the treatment and thereby achieve the effects with less effort.

Further studies are needed to analyze the effects of automation and reducing the feedback intervals. Although there was some variation over the three studies, adherence to find more the intervention protocol was not a big problem for the patients, at least not after the startup period. This may be related to the therapist’s commitment. Demotivated professionals are recognized as an adherence barrier [36]. De Veer and colleagues also analyzed factors which impede or enhance the successful implementation of new technologies in nursing care among potential users. The factors most frequently mentioned as impeding actual use were related to the technology itself, such as malfunctioning, ease of use, relevance for patients and risks to patients.

Here and throughout this article, X^ denote an estimate of X  . Wang and Swail, 2006 and Wang et al., 2010 applied this model to simulate seasonal mean or 12-hourly HsHs in the global oceans and in the North Atlantic, respectively, with spatial resolution of 2°°. Recently, Wang et al. (2012) extended the set of predictors in model (1), adding the principle components (PCs) of P(t,m)P(t,m)

and of G(t,m)G(t,m) over a domain that is larger than the wave field in question to represent the swell component of waves, as well as p  -lagged dependent variables, Hs(t-p,m)Hs(t-p,m), to account for serial correlation in the predictand (dependent variable) HsHs. They also proposed a data adaptive Box–Cox Crizotinib transformation procedure to diminish the departure of HsHs and SLP gradients from a normal distribution. They have shown that their new model is more skillful, resulting in less biased simulations of 6-hourly HsHs, than model (1). The methodological developments we propose below include physical and statistical aspects. On the physical aspects, we modify the way to account for swell waves by using the term ΔswΔsw as defined later in Section 4.2, and the way to account

for serial correlation in HsHs using the term ΔtΔt defined later in Section 4.3. Thus, our new model is of the form: equation(2) H^s(t,m)=aˆ(m)+aˆP(m)P(t,m)+aˆG(m)G(t,m)+Δsw(t,m)+Δt(t,m). The last term makes the statistical model more coherent with ocean wave physics, because it can be interpreted Selumetinib ic50 as a discrete approximation of the first order derivative that appears in the spectral energy balance governing equation (e.g. Holthuijsen, 2007). Such temporal dependence is especially important for high temporal resolution data as in the present study. In fact, it is closely related to the large autocorrelation found in the 3-hourly HsHs time series. More

details about the inclusion of this term are given in Section 4.3. On the statistical aspects, we take into account the data scale and explore the effects of deviation from the Gaussian distribution Interleukin-2 receptor assumption in the multiple linear regression analysis by transforming the data in different ways, as detailed below in Section 4.4. Since different regimes dominate in different seasons (see Section 2.1), waves in different seasons should be modeled, separately. In this study, we focus on the winter (most energetic) season, which is defined here as December–January–February. Swell waves are waves propagating across the ocean, after being generated remotely during a storm. As explained in Section 2.2, the Catalan coast is often affected by an important swell component coming from E. Ignoring swell waves would lead to a significant underestimation of HsHs.

3642; Figure W4C). Although comparable numbers of CD3 + cells were identified in the lamina propria of the normal

colonic mucosa of both untreated control groups ( Figure 4C), the lymphoid follicles of uPA−/− mice had more CD3 + cells than their find more WT counterparts (P = .041; Figure W4C). Having documented these differences in the CD3 + cell colonic mucosa population, we next quantified Foxp3 + Treg in four different areas, including the ulcerative lesions ( Figure W4D), the lamina propria ( Figure 4D), and the gut-associated lymphoid tissue (GALT; Figure W4E) of the colon and the MLN ( Figure 4E). The number of Foxp3 + cells was lower in the uPA−/− + DSS compared to the WT + DSS mice, with difference reaching significance only in the lamina propria (P = .0282; Figure 4D). Interestingly, in the normal colonic mucosa of the non–DSS-treated controls, the same comparison had the opposite outcome ( Figure 4D). Specifically, uPA−/− mice had significantly more Treg

than their WT counterparts in all areas examined (lamina propria, P = .0204; GALT, P = .0015; MLN, STA-9090 clinical trial P = .0433; Figures 4D and W4, D and E). Finally, c-kit + mast cells were practically undetectable both in mice with colitis and in the normal colon of the control groups. To confirm previously published results suggesting that uPA is upregulated in DSS colitis, we assessed uPA protein in the colon mucosa of mice by ELISA. As expected, WT + DSS mice had significantly higher levels of uPA than the WT untreated controls (P = .0023; Figure 5A). Both groups of uPA−/− mice showed no expression of uPA, thus confirming their genetic deficiency. Having shown that deficiency

in uPA affects the inflammatory cell component of DSS colitis, we next quantified the expression of selected cytokines with important roles in colitis-associated colon carcinogenesis by real-time PCR and IHC. We found that the gene expression of the pro-inflammatory cytokines TNF-α ( Figure 5B) and IL-6 ( Figure 5C), as well as the anti-inflammatory cytokine IL-10 ( Figure 5D), was significantly upregulated in uPA−/− + DSS compared to WT + DSS mice (P = .0303, P = .0079, and P = .0082, respectively). With IHC, IL-6 + cells were located at the base of colonic mucosa Tacrolimus (FK506) and within the granular tissue of typical DSS-induced ulcers ( Figure 5E). Morphometric counts of IL-6 + cells were done in these two areas and were in accordance with real-time PCR quantification of IL-6 expression. IL-6 + cells were significantly more in uPA−/− + DSS compared to WT + DSS mice in both areas (ulcerative lesions, P = .0022; lamina propria, P = .0042) ( Figure 5E). Likewise, the pro-inflammatory cytokine IL-17 was also found in higher levels in the colonic mucosa (P = .0065) and the MLN (P = .0015) of uPA−/− + DSS mice by IHC( Figure 5F).

