Lactate modulates microglia polarization via IGFBP6 expression and remodels tumor microenvironment in glioblastoma.

Lactic acidosis has been reported in solid tumor microenvironment (TME) including glioblastoma (GBM). In TME, several signaling molecules, growth factors and metabolites have been identified to induce resistance to chemotherapy and to sustain immune escape. In the early phases of the disease, microglia infiltrates TME, contributing to tumorigenesis rather than counteracting its growth. Insulin-like Growth Factor Binding Protein 6 (IGFBP6) is expressed during tumor development, and it is involved in migration, immune-escape and inflammation, thus providing an attractive target for GBM therapy. Here, we aimed at investigating the crosstalk between lactate metabolism and IGFBP6 in TME and GBM progression. Our results show that microglia exposed to lactate or IGFBP6 significantly increased the Monocarboxylate transporter 1 (MCT1) expression together with genes involved in mitochondrial metabolism. We, also, observed an increase in the M2 markers and a reduction of inducible nitric oxide synthase (iNOS) levels, suggesting a role of lactate/IGFBP6 metabolism in immune-escape activation. GBM cells exposed to lactate also showed increased levels of IGFBP6 and vice-versa. Such a phenomenon was coupled with a IGFBP6-mediated sonic hedgehog (SHH) ignaling increase. We, finally, tested our hypothesis in a GBM zebrafish animal model, where we observed an increase in microglia cells and igfbp6 gene expression after lactate exposure. Our results were confirmed by the analysis of human transcriptomes datasets and immunohistochemical assay from human GBM biopsies, suggesting the existence of a lactate/IGFBP6 crosstalk in microglial cells, so that IGFBP6 expression is regulated by lactate production in GBM cells and in turn modulates microglia polarization.

Anaplastic thyroid cancer cells reduce CD71 levels to increase iron overload tolerance.

BACKGROUND: Follicular thyroid cancer (FTC) is a prevalent form of differentiated thyroid cancer, whereas anaplastic thyroid cancer (ATC) represents a rare, fast-growing, undifferentiated, and highly aggressive tumor, posing significant challenges for eradication. Ferroptosis, an iron-dependent cell death mechanism driven by the excessive production of reactive oxygen species and subsequent lipid peroxidation, emerges as a promising therapeutic strategy for cancer. It has been observed that many cancer cells exhibit sensitivity to ferroptosis, while some other histotypes appear to be resistant, by counteracting the metabolic changes and oxidative stress induced by iron overload. METHODS: Here we used human biopsies and in vitro approaches to analyse the effects of iron-dependent cell death. We assessed cell proliferation and viability through MTT turnover, clonogenic assays, and cytofluorimetric-assisted analysis. Lipid peroxidation assay and western blot were used to analyse molecular mechanisms underlying ferroptosis modulation. Two distinct thyroid cancer cell lines, FTC-133 (follicular) and 8505C (anaplastic), were utilized. These cell lines were exposed to ferroptosis inducers, Erastin and RSL3, while simulating an iron overload condition using ferric ammonium citrate. RESULTS: Our evidence suggests that FTC-133 cell line, exposed to iron overload, reduced their viability and showed increased ferroptosis. In contrast, the 8505C cell line seems to better tolerate ferroptosis, responding by modulating CD71, which is involved in iron internalization and seems to have a role in resistance to iron overload and consequently in maintaining cell viability. CONCLUSIONS: The differential tolerance to ferroptosis observed in our study may hold clinical implications, particularly in addressing the unmet therapeutic needs associated with ATC treatment, where resistance to ferroptosis appears more pronounced compared to FTC.

IGFBP-6: At the Crossroads of Immunity, Tissue Repair and Fibrosis.

Insulin-like growth factors binding protein-6 (IGFBP-6) is involved in a relevant number of cellular activities and represents an important factor in the immune response, particularly in human dendritic cells (DCs). Over the past several years, significant insights into the IGF-independent effects of IGFBP-6 were discovered, such as the induction of chemotaxis, capacity to increase oxidative burst and neutrophils degranulation, ability to induce metabolic changes in DCs, and, more recently, the regulation of the Sonic Hedgehog (SHH) signaling pathway during fibrosis. IGFBP-6 has been implicated in different human diseases, and it plays a rather controversial role in the biology of tumors. Notably, well established relationships between immunity, stroma activity, and fibrosis are prognostic and predictive of response to cancer immunotherapy. This review aims at describing the current understanding of mechanisms that link IGFBP-6 and fibrosis development and at highlighting the multiple roles of IGFBP-6 to provide an insight into evolutionarily conserved mechanisms that can be relevant for inflammation, tumor immunity, and immunological diseases.

