Viral modulation of type II interferon increases T cell adhesion and virus spread.

During primary varicella zoster virus (VZV) infection, infected lymphocytes drive primary viremia, causing systemic dissemination throughout the host, including the skin. This results in cytokine expression, including interferons (IFNs), which partly limit infection. VZV also spreads from skin keratinocytes to lymphocytes prior to secondary viremia. It is not clear how VZV achieves this while evading the cytokine response. Here, we show that VZV glycoprotein C (gC) binds IFN-γ and modifies its activity, increasing the expression of a subset of IFN-stimulated genes (ISGs), including intercellular adhesion molecule 1 (ICAM1), chemokines and immunomodulatory genes. The higher ICAM1 protein level at the plasma membrane of keratinocytes facilitates lymphocyte function-associated antigen 1-dependent T cell adhesion and expression of gC during infection increases VZV spread to peripheral blood mononuclear cells. This constitutes the discovery of a strategy to modulate IFN-γ activity, upregulating a subset of ISGs, promoting enhanced lymphocyte adhesion and virus spread.

Mercury distribution, bioaccumulation, and biomagnification in riparian ecosystems from a neotropical savanna floodplain, Araguaia River, central Brazil.

Litterfall is the main source of dry deposition of mercury (Hg) into the soil in forest ecosystems. The accumulation of Hg in soil and litter suggests the possibility of transfer to terrestrial invertebrates through environmental exposure or ingestion of plant tissues. We quantified total mercury (THg) concentrations in two soil layers (organic: 0-0.2 m; mineral: 0.8-1 m), litter, fresh leaves, and terrestrial invertebrates of the Araguaia River floodplain, aiming to evaluate the THg distribution among terrestrial compartments, bioaccumulation in invertebrates, and the factors influencing THg concentrations in soil and invertebrates. The mean THg concentrations were significantly different between the compartments evaluated, being higher in organic soil compared to mineral soil, and higher in litter compared to mineral soil and fresh leaves. Soil organic matter content was positively related to THg concentration in this compartment. The order Araneae showed significantly higher Hg concentrations among the most abundant invertebrate taxa. The higher Hg concentrations in Araneae were positively influenced by the concentrations determined in litter and individuals of the order Hymenoptera, confirming the process of biomagnification in the terrestrial trophic chain. In contrast, the THg concentrations in Coleoptera, Orthoptera and Hymenoptera were not significantly related to the concentrations determined in the soil, litter and fresh leaves. Our results showed the importance of organic matter for the immobilization of THg in the soil and indicated the process of biomagnification in the terrestrial food web, providing insights for future studies on the environmental distribution of Hg in floodplains.

Mercury in Fish and Human Hair and Estimated Dietary Intake in a Riverside Community of the Madeira River Basin in the Brazilian Amazon.

Mercury (Hg) is a chemical element that poses risks to human health due to its high toxicity and environmental persistence. We determined the total Hg (THg) and methyl Hg (MeHg) concentrations in hair samples from residents of the Demarcação District (Porto Velho, Rondônia) in the Brazilian Amazon, as well as in water and fish samples, to evaluate factors influencing human exposure. The average THg concentration in human hair was 7.86 ± 6.78 mg kg-1 and it was significantly higher in men, with an increasing trend related to age. There was no significant difference between female age groups. Human exposure to Hg through water was negligible compared to fish consumption. The average weekly intake estimates in the community varied between 1.54 and 4.62 µg kg-1, substantially higher than the recommended limit. The fish species with the highest amounts safe for daily consumption were herbivores and detritivores. Our results contribute to an understanding of how exposure to Hg affects the health of riverside populations and provide insights for new research to develop methods to mitigate such exposure and thus improve the quality of life of Amazonian people.

Objective demonstration of eyelid spasm relief with smartphone and custom software in hemifacial spasm patients.

