Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

Analysis of Uncertainty and Sensitivity in Tailings Dam Breach-Runout Numerical Modelling.

Tailings dam breaches (TDBs) and subsequent flows can pose significant risk to public safety, the environment, and the economy. Numerical runout models are used to simulate potential tailings flows and understand their downstream impacts. Due to the complex nature of the breach-runout processes, the mobility and downstream impacts of these types of failures are highly uncertain. We applied the first-order second-moment (FOSM) methodology to a database of 11 back-analyzed historical tailings flows to evaluate uncertainties in TDB runout modelling and conducted a sensitivity analysis to identify key factors contributing to the variability of the HEC-RAS model output, including at different locations along the runout path. The results indicate that prioritizing resources toward advancements in estimating the values of primary contributors to the sensitivity of the selected model outputs is necessary for more reliable model results. We found that the total released volume is among the top contributors to the sensitivity of modelled inundation area and maximum flow depth, while surface roughness is among the top contributors to the sensitivity of modelled maximum flow velocity and flow front arrival time. However, the primary contributors to the sensitivity of the model outputs varied depending on the case study; therefore, the selection of appropriate rheological models and consideration of site-specific conditions are crucial for accurate predictions. The study proposes and demonstrates the FOSM methodology as an approximate probabilistic approach to model-based tailings flow runout prediction, which can help improve the accuracy of risk assessments and emergency response plans. Supplementary Information: The online version contains supplementary material available at 10.1007/s10230-024-00970-w.

Las roturas de presas de relaves (TDBs) y los flujos subsiguientes pueden suponer un riesgo significativo para la seguridad pública, el medio ambiente y la economía. Los modelos numéricos de desbordamiento se utilizan para simular posibles flujos de relaves y comprender su impacto aguas abajo. Debido a la naturaleza compleja de los procesos de rotura-desbordamiento, la movilidad y los impactos aguas abajo de este tipo de fallos tienen mucha incertidumbre. Se aplicó la metodología del segundo-momento de primer-orden (FOSM) a una base de datos de 11 flujos históricos de relaves analizados retrospectivamente para evaluar las incertidumbres en la modelización del desbordamiento de TDB y se realizó un análisis de sensibilidad para identificar los factores clave que contribuyen a la variabilidad de los resultados del modelo HEC-RAS, incluso en diferentes ubicaciones a lo largo de la trayectoria de fuga. Los resultados indican que es necesario priorizar los recursos hacia avances en la estimación de los valores de los principales contribuyentes a la sensibilidad de los resultados del modelo seleccionado para obtener resultados más fiables del modelo. El volumen total liberado se encuentra entre los principales contribuyentes a la sensibilidad del área de inundación modelizada y la profundidad máxima del flujo, mientras que la rugosidad de la superficie se encuentra entre los principales contribuyentes a la sensibilidad de la velocidad máxima del flujo modelizado y el tiempo de llegada del frente de flujo. Sin embargo, los principales factores que contribuyen a la sensibilidad de los resultados del modelo varían dependiendo del caso de estudio; por lo tanto, la selección de modelos reológicos apropiados y la consideración de las condiciones específicas del emplazamiento son cruciales para obtener predicciones precisas. El estudio propone y muestra la metodología FOSM como un enfoque probabilístico aproximado para la predicción de la extensión de flujos de relaves basada en modelos, que puede ayudar a mejorar la precisión de las evaluaciones de riesgos y los planes de respuesta a emergencias.

The ancestral type of the R-RAS protein has oncogenic potential.

