Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in ChlamydomonasEspaña, MINECO BFU2015-68216-PEspaña, Junta de Andalucía CVI-7336 (to JLC), BIO2015-74432-JIN (to MEPP

Phosphorus Availability Regulates TORC1 Signaling via LST8 in Chlamydomonas

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas (Chlamydomonas reinhardtii) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas.España Ministerio de Economía y Competitividad (grants BFU2015-68216-P and PGC2018-099048- B-100 to J.L.C. and grant BIO2015-74432-JIN to M.E.P.-P.)National Science Foundation (CAREER award MCB-1552522 to L.M.H. and grant MCB-1616820 to J.G.U.)European Commission (grant number 750996

Choroidal thickness assessment in keratoconus patients treated with cross-linking compared to healthy population.

Purpose: To analyze the choroidal thickness between patients with keratoconus undergoing cross-linking treatment and a healthy population, as well as to determine the factors that influence choroidal thickness. Methods: This was an observational, analytical, case–control study that was conducted from February 2021 to June 2021. Choroidal thickness was measured at different locations, including the subfoveal, nasal (1000 μm), temporal (1000 μm), superior (1000 μm) and inferior (1000 μm) locations using a Spectral-domain optical coherence tomography with enhanced depth imaging, which allowed us to obtain horizontal and vertical B-scans centered on the fovea. Results: This study included 21 patients with keratoconus (mean age, 21.86 ± 5.28 years) and 28 healthy patients (mean age, 24.21 ± 4.71 years). Choroidal thickness was significantly greater in patients with keratoconus than in healthy patients in each of the following measured locations: subfoveal (P < 0.001); nasal (1000 μm) (P < 0.001), temporal (1000 μm) (P < 0.001), superior (1000 μm) (P < 0.001) and inferior (1000 μm) (P < 0.001) locations. Variables such as age (ρ = − 0.09; P = 0.50) and refraction (ρ = 0.14; P = 0.34) were not found to be associated with choroidal thickness. In a stepwise multiple linear regression, the group was the single variable correlated with choroidal thickness (β = 0.88; P < 0.001). Conclusion: Choroidal thickness is thicker in keratoconus patients treated with cross-linking than in the healthy population. This finding could be associated with inflammatory choroidal mechanisms in keratoconus patients, but more studies are needed. Age and refractive error do not seem to influence choroidal thickness. © 2022, The Author(s), under exclusive licence to Springer Nature B.V

Drying-rewetting cycles in ordinary Portland cement mortars investigated by electrical impedance spectroscopy

[EN] Changes caused in the porous microstructure of ordinary Portland cement (OPC) mortars were studied using electrical impedance spectroscopy (EIS) and equivalent circuit (EqC). Two successive processes, at 20 ºC and 50 °C, consisting of several drying-rewetting cycles, were applied to the mortars. After each cycle, the electrical impedance and the amount of water absorbed were measured. The EIS-EqC methodology allowed to find two distributed impedance relaxations, associated to capillary and gel-C-S-H porosities, respectively. At room temperature any microstructural change was not detected. Nevertheless, at 50 °C two microstructural changes were inferred: 1) the volume of accessible porosity increased (pore coarsening) and 2) the surface of the conductive path through C-S-H gel became more conductive (surface smoothing).The authors would like to thank the Spanish Ministry of Science and Innovation for supporting this research through the project BIA 2011-26947.Fita Fernández, IC.; Cruz González, JM.; Calvo Muñoz, C.; Soriano Martínez, L.; Paya Bernabeu, JJ.; Sánchez Martín, I. (2018). Drying-rewetting cycles in ordinary Portland cement mortars investigated by electrical impedance spectroscopy. Construction and Building Materials. 187:954-963. https://doi.org/10.1016/j.conbuildmat.2018.07.227S95496318

A Novel Approach for the Shape Characterisation of Non-Melanoma Skin Lesions Using Elliptic Fourier Analyses and Clinical Images