PURPOSE: To objectively demonstrate eyelid spasms relief in hemifacial spasm (HFS) patients using a smartphone and a custom-made software. METHODS: Nineteen patients with HFS had standardized videos recorded with a smartphone (iPhone 6S, Apple) camera before and 15 days after receiving onabotulinumtoxinA injections. Nineteen age-matched control subjects were also assessed. The Eye Aspect Ratio (EAR) is an algorithm previously described to determine whether the eye is opened or closed. When the eye is closed, EAR tends to be closer to zero. Analogously, if the eye is wide open, values are greater. A custom-made software using the EAR concept was developed and pre- and post-treatment EARs were analyzed to assess HFS patients. RESULTS: Botulinum toxin (BoNT) injections led to a significant increase in the average EAR of the affected side: + 10.4% (p = 0.0175) of HFS patients, compared to baseline. Mean EAR before BoNT applications were significantly lower (16.2%) on the affected side (0.25 ± 0.05) of HFS patients when compared to controls (0.30 ± 0.05, p = 0.004). After BoNT injections, no statistically significant difference was observed for the average EAR between the affected side of HFS patients (0.27 ± 0.04) and controls (p = 0.20). CONCLUSIONS: Use of a smartphone and custom-made software objectively demonstrated eyelid spasm relief in patients with HFS. Additional refinement of this system could permit more accurate assessments of treatment response rates for each patient, making it possible to be used in clinical practice.

Mercury distribution in organisms, litter, and soils of the Middle Araguaia floodplain in Brazil.

Mercury (Hg) is a chemical element that, depending on its concentration, may become toxic to living organisms due to the ability of Hg to bioaccumulate in food chains. In this study, we collected samples of soil, litter, and organisms in the Middle Araguaia floodplain, Brazil. Total mercury (THg) concentrations in litter were significantly higher (p < 0.0001) than that in soil, ranging from 10.68 ± 0.55 to 48.94 ± 0.13 and 20.80 ± 1.07 to 55 .19 ± 1.59 ng g-1, respectively. Total mercury concentration levels in soil showed a linear, inversely proportional relationship with soil organic matter (SOM) contents and soil pH, consistent with the geochemical behavior of chemical elements in flooded environments. Ten orders of organisms were identified, and the average THg concentrations determined in their bodies were up to 20 times higher than those in soil and litter. We found a significant linear relationship between the levels of THg in litter and those found in soil organisms, thereby allowing the prediction of THg concentration levels in soil organisms through the analysis of litter at the sample units. The different dynamics and feeding habits of soil organisms and the concentration of THg in these organisms may be influenced by the river's course. This study provides evidence of the bioaccumulation of THg in soil organisms in the floodplain of the Middle Araguaia River, an important river basin in the Brazilian savanna.

Mercury Contamination as an Indicator of Fish Species' Trophic Position in the Middle Araguaia River, Brazil.

This study evaluates the use of mercury (Hg) concentrations in fish muscle tissue to determine a species' trophic position (TP) in its environment. A campaign conducted in 2019 along 375 km in the middle Araguaia River basin, Brazil, resulted in 239 organisms from 20 species collected. The highest total mercury (THg) concentrations were found in Pellonacastelnaeana (6.93 µg·g-1, wet weight) and in Triportheus elongatus (3.18 µg·g-1, wet weight), whose TPs were different according to the FishBase database. However, they occupied the same trophic level in this study. The intra-specific comparison showed a difference in Hg concentrations between individuals captured in distinct sites. The study of the biota-sediment accumulation factor (BSAF) showed that spatiality interferes with a species' TP. Statistical analyses revealed that when we used a predicted species' TP based on each individual's size, it explained 72% of the variability in THg concentration across all fish species. Multiple regression analysis confirmed that standard length and FishBase values are positively associated with THg (R2 = 0.943). These results point to Hg as a viable indicator of a fish species' TP since it reflects regional, biological, and environmental factors, as demonstrated here for the middle Araguaia River.

Ecological risk of mercury in bottom sediments and spatial correlation with land use in Neotropical savanna floodplain lakes, Araguaia River, Central Brazil.

