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Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Adsorption process has proven its efficiency in the abatement of pharmaceuticals in liquid media, even if large volumes of wastewater need to be treated. Nevertheless, exhausted adsorbent regeneration is economically and environmentally necessary. For this reason, recent studies are aimed at finding new methods of regeneration. In this study, an on-site adsorption-regeneration method was assessed. Initially, a model pharmaceutical, Antipyrine (Apy), has been adsorbed onto a low-cost biochar. Apy adsorption followed a pseudo-second order kinetic and a Langmuir isotherm. In a second step, spent biochar was regenerated by oxidation using SO4- . To do this, SO4- was generated by activation of persulfate by ultrasound and assisted by the iron inherently into the biochar. To facilitate the availability of this iron, the addition of an enhancing agent such as oxalic acid was evaluated. The regenerated biochar proved its stability and reusability achieving an uptake percentage of around 87% after the third adsorption-regeneration cycle. Therefore, this on-site regeneration method could be promising for treating other kinds of adsorbents and resolving the pollution problems caused by the non-controlled throw of the exhausted adsorbents. Agencia Estatal de Investigación | Ref. PID2020-113667GB-I00 Agencia Estatal de Investigación | Ref. PDC2021-121394-I00 Xunta de Galicia | Ref. ED431C 2021-43

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUG Pharmaceuticals and personal care products (PPCPs) harm ecosystems and human health. The focus is now being put on regulating discharges at wastewater treatment plants (WWTPs). Stressful and lonely lifestyles, accentuated by the COVID-19 outbreak, have led to an increase in the consumption of antidepressants, particularly in the form of Fluoxetine (FLX). A highly effective process for the removal of PPCPs is of key importance for the economic feasibility of the process in the industry. Consequently, a high-capacity adsorption/regeneration system is crucial to solving this problem. In this study, the removal of FLX by an adsorption process and the subsequent in situ regeneration of the adsorbent was evaluated in successive cycles. NQ40 honeycomb 3D carbon aerogel was employed as a high-capacity adsorbent, obtaining uptakes of 125.24 mg/g. A detailed study of NQ40 and the mechanisms governing the adsorption process was conducted, with chemisorption and intra-particle diffusion playing the main role. A Fenton and a Fenton-like process with peroxymonosulfate (PMS) were evaluated for regeneration purposes, finding hydrogen peroxide to be more efficient in removing a high concentration of FLX. A regeneration capacity of 98.85% was achieved in the first NQ40 adsorption-regeneration cycle and 98.04% in the second one, without negatively affecting the structural characteristics of NQ40. Therefore, with a high porosity, low density, high biocompatibility, high chemical stability and 3D structural stability, this aerogel is at the forefront of novel high-capacity adsorbent materials for industrial-scale use in WWTPs. Agencia Estatal de Investigación | Ref. PID2020-113667GB-I00 Agencia Estatal de Investigación | Ref. PDC2021-121394-I00 Xunta de Galicia | Ref. ED431C 2021-43

Very long-chain acyl-CoA dehydrogenase (VLCAD) is an inner mitochondrial membrane enzyme that catalyzes the first and rate-limiting step of long-chain fatty acid oxidation. Point mutations in human VLCAD can produce an inborn error of metabolism called VLCAD deficiency that can lead to severe pathophysiologic consequences, including cardiomyopathy, hypoglycemia, and rhabdomyolysis. Discrete mutations in a structurally-uncharacterized C-terminal domain region of VLCAD cause enzymatic deficiency by an incompletely defined mechanism. Here, we conducted a structure-function study, incorporating X-ray crystallography, hydrogen-deuterium exchange mass spectrometry, computational modeling, and biochemical analyses, to characterize a specific membrane interaction defect of full-length, human VLCAD bearing the clinically-observed mutations, A450P or L462P. By disrupting a predicted α-helical hairpin, these mutations either partially or completely impair direct interaction with the membrane itself. Thus, our data support a structural basis for VLCAD deficiency in patients with discrete mutations in an α-helical membrane-binding motif, resulting in pathologic enzyme mislocalization.

The appearance of pharmaceuticals in the aquatic environments has become a serious problem because of their hazardous effect on the biota. Therefore, great efforts are focussed in the removal to these pollutants from wastewaters. In this study, an innovative technology based on the principles of Zero-Waste for the management of wastewater streams is presented. Hence, adsorption of fluoxetine (FLX), selected as a model pollutant, in an eco-friendly adsorbent, biochar, was followed by an in situ removal of the pharmaceutical in the solid matrix by the action of sulfate radicals. Initially, an in-depth characterisation of the adsorbent and the adsorption process was carried out. The pseudo-second order kinetic and Freundlich isotherm described well the process, and the electrostatic attractions were revealed as the primary adsorption mechanism. Later, the removal of the FLX was studied by the sulfate radicals, in the presence of activators (Fe2+ and citric acid), in liquid and onto the biochar medium. It was concluded that in order to enhance the pollutant removal it is necessary the presence of both activators in liquid media. However, in in situ removal onto biochar, it was not necessary the Fe2+ presence and only the addition of complexing agents was required as a result of biochar's mineral content. Finally, the applicability of the proposed approach was studied in fixed-bed column assays where the adsorption and the removal of the pollutant were efficiently accomplished. This fact confirms the suitability of the developed process as a viable alternative in the treatment of wastewaters.

