A System-wide Approach to Monitor Responses to Synergistic BRAF and EGFR Inhibition in Colorectal Cancer Cells.

Intrinsic and/or acquired resistance represents one of the great challenges in targeted cancer therapy. A deeper understanding of the molecular biology of cancer has resulted in more efficient strategies, where one or multiple drugs are adopted in novel therapies to tackle resistance. This beneficial effect of using combination treatments has also been observed in colorectal cancer patients harboring the BRAF(V600E) mutation, whereby dual inhibition of BRAF(V600E) and EGFR increases antitumor activity. Notwithstanding this success, it is not clear whether this combination treatment is the only or most effective treatment to block intrinsic resistance to BRAF inhibitors. Here, we investigate molecular responses upon single and multi-target treatments, over time, using BRAF(V600E) mutant colorectal cancer cells as a model system. Through integration of transcriptomic, proteomic and phosphoproteomics data we obtain a comprehensive overview, revealing both known and novel responses. We primarily observe widespread up-regulation of receptor tyrosine kinases and metabolic pathways upon BRAF inhibition. These findings point to mechanisms by which the drug-treated cells switch energy sources and enter a quiescent-like state as a defensive response, while additionally compensating for the MAPK pathway inhibition.

PaDuA: A Python Library for High-Throughput (Phospho)proteomics Data Analysis.

The increased speed and sensitivity in mass spectrometry-based proteomics has encouraged its use in biomedical research in recent years. Large-scale detection of proteins in cells, tissues, and whole organisms yields highly complex quantitative data, the analysis of which poses significant challenges. Standardized proteomic workflows are necessary to ensure automated, sharable, and reproducible proteomics analysis. Likewise, standardized data processing workflows are also essential for the overall reproducibility of results. To this purpose, we developed PaDuA, a Python package optimized for the processing and analysis of (phospho)proteomics data. PaDuA provides a collection of tools that can be used to build scripted workflows within Jupyter Notebooks to facilitate bioinformatics analysis by both end-users and developers.

Rearrangement of the Protein Phosphatase 1 Interactome During Heart Failure Progression.

BACKGROUND: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors. METHODS: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%-40%), and severe HF (ejection fraction <30%). Cardiac lysates were subjected to affinity purification with anti-PP1c antibodies followed by high-resolution mass spectrometry. PP1 regulatory subunit 7 (Ppp1r7) was knocked down in mouse cardiomyocytes and HeLa cells with adeno-associated virus serotype 9 and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes. RESULTS: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum. CONCLUSIONS: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.

Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.

Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501), as well as subdominant responses through common class I alleles (e.g. B7 and C*0304). Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.

Robust, Sensitive, and Automated Phosphopeptide Enrichment Optimized for Low Sample Amounts Applied to Primary Hippocampal Neurons.

Because of the low stoichiometry of protein phosphorylation, targeted enrichment prior to LC-MS/MS analysis is still essential. The trend in phosphoproteome analysis is shifting toward an increasing number of biological replicates per experiment, ideally starting from very low sample amounts, placing new demands on enrichment protocols to make them less labor-intensive, more sensitive, and less prone to variability. Here we assessed an automated enrichment protocol using Fe(III)-IMAC cartridges on an AssayMAP Bravo platform to meet these demands. The automated Fe(III)-IMAC-based enrichment workflow proved to be more effective when compared to a TiO2-based enrichment using the same platform and a manual Ti(IV)-IMAC-based enrichment workflow. As initial samples, a dilution series of both human HeLa cell and primary rat hippocampal neuron lysates was used, going down to 0.1 µg of peptide starting material. The optimized workflow proved to be efficient, sensitive, and reproducible, identifying, localizing, and quantifying thousands of phosphosites from just micrograms of starting material. To further test the automated workflow in genuine biological applications, we monitored EGF-induced signaling in hippocampal neurons, starting with only 200 000 primary cells, resulting in ∼50 µg of protein material. This revealed a comprehensive phosphoproteome, showing regulation of multiple members of the MAPK pathway and reduced phosphorylation status of two glutamate receptors involved in synaptic plasticity.

Profound Understanding of Effect of Transition Metal Dopant, Sintering Temperature, and pO2 on the Electrical and Optical Properties of Proton Conducting BaCe0.9Sm0.1O3-δ.

