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Abstract There is cumulative evidence that lipid metabolism plays a key role in the pathogenesis of various neurodegenerative disorders including Alzheimer’s disease (AD). Visualising lipid content in a non-destructive label-free manner can aid in elucidating the AD phenotypes towards a better understanding of the disease. In this study, we combined multiple optical molecular-specific methods, Fourier transform infrared (FTIR) spectroscopic imaging, synchrotron radiation-infrared (SR-IR) microscopy, Raman and stimulated Raman scattering (SRS) microscopy, and optical-photothermal infrared (O-PTIR) microscopy with multivariate data analysis, to investigate the biochemistry of brain hippocampus in situ using a mouse model of tauopathy (rTg4510). We observed a significant difference in the morphology and lipid content between transgenic (TG) and wild type (WT) samples. Immunohistochemical staining revealed some degree of microglia co-localisation with elevated lipids in the brain. These results provide new evidence of tauopathy-related dysfunction in a preclinical study at a subcellular level.

Groin hernia repair is the most common procedure performed by general surgeons. The open mesh technique generally represents the main technique for an inguinal repair, but a different approach is often required. Laparoscopy was found to be the answer to minimizing the impact of the preperitoneal open techniques described by Nyhus and Stoppa. The introduction of the totally extraperitoneal hernia repair (TEP) and transabdominal preperitoneal repair (TAPP) in the early 1990s started a new chapter in groin hernia surgery. The minimally invasive techniques vs. open mesh, and then one against the other, soon became a hot topic among abdominal wall surgeons. With time, the number of procedures and indications increased and are still increasing. This review aims to provide an overview of the two main laparoscopic techniques for groin hernia repair, answering the following questions: Who should perform them? What is the learning curve required to minimize complications and optimize operative time? When is a minimally invasive approach indicated, and which one (both in elective and in emergency setting)? How are they performed? The standard techniques have been described in detail, and personal observations from an abdominal wall surgery referral center were added. The main reviews from the early 2000s up to date, which compared the techniques, were analyzed, and the results reported, confirming the comparable safety and efficacy of both these techniques.

Summary: Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.

Abstract Among evolutionary trends shaping phenotypic diversity over macroevolutionary scales, CREA (CRaniofacial Evolutionary Allometry) describes a tendency, among closely related species, for the smaller-sized of the group to have proportionally shorter rostra and larger braincases. Here, we used a phylogenetically broad cranial dataset, 3D geometric morphometrics, and phylogenetic comparative methods to assess the validity and strength of CREA in extinct and living felids. To test for the influence of biomechanical constraints, we quantified the impact of relative canine height on cranial shape evolution. Our results provided support to CREA at the family level. Yet, whereas felines support the rule, big cats, like Pantherinae and Machairodontinae, conform weakly if not at all with CREA predictions. Our findings suggest that Machairodontinae constitute one of the first well-supported exceptions to this biological rule currently known, probably in response to the biomechanical demands and developmental changes linked with their peculiar rostral adaptations. Our results suggest that the acquisition of extreme features concerning biomechanics, evo-devo constraints, and/or ecology is likely to be associated with peculiar patterns of morphological evolution, determining potential exceptions to common biological rules, for instance, by inducing variations in common patterns of evolutionary integration due to heterochronic changes under ratchet-like evolution.

Abstract Background Lyngbya majuscula CCAP 1446/4 is a N2-fixing filamentous nonheterocystous strain that contains two NiFe-hydrogenases: an uptake (encoded by hupSL) and a bidirectional enzyme (encoded by hoxEFUYH). The biosynthesis/maturation of NiFe-hydrogenases is a complex process requiring several accessory proteins for e.g. for the incorporation of metals and ligands in the active center (large subunit), and the insertion of the FeS clusters (small subunit). The last step in the maturation of the large subunit is the cleavage of a C-terminal peptide from its precursor by a specific endopeptidase. Subsequently, the mature large and small subunits can assemble forming a functional enzyme. Results In this work we demonstrated that, in L. majuscula, the structural genes encoding the bidirectional hydrogenase are cotranscribed, and that hoxW (the gene encoding its putative specific endopeptidase) is in the same chromosomal region but transcribed from a different promoter. The gene encoding the putative specific uptake hydrogenase endopeptidase, hupW, can be cotranscribed with the structural genes but it has its own promoter. hoxH, hupL, hoxW and hupW transcription was followed in L. majuscula cells grown under N2-fixing and non-N2-fixing conditions over a 12 h light/12 h dark cycle. The transcription of hoxH, hoxW and hupW did not vary remarkably in the conditions tested, while the hupL transcript levels are significantly higher under N2-fixing conditions with a peak occurring in the transition between the light and the dark phase. Furthermore, the putative endopeptidases transcript levels, in particular hoxW, are lower than those of the respective hydrogenase structural genes. Conclusion The data presented here indicate that in L. majuscula the genes encoding the putative hydrogenases specific endopeptidases, hoxW and hupW, are transcribed from their own promoters. Their transcript levels do not vary notably in the conditions tested, suggesting that HoxW and HupW are probably constantly present and available in the cells. These results, together with the fact that the putative endopeptidases transcript levels, in particular for hoxW, are lower than those of the structural genes, imply that the activity of the hydrogenases is mainly correlated to the transcription levels of the structural genes. The analysis of the promoter regions indicates that hupL and hupW might be under the control of different transcription factor(s), while both hoxH and xisH (hoxW) promoters could be under the control of LexA.