Glucagon-like peptide 1 (GLP-1) is a natural peptide agonist of the GLP-1 receptor (GLP-1R) found on pancreatic β-cells. Engagement of the receptor stimulates insulin release in a glucose-dependent fashion and increases β-cell mass, two ideal features for pharmacologic management of type 2 diabetes. Thus, intensive efforts have focused on developing GLP-1-based peptide agonists of GLP-1R for therapeutic application. A primary challenge has been the naturally short half-life of GLP-1 due to its rapid proteolytic degradation in vivo. Whereas mutagenesis and lipidation strategies have yielded clinical agents, we developed an alternative approach to preserving the structure and function of GLP-1 by all-hydrocarbon i, i + 7 stitching. This particular "stitch" is especially well-suited for reinforcing and protecting the structural fidelity of GLP-1. Lead constructs demonstrate striking proteolytic stability and potent biological activity in vivo. Thus, we report a facile approach to generating alternative GLP-1R agonists for glycemic control.

The search for efficient and environmentally friendly adsorbents has positioned lignocellulosic materials as attractive and low-cost alternatives instead of synthetic materials. Consequently, the present work investigates the efficacy of untreated lime peel (LM) and pineapple core (PP) as biosorbents for Cr(VI) removal. The maximum adsorption capacities (acquired at 24 h) of these sorbents were 9.20 and 4.99 mg/g, respectively. The use of these sorbents is expected to offer a rapid and efficient solution to treat effluents containing Cr(VI). Pineapple core showed the best biosorption properties and good distribution coefficients (distribution coefficient KD 8.35&ndash, 99.20 mL/g) and the optimization of the adsorption was carried out by a response surface methodology using the Box&ndash, Behnken design. Thus, the effect of pH, biosorbent dosage, and temperature were assessed during the whole procedure. Three different responses were studied&mdash, Cr(VI) removal, Cr biosorption, and distribution coefficient&mdash, and the optimal conditions for maximizing the responses were identified by numerical optimization applying the desirability function. The resulting optimal conditions were: initial solution pH 2.01, biosorbent dosage 30 g/L, and temperature 30.05 &deg, C. Finally, the process scale-up was evaluated by the simulation of the process working with a column of 100 L using the Fixed-bed Adsorption Simulation Tool (FASTv2.1). This research presents the obtained environmental benefits: i) reduction of pineapple waste, ii) Cr(VI) reduction and biosorption, iii) shortest sorption time for Cr, iv) properties that allow the biosorption process on the flow system, and v) low-cost process.

MCL-1 is an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. Like BCL-2, MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, a mechanism harnessed by cancer cells to establish formidable apoptotic blockades. Although drugging the BH3-binding groove has been achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small-molecule covalent modification of C286 at a new interaction site distant from the BH3-binding groove. Our structure-function analyses revealed that the BH3 binding capacity of MCL-1 and its suppression of BAX are impaired by molecular engagement, a phenomenon recapitulated by C286W mutagenic mimicry in vitro and in mouse cells. Thus, we characterize an allosteric mechanism for disrupting the antiapoptotic BH3 binding activity of MCL-1, informing a new strategy for disarming MCL-1 in cancer.

We present the application of cognitive-behavioural therapy in a clinical case diagnosed with delusional dysmorphophobia.The psychometric scales used for evaluation were the Positive and Negative Syndrome Scale for Schizophrenia, Beck Anxiety and Depression inventories, the Rosenberg Self-Esteem Scale along with the degree of conviction in the delusional belief and in alternative explanations, and social functioning measured by patient reporting. The therapy included cognitive and behavioural techniques: evidence analysis, search for alternative explanations, logical and functional analysis, reality testing, progressive relaxation techniques, in vivo and imaginal exposure therapy. Evaluations were performed before and after the treatment and then at follow-up after 12 and 24 months.Progressively, the delusional conviction disappeared. There were significant improvements at an emotional level and the patient recovered social and worknbsp; functioning.The need to use psychological treatments for people with delusional disorder as first choice treatment must be considered.

Summary MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid β-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1 Matrix ) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1 Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼ 30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D(V12) activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.

