Discovery of novel methionine adenosyltransferase 2A (MAT2A) allosteric inhibitors by structure-based virtual screening.

Methionine adenosyltransferase 2A (MAT2A) has been indicated as a drug target for oncology indications. Clinical trials with MAT2A inhibitors are currently on-going. Here, a structure-based virtual screening campaign was performed on the commercially available chemical space which yielded two novel MAT2A-inhibitor chemical series. The binding modes of the compounds were confirmed with X-ray crystallography. Both series have acceptable physicochemical properties and show nanomolar activity in the biochemical MAT2A inhibition assay and single-digit micromolar activity in the proliferation assay (MTAP -/- cell line). The identified compounds and the relating structural data could be helpful in related drug discovery projects.

Creation and evaluation of a participatory child abuse and neglect workshop for medical students.

BACKGROUND: Since child abuse and neglect (CAN) is prevalent worldwide, medical students should acquire basic knowledge, skills, and confidence in identifying and addressing CAN. Although significant educational efforts have been previously described, none has focused on using participatory methods to teach medical students CAN. PURPOSE: To: 1) develop a participatory educational workshop in CAN for medical students, 2) gather, train, and establish a peer-to-peer teaching group, and 3) assess the effectiveness of the workshop in gain of knowledge and improvement of self-confidence for participants. METHODS: A two-hour workshop was created with role-playing, the use of mannikins and peer-to-peer teaching. A 15-item knowledge and a 9-item self-confidence questionnaire were used before, right after, and six months after each workshop. RESULTS: Nine workshops in two academic pediatric departments with a total attendance of 300 6th year medical students were conducted. For the 69 students who completed the questionnaires at all three times, there were statistically significant gains in knowledge right after (p < .001) and six months after (p < .0001) the workshops. Similarly, self-confidence increased right after (p < .0001) and six months after (p < .001) the workshops. Self-selection bias testing indicated that these 69 students who completed all three questionnaires were representative of those who completed the pre-testing and the testing right after. CONCLUSIONS: We successfully established a peer-to-peer teaching group to conduct nine participatory workshops that improved the participants' knowledge and self-confidence in CAN. This feasible and novel active learning approach may help address inadequacies in medical curricula.

MYO10-filopodia support basement membranes at pre-invasive tumor boundaries.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer. During invasion, the encapsulating DCIS basement membrane (BM) is compromised, and tumor cells invade the surrounding stroma. The mechanisms that regulate functional epithelial BMs in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing protein associated with metastasis and poor clinical outcome in invasive breast cancer (IBC). We identify elevated MYO10 expression in human DCIS and IBC, and this suggests links with disease progression. MYO10 promotes filopodia formation and cell invasion in vitro and cancer-cell dissemination from progressively invasive human DCIS xenografts. However, MYO10-depleted xenografts are more invasive. These lesions exhibit compromised BMs, poorly defined borders, and increased cancer-cell dispersal and EMT-marker-positive cells. In addition, cancer spheroids are dependent on MYO10-filopodia to generate a near-continuous extracellular matrix boundary. Thus, MYO10 is protective in early-stage breast cancer, correlating with tumor-limiting BMs, and pro-invasive at later stages, facilitating cancer-cell dissemination.

Sustainable Production of Novel Oleogels Valorizing Microbial Oil Rich in Carotenoids Derived from Spent Coffee Grounds.

Sustainable food systems that employ renewable resources without competition with the food chain are drivers for the bioeconomy era. This study reports the valorization of microwave-pretreated spent coffee grounds (SCGs) to produce oleogels rich in bioactive compounds. Microbial oil rich in carotenoids (MOC) was produced under batch fermentation of Rhodosporidium toruloides using SCG enzymatic hydrolysates. Candelilla wax (CLW) could structure MOC and sunflower oil at a 3.3-fold lower concentration than that of carnauba wax (CBW). MOC-based oleogels with 10% CBW and 3% CLW showed an elastic-dominant and gel-like structure (tan δ ≪ 1), providing gelation and oil binding capacity (>95%). Dendritic structures of CBW-based oleogels and evenly distributed rod-like crystals of CLW-based ones were observed via polarized light microscopy. MOC-based oleogels exhibited similar Fourier-transform infrared spectroscopy spectra. X-ray diffractograms of oleogels were distinguished by the oil type that presented ß'-type polymorphism. MOC-based oleogels could be applied in confectionary products and spreads as substitutes for trans fatty acids, reformulating fat-containing food products.