This study reports the effect of transition metal (TM) substitution on the electrical and optical properties of BaCe0.9Sm0.1O3-δ (BCS). Concentrations of 5-10 mol % of each of Fe and Co have been doped for the B-site of BCS by citric acid autocombustion method. Powder X-ray diffraction has revealed the formation of an orthorhombic perovskite-type structure. FTIR confirmed a distortion in the lattice upon TM-doping in BCS. Scanning electron microscopy (SEM) images of 1400 °C sintered samples have manifested a higher densification in BaCe0.8Sm0.1Co0.1O3-δ (BCSC10) with a grain size ∼11 µm compared to the parent compound BCS (∼2 µm). Thermogravimetric (TG) analysis showed a water uptake in case of BaCe0.85Sm0.1Co0.05O3-δ (BCSC5), while BaCe0.85Sm0.1Fe0.05O3-δ (BCSF5) did not show a noteworthy uptake of water. TG has also proved that the incorporation of Fe and Co in BCS did not improve the chemical stability in CO2 at elevated temperature. The band gap estimated using Kubelka-Munk model based on the diffuse reflectance data was found to be the lowest for BCSC5 (2.47 eV). However, it increases upon lowering oxygen partial pressure (pO2), which was interpreted by a band structure modifications. Among the samples investigated, BCSC10 sintered at 1400 °C showed the highest electrical conductivity of 0.02 S cm(-1) in air at 600 °C, while its proton mobility appears to be negligible under the investigated humidity atmosphere.

Pathomx: an interactive workflow-based tool for the analysis of metabolomic data.

BACKGROUND: Metabolomics is a systems approach to the analysis of cellular processes through small-molecule metabolite profiling. Standardisation of sample handling and acquisition approaches has contributed to reproducibility. However, the development of robust methods for the analysis of metabolomic data is a work-in-progress. The tools that do exist are often not well integrated, requiring manual data handling and custom scripting on a case-by-case basis. Furthermore, existing tools often require experience with programming environments such as MATLAB® or R to use, limiting accessibility. Here we present Pathomx, a workflow-based tool for the processing, analysis and visualisation of metabolomic and associated data in an intuitive and extensible environment. RESULTS: The core application provides a workflow editor, IPython kernel and a HumanCyc™-derived database of metabolites, proteins and genes. Toolkits provide reusable tools that may be linked together to create complex workflows. Pathomx is released with a base set of plugins for the import, processing and visualisation of data. The IPython backend provides integration with existing platforms including MATLAB® and R, allowing data to be seamlessly transferred. Pathomx is supplied with a series of demonstration workflows and datasets. To demonstrate the use of the software we here present an analysis of 1D and 2D (1)H NMR metabolomic data from a model system of mammalian cell growth under hypoxic conditions. CONCLUSIONS: Pathomx is a useful addition to the analysis toolbox. The intuitive interface lowers the barrier to entry for non-experts, while scriptable tools and integration with existing tools supports complex analysis. We welcome contributions from the community.

Metabolic profiling predicts response to anti-tumor necrosis factor α therapy in patients with rheumatoid arthritis.

OBJECTIVE: Anti-tumor necrosis factor (anti-TNF) therapies are highly effective in rheumatoid arthritis (RA) and psoriatic arthritis (PsA), but a significant number of patients exhibit only a partial or no therapeutic response. Inflammation alters local and systemic metabolism, and TNF plays a role in this. We undertook this study to determine if the patient's metabolic fingerprint prior to therapy could predict responses to anti-TNF agents. METHODS: Urine was collected from 16 RA patients and 20 PsA patients before and during therapy with infliximab or etanercept. Urine metabolic profiles were assessed using nuclear magnetic resonance spectroscopy. Discriminating metabolites were identified, and the relationship between metabolic profiles and clinical outcomes was assessed. RESULTS: Baseline urine metabolic profiles discriminated between RA patients who did or did not have a good response to anti-TNF therapy according to European League Against Rheumatism criteria, with a sensitivity of 88.9% and a specificity of 85.7%, with several metabolites contributing (in particular histamine, glutamine, xanthurenic acid, and ethanolamine). There was a correlation between baseline metabolic profiles and the magnitude of change in the Disease Activity Score in 28 joints from baseline to 12 months in RA patients (P = 0.04). In both RA and PsA, urinary metabolic profiles changed between baseline and 12 weeks of anti-TNF therapy. Within the responders, urinary metabolite changes distinguished between etanercept and infliximab treatment. CONCLUSION: The clear relationship between urine metabolic profiles of RA patients at baseline and their response to anti-TNF therapy may allow development of novel approaches to the optimization of therapy. Differences in metabolic profiles during treatment with infliximab and etanercept in RA and PsA may reflect distinct mechanisms of action.

Tumour kinome re-wiring governs resistance to palbociclib in oestrogen receptor positive breast cancers, highlighting new therapeutic modalities.

Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.

Changes in urinary metabolomic profile during relapsing renal vasculitis.

