Ten μL of extract were applied to a Zorbax 300SB-C18 reverse-phase analytical column (4.6 mm ID × 150 mm, Agilent Technologies, Santa Clara, CA, USA) using an Agilent 1200 UPLC system equipped with a diode array detector. The process was performed GSK2656157 research buy as described in Paulo et al. [18], with a flow rate of 1 mL/min. Standard curves were constructed by plotting the area ratio between resveratrol and IS versus resveratrol concentration. All resveratrol analyses were performed in triplicate at each fermentation time. Samples were analyzed on a CyAn ADP (Beckman

Coulter, Brea, CA, USA) flow cytometer equipped with a 20 mW semiconductor laser at 488 nm. Fluorescence (FL1 and FL3 bandpass filters) and light scatter (FSC and SSC) signals were acquired logarithmically. Acquisition was performed with Summit 4.3 (Beckman Coulter, Brea, CA, USA) software. To reduce electronic and small particle noise, threshold levels were set on SSC. For the evaluation of cell viability, a bis-(1,3-dibutylbarbituric acid) trimethine oxonol (BOX, 2.5 μg/mL final concentration) and

propidium iodide (PI, 10 μg/mL final concentration) dual staining was performed as previously described [13]. The fluorescence signals were collected by FL1 (BOX) and FL3 (PI) bandpass filters and Cobimetinib cell line 5000 events/cells were acquired for each sample. Fermentation samples for real-time qPCR were prepared as previously described [13]. Specific primers (Stab Vida, Lisboa, Portugal) for chloramphenicol resistance gene (forward: 5′-ACCGTAACACGCCACATCTT-3′; reverse: 5′-TTCTTGCCCGCCTGATGAAT-3′) and ampicillin resistance gene (forward: 5′-TCCTTGAGAGTTTTCGCCCC-3′; reverse: 5′-TTCATTCAGCTCCGGTTCCC-3′) were used to amplify fragments in each of the two plasmids used. Real-time qPCR efficiency was determined for this primer set using standard solutions of known plasmid

copy number. Real-time qPCR (IQ5 Biorad, Hercules, CA, USA) reactions were performed using 3 μL of sample for a 20 μL reaction containing 10 μL of Maxima™ SYBR Green qPCR Master Mix (Fermentas, Burlington, ON, Canada) and, 400 nM of pAC-4CL1 or 200 nM of pUC-STS primer set. Regarding pUC-STS, reactions Rolziracetam were incubated at 95 °C for 3 min, followed by 30 cycles of 10 s at 95 °C and 30 s at 58 °C. For pAC-4CL1, reactions were incubated at 95 °C for 3 min, followed by 30 cycles of 10 s at 95 °C and 30 s at 60 °C. The amplified PCR fragments were checked by melting curves: reactions were heated from 55 to 95 °C with 10 s holds at each temperature (0.05 °C/s). Bacterial cell concentration was kept constant at 3 × 104 cells/reaction and for each fermentation sample, triplicate measurements were performed. PCN standards for calibration curve were made according to a previously described method [13]. Acquisition and analysis were performed in BioRad IQ 5 Software, Hercules, CA, USA.

RI is a Tertiary aquifer at 41 m depth, RII is a Quaternary aquifer at 15.7 m depth, RIII is a Craterous aquifer at 178 m depth, H1 and W1 are Pleistocene aquifers at 170 m and 122.5 m depth respectively. In July 2013 groundwater samples were collected via push-point lances, in Dabrafenib purchase each of the study sites indicated

in Figure 1. After collection, the water samples for DOC analysis were passed through 0.2 μm pre-combusted glass-fibre filters. A total of 10 ml of the filtrate was acidified with 150 μl of conc. HCl to remove carbonates and to prevent mineralisation of dissolved organic matter ( Pempkowiak 1983), then stored in the dark at 5 °C until analysis. This was carried out by means of a ‘HyPerTOC’ analyser (Thermo Electron Corp., The Netherlands), using the UV/persulphate oxidation method and non-dispersive infrared (NDIR) detection ( Kuliński