The Araguaia River floodplain is an important biogeographic boundary between the two largest South American biomes: the Cerrado (Brazilian Savanna) and the Amazon rainforest. The large-scale degradation due to land use conversion experienced in the Araguaia River watershed represents a potential source of mercury (Hg) transport to aquatic ecosystems. However, more information is needed about the dynamics of Hg distribution in savanna floodplains, including the Araguaia River floodplain. We analyzed total mercury (THg) concentrations in the bottom sediments of 30 lakes connected to the Araguaia River and four tributaries, aiming to evaluate the environment's integrity based on the geoaccumulation index (Igeo) and the ecological risk index (ERI). The principal component analysis was applied to examine associations between Hg concentrations, environmental conditions, and land use intensity among lakes associated with different river systems. We used indicator cokriging to identify areas with a greater probability of Hg pollution and ecological risk associated with land use intensity. The land use data showed the predominance of areas used for pasture in the Araguaia River basin. THg concentrations in the sediments varied between 22.6 and 81.9 ng g-1 (mean: 46.5 ± 17.7 ng g-1). Sediments showed no significant pollution (Igeo: 1.35 - 0.50; Classes 1 and 2) and low to considerable ecological risks (ERI: 23.5-85.1; Classes 1 to 3). THg in bottom sediments was associated with land use, water turbidity and electrical conductivity, and sediment organic matter. The indicator cokriging indicates a moderate to strong spatial dependence between land use intensity and Hg, confirming the contribution of anthropic sources to the increment of ecological risk but also the influence of extrinsic factors (such as environmental conditions, geology, and hydrology). Integrating sediment assessment and land use indices with geostatistical methods proved a valuable tool for identifying priority areas for Hg accumulation at a regional scale.

Land use impact on mercury in sediments and macrophytes from a natural lake in the Brazilian savanna.

Freshwater ecosystems are essential to human and wildlife survival. They harbor a wide biodiversity that contributes to ecosystem services. In the ecosystem of the Brazilian Savanna, anthropic activities related to environmental pollution that includes mercury (Hg) is of concern. We studied total mercury concentrations ([THg]) in bottom sediments and macrophytes to assess its short-term (2012 and 2019) impact on a natural lake. Temporal changes in [Hg] were assessed with the geoaccumulation index (Igeo) and the sediment quality guidelines (SQG). The land use index (LUI) was used to assess differences in anthropogenic activities and the Normalized Difference Aquatic Vegetation Index (NDAVI) was used to assess macrophyte biomass density. The bioaccumulation factor (BAF) was used to assess Hg accumulation in macrophytes relative to sediments. The LUI showed an increase in the intensity of agricultural activities in the vicinities of the lake. The NDAVI indicated an increase in the density of macrophytes in the evaluated period. The Igeo indicated that in all sampling sites, pollution levels in sediments increased in 2019 (Igeo > 0), with concentrations exceeding the SQG in 2019. In 2012, [THg] in sediments ranged from 20.7 to 74.6 ng g-1, and in 2019 they ranged from 129.1 to 318.2 ng g-1. In macrophytes, [THg] ranged from 14.0 to 42.1 ng g-1 in 2012, to 53.0 and 175.3 ng g-1 in 2019. [THg] in bottom sediments and macrophytes were significantly higher in the second collection period (p < 0.05). There was no significant difference in BAF values between the periods and no significant bioaccumulation in macrophytes (BAF <1). Our results demonstrated that the macrophytes are not sensitive indicators of Hg pollution in lentic environments of the Brazilian Savanna; however, the increased land use intensity (agriculture, automotive traffic, and urban infrastructure) could increase Hg accumulation in sediments and macrophytes in a short time interval.

Role of Neural Plasticity of Motor Cortex in Gliomas Evaluated by Brain Imaging and Mapping Techniques in Pre- and Postoperative Period: A Systematic Review.

BACKGROUND: Resection of infiltrative neuroepithelial primary brain tumors, such as low-grade gliomas (LGGs) remains a neurosurgical challenge. Usual lack of clinical deficit despite LGGs growing in eloquent brain areas may be explained by reshaping and reorganization of functional networks. The development of modern diagnostic imaging techniques could disclose better understanding of the rearrangement of the brain cortex; however, mechanisms underlying such compensation and how it occurs in the motor cortex remain unclear. This systematic review aims to analyze the neuroplasticity of motor cortex in patients with LGGs, as determined by neuroimaging and functional techniques. METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, medical subject headings (MeSH) and the following terms related to neuroimaging, LGGs and neuroplasticity were used with the Boolean operators AND and OR to synonymous terms in the PubMed database. Among the 118 results, 19 studies were included in the systematic review. RESULTS: Motor function in patients with LGG was characterized by a compensation in the contralateral and supplementary motor areas and premotor functional networks. Furthermore, ipsilateral activation in these types of gliomas was rarely described. Moreover, some studies did not reveal statistical significance in association between functional reorganization and the postoperative period, which can be explained by the low number of patients. CONCLUSION: Our findings suggest a high pattern of reorganization per different eloquent motor areas and gliomas diagnosis. Understanding this process is useful to guide safe surgical resection and to develop protocols that assess the plasticity, even though functional network rearrangement needs to be better characterized by more studies.

