Transcriptomics Studies Reveal Functions of Transglutaminase 2 in Breast Cancer Cells Using Membrane Permeable and Impermeable Inhibitors.

Transglutaminase 2 (TG2) performs many functions both under physiological and pathological conditions. In cancer, its expression is associated with aggressiveness, propensity to epithelial-mesenchymal transition, and metastasis. Since TG2 performs key functions both outside and inside the cell, using inhibitors with different membrane permeability we analyzed the changes in the transcriptome induced in two triple-negative cell lines (MDA-MB-436 and MDA-MB-231) with aggressive features. By characterizing pathways and gene networks, we were able to define the effects of TG2 inhibitors (AA9, membrane-permeable, and NCEG2, impermeable) in relation to the roles of the enzyme in the intra- and extracellular space within the context of breast cancer. The deregulated genes revealed p53 and integrin signaling to be the common pathways with some genes showing opposite changes in expression. In MDA-MB-436, AA9 induced apoptosis, modulated cadherin, Wnt, gastrin and cholecystokinin receptors (CCKR) mediated signaling, with RHOB and GNG2 playing significant roles, and affected the Warburg effect by decreasing glycolytic enzymes. In MDA-MB-231 cells, AA9 strongly impacted HIF-mediated hypoxia, including AKT and mTOR pathway. These effects suggest an anti-tumor activity by blocking intracellular TG2 functions. Conversely, the use of NCEG2 stimulated the expression of ATP synthase and proteins involved in DNA replication, indicating a potential promotion of cell proliferation through inhibition of extracellular TG2. To effectively utilize these molecules as an anti-tumor strategy, an appropriate delivery system should be evaluated to target specific functions and avoid adverse effects. Additionally, considering combinations with other pathway modulators is crucial.

Metabolic characterisation of transglutaminase 2 inhibitor effects in breast cancer cell lines.

Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1 H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.

Characterization by Gene Expression Analysis of Two Groups of Dopaminergic Cells Isolated from the Mouse Olfactory Bulb.

The olfactory bulb (OB) is one of two regions of the mammalian brain which undergo continuous neuronal replacement during adulthood. A significant fraction of the cells added in adulthood to the bulbar circuitry is constituted by dopaminergic (DA) neurons. We took advantage of a peculiar property of dopaminergic neurons in transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter: while DA neurons located in the glomerular layer (GL) display full electrophysiological maturation, eGFP+ cells in the mitral layer (ML) show characteristics of immature cells. In addition, they also display a lower fluorescence intensity, possibly reflecting different degrees of maturation. To investigate whether this difference in maturation might be confirmed at the gene expression level, we used a fluorescence-activated cell sorting technique on enzymatically dissociated cells of the OB. The cells were divided into two groups based on their level of fluorescence, possibly corresponding to immature ML cells and fully mature DA neurons from the GL. Semiquantitative real-time PCR was performed to detect the level of expression of genes linked to the degree of maturation of DA neurons. We showed that indeed the cells expressing low eGFP fluorescence are immature neurons. Our method can be further used to explore the differences between these two groups of DA neurons.

Machine Learning Algorithms Highlight tRNA Information Content and Chargaff's Second Parity Rule Score as Important Features in Discriminating Probiotics from Non-Probiotics.

Probiotic bacteria are microorganisms with beneficial effects on human health and are currently used in numerous food supplements. However, no selection process is able to effectively distinguish probiotics from non-probiotic organisms on the basis of their genomic characteristics. In the current study, four Machine Learning algorithms were employed to accurately identify probiotic bacteria based on their DNA characteristics. Although the prediction accuracies of all algorithms were excellent, the Neural Network returned the highest scores in all the evaluation metrics, managing to discriminate probiotics from non-probiotics with an accuracy greater than 90%. Interestingly, our analysis also highlighted the information content of the tRNA sequences as the most important feature in distinguishing the two groups of organisms probably because tRNAs have regulatory functions and might have allowed probiotics to evolve faster in the human gut environment. Through the methodology presented here, it was also possible to identify seven promising new probiotics that have a higher information content in their tRNA sequences compared to non-probiotics. In conclusion, we prove for the first time that Machine Learning methods can discriminate human probiotic from non-probiotic organisms underlining information within tRNA sequences as the most important genomic feature in distinguishing them.