Abstract Neutralizing monoclonal antibodies have achieved great efficacy and safety for the treatment of numerous infectious diseases. However, their neutralization potency is often rapidly lost when the target antigen mutates. Instead of isolating new antibodies each time a pathogen variant arises, it can be attractive to adapt existing antibodies, making them active against the new variant. Potential benefits of this approach include reduced development time, cost, and regulatory burden. Here a methodology is described to rapidly evolve neutralizing antibodies of proven activity, improving their function against new pathogen variants without losing efficacy against previous ones. The reported procedure is based on structure‐guided affinity maturation using combinatorial mutagenesis and phage display technology. Its use against the novel severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is demonstrated, but it is suitable for any other pathogen. As proof of concept, the method is applied to CoV‐X2, a human bispecific antibody that binds with high affinity to the early SARS‐CoV‐2 variants but lost neutralization potency against Delta. Antibodies emerging from the affinity maturation selection exhibit significantly improved neutralization potency against Delta and no loss of efficacy against the other viral sequences tested. These results illustrate the potential application of structure‐guided affinity maturation in facilitating the rapid adaptation of neutralizing antibodies to pathogen variants.

Abstract Background Cyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness. Results A 1.7-fold increase of activity (150 µmol min−1 gDCW −1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L−1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions. Conclusions Results show that under photoautotrophic conditions at a specific activity of 90 µmol min−1 gDCW −1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.

Le Cadre européen commun de référence pour les langues (2001) constitue un repère crucial pour toute intervention didactique en français langue seconde (FLS) et en français langue étrangère (FLE). Ainsi peut se mettre en place un projet de contextualisation ou d’adaptation didactique répondant à une conception située de l’Enseignement-Apprentissage (E-A) et ciblé sur un public aux caractéristiques bien spécifiques. Les référentiels constituent l’aboutissement de travaux d’équipe mis sur pied en vue de rendre toute la richesse des savoirs, des savoir-faire et des savoir-être contenus dans un projet de cours. Et ce, grâce à l’intégration congruente que les sciences humaines et non seulement la didactique des langues et des cultures peuvent apporter.

El siguiente artículo presenta una reflexión en torno al estudio etnográfico de prácticas burocráticas en administraciones estatales. Para ello, recupera parte de una investigación sobre la gestión municipal de la diversión nocturna en la ciudad de Córdoba, desarrollada entre 2011 y 2015. Dicha pesquisa propuso indagar procesos de formación del estado municipal a partir de una etnografía de determinados ejercicios de poder para ordenar la noche. Aquí, se presentan unas situaciones etnográficas que (se) configuraron (en) aquel trabajo para elaborar reflexiones situadas en torno a las modalidades del estar allí, el desafío de etnografiar prácticas de poder y la producción de conocimiento socioantropológico sobre el proceso continuo de formación estatal. Con ello, se busca aportar a las discusiones referidas al hacer etnográfico en relación con instancias de poder, los posicionamientos que como etnógrafas podemos ocupar durante el trabajo de campo y las posibles modalidades de acompañar el cotidiano de una administración municipal en pos de aprehender ejercicios de poder a través de los cuales se (re)producen jerarquías sociales, y el estado extiende su presencia ante las y los sujetas/os.