Lactate Rewrites the Metabolic Reprogramming of Uveal Melanoma Cells and Induces Quiescence Phenotype.

Uveal melanoma (UM), the most common primary intraocular cancer in adults, is among the tumors with poorer prognosis. Recently, the role of the oncometabolite lactate has become attractive due to its role as hydroxycarboxylic acid receptor 1 (HCAR1) activator, as an epigenetic modulator inducing lysine residues lactylation and, of course, as a glycolysis end-product, bridging the gap between glycolysis and oxidative phosphorylation. The aim of the present study was to dissect in UM cell line (92.1) the role of lactate as either a metabolite or a signaling molecule, using the known modulators of HCAR1 and of lactate transporters. Our results show that lactate (20 mM) resulted in a significant decrease in cell proliferation and migration, acting and switching cell metabolism toward oxidative phosphorylation. These results were coupled with increased euchromatin content and quiescence in UM cells. We further showed, in a clinical setting, that an increase in lactate transporters MCT4 and HCAR1 is associated with a spindle-shape histological type in UM. In conclusion, our results suggest that lactate metabolism may serve as a prognostic marker of UM progression and may be exploited as a potential therapeutic target.

The Role of Epigenetics in Neuroinflammatory-Driven Diseases.

Neurodegenerative disorders are characterized by the progressive loss of central and/or peripheral nervous system neurons. Within this context, neuroinflammation comes up as one of the main factors linked to neurodegeneration progression. In fact, neuroinflammation has been recognized as an outstanding factor for Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and multiple sclerosis (MS). Interestingly, neuroinflammatory diseases are characterized by dramatic changes in the epigenetic profile, which might provide novel prognostic and therapeutic factors towards neuroinflammatory treatment. Deep changes in DNA and histone methylation, along with histone acetylation and altered non-coding RNA expression, have been reported at the onset of inflammatory diseases. The aim of this work is to review the current knowledge on this field.

Uveal Melanoma Cells Elicit Retinal Pericyte Phenotypical and Biochemical Changes in an in Vitro Model of Coculture.

Vascular pericytes are an important cellular component in the tumor microenvironment, however, their role in supporting cancer invasion is poorly understood. We hypothesized that PDGF-BB could be involved in the transition of human retinal pericytes (HRPC) in cancer-activated fibroblasts (CAF), induced by the 92.1 uveal melanoma (UM) cell line. In our model system, HRPC were conditioned by co-culturing with 92.1UM for 6 days (cHRPC), in the presence or absence of imatinib, to block PDGF receptor-ß (PDGFRß). The effects of the treatments were tested by wound healing assay, proliferation assay, RT-PCR, high-content screening, Western blot analysis, and invasion assay. Results showed profound changes in cHRPC shape, with increased proliferation and motility, reduction of NG2 and increase of TGF-ß1, α-SMA, vimentin, and FSP-1 protein levels, modulation of PDGF isoform mRNA levels, phospho-PDGFRß, and PDGFRß, as well as phospho-STAT3 increases. A reduction of IL-1ß and IFNγ and an increase in TNFα, IL10, and TGF-ß1, CXCL11, CCL18, and VEGF mRNA in cHRPC were found. Imatinib was effective in preventing all the 92.1UM-induced changes. Moreover, cHRPC elicited a significant increase of 92.1UM cell invasion and active MMP9 protein levels. Our data suggest that retinal microvascular pericytes could promote 92.1UM growth through the acquisition of the CAF phenotype.

Proteasome Inhibitors as a Possible Therapy for SARS-CoV-2.

The COVID-19 global pandemic is caused by SARS-CoV-2, and represents an urgent medical and social issue. Unfortunately, there is still not a single proven effective drug available, and therefore, current therapeutic guidelines recommend supportive care including oxygen administration and treatment with antibiotics. Recently, patients have been also treated with off-label therapies which comprise antiretrovirals, anti-inflammatory compounds, antiparasitic agents and plasma from convalescent patients, all with controversial results. The ubiquitin-proteasome system (UPS) is important for the maintenance of cellular homeostasis, and plays a pivotal role in viral replication processes. In this review, we discuss several aspects of the UPS and the effects of its inhibition with particular regard to the life cycle of the coronaviruses (CoVs). In fact, proteasome inhibition by various chemical compounds, such as MG132, epoxomycin and bortezomib, may reduce the virus entry into the eucariotic cell, the synthesis of RNA, and the subsequent protein expression necessary for CoVs. Importantly, since UPS inhibitors reduce the cytokine storm associated with various inflammatory conditions, it is reasonable to assume that they might be repurposed for SARS-CoV-2, thus providing an additional tool to counteract both virus replication as well as its most deleterious consequences triggered by abnormal immunological response.