Dysregulation of Transglutaminase type 2 through GATA3 defines aggressiveness and Doxorubicin sensitivity in breast cancer.

The role of transglutaminase type 2 in cell physiology is related to protein transamidation and signal transduction (affecting extracellular, intracellular and nuclear processes) aided by the expression of truncated isoforms and of two lncRNAs with regulatory functions. In breast cancer TG2 is associated with disease progression supporting motility, epithelial-mesenchymal transition, invasion and drug resistance. The aim of his work is to clarify these issues by emphasizing the interconnections among TGM2 variants and transcription factors associated with an aggressive phenotype, in which the truncated TGH isoform correlates with malignancy. TGM2 transcripts are upregulated by several drugs in MCF-7, but only Doxorubicin is effective in MDA-MB-231 cells. These differences reflect the expression of GATA3, as demonstrated by silencing, suggesting a link between this transcription factor and gene dysregulation. Of note, NC9, an irreversible inhibitor of enzymatic TG2 activities, emerges to control NF-ĸB and apoptosis in breast cancer cell lines, showing potential for combination therapies with Doxorubicin.

A Multidisciplinary Approach Establishes a Link between Transglutaminase 2 and the Kv10.1 Voltage-Dependent K+ Channel in Breast Cancer.

Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.

The Motility and Mesenchymal Features of Breast Cancer Cells Correlate with the Levels and Intracellular Localization of Transglutaminase Type 2.

We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial-mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude to TG2-dependent gene expression modifications. Since enzyme activity can affect both motility and nuclear functions, targeting of this protein could represent a method to improve therapeutic interventions in breast tumors, particularly those to control progression and to limit drug resistance.

Inhibition of the lncRNA Coded within Transglutaminase 2 Gene Impacts Several Relevant Networks in MCF-7 Breast Cancer Cells.

Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular processes and are related to the occurrence of many diseases, including cancer. In this regard, we recently discovered a polyadenylated long non-coding RNA (named TG2-lncRNA) encoded within the first intron of the Transglutaminase type 2 gene (TGM2), which is related to tumour proliferation in human cancer cell lines. To better characterize this new biological player, we investigated the effects of its suppression in MCF-7 breast cancer cells, using siRNA treatment and RNA-sequencing. In this way, we found modifications in several networks associated to biological functions relevant for tumorigenesis (apoptosis, chronic inflammation, angiogenesis, immunomodulation, cell mobility, and epithelial-mesenchymal transition) that were originally attributed only to Transglutaminase type 2 protein but that could be regulated also by TG2-lncRNA. Moreover, our experiments strongly suggest the ability of TG2-lncRNA to directly interact with important transcription factors, such as RXRα and TP53, paving the way for several regulatory loops that can potentially influence the phenotypic behaviour of MCF-7 cells. These considerations imply the need to further investigate the relative relevance of the TG2 protein itself and/or other gene products as key regulators in the organization of breast cancer program.

Involvement of non-coding RNAs and transcription factors in the induction of Transglutaminase isoforms by ATRA.

The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3'UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.

Testing a Combination of Markers of Systemic Redox Status as a Possible Tool for the Diagnosis of Late Onset Alzheimer's Disease.