BACKGROUND: The R-RAS2 is a small GTPase highly similar to classical RAS proteins at the regulatory and signaling levels. The high evolutionary conservation of R-RAS2, its links to basic cellular processes and its role in cancer, make R-RAS2 an interesting research topic. To elucidate the evolutionary history of R-RAS proteins, we investigated and compared structural and functional properties of ancestral type R-RAS protein with human R-RAS2. METHODS: Bioinformatics analysis were used to elucidate the evolution of R-RAS proteins. Intrinsic GTPase activity of purified human and sponge proteins was analyzed with GTPase-GloTM Assay kit. The cell model consisted of human breast cancer cell lines MCF-7 and MDA-MB-231 transiently transfected with EsuRRAS2-like or HsaRRAS2. Biological characterization of R-RAS2 proteins was performed by Western blot on whole cell lysates or cell adhesion protein isolates, immunofluorescence and confocal microscopy, MTT test, colony formation assay, wound healing and Boyden chamber migration assays. RESULTS: We found that the single sponge R-RAS2-like gene/protein probably reflects the properties of the ancestral R-RAS protein that existed prior to duplications during the transition to Bilateria, and to Vertebrata. Biochemical characterization of sponge and human R-RAS2 showed that they have the same intrinsic GTPase activity and RNA binding properties. By testing cell proliferation, migration and colony forming efficiency in MDA-MB-231 human breast cancer cells, we showed that the ancestral type of the R-RAS protein, sponge R-RAS2-like, enhances their oncogenic potential, similar to human R-RAS2. In addition, sponge and human R-RAS2 were not found in focal adhesions, but both homologs play a role in their regulation by increasing talin1 and vinculin. CONCLUSIONS: This study suggests that the ancestor of all animals possessed an R-RAS2-like protein with oncogenic properties similar to evolutionarily more recent versions of the protein, even before the appearance of true tissue and the origin of tumors. Therefore, we have unraveled the evolutionary history of R-RAS2 in metazoans and improved our knowledge of R-RAS2 properties, including its structure, regulation and function.

Mice Overexpressing Wild-Type RRAS2 Are a Novel Model for Preclinical Testing of Anti-Chronic Lymphocytic Leukemia Therapies.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common type of leukemia in the Western world. Mutation in different genes, such as TP53 and ATM, and deletions at specific chromosomic regions, among which are 11q or 17p, have been described to be associated to worse disease prognosis. Recent research from our group has demonstrated that, contrary to what is the usual cancer development process through missense mutations, B-CLL is driven by the overexpression of the small GTPase RRAS2 in its wild-type form without activating mutations. Some mouse models of this disease have been developed to date and are commonly used in B-CLL research, but they present different disadvantages such as the long waiting period until the leukemia fully develops, the need to do cell engraftment or, in some cases, the fact that the model does not recapitulate the alterations found in human patients. We have recently described Rosa26-RRAS2fl/flxmb1-Cre as a new mouse model of B-CLL with a full penetrance of the disease. In this work, we have validated this mouse model as a novel tool for the development of new therapies for B-CLL, by testing two of the most broadly applied targeted agents: ibrutinib and venetoclax. This also opens the door to new targeted agents against R-RAS2 itself, an approach not yet explored in the clinic.

The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

Silent information regulator 2 (Sir2) is a conserved NAD+-dependent histone deacetylase crucial for regulating cellular stress response and the aging process in Saccharomyces cerevisiae. In this study, we investigated the molecular mechanism underlying how the absence of Sir2 can lead to altered stress susceptibilities in S. cerevisiae under different environmental and physiological conditions. In a glucose-complex medium, the sir2Δ strain showed increased sensitivity to H2O2 compared to the wild-type strain during the post-diauxic phase. In contrast, it displayed increased resistance during the exponential growth phase. Transcriptome analysis of yeast cells in the post-diauxic phase indicated that the sir2Δ mutant expressed several oxidative defense genes at lower levels than the wild-type, potentially accounting for its increased susceptibility to H2O2. Interestingly, however, the sir2Δras2Δ double mutant exhibited greater resistance to H2O2 than the ras2Δ single mutant counterpart. We found that the expression regulation of the cytoplasmic catalase encoded by CTT1 was critical for the increased resistance to H2O2 in the sir2Δras2Δ strain. The expression of the CTT1 gene was influenced by the combined effect of RAS2 deletion and the transcription factor Azf1, whose level was modulated by Sir2. These findings provide insights into the importance of understanding the intricate interactions among various factors contributing to cellular stress response.

The Cdc25 and Ras1 Proteins of Candida albicans Influence Epithelial Toxicity in a Niche-Specific Way.

The PKA pathway is a signaling pathway involved in virulence in Candida albicans. This mechanism can be activated via addition of glucose and activation involves at least two proteins, namely Cdc25 and Ras1. Both proteins are involved in specific virulence traits. However, it is not clear if Cdc25 and Ras1 also affect virulence independently of PKA. C. albicans holds a second, atypical, Ras protein, Ras2, but its function in PKA activation is still unclear. We investigated the role of Cdc25, Ras1, and Ras2 for different in vitro and ex vivo virulence characteristics. We show that deletion of CDC25 and RAS1 result in less toxicity towards oral epithelial cells, while deletion of RAS2 has no effect. However, toxicity towards cervical cells increases in both the ras2 and the cdc25 mutants while it decreases in a ras1 mutant compared to the WT. Toxicity assays using mutants of the transcription factors downstream of the PKA pathway (Efg1) or the MAPK pathway (Cph1) show that the ras1 mutant shows similar phenotypes as the efg1 mutant, whereas the ras2 mutant shows similar phenotypes as the cph1 mutant. These data show niche-specific roles for different upstream components in regulating virulence through both signal transduction pathways.

