Continuous Endogenous Exhaled CO Monitoring by Laser Spectrometer in Human EVLP Before Lung Transplantation.

Endogenous production of carbon monoxide (CO) is affected by inflammatory phenomena and ischemia-reperfusion injury. Precise measurement of exhaled endogenous CO (eCO) is possible thanks to a laser spectrometer (ProCeas® from AP2E company). We assessed eCO levels of human lung grafts during the normothermic Ex-Vivo Lung Perfusion (EVLP). ProCeas® was connected in bypass to the ventilation circuit. The surgical team took the decision to transplant the lungs without knowing eCO values. We compared eCO between accepted and rejected grafts. EVLP parameters and recipient outcomes were also compared with eCO values. Over 7 months, eCO was analyzed in 21 consecutive EVLP grafts. Two pairs of lungs were rejected by the surgical team. In these two cases, there was a tendency for higher eCO values (0.358 ± 0.52 ppm) compared to transplanted lungs (0.240 ± 0.76 ppm). During the EVLP procedure, eCO was correlated with glucose consumption and lactate production. However, there was no association of eCO neither with edema formation nor with the PO2/FiO2 ratio per EVLP. Regarding post-operative data, every patient transplanted with grafts exhaling high eCO levels (>0.235 ppm) during EVLP presented a Primary Graft Dysfunction score of 3 within the 72 h post-transplantation. There was also a tendency for a longer stay in ICU for recipients with grafts exhaling high eCO levels during EVLP. eCO can be continuously monitored during EVLP. It could serve as an additional and early marker in the evaluation of the lung grafts providing relevant information for post-operative resuscitation care.

Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy.

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Reuse of medical face masks in domestic and community settings without sacrificing safety: Ecological and economical lessons from the Covid-19 pandemic.

The need for personal protective equipment increased exponentially in response to the Covid-19 pandemic. To cope with the mask shortage during springtime 2020, a French consortium was created to find ways to reuse medical and respiratory masks in healthcare departments. The consortium addressed the complex context of the balance between cleaning medical masks in a way that maintains their safety and functionality for reuse, with the environmental advantage to manage medical disposable waste despite the current mask designation as single-use by the regulatory frameworks. We report a Workflow that provides a quantitative basis to determine the safety and efficacy of a medical mask that is decontaminated for reuse. The type IIR polypropylene medical masks can be washed up to 10 times, washed 5 times and autoclaved 5 times, or washed then sterilized with radiations or ethylene oxide, without any degradation of their filtration or breathability properties. There is loss of the anti-projection properties. The Workflow rendered the medical masks to comply to the AFNOR S76-001 standard as "type 1 non-sanitory usage masks". This qualification gives a legal status to the Workflow-treated masks and allows recommendation for the reuse of washed medical masks by the general population, with the significant public health advantage of providing better protection than cloth-tissue masks. Additionally, such a legal status provides a basis to perform a clinical trial to test the masks in real conditions, with full compliance with EN 14683 norm, for collective reuse. The rational reuse of medical mask and their end-of-life management is critical, particularly in pandemic periods when decisive turns can be taken. The reuse of masks in the general population, in industries, or in hospitals (but not for surgery) has significant advantages for the management of waste without degrading the safety of individuals wearing reused masks.

Gene regulation by voltage-dependent calcium channels.

Ca2+ is the most widely used second messenger in cell biology and fulfills a plethora of essential cell functions. One of the most exciting findings of the last decades was the involvement of Ca2+ in the regulation of long-term cell adaptation through its ability to control gene expression. This finding provided a link between cell excitation and gene expression. In this review, we chose to focus on the role of voltage-dependent calcium channels in mediating gene expression in response to membrane depolarization. We illustrate the different pathways by which these channels are involved in excitation-transcription coupling, including the most recent Ca2+ ion-independent strategies that highlight the transcription factor role of calcium channels.

Large-scale analysis by SAGE reveals new mechanisms of v-erbA oncogene action.

BACKGROUND: The v-erbA oncogene, carried by the Avian Erythroblastosis Virus, derives from the c-erbAalpha proto-oncogene that encodes the nuclear receptor for triiodothyronine (T3R). v-ErbA transforms erythroid progenitors in vitro by blocking their differentiation, supposedly by interference with T3R and RAR (Retinoic Acid Receptor). However, v-ErbA target genes involved in its transforming activity still remain to be identified. RESULTS: By using Serial Analysis of Gene Expression (SAGE), we identified 110 genes deregulated by v-ErbA and potentially implicated in the transformation process. Bioinformatic analysis of promoter sequence and transcriptional assays point out a potential role of c-Myb in the v-ErbA effect. Furthermore, grouping of newly identified target genes by function revealed both expected (chromatin/transcription) and unexpected (protein metabolism) functions potentially deregulated by v-ErbA. We then focused our study on 15 of the new v-ErbA target genes and demonstrated by real time PCR that in majority their expression was activated neither by T3, nor RA, nor during differentiation. This was unexpected based upon the previously known role of v-ErbA. CONCLUSION: This paper suggests the involvement of a wealth of new unanticipated mechanisms of v-ErbA action.