BACKGROUND: Blood-based parameters reflecting systemic abnormalities associated with typical brain physiopathological hallmarks could be a satisfactory answer to the need of less costly/intrusive and widely available biomarkers for late onset Alzheimer's disease (LOAD). Cumulating evidence from ourselves and others suggests that systemic oxidative stress (OxS) is precociously associated with LOAD. On this basis, we aimed to identify a combination of markers of redox status that could aid the diagnosis of LOAD. METHODS: We reexamined and crossed previous data on 9 serum markers of OxS obtained in a cohort including n = 84 controls and n = 90 LOAD patients by multivariate logistic regression analyses. RESULTS: A multimarker panel was identified that included significantly increased (hydroperoxides and uric acid) and decreased (thiols, residual antioxidant power, and arylesterase activity) markers. The multivariate model yielded an area under receiver-operating characteristic curve (AUC) of 0.808 for the discrimination between controls and LOAD patients, with specificity and sensitivity of 64% and 79%, respectively. CONCLUSIONS: This study identified a panel of serum markers that distinguish individuals with LOAD from cognitively healthy control subjects. Replication studies on a larger independent cohort are required to confirm and extend our data.

Spotlight on the transglutaminase 2 gene: a focus on genomic and transcriptional aspects.

The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several intra- and extracellular activities depending on its location (protein modification, modulation of gene expression, membrane signalling and stabilization of cellular interactions with the extracellular matrix) in relation to cell death, survival and differentiation. In contrast with the appreciable knowledge about the regulation of the enzymatic activities, much less is known concerning its inducible expression, which is altered in inflammatory and neoplastic diseases. In this context, we first summarize the gene's basic features including single-nucleotide polymorphism characterization, epigenetic DNA methylation and identification of regulatory regions and of transcription factor-binding sites at the gene promoter, which could concur to direct gene expression. Further aspects related to alternative splicing events and to ncRNAs (microRNAs and lncRNAs) are involved in the modulation of its expression. Notably, this important gene displays transcriptional variants relevant for the protein's function with the occurrence of at least seven transcripts which support the synthesis of five isoforms with modified catalytic activities. The different expression of the TG2 (type 2 transglutaminase) variants might be useful for dictating the multiple biological features of the protein and their alterations in pathology, as well as from a therapeutic perspective.

A long non-coding RNA inside the type 2 transglutaminase gene tightly correlates with the expression of its transcriptional variants.

The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of gene expression. In the case of the transglutaminase 2 gene (TGM2) the databases of genome sequence indicate location of a lncRNA (LOC107987281) within the first intron. This lncRNA is 1000 bp long, arises from 2 exons and starts few nucleotides 3' of the first splicing site of translated TGM2. We have analysed correlations between expression of LOC107987281 lncRNA and TGM2 mRNA by real-time PCR in K562 cell line untreated or treated with the anticancer drugs TPA (12-O-tetradecanoylphorbol-13-acetate), Docetaxel and Doxorubicin. In the treated cells the lncRNA increase follows the trend of TGM2 transcript. To validate this finding we used HumanExon1_0ST Affymetrix; chip data were background-adjusted, quantile-normalized and summarized using robust multi-array average analysis implemented in the R package. The probesets recognize sequences inside each exon, near intronic splicing sites and others located in the untranslated regions of TGM2 gene. The analysis of total RNA samples in GEO datasets from K562, HL-60, THP-1 and U937 cell lines, untreated or treated with TPA in replicated experiments confirmed our earlier results. These demonstrate correlation between LOC107987281 and TGM2 mRNA in the cell lines (K562, HL60 and THP-1) where increased levels of TGM2 mRNA are produced. Additional array study on 358 samples of several normal and paired tumor tissues leads to the same conclusions, indicating a correlation between full-length TGM2 mRNA and LOC107987281 lncRNA in relation to the development of several tumors.

Association between circulatory levels of adipokines and bone mineral density in postmenopausal women.