& Pempkowiak 2008). In order to remove inorganic carbon selleck inhibitor from samples before DOC analysis they were purged with CO2-free air. DOC concentrations in the analysed samples were derived from calibration curves based on the analysis of aqueous solutions of potassium hydrogen phthalate. Quality control for DOC analysis was performed using CRMs seawater (supplied by the Hansell Laboratory, University of Miami) as the accuracy tracer with each series of samples (average recovery was equal to 96 ± 3%). The precision, described as the Relative Standard Deviation (RSD) of triplicate analysis, was no worse than 3%. Samples for DIC analysis were collected in 40 ml glass vials, each poisoned with 150 μl of saturated HgCl2 Histidine ammonia-lyase solution. The analysis was carried out with a ‘HyPer-TOC’ analyser (Thermo Electron Corp., The Netherlands), using a modified method based on sample acidification and detection of the evolving CO2 in

the NDIR detector ( Kaltin et al. 2005). The DIC concentrations in the samples were calculated from the calibration curve obtained using standard aqueous solutions of Na2CO3. The recovery was 97.5 ± 1%. Each sample was analysed in triplicate. The precision assessed as RSD was better than 1.5%. DIC and DOC loads via SGD to the study area were calculated as the product of the measured groundwater fluxes and concentrations of DIC and DOC measured in the groundwater samples. To quantify the annual DIC and DOC loads delivered to the Bay of Puck, the DIC and DOC concentrations measured at the study site in the groundwater samples (salinity ≤ 0.5) and in the groundwater taken from Piekarek-Jankowska et al. (1994) (0.03 km3 yr− 1) were used. The estimate was based on hydrogeological and oceanographic methods and enabled us to evaluate the role of SGD in the water balance of the entire Bay of Puck (Piekarek-Jankowska 1994, Kozerski 2007).

Generally, low molecular mass neurotoxins offer great potential as neurochemical tools to investigate the nervous system. Additionally, they may constitute new models in the drug-screening field for pharmaceutical and agrochemical industries (Palma and Nakajima, 2005). Despite the wide number of LMM compounds already characterised in these venoms, many others remain to be discovered. Some classes of LMM toxins have been reported in spider venoms, including I) acylpolyamines – isolated from the venoms of orb-web-spiders;

some of these are neurotoxic and act as antagonists for different subtypes of ionotropic glutamate receptors, whereas others act on nicotinic acetylcholine receptors (Palma and Nakajima, 2005); II) bis-(agmatine)-oxamide – isolated from the venom of the “fisher-spider”, Plectreurys tristis ( Quistad et al., 1993); III) nucleosides-toxins – mono or disulfated Enzalutamide nmr nucleoside compounds that are able to block kainate receptors and act on type-l calcium channels, such as the toxin HF-6 isolated from the venom of Hololena curta ( Taggi et al., 2004); IV) tetrahydro-β-carbolines – alkaloid compounds isolated from the venom of the social spider Parawixia bistriata ( Cesar et al., 2005) and from the web droplets of the orb-web-spider Nephila CDK inhibitor clavipes ( Marques et al., 2005); these compounds act as reversible inhibitors of monoamine oxidase (MAO) and are very toxic to insects

and are neurotoxic, convulsivant and lethal to rats ( Saidemberg et al., 2009). LMM neurotoxins have been reported in insect venoms, such as the philantho toxins, which are simple types of acylpolyamine toxins isolated