Management of eyelid retraction related to thyroid eye disease.

Eyelid retraction related to thyroid eye disease (TED) is a challenging condition. It is one of the main clinical signs and a major diagnostic criterion in TED. This condition may threaten vision due to exposure keratopathy, in addition to its esthetic alterations, which may lead to psychosocial implications and affect the patient's quality of life. Although it is more commonly observed in the upper eyelid, it may be present on both the upper and lower lids. Numerous surgical and nonsurgical treatment modalities have been described and will be reviewed in this article. Management should be based on an individual patient assessment, taking into consideration the disease stage, severity, and clinician experience.

Virus Propagation and Plaque Assay for Dengue Virus.

This chapter describes the methods of propagation and titration for DENV-1 to -4, which are required for most of the experiments using live viruses. DENV does not reach titers as high as those of other viruses or as high as desired for their use in biological assays. Although DENV grows in many different cell lines derived from both vertebrate and invertebrate cells, the most common cell lines used for virus isolation and propagation are mosquito cells C6/36 from Aedes albopictus. Amongst several methods for virus quantification, plaque assay stands out as being very practical and inexpensive. This technique is carried out essentially to estimate virion density in a particular material, being extremely important when evaluating the effect of an antiviral treatment or antibody neutralization capacity, for example. In this assay, viral particles are serially diluted and added onto confluent cell monolayers immersed in a semisolid medium, which is responsible for limiting virus spread throughout the culture. Therefore, regarding the medium consistency, once a virion successfully infects a cell, the newly produced particles can only infect neighboring cells, eventually leading them to death. This phenomenon can be observed as round gaps or plaques in the culture after staining live cells with a crystal violet solution. Then, plaques are counted and used to determine plaque-forming units per milliliter. Here, we describe a protocol established by our group for dengue virus (DENV) titration with porcine kidney (PS) cells.

Mercury spatiality and mobilization in roadside soils adjacent to a savannah ecological reserve.

Mercury (Hg) is a persistent environmental pollutant of global concern. Recognized anthropic contributions to environmental Hg pollution include fuel fossil emissions, soil erosion, and industrial and mining activities. Environmental Hg that enters water bodies can be methylated before entering the food chain and contaminating man and wildlife. We used a kriging approach for sampling and X-ray crystallography to study the pressure of road-traffic Hg emissions on soil Hg concentrations in an ecological reserve (ESECAE) in Central Brazil' savannah. We took samples of organic (n = 144) and mineral (n = 144) layers from the road-side and from the undisturbed soils at 0.1, 1, and 2 km from traffic, inside the ESECAE. Overall, total mercury (THg) concentrations determined by atomic absorption spectrophotometry were significantly higher in the organic layer than in the mineral layer. The mean soil THg in the organic and mineral layers was highest at the roadside (respectively 19.77 ± 12.01 and 16.18 ± 11.54 µg g-1), gradually decreasing with the distance from the road. At 2 km, the mean soil THg was 0.09 ± 0.30 and 0.029 ± 0.03 µg g-1, respectively, for the organic and mineral layers. X-ray crystallography showed mineralogical similarity of the sampled soils, indicating Hg externality, i.e, it did not originate from existing soil minerals. Co-kriging analysis (n = 288) confirmed Hg hotspots on the roadsides and a faster mobilization occurring up to a distance of 1 km for both layers. The soil reception and retention of traffic Hg emissions are mainly in the organic layer and can impact subsoil and adjacent areas. Thus, traffic soil-Hg pollution is limited to the road proximities; THg concentrations are high up to 100 m with an inflection point at 1 km.