OBJECTIVE: Epidemiological evidence indicates that excess fat may be beneficial for bone health, offering protective effects against the onset of postmenopausal osteoporosis. Experimental data suggest that this link might be due to the direct effect of adipokines on bone tissue. Confirmatory evidence of this association, however, remains limited. METHODS: The levels of a panel of selected adipokines including interleukin (IL)-6, -8, -1ß, adipsin, lipocalin-2/neutrophil gelatinase-associated ipocalin, tumor necrosis factor alpha, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, hepatocyte growth factor, resistin, leptin, and adiponectin in a group of osteopenic and osteoporotic postmenopausal women were compared with those of unaffected women (n = 127). RESULTS: Univariate analysis revealed that leptin and adiponectin were significantly correlated with bone mineral density (BMD). In particular, leptin was positively associated with BMD of the spine (r = 0.22, P < 0.05), femoral neck (r = 0.23, P < 0.05), trochanter (r = 0.20, P < 0.05), and total hip (r = 0.27, P < 0.01), whereas adiponectin was inversely correlated with BMD at the trochanter (r = -0.21, P < 0.05). No correlations were, however, significant after adjusting for body fat variables. Stratification of the sample according to IL-6 levels revealed that adiponectin remained significantly inversely associated with BMD, regardless of fat levels and age (ß=-0.29, P < 0.05; r = 0.198) in the subgroup of participants with low levels of IL-6. CONCLUSIONS: Our data suggest that circulating adiponectin is inversely associated with markers of bone health in postmenopausal women, and that the interaction is influenced by IL-6 levels.

Oxidative damage and the pathogenesis of menopause related disturbances and diseases.

The postmenopausal phase of life is frequently associated in women with subjective symptoms (e.g. vasomotor) and real diseases (atherosclerosis with coronary ischemia, osteoporosis, Alzheimer-type neurodegeneration, urogenital dystrophy), which together determine the post-menopausal syndrome. Observations that oxidative damage by reactive oxygen/nitrogen species in experimental models can contribute to the pathogenesis of these disturbances stimulated research on the relationships between menopause, its endocrine deficiency, oxidative balance and the "wellness" in postmenopausal life. The connection among these events is probably due to the loss of protective actions exerted by estrogens during the fertile life. Most recent studies have revealed that estrogens exert an antioxidant action not by direct chemical neutralization of reactants as it was expected until recently but by modulating the expression of antioxidant enzymes that control levels of biological reducing agents. Also nutritional antioxidants apparently act by a similar mechanism. From this perspective it is conceivable that a cumulative control of body oxidant challenges and biological defenses could help in monitoring between "normal" and "pathological" menopause. However, as clinical studies failed to confirm this scenario in vivo, we have decided to review the existing literature to understand the causes of this discrepancy and whether this was due to methodologic reasons or to real failure of the basic hypothesis.

Higher Urinary Levels of 8-Hydroxy-2'-deoxyguanosine Are Associated with a Worse RANKL/OPG Ratio in Postmenopausal Women with Osteopenia.

Postmenopausal osteoporosis (PO) is a major public health issue which affects a large fraction of elderly women. Emerging in vitro evidence suggests a central role of oxidative stress (OxS) in postmenopausal osteoporosis (PO) development. Contrariwise, the human studies on this topic are still scarce and inconclusive. In the attempt to address this issue, we sought to determine if OxS, as assessed by 8-hydroxy-2-deoxyguanosine (8-OHdG), may influence the level of receptor activator of nuclear factor-κb ligand (RANKL)/osteoprotegerin (OPG) ratio (a central regulator of bone metabolism) in a sample (n = 124), including postmenopausal women with osteoporosis, osteopenia and normal bone mass density (BMD). The most striking result that emerged in our study was the independent and positive (beta = 0.449, p = 0.004, and R(2) = 0.185) association between the OxS marker and RANKL/OPG ratio which was found in osteopenic but not in the other 2 sample groups. If confirmed by longitudinal studies, our findings would suggest that OxS is implicated in the derangement of bone homeostasis which precedes PO development. In line with these considerations, antioxidant treatment of postmenopausal women with moderately low BMD might contribute to preventing PO and related complications.

Oxidative stress and menopause-related hot flashes may be independent events.