from the venom of the solitary wasp Philanthus triangulum. These venoms act at the level of both NMDA-dependent glutamate Masitinib (AB1010) receptors and nicotine acetylcholine receptors ( Tikhonov et al., 2004). Polybioside, a histaminyl glucoside compound, was recently isolated from the venom of the social wasp Polybia paulista and is neuroactive at the level of AMPA/NMDA-glutamate receptors ( Saidemberg et al., 2010). Identifying the neuroactivity of novel natural compounds requires mapping the action of these compounds at the level of the mammalian central nervous system (CNS). Generally, this is done by intracerebroventricular (ICV) application of the compounds in rat brain followed by the use of immunohistochemical methods to detect the expression of c-Fos protein. The expression of c-Fos has been used as a biochemical marker to identify stimulated neurons (Morgan and Curran, 1991). This protein is expressed by the proto-oncogene c-Fos, which is an immediate expression gene and is rapidly activated by neuronal cell stimuli, such as neurotransmitters and trophic factors. The expression of this gene triggers the expression of other specific genes by intracellular secondary messengers, which in turn trigger a series of biochemical events in the cell (Saidemberg et al., 2010).

Wang et al. have discussed these processes in detail [ 3••]. Here we summarise recent advances in both passive and active delivery of platinum-based anticancer complexes. Utilizing nanotechnology

to improve drug delivery is a well-known concept, however innovative designs of nano-vectors to achieve efficient drug delivery and their complexity are emerging [4•]. Carbon nanotubes (CNTs) are the most studied. Pristine CNTs are insoluble in most solvents and bear structural resemblance to carcinogenic asbestos fibres. However, coating CNTs with linear and/or branched poly(ethylene glycol) (PEG) units (1 and 2, Figure 1a) renders them more hydrophilic and more suitable for biomedical applications [5]. The toxic nature of pristine (non-functionalised) multi-walled and single-walled CNTs and ability to induce mesothelioma have been demonstrated. Bianco et al. have shown that mono-functionalisation, Cyclopamine bi-functionalisation, and tri-functionalisation of CNTs (3–5, Figure 1b) give enhanced biocompatibility selleck kinase inhibitor and can be translocated directly into the cytoplasm of cells. Non-biodegradable CNTs have the potential to accumulate in various tissues and organs [ 6], however the oxidative enzyme horseradish peroxidase (HRP) can catalytically degrade f-CNTs [ 7]. Tripisciano et al. have encapsulated CDDP into functionalised single-walled carbon nanotubes (SWCNTs). CDDP-SWCNTs are more cytotoxic than free CDDP towards

PC3 cancer cells, but less potent than CDDP towards DU145 cells [ 8]. Recently, Li et al. capped multi-walled carbon nanotubes (MWCNTs) with functionalized 1-octadecanethiol (ODT) gold nanoparticles (f-GNPs) to facilitate the effective delivery of CDDP (6). The presence

of the f-GNP at the tip of the MWCNTs hinders the encapsulated CDDP from leaving the narrow passage of the MWCNTs. The in vivo activity of CDDP in capped CDDP-MWCNTs towards MCF-7 breast cancer cells was enhanced (IC50 7.7 μM), compared to uncapped CDDP-MWCNTs (IC50 11.7 μM). These results suggest that f-GNP MWCNTs may be effective drug depots [ 9]. Reducing the size of the CNTs renders them more likely to pass into the cell, as seen for SWCNTs of 1–2 nm diameter. Guven et al. have synthesised ultra-short Montelukast Sodium carbon nanotubes (USCNTs) of ca. 1.4 nm diameter in which CDDP was encapsulated (7) and then wrapped with a surfactant. The CDDP-USCNTs were more potent than free CDDP in two breast cancer cell lines (MCF7 and MDA-MB-231) after 24 hours. Wrapping of USCNTs with a surfactant retards release of CDDP resulting in its higher cytotoxicity. For in vivo use, the surfactant molecules could be replaced with a cancer-specific protein [ 10]. Li et al. have entrapped a hydrophobic PtIV complex (8) within the inner cavity of MWCNTs. Chemical reduction converted the PtIV prodrug to its hydrophilic and cytotoxic PtII form triggering its release from the MWCNTs.