Development of biodegradable films using sunflower protein isolates and bacterial nanocellulose as innovative food packaging materials for fresh fruit preservation.

This study presents the valorization of side streams from the sunflower-based biodiesel industry for the production of bio-based and biodegradable food packaging following circular economy principles. Bacterial cellulose (BC) was produced via fermentation in 6 L static tray bioreactors using nutrient-rich supplements derived from the enzymatic hydrolysis of sunflower meal (SFM) combined with crude glycerol as carbon source. Novel biofilms were produced using either matrices of protein isolates extracted from sunflower meal (SFMPI) alone or SFMPI matrices reinforced with nanocellulose biofillers of commercial or bacterial origin. Acid hydrolysis was employed for ex-situ modification of BC to nanostructures (56 nm). The biofilms reinforced with bacterial nanocellulose structures (SFMPI-BNC) showed 64.5% higher tensile strength, 75.5% higher Young's modulus, 131.5% higher elongation at break, 32.5% lower water solubility and 14.1% lower water vapor permeability than the biofilms produced only with SFMPI. The biofilms were evaluated on fresh strawberries packaging showing that the SFMPI-BNC-based films lead to effective preservation at 10 °C considering microbial growth and physicochemical profile (weight loss, chemical characterization, color, firmness and respiration activity). The SFMPI-BNC-based films could be applied in fresh fruit packaging applications.

Circulating tumor necrosis factor-α levels in non-alcoholic fatty liver disease: A systematic review and a meta-analysis.

BACKGROUND AND AIM: To synthesize data on circulating tumor necrosis factor (TNF)-α levels between patients with histologically confirmed non-alcoholic fatty liver disease (NAFLD) (simple steatosis or non-alcoholic fatty liver [NAFL] and/or non-alcoholic steatohepatitis [NASH]) and controls. METHODS: We performed a systematic search in PubMed, Scopus, and Cochrane Library. Fifty-six studies, published between 2003 and 2019, were finally included, reporting data from 5848 individuals (1634 controls and 4214 NAFLD patients). RESULTS: Higher circulating TNF-α levels were observed in NAFLD patients than controls (standardized mean difference [SMD] 0.84; 95% confidence interval [95% CI] 0.59-1.09), NAFL patients than controls (SMD 0.56; 95% CI 0.27-0.85), NASH patients than controls (SMD 0.93; 95% CI 0.64-1.22), and NASH than NAFL patients (SMD 0.31; 95% CI 0.16-0.46). There were only minimal changes in the comparisons between groups after excluding studies with morbidly obese populations (n = 11), or pediatric/adolescent populations (n = 6), or other than enzyme-linked immunosorbent assay method of TNF-α measurement (n = 8). There was high heterogeneity among studies in all comparisons, which was not essentially affected after sensitivity analyses. The meta-regression analysis revealed that the male ratio was positively associated with TNF-α SMD in the comparison between patients with NASH and NAFL (beta = 0.809; 95% CI 0.052-1.566) and accounted for 36% (P = 0.037) of the heterogeneity in this pair of comparison. TNF-α SMD was not associated with age, body mass index, and alanine aminotransferase in any pair of comparisons. CONCLUSIONS: Circulating TNF-α levels were higher in patients with NAFLD compared with controls. Higher levels of circulating TNF-α were also associated with the severity of NAFLD.

Effect of Processed Beverage By-Product-Based Diets on Biological Parameters, Conversion Efficiency and Body Composition of Hermetia illucens (L) (Diptera: Stratiomyidae).

The effect of spent coffee grounds (SCG), brewer's spent grains (BSG) and their mixtures with the addition of brewer's yeast (BY) were tested in two rearing densities of the Black Soldier Fly, Hermetia illucens (L.). Different treatments were investigated on larval development, survival, yield, protein conversion (PrCR) and bioconversion rate (BCR), substrate mass reduction and body composition of the insect. BSF larvae were able to develop sufficiently in all diets, except on sole SCG. The addition of BY enhanced the performance properties of diets, especially in the case of SCG, where larvae underperformed. Substrate mass reduction, PrCR and BCR were affected only by feed and exhibited higher values on reference feed, followed by BSG and SCG+BSG enriched with BY. Density did not have a significant effect on various larval nutrients, except for fat, which was higher on larvae fed enriched feeds with BY and in the 300 larval density. The interaction between feed and density strongly affected the nitrogen and protein levels, larval yield and ash. Generally, diets which contained SCG exhibited high larval crude protein levels. Our results illustrate that low value beverage by-products can be successfully utilized as constituents of a successful BSF diet.