OBJECTIVE: At present, there is growing demand for alternative, or additional, treatments to hormone replacement therapy for menopause-related hot flashes (HF). Antioxidant supplements have been recently proposed as possible candidates for this purpose, regardless of the absence of clear evidence in support of a link between these vasomotor symptoms and oxidative stress (OxS). The aim of our study was to evaluate the association between HF and OxS serum markers in a large sample of middle-aged women. MATERIALS AND METHODS: We conducted a cross-sectional study on 245 perimenopausal and early postmenopausal women (age 45-60 years). The variables examined were presence of self-reported HF and levels of 8-iso-prostaglandin F2α, 8-OH-deoxy-2'-guanosine, advanced oxidation protein products, total antioxidant power, uric acid, thiols, and paroxonase-1. RESULTS: Seventy-six women (31%) reported to suffer from HF (either medium or high intensity). None of the peripheral markers of OxS examined was found to be significantly associated with the presence of HF. CONCLUSION: Taken together, our data suggest that systemic OxS might not be implicated with the onset of the climacteric vasomotor symptoms that most commonly affect women experiencing perimenopause and early postmenopause.

Serum levels of hydroperoxides and multimorbidity among older patients with mild cognitive impairment or late-onset Alzheimer's disease.

BACKGROUND: Oxidative stress (OxS) might be involved in the pathogenesis of late-onset Alzheimer disease (LOAD); noteworthy, the presence of multimorbidity is regarded as a common denominator of OxS and dementia. AIM: To evaluate the contribution of multimorbidity to OxS in LOAD and mild cognitive impairment (MCI). METHODS: Serum hydroperoxides and multimorbidity (CIRS-CI scale) were evaluated in 46 Controls, 104 MCI and 75 LOAD. RESULTS: A trend toward an increase of hydroperoxides from Controls to MCI to LOAD was observed (LOAD vs Controls p = 0.01). This OxS marker was positively and significantly correlated with CIRS-CI in Controls (p = 0.002) and patients with MCI (p = 0.005) but not in those with LOAD (p = 0.104). CONCLUSIONS: Multimorbidity is associated with systemic OxS but only in elderly people with either no or mild cognitive impairment. Although OxS is elevated in LOAD patients, its association with multimorbidity seems to be negligible, confirming the existence of strong disease-specific pro-oxidant mechanisms.

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids in vitro.

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.

PON-1 and ferroxidase activities in older patients with mild cognitive impairment, late onset Alzheimer's disease or vascular dementia.

BACKGROUND: A large body of evidence suggests that not only cerebral but also systemic oxidative stress (OxS) might be involved in the pathogenesis of late onset Alzheimer's disease (LOAD) and vascular dementia (VAD), as well as of the prodromal phase of dementia, the so-called mild cognitive impairment (MCI). In the present study, we evaluated whether paraoxonase 1 (PON-1) and ferroxidase (FeOx) activities, because of their well acknowledged effectiveness as systemic antioxidants, might be associated with dementia and/or MCI. METHODS: Serum arylesterase and paraoxonase of PON-1, along with FeOx I (ceruloplasmin-related) and II activities were assessed in 223 MCI, 162 LOAD, 65 VAD patients, and in 143 older normal cognitive controls. RESULTS: Among the enzymatic activities examined, only arylesterase significantly changed across the groups (ANOVA: p<0.001), with similar lower levels in MCI, LOAD, and VAD compared to controls. By multivariate logistic regression analysis we showed that, in respect to controls, low levels (under the median value) of serum arylesterase were independently associated with an increase in the likelihood of being affected by LOAD [odds ratio (OR) 2.8, 95% confidence interval (CI) 1.5-5.0], VAD (OR 2.7, 95% CI 1.2-6.2), or MCI (OR 2.3, 95% CI 1.3-3.8). CONCLUSIONS: Overall, our results suggest that depression of PON-1, and in particular, of arylesterase activity, in serum might be an early feature of dementia-related diseases. Further longitudinal exploration of the role of this enzyme in the onset and progression of these disorders are required.