Specific Disruption of Ras2 CAAX Proteolysis Alters Its Localization and Function.

Many CAAX proteins, such as Ras GTPase, undergo a series of posttranslational modifications at their carboxyl terminus (i.e., cysteine prenylation, endoproteolysis of AAX, and carboxylmethylation). Some CAAX proteins, however, undergo prenylation-only modification, such as Saccharomyces cerevisiae Hsp40 Ydj1. We previously observed that altering the CAAX motif of Ydj1 from prenylation-only to canonical resulted in altered Ydj1 function and localization. Here, we investigated the effects of a reciprocal change that altered the well-characterized canonical CAAX motif of S. cerevisiae Ras2 to prenylation-only. We observed that the type of CAAX motif impacted Ras2 protein levels, localization, and function. Moreover, we observed that using a prenylation-only sequence to stage hyperactive Ras2-G19V as a farnesylated and nonproteolyzed intermediate resulted in a different phenotype relative to staging by a genetic RCE1 deletion strategy that simultaneously affected many CAAX proteins. These findings suggested that a prenylation-only CAAX motif is useful for probing the specific impact of CAAX proteolysis on Ras2 under conditions where other CAAX proteins are normally modified. We propose that our strategy could be easily applied to a wide range of CAAX proteins for examining the specific impact of CAAX proteolysis on their functions. IMPORTANCE CAAX proteins are subject to multiple posttranslational modifications: cysteine prenylation, CAAX proteolysis, and carboxylmethylation. For investigations of CAAX proteolysis, this study took the novel approach of using a proteolysis-resistant CAAX sequence to stage Saccharomyces cerevisiae Ras2 GTPase in a farnesylated and nonproteolyzed state. Our approach specifically limited the effects of disrupting CAAX proteolysis to Ras2. This represented an improvement over previous methods where CAAX proteolysis was inhibited by gene knockout, small interfering RNA knockdown, or biochemical inhibition of the Rce1 CAAX protease, which can lead to pleiotropic and unclear attribution of effects due to the action of Rce1 on multiple CAAX proteins. Our approach yielded results that demonstrated specific impacts of CAAX proteolysis on the function, localization, and other properties of Ras2, highlighting the utility of this approach for investigating the impact of CAAX proteolysis in other protein contexts.

Does flooding get worse with subsiding land? Investigating the impacts of land subsidence on flood inundation from Hurricane Harvey.

As one of the most devastating tropical storms, 2017 Hurricane Harvey caused severe flooding and damage in Houston, Texas. Besides enormous rainfall amount, land subsidence might be another contributing factor to the Harvey flood. However, few studies have numerically quantified the evolvement of land subsidence over decades, largely due to the lack of reliable methods to realistically estimate land subsidence both continuously and at high spatial resolution. Therefore, this study aims to investigate retrospective changes of regional topology due to 117 years (1900 to 2017) of land subsidence and the consequent impacts on flood inundation. Based on continuous land subsidence, we conduct a series of simulations on the 2017 Hurricane Harvey in Brays Bayou, Texas using a hydrodynamic/hydraulic model. The results indicate that the overall change of flood depth caused by land subsidence is relatively minor with the flood water deepened by six centimeters per one meter of subsided land at the worst impacted location. The impact from land subsidence on flood depth exhibits strong nonlinearity in time, where effects from previous land subsidence hotspots could be altered by later continuing land subsidence. Spatially, changes in flood depth due to the land subsidence are not only heterogeneous but mixed with coexisting increased and reduced flood depths. The results of this study improve the understanding of the dynamic evolvement of flood inundation due to continuous land subsidence so that better planning can be initiated for sustainable urban development for coastal communities, which is imperative under ongoing climate change and sea level rise.

Corrigendum: The intricate role of Sir2 in oxidative stress response during the post-diauxic phase in Saccharomyces cerevisiae.