Mulder et al3 and Ishioka et al4 initially described diverticulotomy by using freehand endoscope manipulation. Sakai et al5 demonstrated that using a cap at the tip of the endoscope offers SCH772984 research buy better visualization of the septum. Evrard et al6 showed better exposition of the septum with the use of a soft diverticuloscope. The fear of bleeding during treatment prompted Mulder et al3 and 7 to use argon plasma coagulation (APC) instead of conventional current, requiring multiple sessions with the risk of inducing fibrosis.

The availability of a soft, plastic diverticuloscope that mimics the Van Overbeek diverticuloscope8 has allowed better septum exposure and endoscope stability without the risk of trauma associated with the use of the rigid instrument. Moreover, it allows an extended section of the septum and protects the airway from aspiration in nonintubated patients. A previous study showed that diverticulotomy with a flexible endoscope and soft diverticuloscope is an effective treatment for ZD,6 and another suggested that this treatment was safer and more effective than freehand

treatment.9 This initial experience click here prompted us to modify the technique with systematic clipping of the bottom section at the end of the procedure to reduce the risk of perforation and improve hemostasis. We report our long-term results of ZD treatment by using flexible endoscopy assisted by the use of a soft diverticuloscope. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki, in compliance with good clinical

practice and according to local regulations. This work was not supported financially or otherwise by any external sources. All patients gave informed consent after explanation of the technique. Ethical approval for the study was obtained from the Institutional Review Board from our center, reference P2010/353. All patients with ZD who were treated in our medical-surgical department between July 2002 and June 2011 were included in the study. None of them very were included in our previous report.6 Files were reviewed retrospectively, and clinical data were recorded (demographics, symptoms, dysphagia score, endoscopic treatment technique, adverse events, and outcome). Adverse events are defined by the Cotton et al10 severity scoring system. Patients were asked to fill in a questionnaire to describe their symptoms and quantify dysphagia before and after treatment. Dysphagia scores 1 month after treatment were available only for patients who were seen in the outpatient clinic at that time. Because a significant number of patients came from abroad, early follow-up in them was performed by the referring physician, and results were not available at the time of data collection. The Dakkak and Bennett11 score of dysphagia (score 0, no dysphagia; score 1, dysphagia to solids; score 2, dysphagia to semi-solids; score 3, dysphagia to liquids; score 4, aphagia) was used to quantify dysphagia.

Balb/c 3T3-A31 fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM – D5648 Sigma–Aldrich), CHIR-99021 cost supplemented with 10% fetal bovine serum (FBS-GIBCO) at 37 ± 1 °C, 90 ± 10% humidity, 5.0 ± 1.0% CO2/air. The cells were removed from the culture flasks using trypsinization (trypsin:EDTA solution at a 0.25%:0.02% ratio) when they exceeded 50% confluence but prior to reaching 80% confluence. Cell viability was evaluated using the Trypan blue exclusion method with a Neubauer chamber. A cell suspension containing 3 × 104 cells/mL was prepared on the day of plate seeding using culture medium supplemented with 10% FBS. The peripheral wells (blanks) of the 96-well microtiter plates

were seeded with 100 μL of routine culture medium and the remaining wells received 100 μL of a suspension containing 3 × 104 cells/mL (3 × 103 cells/well). The plates were incubated for 24 ± 2 h (37 ± 1 °C; 90 ± 10% humidity, 5.0 ± 1.0% CO2/air) to allow the cells to form a monolayer of less than 50% confluence. This incubation period assured cell recovery, adherence and progression to the exponential growth phase. Each plate was examined under a phase contrast microscope to identify experimental and systemic cell seeding errors. For in vitro assays, the terpenes (nerolidol, α-terpineol, L(−)-carvone, (+)-limonene, L-menthone, DL-menthol, PD0325901 order pulegone or 1,8-cineole) were prepared individually as

a micellar suspension to allow dissolution in water. The micelles were prepared as follows: 10 mg of phosphatidylcholine (PC) and 50 μL of the terpenes to be tested were dissolved in 50 μL of ethanol. The mixture was sonicated for 10 min in a Ti-probe sonicator to obtain a homogeneous dispersion of small micelles. The micellar suspension was prepared without terpenes for control groups. The experimental samples were directly diluted Hydroxychloroquine in culture medium (DMEM) to obtain the concentration of use and filtered through a syringe-filter with a PES TPP® membrane (0.22 μm pore size)

to assure sterility. The final concentration of ethanol in all cultures was lower than 0.05%. A Balb/c 3T3-A31 cell suspension containing 3 × 104 cells/well was seeded in 96-well plates, and after a 24 h recovery period, the plates were treated with eight different concentrations of freshly prepared test compounds in complete medium (six wells per concentration) and incubated for an additional 48 h. The control wells (blanks) received complete culture medium supplemented with 10% FBS. Subsequently, 250 μL of neutral red (NR) medium was added to all wells, including the blanks, and incubated (37 ± 1 °C, 90 ± 10% humidity, 5.0 ± 1.0% CO2/air) for 3.0 ± 0.1 h. The cells were briefly observed 2–3 h after incubation for NR crystal formation. After 3 h, the NR medium was removed and the cells were carefully rinsed with 250 μL/well of pre-warmed PBS.