ApoA1 Neutralizes Proinflammatory Effects of Dengue Virus NS1 Protein and Modulates Viral Immune Evasion.

Dengue is a mosquito-borne infectious disease that is highly endemic in tropical and subtropical countries. Symptomatic patients can rapidly progress to severe conditions of hemorrhage, plasma extravasation, and hypovolemic shock, which leads to death. The blood tests of patients with severe dengue typically reveal low levels of high-density lipoprotein (HDL), which is responsible for reverse cholesterol transport (RCT) and regulation of the lipid composition in peripheral tissues. It is well known that dengue virus (DENV) depends on membrane cholesterol rafts to infect and to replicate in mammalian cells. Here, we describe the interaction of DENV nonstructural protein 1 (NS1) with apolipoprotein A1 (ApoA1), which is the major protein component of HDL. NS1 is secreted by infected cells and can be found circulating in the serum of patients with the onset of symptoms. NS1 concentrations in plasma are related to dengue severity, which is attributed to immune evasion and an acute inflammatory response. Our data show that the DENV NS1 protein induces an increase of lipid rafts in noninfected cell membranes and enhances further DENV infection. We also show that ApoA1-mediated lipid raft depletion inhibits DENV attachment to the cell surface. In addition, ApoA1 is able to neutralize NS1-induced cell activation and to prevent NS1-mediated enhancement of DENV infection. Furthermore, we demonstrate that the ApoA1 mimetic peptide 4F is also capable of mediating lipid raft depletion to control DENV infection. Taken together, our results suggest the potential of RCT-based therapies for dengue treatment. These results should motivate studies to assess the importance of RCT in DENV infection in vivo. IMPORTANCE DENV is one of the most relevant mosquito-transmitted viruses worldwide, infecting more than 390 million people every year and leading to more than 20 thousand deaths. Although a DENV vaccine has already been approved, its potential side effects have hampered its use in large-scale immunizations. Therefore, new treatment options are urgently needed to prevent disease worsening or to improve current clinical management of severe cases. In this study, we describe a new interaction of the NS1 protein, one of the major viral components, with a key component of HDL, ApoA1. This interaction seems to alter membrane susceptibility to virus infection and modulates the mechanisms triggered by DENV to evade the immune response. We also propose the use of a mimetic peptide named 4F, which was originally developed for atherosclerosis, as a potential therapy for relieving DENV symptoms.

Brain abnormalities on neuroimaging in Children with Congenital Zika Syndrome in Salvador, Brazil, and its possible implications on neuropsychological development.

OBJECTIVE: To characterize the spectrum of brain damages presented in children affected by Congenital Zika Syndrome (CZS), verify the existence of a co-occurrence pattern of these damages and discuss possible implications for the neuropsychological development. METHODS: Descriptive, quantitative, individualized, and cross-sectional study using secondary sources. We selected 136 children with CZS from the database of the Center of Strategic Information on Health Vigilance of the Municipal Office of Salvador, Brazil. We conducted descriptive and multiple correspondence analyses. RESULTS: Among the set of analyzed variables, microcephaly (51.5%), ventriculomegaly (57.4%), and brain calcifications (77.2%) were identified as the most frequent. The multiple correspondence analysis showed that the combination of these three variables (32.4%) was what better represented the spectrum of brain damages in the Central Nervous System. INTERPRETATION: Damage in the sensory-motor, cognitive and language development, as well as neurodevelopmental disorders, are described in the literature as impairments associated, either isolated or combined, with these damages, and it is worth highlighting that, in combined brain damages, impairments tend to be more severe. The findings of this study may contribute to understanding the repercussions of CZS on the neuropsychological development of children affected by the epidemic.

Proteomic and Interactome Approaches Reveal PAK4, PHB-2, and 14-3-3η as Targets of Overactivated Cdc42 in Cellular Responses to Genomic Instability.

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein-protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3η, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.

Network analysis of DUSP12 partners in the nucleus under genotoxic stress.