Rapid screening on aflatoxins' presence in Pistachia vera nuts using diffuse reflectance infrared Fourier transform spectroscopy and chemometrics.

Aflatoxin contamination in pistachios has been analyzed in this work, using Diffuse Reflectance Infrared Spectroscopy (DRIFTS) with chemometrics. Forty-nine Greek pistachio samples of different aflatoxin concentrations were classified into aflatoxin and non-aflatoxin groups using the 3035-2821, 1770-1721, 1570-1481 and 1260-1061 cm-1 spectral regions in Kubelka-Munk conversion and first derivative form. A chemometric model was developed using twenty-eight samples as calibration, 11 as validation and 10 as test set. The discrimination analysis separated correctly the 100% of the calibration and the validation set and the 80% of the test set. The proposed chemometric model is simple, rapid, economical and environmentally friendly since it does not require chemical pre-treatment of the samples. It is expected that the present method may be proved useful in food industry and the inspection authorities as a rapid decision-making tool to detect batches that must be rejected and enhance consumers' protection from aflatoxin contaminated pistachios.

Erratum: Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

[This corrects the article DOI: 10.1016/j.isci.2020.100907.].

Mechano-responsiveness of fibrillar adhesions on stiffness-gradient gels.

Fibrillar adhesions are important structural and adhesive components in fibroblasts, and are required for fibronectin fibrillogenesis. While nascent and focal adhesions are known to respond to mechanical cues, the mechanoresponsive nature of fibrillar adhesions remains unclear. Here, we used ratiometric analysis of paired adhesion components to determine an appropriate fibrillar adhesion marker. We found that active α5ß1-integrin exhibits the most definitive fibrillar adhesion localization compared to other proteins, such as tensin-1, reported to be in fibrillar adhesions. To elucidate the mechanoresponsiveness of fibrillar adhesions, we designed a cost-effective and reproducible technique to fabricate physiologically relevant stiffness gradients on thin polyacrylamide (PA) hydrogels, embedded with fluorescently labelled beads. We generated a correlation curve between bead density and hydrogel stiffness, thus enabling a readout of stiffness without the need for specialized knowhow, such as atomic force microscopy (AFM). We find that stiffness promotes growth of fibrillar adhesions in a tensin-1-dependent manner. Thus, the formation of these extracellular matrix-depositing structures is coupled to the mechanical parameters of the cell environment and may enable cells to fine-tune their matrix environment in response to changing physical conditions.

Study of the Quality Parameters and the Antioxidant Capacity for the FTIR-Chemometric Differentiation of Pistacia Vera Oils.

The aim of this work was to characterize the pistachio oil of the Greek variety, "Aegina", evaluate its various quality indices, and investigate the potential use of FTIR as a tool to discriminate different oil qualities. For this purpose, the antioxidant capacity, the tocopherol content and the oxidation and degradation of fatty acids, as described by k, Δk, R-values, and free acidity were evaluated using 45 samples from eight different areas of production and two subsequent years of harvesting. The antioxidant capacity was estimated using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS) and 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazine (DPPH) assays, and the tocopherol content was quantified through HPLC analysis. FTIR spectra were recorded for all samples and multivariate analysis was applied. The results showed significant differences between the oil samples of different harvesting years, which were successfully discriminated by a representative FTIR spectral region based on R-value, total antioxidant capacity, and scavenging capacity, through ABTS. A similar approach could not be confirmed for the other quality parameters, such as the free acidity and the tocopherol content. This research highlighted the possibility of developing a simple, rapid, economic, and environment friendly method for the discrimination of pistachio oils according to their quality profile, through FTIR spectroscopy and multivariate analysis.

Integrin Binding Dynamics Modulate Ligand-Specific Mechanosensing in Mammary Gland Fibroblasts.

The link between integrin activity regulation and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently, generate high forces, and display nuclear YAP on soft collagen-coated substrates, resembling the soft mammary gland tissue. We describe that loss of the integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modeling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of collagen-binding integrin α11ß1. Thus integrin activity regulation and α11ß1 play a role in collagen-specific mechanosensing in MSFs.

Dual Transcriptional Profile of Aspergillus flavus during Co-Culture with Listeria monocytogenes and Aflatoxin B1 Production: A Pathogen-Pathogen Interaction.