[This corrects the article DOI: 10.3389/fmicb.2023.1285559.].

Genome-wide CRISPR-Cas9 screen reveals a persistent null-hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica.

Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress-induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome-wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null-hyphal phenotype-ΔRAS2, ΔRHO5, ΔSFL1, ΔSNF2, and ΔPAXIP1. Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L-1 OD-1 from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

Trehalose biosynthetic pathway regulates filamentation response in Saccharomyces cerevisiae.

BACKGROUND: Diploid cells of Saccharomyces cerevisiae undergo either pseudohyphal differentiation or sporulation in response to depletion of carbon and nitrogen sources. Distinct signaling pathways regulate filamentation and sporulation in response to nutrient limitation. How these pathways are coordinated for implementing distinct cell fate decisions in response to similar nutritional cues is an enigma. Although the role of trehalose pathway in sporulation has been extensively studied, it's possible role in pseudohyphal differentiation has been unexplored. METHODS AND RESULTS: Briefly, tps1 and tps2 mutants were tested for their ability to form pseudohyphae independently as well as in the background of GPR1 and RAS2 mutations. Here, we demonstrate that disruption of TPS1 but not TPS2 inhibits pseudohyphae formation. Interestingly, deletion of GPR1 suppresses the above defect. Further genetic analysis revealed that TPS1 and TPS2 exert opposing effects in triggering filamentation. CONCLUSION: We provide new insights into the role of an otherwise well-known pathway of trehalose biosynthesis in pseudohyphal differentiation. Based on additional data we propose that downstream signaling, mediated by cAMP may be modulated by nutrient mediated differential regulation of RAS2 by TPS1 and TPS2.

Application of Unmanned Aerial Vehicle DEM in flood modeling and comparison with global DEMs: Case study of Atrak River Basin, Iran.

Digital Elevation Models (DEMs) play a significant role in hydraulic modeling and flood risk management. This study initially investigated the effect of Unmanned Aerial Vehicle (UAV) DEM resolutions, ranging from 1 m to 30 m, on flood characteristics, including the inundation area, mean flow depth, and mean flow velocity. Then, the errors of flood characteristics for global DEMs, comprising ALOS (30 m), ASTER (30 m), SRTM (30 m), and TDX (12 m) were quantified using UAV DEM measurements. For these purposes, the HEC-RAS 2D model in steady-state conditions was used to simulate the flood with return periods of 5- to 200 years along 20 km reach of Atrak River located in northeastern Iran. Results indicated when UAV DEM resolution decreased from 1 m to 30 m, inundation area and mean flow depth increased 17.0% (R2 = 0.94) and 10.2% (R2 = 0.96) respectively, while mean flow velocity decreased 16.8% (R2 = -0.94). Validation of the hydraulic modeling using the modified normalized difference water index demonstrated that the HEC-RAS 2D model in conjunction with UAV DEM simulates the flood with ⁓92% accuracy. Comparing the global DEMs with UAV DEM showed that the root mean square error (RMSE) values of the flow depth for ASTER, SRTM, ALOS, and TDX DEMs were 1.77, 1.12, 1.02, and 0.93 m, and the RMSE values of the flow velocity for the same DEMs were 0.81, 0.66, 0.55, and 0.47 m/s, respectively. Furthermore, TDX DEM with a 6.15% error in the inundation area was the nearest to UAV measurements. Overall, TDX DEM revealed a better performance in hydraulic modeling of the fluvial flood characteristics. Hence, it is recommended for environments where high-resolution topography data is scarce. The results of this study could potentially serve as a guideline for selecting global DEMs for hydraulic simulations.

Targeting Oncogenic KRAS in Non-Small-Cell Lung Cancer.

Recent advances in molecular biology and the resultant identification of driver oncogenes have achieved major progress in precision medicine for non-small-cell lung cancer (NSCLC). v-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS) is the most common driver in NSCLC, and targeting KRAS is considerably important. The recent discovery of covalent KRAS G12C inhibitors offers hope for improving the prognosis of NSCLC patients, but the development of combination therapies corresponding to tumor characteristics is still required given the vast heterogeneity of KRAS-mutated NSCLC. In this review, we summarize the current understanding of KRAS mutations regarding the involvement of malignant transformation and describe the preclinical and clinical evidence for targeting KRAS-mutated NSCLC. We also discuss the mechanisms of resistance to KRAS G12C inhibitors and possible combination treatment strategies to overcome this drug resistance.

