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Mutations in SWI/SNF genes are amongst the most common across all human cancers, but efficient therapeutic approaches that exploit vulnerabilities caused by SWI/SNF mutations are currently lacking. Here, we show that the SWI/SNF ATPases BRM/SMARCA2 and BRG1/SMARCA4 promote the expression of p62/GTF2H1 , a core subunit of the transcription factor IIH (TFIIH) complex. Inactivation of either ATPase subunit downregulates GTF2H1 and therefore compromises TFIIH stability and function in transcription and nucleotide excision repair (NER). We also demonstrate that cells with permanent BRM or BRG1 depletion have the ability to restore GTF2H1 expression. As a consequence, the sensitivity of SWI/SNF-deficient cells to DNA damage induced by UV irradiation and cisplatin treatment depends on GTF2H1 levels. Together, our results expose GTF2H1 as a potential novel predictive marker of platinum drug sensitivity in SWI/SNF-deficient cancer cells. SWI/SNF genes are commonly found to be mutated in different cancers. Here the authors report that the remodelers BRM and BRG1 are necessary for efficient nucleotide excision repair by promoting the expression of TFIIH subunit GTF2H1.

Extracellular vesicles (EVs) are secreted by all cells in the CNS, including neurons and astrocytes. EVs are lipid membrane enclosed particles loaded with various bioactive cargoes reflecting the dynamic activities of cells of origin. In contrast to neurons, the specific role of EVs released by astrocytes is less well understood, partly due to the difficulty in maintaining primary astrocyte cultures in a quiescent state. The aim of this study was to establish a human serum-free astrocyte culture system that maintains primary astrocytes in a quiescent state to study the morphology, function, and protein cargoes of astrocyte-derived EVs. Serum-free medium with G5 supplement and serum-supplemented medium with 2% FBS were compared for the culture of commercially available human primary fetal astrocytes. Serum-free astrocytes displayed morphologies similar to in vivo astrocytes, and surprisingly, higher levels of astrocyte markers compared to astrocytes chronically cultured in FBS. In contrast, astrocyte and inflammatory markers in serum-free astrocytes were upregulated 24 h after either acute 2% FBS or cytokine exposure, confirming their capacity to become reactive. Importantly, this suggests that distinct signaling pathways are involved in acute and chronic astrocyte reactivity. Despite having a similar morphology, chronically serum-cultured astrocyte-derived EVs (ADEVs) were smaller in size compared to serum-free ADEVs and could reactivate serum-free astrocytes. Proteomic analysis identified distinct protein datasets for both types of ADEVs with enrichment of complement and coagulation cascades for chronically serum-cultured astrocyte-derived EVs, offering insights into their roles in the CNS. Collectively, these results suggest that human primary astrocytes cultured in serum-free medium bear similarities with in vivo quiescent astrocytes and the addition of serum induces multiple morphological and transcriptional changes that are specific to human reactive astrocytes and their ADEVs. Thus, more emphasis should be made on using multiple structural, molecular, and functional parameters when evaluating ADEVs as biomarkers of astrocyte health.

Marine bioadhesives perform in ways that manmade products simply cannot match, especially in wet environments. Despite their technological potential, bioadhesive molecular mechanisms are still largely understudied, and sea urchin adhesion is no exception. These animals inhabit wave-swept shores, relying on specialized adhesive organs, tube feet, composed by an adhesive disc and a motile stem. The disc encloses a duo-gland adhesive system, producing adhesive and deadhesive secretions for strong reversible substratum attachment. The disclosure of sea urchin Paracentrotus lividus tube foot disc proteome led to the identification of a secreted adhesion protein, Nectin , never before reported in adult adhesive organs but, that given its adhesive function in eggs/embryos, was pointed out as a putative substratum adhesive protein in adults. To further understand Nectin involvement in sea urchin adhesion, Nectin cDNA was amplified for the first time from P. lividus adhesive organs, showing that not only the known Nectin mRNA, called Nectin-1 (GenBank AJ578435), is expressed in the adults tube feet but also a new mRNA sequence, called Nectin-2 (GenBank KT351732), differing in 15 missense nucleotide substitutions. Nectin genomic DNA was also obtained for the first time, indicating that both Nectin-1 and Nectin-2 derive from a single gene. In addition, expression analysis showed that both Nectins are overexpressed in tube feet discs, its expression being significantly higher in tube feet discs from sea urchins just after collection from the field relative to sea urchin from aquarium. These data further advocate for Nectin involvement in sea urchin reversible adhesion, suggesting that its expression might be regulated according to the hydrodynamic conditions.

