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The COVID-19 pandemic exacerbated the use of medical protective equipment, including face masks, to protect the individual from the virus. This work studies the feasibility of using these materials as fuel for thermochemical processes for the production of syngas. A preliminary physic-chemical characterization was made by means of moisture and ash determination, thermogravimetric analysis, X-ray fluorescence. Afterward, pyrolysis and gasification tests were executed in a laboratory-scale fluidized bed reactor with chirurgical and FFP2 masks investigating four temperature levels and three different operating conditions (fluidizing agents and dry/wet sample). A qualitative and quantitative analysis of condensable aromatic hydrocarbons in the produced gas, collected during the test campaign, was performed employing a gas chromatograph-mass spectrometer. The experimental data from the tests were used to propose a hybrid approach to simulate the gasification process, based on experimental laws for the devolatilization step and a thermodynamic equilibrium approach for char gasification. The resulting data were compared with a thermodynamic equilibrium model, showing that the new approach captures non-equilibrium effects always present in real gasifiers operation.

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.

Cullin-3 (Cul3) functions as a scaffolding protein in the Bric-a-brac, Tramtrack, Broad-complex (BTB)–Cul3–Rbx1 ubiquitin E3 ligase complex. Here, we report a previously undescribed role for Cul3 complexes in late endosome (LE) maturation. RNAi-mediated depletion of Cul3 results in a trafficking defect of two cargoes of the endolysosomal pathway, influenza A virus (IAV) and epidermal growth factor receptor (EGFR). IAV is able to reach an acidic endosomal compartment, coinciding with LE/lysosome (LY) markers. However, it remains trapped or the capsid is unable to uncoat after penetration into the cytosol. Similarly, activation and subsequent ubiquitination of EGFR appear normal, whereas downstream EGFR degradation is delayed and its ligand EGF accumulates in LE/LYs. Indeed, Cul3-depleted cells display severe morphological defects in LEs that could account for these trafficking defects; they accumulate acidic LE/LYs, and some cells become highly vacuolated, with enlarged Rab7-positive endosomes. Together, these results suggest a crucial role of Cul3 in regulating late steps in the endolysosomal trafficking pathway.

The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.

It is unclear whether viral particles can induce membrane curvature. Binding of Simian virus 40 (SV40) to the GM1 ganglioside on host plasma membranes leads to membrane curvature and the formation of invaginations in cells and in giant unilamellar vesicles, an effect required for viral infection. Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.

Recent advances in live Ca 2+ imaging with increasing spatial and temporal resolution offer unprecedented opportunities, but also generate an unmet need for data processing. Here we developed SICT, a MATLAB program that automatically identifies rapid Ca 2+ rises in time-lapse movies with low signal-to-noise ratios, using fluorescent indicators. A graphical user interface allows visual inspection of automatically detected events, reducing manual labour to less than 10% while maintaining quality control. The detection performance was tested using synthetic data with various signal-to-noise ratios. The event inspection phase was evaluated by four human observers. Reliability of the method was demonstrated in a direct comparison between manual and SICT-aided analysis. As a test case in cultured neurons, SICT detected an increase in frequency and duration of spontaneous Ca 2+ transients in the presence of caffeine. This new method speeds up the analysis of elementary Ca 2+ transients.

Alport syndrome (ATS) is a hereditary nephropathy often associated with sensorineural hypoacusis and ocular abnormalities. Mutations in the COL4A5 gene cause X-linked ATS. Mutations in COL4A4 and COL4A3 genes have been reported in both autosomal recessive and autosomal dominant ATS. The conventional mutation screening, performed by DHPLC and/or Sanger sequencing, is time-consuming and has relatively high costs because of the absence of hot spots and to the high number of exons per gene: 51 ( COL4A5 ), 48 ( COL4A4 ) and 52 ( COL4A3 ). Several months are usually necessary to complete the diagnosis, especially in cases with less informative pedigrees. To overcome these limitations, we designed a next-generation sequencing (NGS) protocol enabling simultaneous detection of all possible variants in the three genes. We used a method coupling selective amplification to the 454 Roche DNA sequencing platform (Genome Sequencer junior). The application of this technology allowed us to identify the second mutation in two ATS patients (p.Ser1147Phe in COL4A3 and p.Arg1682Trp in COL4A4 ) and to reconsider the diagnosis of ATS in a third patient. This study, therefore, illustrates the successful application of NGS to mutation screening of Mendelian disorders with locus heterogeneity.

Recent advances in live Ca imaging with increasing spatial and temporal resolution offer unprecedented opportunities, but also generate an unmet need for data processing. Here we developed SICT, a MATLAB program that automatically identifies rapid Ca rises in time-lapse movies with low signal-to-noise ratios, using fluorescent indicators. A graphical user interface allows visual inspection of automatically detected events, reducing manual labour to less than 10% while maintaining quality control. The detection performance was tested using synthetic data with various signal-to-noise ratios. The event inspection phase was evaluated by four human observers. Reliability of the method was demonstrated in a direct comparison between manual and SICT-aided analysis. As a test case in cultured neurons, SICT detected an increase in frequency and duration of spontaneous Ca transients in the presence of caffeine. This new method speeds up the analysis of elementary Ca transients.

The implementation of next-generation sequencing (NGS) technologies in clinical diagnostics requires rigorous validation of sequencing platforms and analytical workflows. In this study, we validated the performance of the Illumina NovaSeq6000 Research Use Only (RUO) platform, combined with automated library preparation using the Hamilton Microlab STAR system, by comparison to the CE-IVD certified NovaSeq6000Dx platform, which currently relies on manual library preparation. A total of 96 clinical samples underwent whole-exome sequencing (WES) on both platforms. Variant detection performance was assessed for single nucleotide variants (SNVs) and copy number variants (CNVs). The RUO platform demonstrated 100% concordance with the CE-IVD system for clinically relevant SNVs, with full agreement across positive, negative, and overall percent agreement metrics. For CNVs larger than 150 kb, the positive percent agreement was 79%, rising to 91.7% for CNVs >900 kb. Coverage uniformity and autosomal callability were consistently high across platforms. These results confirm the analytical equivalence of the NovaSeq6000 RUO configuration with automated library preparation for clinical-grade WES. This validation framework supports the adoption of scalable, cost-effective workflows that can achieve diagnostic performance comparable to CE-IVD certified systems and may facilitate routine implementation of exome sequencing in clinical laboratories.

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that upon glucose withdrawal the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and to control gene expression during stress. The nuclear poly(A)-binding protein Nab2 forms RNA-containing condensate-like structures upon glucose starvation and upon acute cellular depletion of the DEAD-box ATPase Dbp5 The Nab2 multimerization interface but not the intrinsically disordered regions (IDRs) are essential for condensation in vitro and in vivo Glucose stress leads to poly(A) RNA retention in the nucleus, which is affected by the state of the Nab2 condensate