Application of different building representation techniques in HEC-RAS 2-D for urban flood modeling using the Toce River experimental case.

This paper presents the impact of the choice of building representation techniques and hydrodynamic models on urban flood simulations using HEC-RAS 2-D for the Toce River physical model. To this end, eight numerical models based on previous laboratory experiments were prepared to simulate unsteady urban flooding on each side of building units. Two simplified building layouts (aligned and staggered) were examined, where models were prepared for two different building representation techniques: Building Block (BB) and Building Resistance (BR). Water depth variation computations using the BR and BB techniques were compared to the laboratory measurements and previous studies in the literature. A statistical analysis was performed using both the Root Mean Square Error (RMSE) and the Pearson Product-Moment Correlation Coefficient (PPMCC) in order to evaluate the performance of the models. A sensitivity analysis showed that the proper mesh resolution and model parameter values were obtained. As far as the BR technique is concerned, it is well-suited for representing building units in numerical simulations using high Manning coefficients. Furthermore, this study confirms the importance of the BR technique, which should help researchers in using low-resolution Digital Elevation Models (DEMs) along with open-source programs. Moreover, the study aims to produce a deeper comprehension of numerical modeling and urban flooding.

Seasonal Variations of Rosmarinic Acid and Its Glucoside and Expression of Genes Related to Their Biosynthesis in Two Medicinal and Aromatic Species of Salvia subg. Perovskia.

Salvia abrotanoides Kar. and Salvia yangii B.T. Drew are medicinal and aromatic plants belonging to the subgenus Perovskia and used as herbal medicines in Asia. Derivatives of caffeic acid, mainly rosmarinic acid (RA), are the major phenolic compounds identified in these plants. Understanding the factors and molecular mechanisms regulating the accumulation of pharmacologically and ecologically relevant phenolic metabolites is essential for future biotechnological and medical applications. Up to date, no studies of phenylpropanoid biosynthetic pathway at the transcriptional level has been performed in the Perovskia subgenus. Using a combined qRT-PCR transcriptional activity analysis with LC-MS based metabolic profiling of roots and leaves at the beginning, in the middle and at the end of vegetation season, we have identified the following gene candidates with properties correlating to phenolic acid biosynthesis in S. abrotanoides and S. yangii: PAL, C4H, 4CL, TAT, HPPR, RAS1, RAS2 and Cyp98A14. A comparison of phenolic acid profiles with gene transcript levels revealed the transcriptional regulation of RA biosynthesis in the roots but not the leaves of the studied species. Additionally, RAS1 and Cyp98A14 were identified as rate-limiting steps regulating phenylpropanoid biosynthesis on a transcription level. In the future, this will facilitate the gene-based metabolic enhancement of phenolic compounds production in these promising medicinal herbs.

Tyrosine phosphatase SHP2 inhibitors in tumor-targeted therapies.

Src homology containing protein tyrosine phosphatase 2 (SHP2) represents a noteworthy target for various diseases, serving as a well-known oncogenic phosphatase in cancers. As a result of the low cell permeability and poor bioavailability, the traditional inhibitors targeting the protein tyrosine phosphate catalytic sites are generally suffered from unsatisfactory applied efficacy. Recently, a particularly large number of allosteric inhibitors with striking inhibitory potency on SHP2 have been identified. In particular, few clinical trials conducted have made significant progress on solid tumors by using SHP2 allosteric inhibitors. This review summarizes the development and structure-activity relationship studies of the small-molecule SHP2 inhibitors for tumor therapies, with the purpose of assisting the future development of SHP2 inhibitors with improved selectivity, higher oral bioavailability and better physicochemical properties.

Ras2 is important for growth and pathogenicity in Fusarium circinatum.

In this study, we investigated to possible role of Ras2 in Fusarium circinatum- a fungus that causes pine pitch canker disease on many different pine species and has a wide geographic distribution. This protein is encoded by the RAS2 gene and has been shown to control growth and pathogenicity in a number of fungi in a mitogen-activated protein kinase- and/or cyclic adenosyl monophosphate pathway-dependent manner. The aim was therefore to characterize the phenotypes of RAS2 gene knockout and complementation mutants of F. circinatum. These mutants were generated by transforming protoplasts of the fungus with suitable split-marker constructs. The mutant strains, together with the wild type strain, were used in growth studies as well as pathogenicity assays on Pinus patula seedlings. Results showed that the knockout mutant strain produced significantly smaller lesions compared to the complementation mutant and wild type strains. Growth studies also showed significantly smaller colonies and delayed conidial germination in the knockout mutant strain compared to the complement mutant and wild type strains. Interestingly, the knockout mutant strain produced more macroconidia than the wild type strain. Collectively, these results showed that Ras2 plays an important role in both growth and pathogenicity of F. circinatum. Future studies will seek to determine the pathway(s) through which Ras2 controls these traits in F. circinatum.