The objective of this study was to investigate the effect of growth temperature and co-culture of Aspergillus flavus with Listeria monocytogenes on the production of Aflatoxin B1 (AFB1) and the transcriptional profile of associated regulatory and biosynthetic genes. The transcription of virulence- and homeostasis-associated genes of L. monocytogenes was also assessed. For this purpose, mono- and co-cultures of L. monocytogenes strain LQC 15257 and A. flavus strain 18.4 were inoculated into Malt Extract broth and allowed to grow for seven days at 25 °C and 30 °C. AFB1 quantification was performed by HPLC analysis and gene expression assessment by RT-qPCR. AFB1 production was lower at 30 °C compared to 25 °C during monoculture and also lower during co-cultures at both temperatures. This was accompanied by downregulation of aflM, aflR, aflP, and aflS during monoculture and aflM and aflS during co-culture at 30 °C. On the other hand, transcription of prfA, plcA, plcB, inlA, inlB, inlJ, murE, accA, acpP, as well as fapR, was not affected. sigB gene was downregulated after co-culture with the fungus at 25 °C and hly was downregulated after monoculture at 30 °C compared to 25 °C. In this work, the molecular interactions between A. flavus and L. monocytogenes were studied for the first time, offering a novel insight into their co-occurrence. Monitoring of their toxigenic and virulence potential at the molecular level revealed a complex dynamic in natural ecosystems.

SORLA regulates endosomal trafficking and oncogenic fitness of HER2.

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

Tensins: Bridging AMP-Activated Protein Kinase with Integrin Activation.

Integrin activation is essential for cell adhesion and for connecting the extracellular matrix to the actin cytoskeleton. Thus, inappropriate integrin activation has been linked to several diseases, including cancer. Recent insights demonstrate that the main fibrillar adhesion component tensin maintains ß1-integrin active in these mature adhesions. Depletion or silencing of AMP-activated protein kinase (AMPK), the energy sensor involved in maintaining the energy balance of the cell, enhances integrin activity by increasing the expression of tensin and thereby promoting cell adhesion, matrix formation, and mechanotransduction. Here, we discuss the role of tensin and AMPK in the regulation of integrin activity and integrin-dependent processes and their implication in diseases such as cancer and tissue fibrosis.

AMPK negatively regulates tensin-dependent integrin activity.

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a ß1-integrin inhibitor in fibroblasts. Loss of AMPK promotes ß1-integrin activity, the formation of centrally located active ß1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases ß1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind ß1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

SHANK proteins limit integrin activation by directly interacting with Rap1 and R-Ras.

SHANK3, a synaptic scaffold protein and actin regulator, is widely expressed outside of the central nervous system with predominantly unknown function. Solving the structure of the SHANK3 N-terminal region revealed that the SPN domain is an unexpected Ras-association domain with high affinity for GTP-bound Ras and Rap G-proteins. The role of Rap1 in integrin activation is well established but the mechanisms to antagonize it remain largely unknown. Here, we show that SHANK1 and SHANK3 act as integrin activation inhibitors by sequestering active Rap1 and R-Ras via the SPN domain and thus limiting their bioavailability at the plasma membrane. Consistently, SHANK3 silencing triggers increased plasma membrane Rap1 activity, cell spreading, migration and invasion. Autism-related mutations within the SHANK3 SPN domain (R12C and L68P) disrupt G-protein interaction and fail to counteract integrin activation along the Rap1-RIAM-talin axis in cancer cells and neurons. Altogether, we establish SHANKs as critical regulators of G-protein signalling and integrin-dependent processes.

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial-stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.

L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.

Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.

Normal stroma suppresses cancer cell proliferation via mechanosensitive regulation of JMJD1a-mediated transcription.

Tissue homeostasis is dependent on the controlled localization of specific cell types and the correct composition of the extracellular stroma. While the role of the cancer stroma in tumour progression has been well characterized, the specific contribution of the matrix itself is unknown. Furthermore, the mechanisms enabling normal-not cancer-stroma to provide tumour-suppressive signals and act as an antitumorigenic barrier are poorly understood. Here we show that extracellular matrix (ECM) generated by normal fibroblasts (NFs) is softer than the CAF matrix, and its physical and structural features regulate cancer cell proliferation. We find that normal ECM triggers downregulation and nuclear exit of the histone demethylase JMJD1a resulting in the epigenetic growth restriction of carcinoma cells. Interestingly, JMJD1a positively regulates transcription of many target genes, including YAP/TAZ (WWTR1), and therefore gene expression in a stiffness-dependent manner. Thus, normal stromal restricts cancer cell proliferation through JMJD1a-dependent modulation of gene expression.