[EN] The early detection of Non-Melanoma Skin Cancer (NMSC) is crucial to achieve the best treatment outcomes. Shape is considered one of the main parameters taken for the detection of some types of skin cancer such as melanoma. For NMSC, the importance of shape as a visual detection parameter is not well-studied. A dataset of 993 standard camera images containing different types of NMSC and benign skin lesions was analysed. For each image, the lesion boundaries were extracted. After an alignment and scaling, Elliptic Fourier Analysis (EFA) coefficients were calculated for the boundary of each lesion. The asymmetry of lesions was also calculated. Then, multivariate statistics were employed for dimensionality reduction and finally computational learning classification was employed to evaluate the separability of the classes. The separation between malignant and benign samples was successful in most cases. The best-performing approach was the combination of EFA coefficients and asymmetry. The combination of EFA and asymmetry resulted in a balanced accuracy of 0.786 and an Area Under Curve of 0.735. The combination of EFA and asymmetry for lesion classification resulted in notable success rates when distinguishing between benign and malignant lesions. In light of these results, skin lesions’ shape should be integrated as a fundamental part of future detection techniques in clinical screening.SIJunta de Castilla y Leó

Chemical Modification of a Dehydratase Enzyme Involved in Bacterial Virulence by an Ammonium Derivative: Evidence of its Active Site Covalent Adduct

The first example of an ammonium derivative that causes a specific modification of the active site of type I dehydroquinase (DHQ1), a dehydratase enzyme that is a promising target for antivirulence drug discovery, is described. The resolution at 1.35 Å of the crystal structure of DHQ1 from Salmonella typhi chemically modified by this ammonium derivative revealed that the ligand is covalently attached to the essential Lys170 through the formation of an amine. The detection by mass spectroscopy of the reaction intermediates, in conjunction with the results of molecular dynamics simulations, allowed us to explain the inhibition mechanism and the experimentally observed differences between S. typhi and Staphylococcus aureus enzymes. The results presented here reveal that the replacement of Phe225 in St-DHQ1 by Tyr214 in Sa-DHQ1 and its hydrogen bonding interaction with the conserved water molecule observed in several crystal structures protects the amino adduct against further dehydration/aromatization reactions. In contrast, for the St-DHQ1 enzyme, the carboxylate group of Asp114, with the assistance of this water molecule, would trigger the formation of a Schiff base that can undergo further dehydration reactions until full aromatization of the cyclohexane ring is achieved. Moreover, in vitro antivirulence studies showed that the reported compound is able to reduce the ability of Salmonella Enteritidis to kill A459 respiratory cells. These studies have identified a good scaffold for the design of irreversible inhibitors that can be used as drugs and has opened up new opportunities for the development of novel antivirulence agents by targeting the DHQ1 enzymeFinancial support from the Spanish Ministry of Science and Innovation (SAF2013-42899-R), Xunta de Galicia (GRC2013-041), and the European Regional Development Fund (ERDF) is gratefully acknowledged. E.L. thanks the Xunta de Galicia for his postdoctoral fellowship. A.B. thanks the Miguel Servet Programme ISCIII-FEDER (CP13/00226) and the ISCIIIGeneral Subdirection of Assesment and Promotion of the Research (PI14/00059) for financial supportS

Targeting PDE10A GAF Domain with Small Molecules: A Way for Allosteric Modulation with Anti-Inflammatory Effects

Phosphodiesterase (PDE) enzymes regulate the levels of cyclic nucleotides, cAMP, and/or cGMP, being attractive therapeutic targets. In order to modulate PDE activity in a selective way, we focused our efforts on the search of allosteric modulators. Based on the crystal structure of the PDE10A GAF-B domain, a virtual screening study allowed the discovery of new hits that were also tested experimentally, showing inhibitory activities in the micromolar range. Moreover, these new PDE10A inhibitors were able to decrease the nitrite production in LPS-stimulated cells, thus demonstrating their potential as anti-inflammatory agentsFinancial support from MINECO and FEDER founds (UE program) (project SAF2012-33600) is acknowledged. A.M.G. acknowledges pre-doctoral grants to the CSIC (JAEPre program)S

In silico simulations and functional cell studies evidence similar potency and distinct binding of pacific and caribbean ciguatoxins

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scaleOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The research leading to these results has received funding from the following FEDER-co-funded grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01, ED431C 2021/29, and the Centro singular de investigación de Galicia accreditation 2019–2022 ED431G 2019/03). From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX, and the EUROCIGUA project: “Risk Characterization of Ciguatera Fish Poisoning in Europe” GP/EFSA/AFSCO/2015/03, co-funded by the European Food Safety Authority (EFSA). From Ministerio de Ciencia e Innovación PID2020-115010RB-I00/AEI/10.13039/501100011033. David Castro (D.C.) financial support for the PhD studies was obtained through EUROCIGUA project: Risk characterization of Ciguatera Fish Poisoning in Europe, framework partnership agreement GP/EFSA/AFSCO/2015/03, co-funded by the EFSA. Pablo Estevez (P.E.) acknowledges financial support from the Xunta de Galicia (Regional Government, Spain) under grant ED481A-2018/207S