Autophagy Stimulus-Dependent Role of the Small GTPase Ras2 in Peroxisome Degradation.

The changing accessibility of nutrient resources induces the reprogramming of cellular metabolism in order to adapt the cell to the altered growth conditions. The nutrient-depending signaling depends on the kinases mTOR (mechanistic target of rapamycin), which is mainly activated by nitrogen-resources, and PKA (protein kinase A), which is mainly activated by glucose, as well as both of their associated factors. These systems promote protein synthesis and cell proliferation, while they inhibit degradation of cellular content by unselective bulk autophagy. Much less is known about their role in selective autophagy pathways, which have a more regulated cellular function. Especially, we were interested to analyse the central Ras2-module of the PKA-pathway in the context of peroxisome degradation. Yeast Ras2 is homologous to the mammalian Ras proteins, whose mutant forms are responsible for 33% of human cancers. In the present study, we were able to demonstrate a context-dependent role of Ras2 activity depending on the type of mTOR-inhibition and glucose-sensing situation. When mTOR was inhibited directly via the macrolide rapamycin, peroxisome degradation was still partially suppressed by Ras2, while inactivation of Ras2 resulted in an enhanced degradation of peroxisomes, suggesting a role of Ras2 in the inhibition of peroxisome degradation in glucose-grown cells. In contrast, the inhibition of mTOR by shifting cells from oleate-medium, which lacks glucose, to pexophagy-medium, which contains glucose and is limited in nitrogen, required Ras2-activity for efficient pexophagy, strongly suggesting that the role of Ras2 in glucose sensing-associated signaling is more important in this context than its co-function in mTOR-related autophagy-inhibition.

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The clinical application of immune checkpoint inhibitors (ICIs) lead to dramatic changes in the treatment strategy for patients with advanced non-small cell lung cancer (NSCLC), but the efficacy of ICIs in oncogene-driven NSCLC is controversial. Existing research shows that the efficacy of ICIs may be related to different types of driver genes, programmed cell death-ligand 1 (PD-L1) level, and tumor mutational burden (TMB). It may involved in other factors, such as clinical characteristics, and immune cell density. ICIs monotherapy or combination therapy may play a role in a subset of oncogene-driven NSCLC patients, but further studies are needed to select these patients, which may be an important direction for the future development of advanced NSCLC.

In S. cerevisiae hydroxycitric acid antagonizes chronological aging and apoptosis regardless of citrate lyase.

Caloric restriction mimetics (CRMs) are promising molecules to prevent age-related diseases as they activate pathways driven by a true caloric restriction. Hydroxycitric acid (HCA) is considered a bona fide CRM since it depletes acetyl-CoA pools by acting as a competitive inhibitor of ATP citrate lyase (ACLY), ultimately repressing protein acetylation and promoting autophagy. Importantly, it can reduce inflammation and tumour development. In order to identify phenotypically relevant new HCA targets we have investigated HCA effects in Saccharomyces cerevisiae, where ACLY is lacking. Strikingly, the drug revealed a powerful anti-aging effect, another property proposed to mark bona fide CRMs. Chronological life span (CLS) extension but also resistance to acetic acid of HCA treated cells were associated to repression of cell apoptosis and necrosis. HCA also largely prevented cell deaths caused by a severe oxidative stress. The molecule could act widely by negatively modulating cell metabolism, similarly to citrate. Indeed, it inhibited both growth reactivation and the oxygen consumption rate of yeast cells in stationary phase. Genetic analyses on yeast CLS mutants indicated that part of the HCA effects can be sensed by Sch9 and Ras2, two conserved key regulators of nutritional and stress signal pathways of primary importance. Our data together with published biochemical analyses indicate that HCA may act with multiple mechanisms together with ACLY repression and allowed us to propose an integrated mechanistic model as a basis for future investigations.