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Purpose: Argininosuccinate Synthetase 1 (ASS1) silencing in carcinomas and sarcomas leads to a dependence on extracellular arginine for survival. Arginine deprivation therapies, like PEGylated arginine deiminase (ADI-PEG20), have shown limited effectiveness, which may be due to underlying mechanisms that inhibit apoptosis., Experimental Design: The effects of ADI-PEG20 on cell cycle regulation, apoptosis, and Bcl-xL-mediated survival pathways in ASS1-deficient cancer cells were determined. The mechanism of cell death protection was determined by assessing caspase and PARP cleavage, CDK2 activity, MCL1 expression, and the interactions between Bcl-xL, Bax, and Bak. In vitro synergy was determined, and in vivo efficacy was modeled., Results: Treatment with ADI-PEG20 led to reduced CDK2 activity and inhibited cell cycle progression but did not induce significant cell death. Bcl-xL was found to bind to Bax and Bak, preventing the initiation of apoptosis despite arginine starvation. Inhibition of Bcl-xL allowed proapoptotic Bax and Bak to initiate the intrinsic apoptosis pathway, leading to increased cell death. This was found to be synergistic in vitro and efficacious in combination in vivo., Conclusions: The study identifies Bcl-xL as a key factor limiting the efficacy of arginine starvation therapies. Combining Bcl-xL inhibitors with arginine deprivation strategies may overcome this resistance and enhance therapeutic outcomes. These findings provide a strong preclinical rationale for testing this combination approach in Phase 1 clinical trials for ASS1-deficient cancers.

Purpose: Many cancers lack argininosuccinate synthetase 1 (ASS1), the rate-limiting enzyme of arginine biosynthesis. This deficiency causes arginine auxotrophy, targetable by extracellular arginine-degrading enzymes such as ADI-PEG20. Long-term tumor resistance has thus far been attributed solely to ASS1 reexpression. This study examines the role of ASS1 silencing on tumor growth and initiation and identifies a noncanonical mechanism of resistance, aiming to improve clinical responses to ADI-PEG20., Experimental Design: Tumor initiation and growth rates were measured for a spontaneous Ass1 knockout (KO) murine sarcoma model. Tumor cell lines were generated, and resistance to arginine deprivation therapy was studied in vitro and in vivo., Results: Conditional Ass1 KO affected neither tumor initiation nor growth rates in a sarcoma model, contradicting the prevalent idea that ASS1 silencing confers a proliferative advantage. Ass1 KO cells grew robustly through arginine starvation in vivo, while ADI-PEG20 remained completely lethal in vitro, evidence that pointed toward a novel mechanism of resistance mediated by the microenvironment. Coculture with Ass1-competent fibroblasts rescued growth through macropinocytosis of vesicles and/or cell fragments, followed by recycling of protein-bound arginine through autophagy/lysosomal degradation. Inhibition of either macropinocytosis or autophagy/lysosomal degradation abrogated this growth support effect in vitro and in vivo., Conclusions: Noncanonical, ASS1-independent tumor resistance to ADI-PEG20 is driven by the microenvironment. This mechanism can be targeted by either the macropinocytosis inhibitor imipramine or the autophagy inhibitor chloroquine. These safe, widely available drugs should be added to current clinical trials to overcome microenvironmental arginine support of tumors and improve patient outcomes., (©2023 The Authors; Published by the American Association for Cancer Research.)