TGF-β1 also plays an important role as a modulator of the immune system and

is one of the hallmarks of CD4 + CD25 + regulatory T cells that primarily display suppressive effects (Wahl et al., 2006). Humans and other mammals have three isoforms of TGF-β that are translated as pro-proteins linked to a Latency Associated Protein (LAP) which is unique for each isoform. TGF-β isoforms are proteolytically cleaved from LAP but the two proteins remain together and are secreted in a latent complex (Latent TGF-β) comprising a dimer of TGF-β non-covalently associated with a dimer of LAP (Fig. 1) (Koli et al., 2001 and Lawrence, 2001). Yet another family of proteins, Latent TGF-β Binding Protein (LTBP)-1, − 3 and − 4, can bind to LAP and form a large Latent TGF-β complex which increases the Hydroxychloroquine research buy secretion efficiency and targets the complex to the extracellular matrix (Saharinen et al., 1999 and Saharinen and

Keski-Oja, learn more 2000). Extracellular activation of Latent TGF-β resulting in the release of LAP is required for TGF-β binding to its receptor. Mechanisms involved in TGF-β activation under physiological conditions most likely involve enzymatic as well as pH-dependent processes (Lawrence, 2001). Given its importance, human TGF-β1 is measured in serum and plasma to investigate its potential dysregulation in various diseases (Hellmich et al., 2000, Juraskova et al., 2010, Lawrence, 2001, Malaguarnera et al., 2002, Mieliauskaite et al., 2009, Szkaradkiewicz et al., 2010 and Yang et al., 1999) and in cell supernatants for research on physiological or immunological processes (Kropf et al., 1997 and Jurukovski et al., 2005). Since TGF-β1 is secreted in a latent form and primarily is found as such, analysis of the latent form by TGF-β1 ELISA commonly includes acidification of samples to dissociate TGF-β1 from LAP, a prerequisite for the recognition by the ELISA. Analysis is made immediately after neutralization of the acidified sample as TGF-β1 and LAP1 (from here on LAP from

Latent TGF-β1, 2 and 3 is termed LAP1, 2 and 3, respectively) otherwise can re-associate (Kropf et al., 1997). The total TGF-β1 measured corresponds to TGF-β1 dissociated from its latent form plus any free bioactive TGF-β1 potentially present Progesterone in the samples prior to the dissociation; the level of bioactive TGF-β1 generally represents a minor fraction of the total TGF-β1 (Hellmich et al., 2000 and Walther et al., 2009). Evolutionary conservation of TGF-β1 in mammals adds to the complexity when cell supernatants are analyzed. The common use of fetal bovine serum (FBS) in human cell cultures is an issue since FBS contains significant levels of Latent TGF-β1 and human TGF-β1 ELISA systems inevitably cross-react with bovine TGF-β1. Because of the issues involved in the analysis of Latent TGF-β1 by TGF-β1 ELISA, an assay allowing a more straight-forward measurement of Latent TGF-β1 was developed.

, 2005) have shown how the wave propagated and are in reasonable agreement with run-up heights inferred from geological observations. However, previous models have been limited by two important technical constraints. First, they used relatively low spatial resolution along coastlines due to the large region simulated. This means that wave propagation along the complex Norwegian coast, for example, may not be properly simulated. Second, all previous studies used modern bathymetry, as opposed to the inferred bathymetry from 8150 years ago, which has likely changed by tens of metres as a result of non-uniform isostatic relative sea-level changes. Numerical simulations are a useful

tool for studying tsunamis. A number of previous studies have used numerical models to study land- and submarine-slide generated tsunamis (e.g. Abadie et al., 2012 and Assier-Rzadkieaicz et al., 2000). They allow learn more some quantification of the hazard posed by such events, which is uncertain (Masson et al., 2006). A number of these studies have used nested models