Immunoproteasome Genes Are Modulated in CD34+ JAK2V617F Mutated Cells from Primary Myelofibrosis Patients.

Primary myelofibrosis (PMF) is a rare myeloproliferative neoplasm characterized by stem-cell-derived clonal over-proliferation of mature myeloid lineages, bone marrow fibrosis, osteosclerosis, defective erythropoiesis, and pro-inflammatory cytokine over-expression. The aim of the present study was to highlight possible differences in the transcriptome among CD34+ cells from peripheral blood (PB) of PMF patients. Therefore, we merged two microarray datasets of healthy control subjects and PMF (34 JAK2V617F MUTATED and 28 JAK2 wild-type). The GO analysis of upregulated genes revealed enrichment for JAK2/STAT1 pathway gene set in PB CD34+ cells of PMF patients with and without the JAK2V617F mutation comparing to the healthy control subjects, and in particular a significant upregulation of immunoproteasome (IP)-belonging genes as PSMB8, PSMB9, and PSMB10. A more detailed investigation of the IFN-gamma (IFNG) pathway also revealed that IFNG, IRF1, and IFNGR2 were significantly upregulated in PB CD34+ cells of PMF patients carrying the mutation for JAK2V617F compared to JAK2 wild-type PMF patients. Finally, we showed an upregulation of HLA-class I genes in PB CD34+ cells from PMF JAK2V617F mutated patients compared to JAK2 wild-type and healthy controls. In conclusion, our results demonstrate that IPs and IFNG pathways could be involved in PMF disease and in particular in patients carrying the JAK2V617F mutation.

Immune off-target effects of Brentuximab Vedotin in relapsed/refractory Hodgkin Lymphoma.

Hodgkin Lymphoma (HL) is associated with deep microenvironment re-shaping and myeloid dysfunction. Given that only limited data are available regarding the role of Brentuximab Vedotin (BV) as single agent in transplant-naive relapsed/refractory (R/R) patients and its off-target effects on immune system, we evaluated the amount of regulatory T-cells (T-regs), myeloid-derived suppressor cells (MDSC) subpopulations, and their functional marker, serum arginase-1 (s-Arg-1), in peripheral blood of 15 consecutive R/R HL patients. After a median of four BV cycles, the overall response rate (complete response + partial response) was 47%, with 4 (27%) complete metabolic remissions. BV reduced the absolute number of three MDSC subtypes and s-Arg-1 levels. Patients with baseline s-Arg-1 ≥200 ng/ml had inferior progression-free survival at 36 months compared to those with low s-Arg-1. T-regs dysfunction was recovered by BV: absolute T-regs count was increased after treatment with BV, independently of metabolic response achieved, with a significant reduction of CD30+ T-regs. Our data disclose off-target effects of BV in the microenvironment that could explain its deep and durable clinical efficacy.

Monocytic Myeloid Derived Suppressor Cells in Hematological Malignancies.

In the era of novel agents and immunotherapies in solid and liquid tumors, there is an emerging need to understand the cross-talk between the neoplastic cells, the host immune system, and the microenvironment to mitigate proliferation, survival, migration and resistance to drugs. In the microenvironment of hematological tumors there are cells belonging to the normal bone marrow, extracellular matrix proteins, adhesion molecules, cytokines, and growth factors produced by both stromal cells and neoplastic cells themselves. In this context, myeloid suppressor cells are an emerging sub-population of regulatory myeloid cells at different stages of differentiation involved in cancer progression and chronic inflammation. In this review, monocytic myeloid derived suppressor cells and their potential clinical implications are discussed to give a comprehensive vision of their contribution to lymphoproliferative and myeloid disorders.

Pericytes in Microvessels: From "Mural" Function to Brain and Retina Regeneration.

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFß, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine.

Clobetasol Modulates Adult Neural Stem Cell Growth via Canonical Hedgehog Pathway Activation.

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.

Plasticity of High-Density Neutrophils in Multiple Myeloma is Associated with Increased Autophagy Via STAT3.