In this study, third in a series of studies of the relationships between the dominant type of the Word Meaning Structure (WMS) and various psychic processes, response patterns on personality questionnaires with Likert-type response format of individuals with different levels of education (including adult illiterates) in Brazil (N = 102) and in Estonia (N = 520) were assessed with person oriented methods of data analysis. We found that responses to two personality questionnaires (International Personality Item Pool Questionnaire, IPIP-Q60 and Estonian Collectivism Scale, ESTCOL) are inconsistent and do not correspond to theories that underlie construction and interpretation of such assessment tools. Two novel ways to assess inconsistent response patterns were developed. The Consistency Index (CI) characterizes between-item inconsistency and the Determinacy Index (DI) characterizes within-item inconsistency. The dominant type of the WMS and the level of education were related to both CI and DI. Higher level of between-item inconsistency characterizes everyday conceptual thinkers with lower levels of education and higher level of within-item inconsistency was observed among logical conceptual thinkers with higher levels of education. Systematic relationships between WMS and inconsistent patterns of responses indicate that responses on personality questionnaires cannot be interpreted in terms of personality characteristics. The results of our study also provide further support to the idea that dominant type of the WMS is a pervasive characteristic of the psyche and determines qualitatively possibilities and limits of the psychic processes. The results of this study are in agreement with the idea that WMS defines the "Great Divide.", (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Loss of TDP-43 protein homeostasis and dysfunction, in particular TDP-43 aggregation, are tied to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 is an RNA binding protein tightly controlling its own expression levels through a negative feedback loop, involving TDP-43 recruitment to the 3' untranslated region of its own transcript. Aberrant TDP-43 expression caused by autoregulation defects are linked to TDP-43 pathology. Therefore, interactions between TDP-43 and its own transcript are crucial to prevent TDP-43 aggregation and loss of function. However, the mechanisms that mediate this interaction remain ill-defined. We find that a central RNA sequence in the 3' UTR, which mediates TDP-43 autoregulation, increases the liquid properties of TDP-43 phase separation. Furthermore, binding to this RNA sequence induces TDP-43 condensation in human cell lysates, suggesting that this interaction promotes TDP-43 self-assembly into dynamic ribonucleoprotein granules. In agreement with these findings, our experiments show that TDP-43 oligomerization and phase separation, mediated by the amino and carboxy-terminal domains, respectively, are essential for TDP-43 autoregulation. According to our additional observations, CLIP34-associated phase separation and autoregulation may be efficiently controlled by phosphorylation of the N-terminal domain. Importantly, we find that specific ALS-associated TDP-43 mutations, mainly M337V, and a shortened TDP-43 isoform recently tied to motor neuron toxicity in ALS, disrupt the liquid properties of TDP-43-RNA condensates as well as autoregulatory function. In addition, we find that M337V decreases the cellular clearance of TDP-43 and other RNA binding proteins associated with ALS/FTD. These observations suggest that loss of liquid properties in M337V condensates strongly affects protein homeostasis. Together, this work provides evidence for the central role of TDP-43 oligomerization and liquid-liquid phase separation linked to RNA binding in autoregulation. These mechanisms may be impaired by TDP-43 disease variants and controlled by specific cellular signaling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Koehler, Grese, Bastos, Mamede, Heyduk and Ayala.)

The glutaminase (GLS) enzyme hydrolyzes glutamine into glutamate, an important anaplerotic source for the tricarboxylic acid cycle in rapidly growing cancer cells under the Warburg effect. Glutamine-derived α-ketoglutarate is also an important cofactor of chromatin-modifying enzymes, and through epigenetic changes, it keeps cancer cells in an undifferentiated state. Moreover, glutamate is an important neurotransmitter, and deregulated glutaminase activity in the nervous system underlies several neurological disorders. Given the proven importance of glutaminase for critical diseases, we describe the development of a new coupled enzyme-based fluorescent glutaminase activity assay formatted for 384-well plates for high-throughput screening (HTS) of glutaminase inhibitors. We applied the new methodology to screen a ∼30,000-compound library to search for GLS inhibitors. The HTS assay identified 11 glutaminase inhibitors as hits that were characterized by in silico , biochemical, and glutaminase-based cellular assays. A structure-activity relationship study on the most promising hit (C9) allowed the discovery of a derivative, C9.22, with enhanced in vitro and cellular glutaminase-inhibiting activity. In summary, we discovered a new glutaminase inhibitor with an innovative structural scaffold and described the molecular determinants of its activity., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