MCL-1 is an anti-apoptotic BCL-2 family protein that has emerged as a major pathogenic factor across a broad range of human cancers. Thus, pharmacologic inhibition of MCL-1 has been the focus of intensive drug development efforts. Like BCL-2, MCL-1 bears a C-terminal surface groove whose function is to bind and sequester the essential BH3 killer domains of pro-apoptotic BCL-2 family proteins, a mechanism harnessed by cancer cells to create formidable apoptotic blockades. Whereas drugging the BH3-binding groove has been successfully achieved for BCL-2, translating this approach to MCL-1 has been challenging. Here, we report an alternative mechanism for MCL-1 inhibition by small molecule covalent modification of an interaction site that is distant from the BH3-binding groove. Biochemical and hydrogen-deuterium exchange mass spectrometry analyses revealed that both the BH3-binding capacity of MCL-1 and its functional suppression of BAX-mediated membrane poration is impaired by molecular engagement, a phenomenon recapitulated by mutagenic mimicry at the novel regulatory region. This allosteric mechanism for disrupting the anti-apoptotic, BH3-binding activity of MCL-1 could inform a new therapeutic strategy for disarming MCL-1 in cancer. Citation Format: Susan Lee, Thomas E. Wales, Catherine Gallagher, Daniel T. Cohen, Silvia Escudero, James Luccarelli, Annissa J. Huhn, Nicole A. Cohen, Gregory H. Bird, John R. Engen, Loren D. Walensky. A novel allosteric mechanism for molecular inhibition of anti-apoptotic MCL-1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3553.

Lifelong self-renewal of the adult intestinal epithelium requires the activity of stem cells located in mucosal crypts. Lgr5 and Bmi1 are two molecular markers of crypt-cell populations that replenish all lineages over time and hence function as stem cells. Intestinal stem cells require Wnt signaling, but the understanding of their cellular niche is incomplete. Lgr5-expressing crypt base columnar cells (CBCs) reside deep in the crypt, mingled among mature Paneth cells that are well positioned for short-range signaling. Partial lineage ablation previously had implied that Paneth cells are nonessential constituents of the stem-cell niche, but recently their absence was reported to interfere with Lgr5 + CBCs, resurrecting an appealing idea. However, previous mouse models failed to remove Paneth cells completely or permanently; defining the intestinal stem-cell niche requires clarity with respect to the Paneth cell role. We find that Lgr5 + cells with stem-cell activity cluster in future crypts early in life, before Paneth cells develop. We also crossed conditional Atoh1 − / − mice, which lack Paneth cells entirely, with Lgr5 GFP mice to visualize Lgr5 + CBCs and to track their stem-cell function. In the sustained absence of Paneth cells, Lgr5 + CBCs occupied the full crypt base, proliferated briskly, and generated differentiated progeny over many months. Gene expression in fluorescence-sorted Lgr5 + CBCs reflected intact Wnt signaling despite the loss of Paneth cells. Thus, Paneth cells are dispensable for survival, proliferation, and stem-cell activity of CBCs, and direct contact with Lgr5-nonexpressing cells is not essential for CBC function.

Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here, we report that p38γ motogen-activated protein kinase regulates breast cancer cell motility and metastasis, in part, by controlling expression of the metastasis-associated small GTPase RhoC. This p38γ-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we showed that p38γ-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38γ is associated with lower overall survival of patients with breast cancer. Taken together, our results offer a detailed characterization of how p38γ contributes to breast cancer progression. Herein we present a new mechanics-based analysis of cell motility, and report on the discovery of a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38γ as a candidate therapeutic target. Fil: Rosenthal, Devin T.. University of Michigan; Estados Unidos Fil: Iyer, Harish. University of Michigan; Estados Unidos Fil: Escudero, Silvia. Harvard Medical School; Estados Unidos Fil: Bao, Liwei. University of Michigan; Estados Unidos Fil: Wu, Zhifen. University of Michigan; Estados Unidos Fil: Ventura, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina Fil: Kleer, Celina G.. University of Michigan; Estados Unidos Fil: Arruda, Ellen M.. University of Michigan; Estados Unidos Fil: Garikipati, Krishna. University of Michigan; Estados Unidos Fil: Merajver, Sofia D.. University of Michigan; Estados Unidos

The complexity of biological systems is often prohibitive in testing specific hypotheses from first physical principles. To circumvent these limitations we used biological data to inform a mathematical model of breast cancer cell motility. Using this in silico model we were able to accurately assess the influence of actin cytoskeletal architecture on the motility of a genetically modified breast cancer cell line. Furthermore, using the in silico model revealed a biological phenomenon that has not been previously described in live cell movement. Fusing biology and mathematics as presented here represents a new direction for biomedical research in which advances in each field synergistically drive discoveries in the other.