(multiple, coupled models with different spatial resolutions, using one or more codes) to simultaneously simulate both the large region and local details (Allgeyer et al., 2013, Kirby et al., 2013 and Horsburgh et al., 2008). In particular, Bondevik et al. (2005) simulated the Storegga slide as a series of retrogressive blocks on Trichostatin A mouse a 2.08 ×× 2.08 km grid for the Norwegian-Greenland sea, with a nested 500 ×× 500 m grid focused on a limited region of the Norwegian coast. This work was extended by Løvholt et al. (2005) to include ideas about how the slide may have moved. A major limitation of these studies was an inability to resolve complex coastlines in the regional models, hence the use of nested models. In particular, no study to date has quantified the effect of increasing coastline

resolution on the numerical simulations. An alternative to nested models is to use a multiscale simulation, where grid resolution varies Cediranib (AZD2171) spatially, often by orders of magnitude (Piggott et al., 2008). Multiscale models often use an unstructured mesh, so in addition can accurately represent complex coastlines and bathymetry without “staircase” effects (Wells et al., 2005). Multiscale modelling then also allows more complex coastal morphologies to be included in the simulation. Here, we use Fluidity—a 3D finite element, non-hydrostatic, numerical model that makes use of unstructured triangular/tetrahedral meshes to enable accurate representations of the domain and allow multiscale simulations of large regions. Fluidity has previously been used to simulate earthquake-generated tsunami (Shaw et al., 2008, Mitchell et al., 2010 and Oishi et al., 2013). Oishi et al. (2013) showed that Fluidity could accurately simulate the 2011 Japanese tsunami and, in particular, was able to represent the dispersive effects of the tsunami by using multiple vertical layers.

With the aim of targeting prostate cancer (PCa), a PtIV prodrug (for CDDP) has been encapsulated into aptamer (Apt)-targeted poly

(d,l-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles (NPs) forming a Pt-PLGA-b-PEG-Apt-NP conjugate (40) engineered to target the prostate-specific membrane antigen (PSMA). These nanoparticles demonstrated enhanced in vivo pharmacokinetics (PK), biodistribution, tolerability and efficacy. The maximum tolerated dose (MTD) for Pt-PLGA-b-PEG-NP was 40 mg/kg while that of CDDP and the prodrug alone was 20 mg/kg. The Pt in 40 remained in systemic circulation one hour post-administration [ 41••], longer than for cisplatin itself. Since the androgen receptor (AR) is upregulated in breast, mTOR inhibitor ovarian and prostate tumour cells, Huxley et al. have designed multiple androgenic steroidal ligands with various nitrogen-containing heterocyclic rings conjugated to either cis-platin or trans-platin (41 and 42, Figure 3d) as platinum drug delivery vectors. These [PtII(NH3)2Cl(steroid)] conjugates were 2–12-fold more cytotoxic than the non-steroidal complexes, but with a similar activity range as CDDP. Interestingly, the cis-complex conjugates displayed two to threefold higher activity than their trans analogues. Conjugation to lipophilic testosterone appears to help the cationic complexes through the cell membrane [ 42]. Many proliferating cells have a high

demand for cobalamin Z-VAD-FMK (Cbl, coenzyme vitamin B12) making it an attractive carrier. Enzymatic reduction of complexes of the type B12-CN-PtII (Figure 3g) releases PtII diammine complexes. Complex 43 was the most active but still with an IC50 ca. 27-fold