Current biomarkers of renal disease in systemic vasculitis lack predictive value and are insensitive to early damage. To identify novel biomarkers of renal vasculitis flare, we analysed the longitudinal urinary metabolomic profile of a rat model of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Wistar-Kyoto (WKY) rats were immunised with human myeloperoxidase (MPO). Urine was obtained at regular intervals for 181 days, after which relapse was induced by re-challenge with MPO. Urinary metabolites were assessed in an unbiased fashion using nuclear magnetic resonance (NMR) spectroscopy, and analysed using partial least squares discriminant analysis (PLS-DA) and partial least squares regression (PLS-R). At 56 days post-immunisation, we found that rats with vasculitis had a significantly different urinary metabolite profile than control animals; the observed PLS-DA clusters dissipated between 56 and 181 days, and re-emerged with relapse. The metabolites most altered in rats with active or relapsing vasculitis were trimethylamine N-oxide (TMAO), citrate and 2-oxoglutarate. Myo-inositol was also moderately predictive. The key urine metabolites identified in rats were confirmed in a large cohort of patients using liquid chromatography-mass spectrometry (LC-MS). Hypocitraturia and elevated urinary myo-inositol remained associated with active disease, with the urine myo-inositol:citrate ratio being tightly correlated with active renal vasculitis.

Evidence of detrimental effects of environmental contaminants on growth and reproductive physiology of white sturgeon in impounded areas of the Columbia River.

This study sought to determine whether wild white sturgeon from the Columbia River (Oregon) were exhibiting signs of reproductive endocrine disruption. Fish were sampled in the free-flowing portion of the river (where the population is experiencing reproductive success) and from three reservoirs behind hydroelectric dams (where fish have reduced reproductive success). All of the 18 pesticides and almost all of the 28 polychlorinated biphenyls (PCBs) that were analyzed in livers and gonads were detected in at least some of the tissue samples. Metabolites of p,p -dichlorodiphenyltrichloroethane (DDT) [p,p -dichlorodiphenyldichloroethylene (DDE) and p,p -1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD)]were consistently found at relatively high levels in fish. Some males and immature females showed elevated plasma vitellogenin; however, concentrations were not correlated with any of the pesticides or PCBs analyzed. Negative correlations were found between a number of physiologic parameters and tissue burdens of toxicants. Plasma triglycerides and condition factor were negatively correlated with total DDT (DDD + DDE + DDT), total pesticides (all pesticides detected - total DDT), and PCBs. In males, plasma androgens and gonad size were negatively correlated with total DDT, total pesticides, and PCBs. Fish residing in the reservoir behind the oldest dam had the highest contaminant loads and incidence of gonadal abnormalities, and the lowest triglycerides, condition factor, gonad size, and plasma androgens. These data suggest that endocrine-disrupting chemicals may be accumulating behind dams over time. Overall, results of this study indicate that exposure to environmental contaminants may be affecting both growth and reproductive physiology of sturgeon in some areas of the Columbia River.

Short-term exposure of Chinook salmon (Oncoryhnchus tshawytscha) to o,p-DDE or DMSO during early life-history stages causes long-term humoral immunosuppression.

We evaluated the effect of short-term exposures to a xenobiotic chemical during early life-history stages on the long-term immune competence of chinook salmon (Oncoryhnchus tshawytscha). Immersion of chinook salmon eggs in a nominal concentration of o,p-dichlorodiphenyldichloroethylene (o,p-DDE; 10 ppm) for 1 hr at fertilization followed by immersion in the same dose for 2 hr at hatch resulted in a significant reduction in the ability of splenic leukocytes from fish 1 year after treatment to undergo blastogenesis upon in vitro stimulation with lipopolysaccharide. We also observed that the vehicle, dimethyl sulfoxide (DMSO), caused a significant reduction in the ability of the splenic leukocytes to express surface immunoglobin M (SIgM) at this time. The concentration of o,p-DDE in a pooled sample of whole fry from this treatment was 0.53 microg/g lipid 1 month after first feeding but was undetectable in all other treatments. Mortality rate, time to hatch, fish length, and weight were unaffected by treatment with o,p-DDE. Similarly, sex ratios, gonadal development, and concentrations of plasma estradiol and 11-ketotestosterone were not affected by the treatment. In addition, we found no evidence that plasma lysozyme concentrations or the mitogenic responses of splenic leukocytes to concanavalin A or polyinosinic-polycytidylic acid were influenced by the treatment. In this experiment, a brief period of exposure to o,p-DDE or DMSO during early development was able to induce long-term effects on humoral immune competence of chinook salmon. Such immunosuppression may increase susceptibility to disease, which may in turn be critical to regulating the population.

Metabolomics--a novel window into inflammatory disease.