In both monoclonal gammopathy of uncertain significance (MGUS) and multiple myeloma (MM) patients, immune functions are variably impaired, and there is a high risk of bacterial infections. Neutrophils are the most abundant circulating leukocytes and constitute the first line of host defense. Since little is known about the contribution of autophagy in the neutrophil function of MGUS and MM patients, we investigated the basal autophagy flux in freshly sorted neutrophils of patients and tested the plastic response of healthy neutrophils to soluble factors of MM. In freshly sorted high-density neutrophils obtained from patients with MGUS and MM or healthy subjects, we found a progressive autophagy trigger associated with soluble factors circulating in both peripheral blood and bone marrow, associated with increased IFNγ and pSTAT3S727. In normal high-density neutrophils, the formation of acidic vesicular organelles, a morphological characteristic of autophagy, could be induced after exposure for three hours with myeloma conditioned media or MM sera, an effect associated with increased phosphorylation of STAT3-pS727 and reverted by treatment with pan-JAK2 inhibitor ruxolitinib. Taken together, our data suggest that soluble factors in MM can trigger contemporary JAK2 signaling and autophagy in neutrophils, targetable with ruxolitinib.

α-Lipoic Acid Reduces Iron-induced Toxicity and Oxidative Stress in a Model of Iron Overload.

Iron toxicity is associated with organ injury and has been reported in various clinical conditions, such as hemochromatosis, thalassemia major, and myelodysplastic syndromes. Therefore, iron chelation therapy represents a pivotal therapy for these patients during their lifetime. The aim of the present study was to assess the iron chelating properties of α-lipoic acid (ALA) and how such an effect impacts on iron overload mediated toxicity. Human mesenchymal stem cells (HS-5) and animals (zebrafish, n = 10 for each group) were treated for 24 h with ferric ammonium citrate (FAC, 120 µg/mL) in the presence or absence of ALA (20 µg/mL). Oxidative stress was evaluated by reduced glutathione content, reactive oxygen species formation, mitochondrial dysfunction, and gene expression of heme oxygenase-1b and mitochondrial superoxide dismutase; organ injury, iron accumulation, and autophagy were measured by microscopical, cytofluorimetric analyses, and inductively coupled plasma‒optical mission Spectrometer (ICP-OES). Our results showed that FAC results in a significant increase of tissue iron accumulation, oxidative stress, and autophagy and such detrimental effects were reversed by ALA treatment. In conclusion, ALA possesses excellent iron chelating properties that may be exploited in a clinical setting for organ preservation, as well as exhibiting a good safety profile and low cost for the national health system.

Monocytic myeloid-derived suppressor cells as prognostic factor in chronic myeloid leukaemia patients treated with dasatinib.

Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in patients with chronic myeloid leukaemia (CML) inducing T cell tolerance. In this study, we found that therapy with tyrosine kinase inhibitors (TKI) decreased the percentage of granulocytic MDSC, but only patients treated with dasatinib showed a significant reduction in the monocytic subset (M-MDSC). Moreover, a positive correlation was observed between number of persistent M-MDSC and the value of major molecular response in dasatinib-treated patients. Serum and exosomes from patients with CML induced conversion of monocytes from healthy volunteers into immunosuppressive M-MDSC, suggesting a bidirectional crosstalk between CML cells and MDSC. Overall, we identified M-MDSC as prognostic factors in patients treated with dasatinib. It might be of interest to understand whether MDSC may be a candidate predictive markers of relapse risk following TKI discontinuation, suggesting their potential significance as practice of precision medicine.

Biochemical and clinical relevance of alpha lipoic acid: antioxidant and anti-inflammatory activity, molecular pathways and therapeutic potential.

BACKGROUND: The molecular nature of lipoic acid (LA) clarifies its capability of taking part to a variety of biochemical reactions where redox state is meaningful. The pivotal action of LA is the antioxidant activity due to its ability to scavenge and inactivate free radicals. Furthermore, LA has been shown to chelate toxic metals both directly and indirectly by its capability to enhance intracellular glutathione (GSH) levels. This last property is due to its ability to interact with GSH and recycle endogenous GSH. LA exhibits significant antioxidant activity protecting against oxidative damage in several diseases, including neurodegenerative disorders. Interestingly, LA is unique among natural antioxidants for its capability to satisfy a lot of requirements, making it a potentially highly effective therapeutic agent for many conditions related with oxidative damage. In particular, there are evidences showing that LA has therapeutic activity in lowering glucose levels in diabetic conditions. Similarly, LA supplementation has multiple beneficial effects on the regression of the mitochondrial function and on oxidative stress associated with several diseases and aging. AIM: The aim of the present review is to describe the molecular mechanisms underlying the beneficial effects of LA under various experimental conditions and disease and how to exploit such effect for clinical purposes. CONCLUSION: LA has pleiotropic effects in different pathways related with several diseases, its use as a potential therapeutic agent is very promising.