higher than free CDDP; conjugates 44 and 45 were ca. 180-fold less active than free cisplatin towards A2780 ovarian and MCF-7 breast cancer cell lines. The reduced cytotoxicity was attributed to a low receptor-mediated response [43]. Nowotnik et al. have reviewed the nano-polymer, ProLindac™ (46), consisting of the active Pt(R,R-dach)2+ fragment of oxaliplatin bound to hydroxypropylmethacryl-amide (HMPA). Release of the active platinum pharamacophore Ponatinib chemical structure was ca. seven-times greater at pH 5.4 in comparison to pH 7.4 after 24 hours. The superior activity of ProLindac™ over oxaliplatin was shown in both human and mouse xenograft models, while the cytotoxicity profile of 46 was similar to oxaliplatin [ 2•• and 44]. Release of nitric oxide (NO) from prodrugs is usually activated by glutathione reductase in tumour cells resulting in growth inhibition of cancerous tissues. Duan et al. have synthesised both hydrophilic poly(acrylic)-cis-[Pt(NH3)2(carboxylate)2] (47 and 48) and hydrophobic NO-donating (49) prodrugs ( Figure 3h) combining NO prodrug therapy with Pt based-therapy. The extended life-times of both prodrugs suggest potential future use in combination therapy.

, 2011). The olfactory system has attracted considerable interest as a promising source of cells for transplantation after SCI, because of its capacity for lifelong regeneration (Lindsay et al., 2010). The main focus of attention in the olfactory tissue has been a unique type of glia, known as the olfactory ensheathing cells (OECs) (Doucette, 1991, Raisman, 2001 and Ramón-Cueto and Muñoz-Quiles, 2011). These cells reside within the two find more main regions of the olfactory axis: peripherally, in the lamina propria and centrally, along the nerve fiber layer of the olfactory bulb (OB) (Au and Roskams, 2003). The OECs are responsible for maintaining an environment which favors neurite

outgrowth and the creation of new functional synapses

in the central nervous system (Au and Roskams, 2003 and Franssen et al., 2007). Due to their supposed axon regenerative properties, OECs have been extensively studied in animal models of SCI. Although some research has shown locomotor and axonal regeneration improvements, a consensus on the efficacy of this cellular transplantation and mode of action has yet to be reached (Barnett and Riddell, 2007, Boyd et al., 2004, Franssen et al., 2008, Kubasak et al., 2008, Raisman and Li, 2007, Ramón-Cueto and Avila, 1998, Ramón-Cueto et al., 1998, Ramón-Cueto et al., 2000 and Tetzlaff et al., KRX-0401 2011). The source of OECs for transplantation into injured spinal cord is also subject of debate (Richter et al., 2005). However, the use of olfactory lamina propria (OLP) grafts, which is a more accessible source of OECs in humans, could enable a safer approach for autologous transplantation (Bianco et al., 2004, Féron et al., 1998 and Franklin, 2002). The devastating prognosis associated with the social and economic impacts, has led to increased efforts to find therapies that provide functional recovery for people who undergo severe SCI (Blight, 2002 and van den Berg et al., 2010). According to previous studies, the use of OLP transplantation is a promising, though controversial,

repair strategy (Lu et al., 2001, Lu et al., 2002 and Steward Non-specific serine/threonine protein kinase et al., 2006). In the present study we hypothesized that the OECs present in OLP grafts could create a favorable glial environment that would favor neurite and axonal outgrowth after thoracic spinal cord transection in rats. Thus, OLP transplantation could produce higher levels of hindlimb motor recovery when compared to respiratory lamina propria (RLP), which is a graft devoid of OECs. Additionally, we tested the efficacy of OLP transplantation in three different therapeutic windows (acutely, 2 weeks and 4 weeks post-injury), since another key aspect in the translation of this therapy to clinical practice is their potential to produce axonal regeneration even when transplantation is delayed after SCI. Fig.

In contrast to the perceived negative impacts the activities were seen to have on the environment, all activities were seen to be beneficial to visitors, such as leaving the shore happier than when they arrived. All activities were seen to improve visitor mood, with wildlife watching consistently being a more beneficial one. Some activities were also seen to be calming and others more exciting. These findings agree with White et al. (2010) that the aquatic environment is perceived to be beneficial, as, regardless

of the activity performed, Selleckchem Erastin visitors are seen to leave the shore in a happier mood. However, this research supplements past work as it has started to explore the differences between activities. As participants perceived that activities would have different effects