Dual Specificity Phosphatase 12 is a member of the Atypical DUSP Protein Tyrosine Phosphatase family, meaning that it does not contain typical MAP kinase targeting motifs, while being able to dephosphorylate tyrosine and serine/threonine residues. DUSP12 contains, apart from its catalytic domain, a zinc finger domain, making it one of the largest DUSPs, which displays strong nuclear expression in several tissues. In this work we identified nuclear targets of DUSP12 in two different cancer cell lines (A549 and MCF-7), challenging them with genotoxic stimuli to observe the effect on the networks and to link existing information about DUSP12 functions to the data obtained though mass spectrometry. We found network connections to the cytoskeleton (e.g. IQGAP1), to the chromatin (e.g. HP1BP3), to the splicing machinery and to the previously known pathway of ribosome maturation (e.g. TCOF1), which draw insight into many of the functions of this phosphatase, much likely connecting it to distinct, previously unknown genomic stability mechanisms.

DUSP3/VHR: A Druggable Dual Phosphatase for Human Diseases.

Protein tyrosine kinases (PTK), discovered in the 1970s, have been considered master regulators of biological processes with high clinical significance as targets for human diseases. Their actions are countered by protein tyrosine phosphatases (PTP), enzymes yet underrepresented as drug targets because of the high homology of their catalytic domains and high charge of their catalytic pocket. This scenario is still worse for some PTP subclasses, for example, for the atypical dual-specificity phosphatases (ADUSPs), whose biological functions are not even completely known. In this sense, the present work focuses on the dual-specificity phosphatase 3 (DUSP3), also known as VH1-related phosphatase (VHR), an uncommon regulator of mitogen-activated protein kinase (MAPK) phosphorylation. DUSP3 expression and activities are suggestive of a tumor suppressor or tumor-promoting enzyme in different types of human cancers. Furthermore, DUSP3 has other biological functions involving immune response mediation, thrombosis, hemostasis, angiogenesis, and genomic stability that occur through either MAPK-dependent or MAPK-independent mechanisms. This broad spectrum of actions is likely due to the large substrate diversity and molecular mechanisms that are still under scrutiny. The growing advances in characterizing new DUSP3 substrates will allow the development of pharmacological inhibitors relevant for possible future clinical trials. This review covers all aspects of DUSP3, since its gene cloning and crystallographic structure resolution, in addition to its classical and novel substrates and the biological processes involved, followed by an update of what is currently known about the DUSP3/VHR-inhibiting compounds that might be considered potential drugs to treat human diseases.

Proteomic and interactome approaches reveal PAK4, PHB-2, and 14-3-3n as targets of overactivated Cdc42 in cellular responses to genomic instability

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein–protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3?, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.

Proteomic and interactome approaches reveal PAK4, PHB-2, and 14-3-3n as targets of overactivated Cdc42 in cellular responses to genomic instability

Cdc42, a member of the Rho GTPase family, is an intracellular signaling protein known for its roles in cytoskeleton rearrangements and, more recently, in apoptosis/senescence triggered by genotoxic stress. In some tumor cells, the overactivation of Cdc42 through the expression of constitutively active mutants (G12V or Q61L), GEF activation, or GAP downregulation functions as an antiproliferative or pro-aging mechanism. In this study, human cell lines with different P53 protein profiles were exposed to UV radiation, and the interactions between Cdc42 and proteins that are putatively involved in the DNA damage response and repair mechanisms were screened. The affinity-purified proteins obtained through pull-down experiments of the cell lysates using the recombinant protein baits GST, GST-Cdc42-WT, or GST-Cdc42-G12V were identified by mass spectrometry. The resulting data were filtered and used for the construction of protein–protein interaction networks. Among several promising proteins, three targets, namely, PAK4, PHB-2, and 14-3-3?, which are involved in the cell cycle, apoptosis, DNA repair, and chromatin remodeling processes, were identified. Biochemical validation experiments showed physical and proximal interactions between Cdc42 and the three targets in the cells, particularly after exposure to UV. The results suggest that the molecular mechanisms coordinated by overactivated Cdc42 (with the G12V mutation) to increase the cellular sensitivity to UV radiation and the susceptibility to cell death are collectively mediated by these three proteins. Therefore, the Cdc42 GTPase can potentially be considered another player involved in maintenance of the genomic stability of human cells during exposure to genotoxic stress.