Inflammation is an important component of normal responses to infection and injury. However, chronic activation of the immune system, due to aberrant responses to normal stimuli, can lead to the establishment of a persistent inflammatory state. Such inflammatory conditions are often debilitating, and are associated with a number of important co-morbidities including cardiovascular disease. Resting non-proliferative tissues have distinctive metabolic activities and requirements, which differ considerably from those in infiltrating immune cells, which are undergoing proliferation and differentiation. Immune responses in tissues may therefore be modulated by the relative abundance of substrates in the inflamed site. In turn immune cell activity can feed back and affect metabolic behaviour of the tissues, as most clearly demonstrated in cachexia - the loss of cellular mass driven by tumour necrosis factor-alpha (TNF-α) a key mediator of the inflammatory response. Here we discuss the potential for metabolomic analysis to clarify the interactions between inflammation and metabolic changes underlying many diseases. We suggest that an increased understanding of the interaction between inflammation and cellular metabolism, energy substrate use, tissue breakdown markers, the microbiome and drug metabolites, may provide novel insight into the regulation of inflammatory diseases.

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry.

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM). Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.

Ovarian steroidogenesis in white sturgeon (Acipenser transmontanus) during oocyte maturation and induced ovulation.

Ovarian follicles and plasma were collected from two female white sturgeon, Acipenser transmontanus, injected with sturgeon pituitary homogenate followed 12h later with GnRHa to induce ovulation. The oocytes of one female underwent germinal vesicle breakdown (GVBD) but ovulation did not occur in response to hormonal stimulation (Female 1), while the oocytes of the other female underwent GVBD and ovulation (Female 2). Follicles collected 12h after the first injection to induce ovulation were incubated with radioinert pregnenolone (P5) or tritiated-P5 ([3H]P5) plus radioinert P5. Steroids were extracted from media and intact follicles, and the extracts were fractionated by high performance liquid chromatography (HPLC). Fractions from the incubation with radioinert precursor were used in a bioassay to determine the potency of the steroid products to induce GVBD. Plasma levels of testosterone (T), estradiol, and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were measured by radioimmunoassay during induced ovulation, and plasma collected at the time of ovulation (actual or expected) was analyzed by HPLC. A peak in plasma 17,20beta-P was detected at the time of the second injection to induce ovulation in Female 2 (the time at which follicles were collected for incubation with [3H]P5). The HPLC analysis revealed several progestins in the plasma of Female 2 at ovulation that were not present in Female 1 at the time of expected ovulation. A variety of C19 and C21 steroids were produced in vitro by ovarian follicles from both females. The "suggestive" identities of the major metabolites were 11-deoxycortisol, androstenedione, 17-hydroxyprogesterone (17OHP), and 17,20beta-P in Female 1 and cortisol, 17,20beta, 21-trihydroxyprogesterone (20beta-S), 11-deoxycortisol, T, 17OHP, and 17,20beta-P in Female 2. Several of the steroids were active in a GVBD bioassay, but the fractions that contained the steroid coeluting the authentic 11-deoxycortisol on the HPLC and 17,20beta-P (positively identified by gas chromatography-mass spectrometry) were found to be the most potent. The results from this study combined with the results of Webb et al. (2001b) suggest the potential roles of 11-deoxycortisol, 17,20beta-P, 20beta-S, and P4 as maturation-inducing steroids in sturgeon.

Environmental health services in Europe 6: the development of professional associations

In the European Region, there is a diverse range of professionals engaged in promoting environmental health issues for the benefit of the public’s health. They work in state authorities, local government, nongovernmental organizations and the private sector. All have a common purpose and very often have common or complementary knowledge and skills. In some European countries, environmental health professionals have organized themselves into associations, but in the majority such associations are not well established and have not realized their full potential in civil society. This publication strives to bring together the collective experience of a range of existing associations of environmental health, while also providing the basic information that will be of particular value to an emerging association or to groups of professionals aspiring to develop such associations. The book attempts to provide a means by which groups of environmental health professionals can formulate their own template for developing associations that clearly represent their particular interests and ethos, within a framework whereby they can find common purpose with other professionals at national and international level.

Environmental health services in Europe 3: professional profiles

Since the adoption of the European Charter on Environment and Health in Frankfurt-am-Main in 1989, the countries of the WHO European Region have been placing greater emphasis on the role of their environmental health services in the protection of public health and the environment. The introduction of the Environmental Health Action Plan for Europe, in Helsinki in 1994, further enhanced the need for strong national and local environmental health services. The aim of this book is to provide guidance on key issues relating specifically to staffing that should be considered in the reform of environmental health services in the WHO European Region. The development of staffing policy for these services must be considered within the wider context of a country’s political, social and economic situation, as well as national environmental health needs. This book is intended to assist Member States in adapting the general principles of environmental health to addressing these political, social and economic needs.