on the individual, it shows that this is an important aspect in need of further investigation. This suggests that a comparative analysis of the different activities taking place in coastal environments is an important addition to research that studies the effects of visits in general (e.g. White et al., 2010) and research that focuses on one particular activity (e.g. walking, Hartig et al., 2003). As well as the perceived psychological benefits on visitors’ mood, these two studies also found that marine awareness is seen to increase with a visit to the shore. Previous literature highlights that experiencing nature is beneficial to people’s awareness in

combination Atezolizumab Sitaxentan with educational sessions (Cummins and Snively, 2000, Duerden and Witt, 2010 and Zeppel and Muloin, 2007). However, even without formal teaching, a general leisurely visit to a rocky shore was perceived to increase visitors’ marine awareness significantly. This is consistent with Steel’s (2005) finding that people who live close to the coast had higher levels of marine awareness as they may have more opportunities to visit the shore. Therefore, regardless of whether visitors seek additional information, a general visit to the shore is seen to be beneficial to the visitor by increasing their marine awareness. Consequently, this may be beneficial for the environment as higher levels of awareness has been associated with more pro-environmental behaviour (Norm Activation Theory, Schwartz, 1977; as cited in Jackson, 2005, Stern and Oskamp, 1987 and Wildlife Trusts, 2005). So, as marine awareness increases, people may feel more personally responsible thus adjusting their behaviours accordingly. This was found in a field study by Alessa and colleagues focussing specifically on a coastal area (Alessa et al., 2003). As well as examining the impacts on the environment and on the visitor independently, a key contribution of this paper was to examine these two components together.

Item 5 (‘How likely is the Checklist to encourage clinicians to pursue further neuropsychiatric work-up or referral to relevant specialists?’) had a median score of 4. Statistical comparison between expert professional and expert parent scores showed no significant differences (see table 4) For qualitative analysis all comments made by the expert professionals and expert parents (n = 69) were

used. Summative analysis revealed 6 key themes (see figure 1). The first theme related to administration, such as where the TAND Checklist should be administered and by whom. The second theme that emerged centered around intellectual ability/disability (ID). Respondents felt it was important

to establish the level of intellectual ability FK228 of a participant at the start of the TAND Checklist as it may influence administration of the remaining questions. Both expert professionals and parents/caregivers suggested including examples that would make it easier for parents to understand specific technical/medical terms such as ‘visuo-spatial skills’. There was a total of 22 comments on missing items where experts suggested the inclusion of additional items. Nine comments find more proposed that the TAND Checklist also be used for other purposes such as research or training. The last theme that emerged, overwhelmingly from the parent group (13 comments), highlighted the need for parents to drive clinical usage of the TAND Checklist. Feedback from Stage 1 was used to revise the TAND Checklist and the revised TAND Checklist was used in stage 2 of the study. The total number of behavioral items (Question 3) on the TAND Checklist showed Nutlin-3 order good internal consistency (α = 0.884). The hyperactivity subdomain items (Question 3n-3q) also generated a high Cronbach alpha (α = 0.751) and the social communication subdomain (Question 3h-3m) showed an acceptable level of internal consistency

(α = 0.682). The four components in the academic domain (Question 6) showed excellent internal consistency (α = 0.954). Both the overall neuropsychological domain items (Question 7) and executive function subdomain items (Question 7b-7e) showed good internal consistency (overall α = 0.783; executive subdomain α = 0.792). Internal consistency of the psycho-social domain (Question 8) was relatively poor (α = 0.365). A total of 20 parents, caregivers or individuals with TSC were recruited for stage 2. The mean age of our TSC population of 20 patients was 14.25 years (range: 3-42 years). The gender ratio was 12:8 male and female. The median scores assigned across the five questions were 5 for items 1, 2 and 5, and 4 for items 3 and 4. Scores on items 1 and 3 ranged between 3-5, item 2 was scored either 4 or 5, and items 4 and 5 had a slightly broader range between 2-5.