Tumor quantification of several fluoropyrimidines resistance gene expression with a unique quantitative RT-PCR method. Implications for pretherapeutic determination of tumor resistance phenotype.

Pretherapeutic determination of tumor resistance to chemotherapy is a main challenge, hindered by the low number of mechanisms characterized at the same time, the small size of the clinical specimens and the heterogeneity of the techniques or the lack of true quantification. The aim of the present study was to determine in real time quantitative RT-PCR, tumor cell expression of several transcripts involved in cancer cell resistance with a unique cDNA sample from a tumor biopsy. The technique had to be suitable in clinical practice for determination of several factors involved in resistance to a given drug family, for example, fluoropyrimidines resistance factors: thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine kinase (TK), dihydrofolate reductase (DHFR), folylpolyglutamate synthetase (FPGS). A frame-shifted artificial construct was designed specifically to work within the same conditions. We validated our technique by quantifying expressions of these 5 genes starting from tissue samples of colorectal carcinoma and the surrounding normal mucosa of 33 different patients. That real time quantitative RT-PCR technique using the frame-shifted artificial construct as a standard provided a real comparison and quantification of different resistance factors. Tumor resistance phenotype determination based on that approach will be investigated in a control study.

The Yin and Yang of Microrna Assay Methods.

OBJECTIVE: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. METHODS: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to AffymetrixTM arrays, qPCR and digital droplet qPCR (BioradTM) based on Taqman microRNA assays (Life TechnologiesTM). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. RESULTS: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. CONCLUSION: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

The Yin and Yang of microRNA Assay Methods.

OBJECTIVE: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. METHODS: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to AffymetrixTM arrays, qPCR and digital droplet qPCR (BioradTM) based on Taqman microRNA assays (Life TechnologiesTM). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. RESULTS: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. CONCLUSION: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

Nuclear life of the voltage-gated Cacnb4 subunit and its role in gene transcription regulation.

The pore-forming subunit of voltage-gated calcium channels is associated to auxiliary subunits among which the cytoplasmic ß subunit. The different isoforms of this subunit control both the plasma membrane targeting and the biophysical properties of the channel moiety. In a recent study, we demonstrated that the Cacnb4 (ß 4) isoform is at the center of a new signaling pathway that connects neuronal excitability and gene transcription. This mechanism relies on nuclear targeting of ß 4 triggered by neuronal electrical stimulation. This re-localization of ß 4 is promoted by its interaction with Ppp2r5d a regulatory subunit of PP2A in complex with PP2A itself. The formation, as well as the nuclear translocation, of the ß 4/ Ppp2r5d/ PP2A complex is totally impaired by the premature R482X stops mutation of ß 4 that has been previously associated with juvenile epilepsy. Taking as a case study the tyrosine hydroxylase gene that is strongly upregulated in brain of lethargic mice, deficient for ß 4 expression, we deciphered the molecular steps presiding to this signaling pathway. Here we show that expression of wild-type ß 4 in HEK293 cells results in the regulation of several genes, while expression of the mutated ß 4 (ß 1-481) produces a different set of gene regulation. Several genes regulated by ß 4 in HEK293 cells were also regulated upon neuronal differentiation of NG108-15 cells that induces nuclear translocation of ß 4 suggesting a link between ß 4 nuclear targeting and gene regulation.

Neuropoietin, a new IL-6-related cytokine signaling through the ciliary neurotrophic factor receptor.

A structural profile-based computational screen was used to identify neuropoietin (NP), a new cytokine. The np gene is localized in tandem with the cardiotrophin-1 gene on mouse chromosome 7. NP shares structural and functional features with ciliary neurotrophic factor (CNTF), cardiotrophin-1, and cardiotrophin-like cytokine. It acts through a membrane receptor complex comprising CNTF receptor-alpha component (CNTFRalpha), gp130, and leukemia inhibitory factor receptor to activate signal transducer and activator of transcription 3 signaling pathway. NP is highly expressed in embryonic neuroepithelia. Strikingly, CNTFRalpha, but not its alternate ligands, CNTF and cardiotrophin-like cytokine, is expressed at the same developmental stages. NP is also observed in retina and to a lesser extent in skeletal muscle. Moreover, NP could sustain the in vitro survival of embryonic motor neurons and could increase the proliferation of neural precursors when associated to epidermal growth factor and fibroblast growth factor 2. Thus, NP is a new ligand for CNTFRalpha, with important implications for murine nervous system development.