Antimicrobial and Anti-Proliferative Effects of Skin Mucus Derived from Dasyatis pastinaca (Linnaeus, 1758).

Resistance to chemotherapy occurs in various diseases (i.e., cancer and infection), and for this reason, both are very difficult to treat. Therefore, novel antimicrobial and chemotherapic drugs are needed for effective antibiotic therapy. The aim of the present study was to assess the antimicrobial and anti-proliferative effects of skin mucus derived from Dasyatis pastinaca (Linnaeus, 1758). Our results showed that skin mucus exhibited a significant and specific antibacterial activity against Gram-negative bacteria but not against Gram-positive bacteria. Furthermore, we also observed a significant antifungal activity against some strains of Candida spp. Concerning anti-proliferative activity, we showed that fish mucus was specifically toxic for acute leukemia cells (HL60) with an inhibition of proliferation in a dose dependent manner (about 52% at 1000 µg/mL of fish skin mucous, FSM). Moreover, we did not observe effects in healthy cells, in neuroblastoma cells (SH-SY5Y), and multiple myeloma cell lines (MM1, U266). Finally, it exhibited strong expression and activity of chitinase which may be responsible, at least in part, for the aforementioned results.

Neuroactive molecules and growth factors modulate cytoskeletal protein expression during astroglial cell proliferation and differentiation in culture.

Steroid hormones and neurotrophic factors regulate astroglial cell survival, proliferation, and differentiation in culture. The present study examines the interaction between glucocorticoids and growth factors (GFs) on cytoskeletal proteins and extracellular signal-regulated kinase 2 (ERK2) expression in stressed astroglial cultures at 25 days in vitro, according to the following experimental condition. Pretreatment with basic fibroblast growth factor alone or in combination with dexamethasone 10(-9) M for 48 hr induced an enhancement of glial fibrillary acidic protein, vimetin, and ERK2 expression. Treatment with "progression" GFs alone and in the last 12 hr significantly increased the above-mentioned markers' expression. The present study shows that glucocorticoids may cooperate with GFs or may abrogate their effects, depending on the experimental culture conditions used as well as the exposure time and the types of GFs added. Our findings provide evidence of interactive dialogue between GFs and neurosteroids in cultured astrocytes. This may have implications in the therapeutic approach to neurologic disorders associated with astrogliosis.

Modulation of extracellular signal-related kinase, cyclin D1, glial fibrillary acidic protein, and vimentin expression in estradiol-pretreated astrocyte cultures treated with competence and progression growth factors.

The present study seeks to elucidate the interactions between the "competence" growth factor basic fibroblast growth factor (bFGF) and/or estrogen 17ß-estradiol and the "progression" growth factors epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and insulin (INS) on DNA labeling and also cyclin D1, extracellular signal-related kinase 1/2 (ERK1/2), glial fibrillary acidic protein (GFAP), and vimentin expression in astroglial cultures under different experimental conditions. Pretreatment for 24 hr with bFGF and subsequent exposure for 36 hr to estradiol (E2 ) and EGF, IGF-I, or INS stimulated DNA labeling in the last 12 hr, especially when the cultures were treated with progression growth factors. bFGF pretreatment and subsequent treatment with E2 for 36 hr stimulated DNA labeling. The 36-hr E2 treatment alone did not significantly decrease DNA labeling, but contemporary addition of E2 with two or three growth factors stimulated DNA labeling remarkably. When E2 was coadded with growth factors, a significantly increased DNA labeling was observed, demonstrating an astroglial synergistic mitogenic effect evoked by contemporary treatment with growth factors in the presence of estrogens. Cyclin D1 expression was markedly increased when astrocyte cultures were pretreated for 36 hr with E2 and subsequently treated with two or three competence and progression growth factors. A highly significant increase of ERK1/2 expression was observed after all the treatments (EGF, bFGF, INS, IGF-I alone or in combination with two or three growth factors). GFAP and vimentin expression was markedly increased when the cultures were treated with two or three growth factors. In conclusion, our data demonstrate estradiol-growth factor cross-talk during astroglial cell proliferation and differentiation in culture.