Coffee consumption has been investigated as a protective factor against prostate cancer. Coffee may be related to prostate cancer risk reduction due to its phytochemical compounds, such as caffeine, chlorogenic acids, and trigonelline. The roasting process affects the content of the phytochemicals and undesired compounds can be formed. Microwave-assisted extraction is an alternative to conventional extraction techniques since it preserves more bioactive compounds. Therefore, this study aimed to evaluate the phytochemical composition and the putative preventive effects in prostate cancer development of coffee beans submitted to four different coffee-roasting degrees extracted using microwave-assisted extraction. Coffea arabica green beans (1) were roasted into light (2), medium (3) and dark (4) and these four coffee samples were submitted to microwave-assisted extraction. The antioxidant capacity of these samples was evaluated by five different methods. Caffeine, chlorogenic acid and caffeic acid were measured through HPLC. Samples were tested against PC-3 and DU-145 metastatic prostate cancer cell lines regarding their effects on cell viability, cell cycle progression and apoptotic cell death. We found that green and light roasted coffee extracts had the highest antioxidant activity. Caffeine content was not affected by roasting, chlorogenic acid was degraded due to the temperature, and caffeic acid increased in light roasted and decreased in medium and dark roasted. Green and light roasted coffee extracts promoted higher inhibition of cell viability, caused greater cell cycle arrest in S and G 2 /M and induced apoptosis more compared to medium and dark roasted coffee extracts and the control samples. Coffee extracts were more effective against DU-145 than in PC-3 cells. Our data provide initial evidence that among the four tested samples, the consumption of green and light coffee extracts contributes to inhibit prostate cancer tumor progression features, potentially preventing aspects related to advanced prostate cancer subtypes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We studied cognitive inhibitory processes and verbal regulation of behavior of individuals with different levels of education (including adult illiterates) in Brazil (N = 136) and in Estonia (N = 560) with person-oriented methods of data analysis. Our aim was to discover whether dominant type of word meaning structure (WMS) can define the "Great Divide", the single breaking point that universally defines certain direction of subsequent to it cultural evolution. We found that both cognitive inhibition of irrelevant for the task at hand information or actions as well as correct activation of relevant information or actions is significantly more common in individuals who rely predominantly on logical concepts in their thinking. The higher level of education was also associated with more efficient cognitive inhibition and activation. The patterns of test performance also suggest that there can be a qualitative difference in the efficiency of cognitive inhibition-activation processes between everyday conceptual and logical conceptual thinkers. The former group of individuals may achieve much higher performance levels than any individual in the former group. We also discuss cognitive similarities and differences between adults with low or no formal education on the one hand and children and educated adults with brain damage on the other. The results are in agreement with the theory of unilineal hierarchic cultural evolution. Individual psychic development and cultural evolution can be both understood in terms of the WMS development.

We studied visual-perceptual abilities of individuals with different levels of education (including adult illiterates) in Brazil (N = 136) and in Estonia (N = 560) with person-oriented methods of data analysis. Our aim was to discover whether dominant type of word meaning structure (WMS) can define the "Great Divide", the single breaking point that universally defines certain direction of subsequent to it cultural evolution. We particularly focused on the everyday concept-logical concept shift that takes place in the formal education system. We found that logical concepts were rarely available for illiterates; availability of logical conceptual thought increases together with the level of education. Most illiterates were able to find figures of concrete objects from complex overlapping and embedded contour figures but none of them could find all abstract figures from the same complex figures. Also none of the illiterates could perform beyond chance level in both mental rotation tasks together. Ability to perform correctly on all visual-spatial tasks increased with the increase in logical concepts and with the increasing level of education. The distribution of respondents according to the WMS level, level of education, and performance on the visual-spatial tasks indicated that individuals are developmentally heterogeneous: achievement of the tertiary level of education and logical conceptual thinking mechanisms does not guarantee high level performance on the visual-spatial tasks. The results are in agreement with the theory of unilineal hierarchic cultural evolution. Individual psychic development and cultural evolution can be both understood in terms of the WMS development.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle. However, the splicing event replaces a well-structured three-repeat ankyrin domain in KGA with a shorter, unordered C-terminal stretch in GAC. The multidomain architecture, which contains putative protein-protein binding motifs, has led to speculation that glutaminases are involved in cellular processes other than glutamine metabolism; in fact, some proteins have been identified as binding partners of KGA and the isoforms of its paralogue gene, GLS2. Here, a yeast two-hybrid assay identified nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) as a new binding partner of the glutaminase. We show that KGA and GAC directly bind PPARγ with a low-micromolar dissociation constant; the interaction involves the N-terminal and catalytic domains of glutaminases as well as the ligand-binding domain of the nuclear receptor. The interaction occurs within the nucleus, and by sequestering PPARγ from its responsive element DR1, the glutaminases decreased nuclear receptor activity as assessed by a luciferase reporter assay. Altogether, our findings reveal an unexpected glutaminase-binding partner and, for the first time, directly link mitochondrial glutaminases to an unanticipated role in gene regulation.

This paper develops a critical analysis of the implications of defining the family as an object of intervention in health, taking as reference case the Family Health Program and proposing an evaluation of its socio-cultural impact. As a strategic space for manifestation, confrontation, and therefore observation of the health-illness process, the family requires a multidisciplinary approach to its structure, dynamics, and behavior in the face of health-related problems, determinants, and actions. We present a proposal for assessment of the Family Health Program based on the premise that problems and practices in the health field are socio-culturally determined.