Flora bacteriana asociada al Octopus maya comercial capturado en la Península de Yucatán, México

Background: Octopus is a fishery product of economic importance worldwide, the main species caught on the coast of the Gulf of Mexico and the Caribbean Sea are Octopus maya and O. vulgaris, the first represents up to 95 % of national production. Goals: Identify the bacterial flora associated with commercial Octopus maya captured in the Yucatan Peninsula, using PCR-DGGE. Methods: From the metagenomic DNAs (mDNAs) extracted from samples representative of the octopus muscle, PCR products were synthesized with universal primers for bacteria (gc338F and 518R) and specific primers for Phylum Firmicutes (FirF: 369 and gcFirR: 1244). They were separated by electrophoresis in denaturing gradient gels (DGGE). The fragmented DNAs were recovered by elution, amplified (338F / 518R and FirF: 369 / FirR: 1244), sequenced and analyzed phylogenetically. Results: The sequences amplified with universal primers, after the DNA fragmentation by DGGE were associated with Psychrobacter urativorans, Psychrobacter sp, Pseudomonas sp, Pseudoalteromonas sp, Shewanella sp, Shewanella baltica, Klebsiella oxytoca, Vibrio aestuarianus, Photobacterium sp, Flavobacterium sp, F. antarcticum, Bizionia sp, Flavobacteriaceae bacterium, Bacillus sp, C. divergens, Cetobacterium somerae, Psychrilyobacter atlanticus, Salinimicrobium sp as well as, Flavobacteriaceae not yet classified. In the sequences amplified with specific primers (Phylum Firmicutes) were identified: Carnobacterium sp, Lactococcus piscium Lactococcus sp, and Vagococcus sp Conclusion: The bacterial genus detected have been reported in samples from marine environments; therefore, can be part of the native microbial diversity associated with commercial O. maya captured in the Yucatan Peninsula, Mexico.Antecedentes: El pulpo es un producto pesquero de importancia económica a nivel mundial, las principales especies capturadas en el litoral del Golfo de México y el mar Caribe son Octopus maya y O. vulgaris, el primero representa hasta el 95 % de la producción nacional. Objetivo: Identificar la flora bacteriana asociada al Octopus maya comercial capturado en la Península de Yucatán, utilizando PCR-DGGE. Métodos: A partir de los ADN metagenómicos (ADNmg) extraídos de muestras representativas del músculo del pulpo, se sintetizaron productos de PCR con iniciadores universales para bacterias (gc338F y 518R) e iniciadores específicos para el filo Firmicutes (FirF:369 y gcFirR: 1244). Mismos que fueron separados por electroforesis en geles de gradiente desnaturalizante (DGGE). Los ADN fragmentados se recuperaron por elución, se amplificaron (338F / 518R y FirF: 369 / FirR: 1244), se secuenciaron y analizaron filogenéticamente. Resultados: Las secuencias amplificadas con iniciadores universales, después de la fragmentación del ADN por DGGE se asociaron con Psychrobacter urativorans, Psychrobacter sp, Pseudomonas sp, Pseudoalteromonas sp, Shewanella sp, Shewanella baltica, Klebsiella oxytoca, Vibrio aestuarianus, Photobacterium sp, Flavobacterium sp, F. antarcticum, Bizionia sp, Flavobacteriaceae bacterium, Bacillus sp, Carnobacterium divergens, Cetobacterium somerae, Psychrilyobacter atlanticus, Salinimicrobium sp, así como, Flavobacteriaceae aún no clasificada. En las secuencias amplificadas con iniciadores específicos (filo Firmicutes) se identificaron: Carnobacterium sp, Lactococcus piscium, Lactococcus sp y Vagococcus sp. Conclusión: Los géneros bacterianos detectados han sido reportados en muestras de ambientes marinos, por lo cual, pueden ser parte de la diversidad microbiana nativa asociada al O. maya comercial capturado en la Península de Yucatán, México