Cognitive brain function requires the establishment of neuronal networks, which rely on the formation and elongation and branching of axonal projections and the differentiation of presynaptic terminals during development. The cellular events involved in these processes are dependent on protein translation locally in the axon compartment, which enables rapid changes of the axonal proteome in response to neurotrophic cues to regulate axon growth and patterning. How these dynamic changes of the axonal proteome are regulated locally in the axon has been a topic of intensive investigation in the past decade. MicroRNAs are small RNAs known to regulate protein expression by controlling mRNA translation repression/degradation. Recently, these regulatory RNAs have emerged as key players in the modulation of several molecular pathways underlying neuronal differentiation in early stages of development, making this class of non-coding RNAs interesting candidate regulators of local protein translation in the axon during development. However, the role of microRNAs in the axon, in particular axonal outgrowth and presynaptic differentiation, is only beginning to be unravelled. The work described here used next generation sequencing to identify a set of microRNAs enriched in the axonal fraction of primary cortical neurons cultured in compartmentalised microfluidic devices. Following the characterization of axonal microRNA expression levels, two microRNAs, miR-3470b and miR-99a, were selected for subsequent functional studies. The miR-3470 family are mouse-specific repeat-derived microRNAs originating from the B1-element Mus1. Repetitive elements have a major role in shaping the structure and function of the genome, but evidence on the functional relevance of lineage-specific repeat-derived microRNAs is still limited. Inhibition of miR-3470b during early development of primary cortical neurons produced a significant decrease in axonal growth. Moreover, we discovered a significant association between miR-3470b targets and proteins involved in cell-to-cell contact/synaptic pathways. To investigate the role of miR-3470b in the formation of neuronal networks we used a microelectrode array cortical culture model, in which spontaneous electrical activity shows a progressive increase from day 9 in vitro, reflecting the establishment of functional synaptic connections. Inhibition of miR-3470b from the beginning of the third week in culture, when its endogenous levels are high, produced a marked decrease in network activity, suggesting its role in neuron connectivity during cortical neuron development. Functional studies in cortical cultures showed that inhibition of miR-99a in primary cortical neurons produced a significant decrease in axonal growth, with overexpression of a miR-99a mimic increasing axonal length. By using bioinformatics, luciferase reporter assays and functional rescue experiments we could identify a new target for miR-99a in axonal development, heparan sulphate 3-O-sulphotransferase 2 (Hs3st2), an enzyme part of the biosynthesis of heparan sulphate proteoglycans, key players in axon-extracellular matrix interactions. To investigate the effect of axonal miR-99a in the emergence of neuron connectivity and the functioning of neuronal networks we used imaging of calcium transients to assess the spontaneous rhythmic activity of developing neuronal cultures, demonstrating how inhibition of miR-99a alters the patterns of calcium oscillation frequency and synchronicity, suggesting its relevance for network development and maturation. Overall, this work identified two microRNAs capable of regulating axonal outgrowth in the development of mouse cortical neurons in vitro. Both microRNAs were found to exert growth promoting actions in developing axons. Furthermore, this work demonstrated the ability of mR-99a and miR-3470b to act as regulators of neuronal network formation in vitro, raising potential implications to the development of neuronal connectivity in vivo.

The jumbo squid, Dosidicus gigas, is an oceanic top predator in the eastern tropical Pacific that undergoes diel vertical migrations into mesopelagic oxygen minimum zones (OMZs). Besides glycogen breakdown, the pathways of the squid’s metabolic (suppression) strategy are poorly understood. Here, juvenile D. gigas were exposed to oxygen levels found in the OMZ off Gulf of California (1 % O₂, 1 kPa at 10 °C) with the aim to identify, via proteomic tools, eventual anaerobic protein degradation as potential energy source at such depths. Under hypoxia, total protein concentration decreased nonsignificantly from 79.2 ± 12.4 mg g⁻¹ wet mass to 74.7 ± 11.7 mg g⁻¹ wet mass (p > 0.05). Yet, there was a significant decrease in heat-shock protein (Hsp) 90 and α-actinin contents (p < 0.05). The lower α-actinin concentration at late hypoxia was probably related to decreased protection of the Hsp90 chaperon machinery resulting in increased ubiquitination (p < 0.05) and subsequent degradation. Thus, the present findings indicate that D. gigas might degrade, at least under progressing hypoxia, specific mantle proteins anaerobically to increase/maintain anaerobic ATP production and extend hypoxia exposure time. Moreover, the ubiquitin–proteasome system seems to play an important role in hypoxia tolerance, but further investigations are necessary to discover its full potential and pathways.