Understanding the molecular alterations that confer metastatic properties to otherwise benign cells is essential to controlling breast cancer and significantly improving patient survival. p38γ is a member of the p38 MAPK family and is normally expressed predominantly in muscle tissue. Developmentally, the primary role of p38γ is to promote myoblast differentiation into myotubes. Viewing the function of p38γ through the perspective of its developmental role led us to hypothesize that p38γ is involved in enabling mesenchymal-like behavior in breast cancer cells by controlling their motility properties. To elucidate the role of p38γ in breast cancer progression we used RNAi to knock down (KD) p38γ in three aggressive breast cancer cell lines. We then subjected the KD cells to a panel of in vitro assays targeting the hallmarks of cancer progression. We investigated in detail the relationship between cytoskeletal defects and motility in p38γ KD cells using an innovative fusion of mathematical modeling and experimental cell biology. To evaluate the in vivo relevance of our in vitro observations, stable p38γ KD cells were orthotopically xenografted into athymic nude mice. Finally, we analyzed p38γ and RhoC expression in over 300 clinical breast cancer samples. We found that p38γ phosphorylation is elevated in all three breast cancer cell lines compared to two control cell lines. p38γ KD also significantly impairs cell motility and invasion while increasing proliferation, and also drastically alters actin cytoskeletal architecture. By developing an in silico mechanical model of cell motility we show that the modified cytoskeletal architecture of p38γ KD cells is a driving force behind the observed altered motility. Our analysis also uncovered a novel leading edge behavior used by both p38γ KD and control cells to accommodate varying cytoskeletal architectures. Biochemically, p38γ concurrently alters RhoC expression, and rescuing RhoC expression restores aggressiveness to p38γ KD cells. When xenografted, p38γ KD cells are significantly less metastatic than scrambled control cells despite forming larger primary tumors. p38γ and RhoC expression correlate across clinical breast cancer specimens and may be predictive of disease severity. We demonstrate for the first time that p38γ is a metastatic oncogene that specifically acts as a switch between primary tumor proliferation and metastatic cell motility and thus presumably between ductal carcinoma in situ and invasive breast cancer. p38γ elicits its effects, at least in part, through cytoskeletal remodeling and by modulating expression of another known metastatic oncogene, RhoC. These findings have important therapeutic implications as p38γ and RhoC expression were found to be concurrently altered in patient tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1485. doi:10.1158/1538-7445.AM2011-1485

Breast cancer is the second leading cause of cancer-related deaths in women in the US each year. The vast majority of these fatalities are not caused by primary tumor burden but rather by metastases to vital organs. The clinical shift from localized to metastatic breast cancer entails a requirement that cancer cells activate an invasive program and be able to adapt to changing extracellular stimuli. The p38 mitogen activated protein kinase (MAPK) pathway represents a potential signaling switch for the transition from primary to metastatic cancer. p38 is a member of the MAPK family of stress and mitogen-responsive protein kinases and consists of four closely related isoforms: alpha, beta, gamma, and delta. p38 serves as a major signaling hub in the cell, integrating signals from a variety of signaling pathways and channeling these stimuli into cellular responses through an array of effector proteins. The four isoforms show unique expression patterns in normal tissue: alpha, beta, and delta are the most ubiquitously expressed, while gamma is restricted primarily to skeletal muscle. Surprisingly, we found high levels of phosphorylated p38 gamma in a myoepithelial-derived aggressive breast cancer cell line, MDA-MB-231, compared to the non-tumorigenic mammary epithelial cell line MCF-10A, despite both cell lines having similar levels of total p38 gamma. This led us to hypothesize that, based on its normal function in skeletal muscle, p38 gamma may play a role in mediating metastatic behavior in basally-derived breast cancers. To test this hypothesis, we used isoform-specific shRNA to knock down p38 gamma expression in MDA-MB-231. Inhibition of p38 gamma (referred to as shGamma) resulted in decreased cell motility and invasion, two hallmarks of cancer metastasis. Analysis of individual cells revealed a change in the mode of motility from mesenchymal-like in control cells to lamellipodia-driven motility in the shGamma cells. This altered motility appears to be the result of a dramatically modified actin cytoskeleton, characterized by loss of lengthwise stress fibers and bundled, rather than branched, actin in the lamellipodia. When we investigated gene expression changes responsible for this phenotype we discovered that expression of the metastatic oncogene RhoC GTPase is significantly reduced at the protein, but not mRNA, level in shGamma cells due to decreased RhoC protein stability. These findings have great potential clinical significance, as early evidence indicates that p38 gamma is overexpressed in cases of invasive breast cancer, and appears to correlate with RhoC expression in these samples. Our current and future work includes determining which breast cancer subtypes utilize p38 gamma for invasion and metastasis, and characterizing in more detail the dynamic actin cytoskeletal changes affected by p38 gamma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5193.