The integrated stress response in pulmonary disease.

The respiratory tract and its resident immune cells face daily exposure to stress, both from without and from within. Inhaled pathogens, including severe acute respiratory syndrome coronavirus 2, and toxins from pollution trigger a cellular defence system that reduces protein synthesis to minimise viral replication or the accumulation of misfolded proteins. Simultaneously, a gene expression programme enhances antioxidant and protein folding machineries in the lung. Four kinases (PERK, PKR, GCN2 and HRI) sense a diverse range of stresses to trigger this "integrated stress response". Here we review recent advances identifying the integrated stress response as a critical pathway in the pathogenesis of pulmonary diseases, including pneumonias, thoracic malignancy, pulmonary fibrosis and pulmonary hypertension. Understanding the integrated stress response provides novel targets for the development of therapies

Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

Background/Aims: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. Methods: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. Results: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. Conclusion: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy

Functional validation of EIF2AK4 (GCN2) missense variants associated with pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1½ GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.MRC (MCMB MR/V028669/1 and MR/R009120/1), EPSRC (EP/R03558X/1), Cambridge Biomedical Research Centre (BRC-1215-20014); British Lung Foundation (BLF), Asthma+Lung UK (ALUK), Royal Papworth Hospital, Victor Philip Dahdaleh Foundation philanthropic funding from Rick Medlock EPSRC (EP/R03558X/1), BHF (RE/18/1/34212), Evelyn Trust (Grant 22/03), Cambridge-Tsinghua collaborative programme on sustainability and emerging technologies Cambridge Trust)

Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2.

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart

Geomorphology of the Castel de' Britti area (Northern Apennines, Italy): an example of teaching geomorphological mapping in a traditional and practical way

International audienceTeaching how to map the geomorphology of an area cannot be performed in a satisfying manner only in the lecture room, but requires practical exercises both in the laboratory and in the field. A preliminary study of existing geological maps, geomorphological legends and symbols used in Italy and of the landslide inventory preceded a detailed four-day-long field mapping campaign carried out by students in the framework of their MSc course on ‘Geomorphological Mapping’ at Bologna University. The Geomorphological Map at 1:5000 produced by some of these students is presented in this paper.The study area is located in the northern Apennines, a few kilometres East of Bologna, along the Idice Valley (N-Italy). Lithologies are mainly composed of clayey and marly sequences ranging in age from Cretaceous to Plio-Pleistocene, sands and sandstones of Pleistocene age, and Messinian gypsum, these last being the most resistant rocks.Besides the greater scale used in this map, allowing for a more detailed representation of the mapped features, this map also shows the recent evolution of landslides in this actively mass wasting area