Summary Aquaculture has been one of the fastest‐growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP‐loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

In the early stages of Alzheimer's disease (AD), the accumulation of the peptide amyloid-β (Aβ) damages synapses and disrupts neuronal activity, leading to the disruption of neuronal oscillations associated with cognition. This is thought to be largely due to impairments in CNS synaptic inhibition, particularly via parvalbumin (PV)-expressing interneurons that are essential for generating several key oscillations. Research in this field has largely been conducted in mouse models that over-express humanised, mutated forms of AD-associated genes that produce exaggerated pathology. This has prompted the development and use of knock-in mouse lines that express these genes at an endogenous level, such as the AppNL-G-F/NL-G-F mouse model used in the present study. These mice appear to model the early stages of Aβ-induced network impairments, yet an in-depth characterisation of these impairments in currently lacking. Therefore, using 16 month-old AppNL-G-F/NL-G-F mice, we analysed neuronal oscillations found in the hippocampus and medial prefrontal cortex (mPFC) during awake behaviour, rapid eye movement (REM) and non-REM (NREM) sleep to assess the extent of network dysfunction. No alterations to gamma oscillations were found to occur in the hippocampus or mPFC during either awake behaviour, REM or NREM sleep. However, during NREM sleep an increase in the power of mPFC spindles and decrease in the power of hippocampal sharp-wave ripples was identified. The latter was accompanied by an increase in the synchronisation of PV-expressing interneuron activity, as measured using two-photon Ca2+ imaging, as well as a decrease in PV-expressing interneuron density. Furthermore, although changes were detected in local network function of mPFC and hippocampus, long-range communication between these regions appeared intact. Altogether, our results suggest that these NREM sleep-specific impairments represent the early stages of circuit breakdown in response to amyloidopathy.

There is strong evidence that the omega-3 polyunsaturated fatty acids (n-3 PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have cardioprotective effects. n-3 PUFAs cause vasodilation in hypertensive patients, in part controlled by increased membrane conductance to potassium. As KATP channels play a major role in vascular tone regulation and are involved in hypertension, we aimed to verify whether n-3 PUFA-mediated vasodilation involved the opening of KATP channels. We used a murine model in which the KATP channel pore subunit, Kir6.1, is deleted in vascular smooth muscle. The vasomotor response of preconstricted arteries to physiologically relevant concentrations of DHA and EPA was measured using wire myography, using the channel blocker PNU-37883A. The effect of n-3 PUFAs on potassium currents in wild-type native smooth muscle cells was investigated using whole-cell patch clamping. DHA and EPA induced vasodilation in mouse aorta and mesenteric arteries; relaxations in the aorta were sensitive to KATP blockade with PNU-37883A. Endothelium removal didn’t affect relaxation to EPA and caused a small but significant inhibition of relaxation to DHA. In the knock-out model, relaxations to DHA and EPA were unaffected by channel knockdown but were still inhibited by PNU-37883A, indicating that the action of PNU-37883A on relaxation may not reflect inhibition of KATP. In native aortic smooth muscle cells DHA failed to activate KATP currents. We conclude that DHA and EPA cause vasodilation in mouse aorta and mesenteric arteries. Relaxations in blocker-treated arteries from knock-out mice demonstrate that KATP channels are not involved in the n-3 PUFA-induced relaxation.

This contribution addresses the problem of optimizing the operation of a batch process where energy is supplied electrically from mixed traditional and renewable sources. The case study considered here is that of an evaporator for the synthesis of titanium dioxide nanoparticles, where an energy-demanding distillation step is optimized and scheduled taking into account the forecasted availability of energy from renewables. The goal is to optimize the utilization of the energy from the renewable sources, without violating process and product constraints. A method is outlined to assess whether predictive demand-side management is a viable option and how to integrate it into the operation of a batch process based on the process characteristics.

Disease outbreaks are a common problem in aquaculture, with serious economic consequences to the sector. Some of the most important bacterial diseases affecting aquaculture are caused by Gram-negative bacteria including Vibrio spp. (vibriosis), Photobacterium damselae (photobacteriosis), Aeromonas spp. (furunculosis; haemorrhagic septicaemia) or Tenacibaculum maritimum (tenacibaculosis). Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria and have been linked to strong immunogenic responses in terrestrial vertebrates, playing a role in disease development. To evaluate LPS effects in fish, we used a hot-phenol procedure to extract LPS from common fish pathogens. A. hydrophila, V. harveyi, T. maritimum and P. damselae purified LPS were tested at different concentrations (50, 100, 250 and 500 µg mL−1) at 3 days post-fertilisation (dpf) Danio rerio larvae, for 5 days. While P. damselae LPS did not cause any mortality under all concentrations tested, A. hydrophila LPS induced 15.5% and V. harveyi LPS induced 58.3% of zebrafish larvae mortality at 500 µg mL−1. LPS from T. maritimum was revealed to be the deadliest, with a zebrafish larvae mortality percentage of 80.6%. Analysis of LPS separated by gel electrophoresis revealed differences in the overall LPS structure between the bacterial species analysed that might be the basis for the different mortalities observed.

El presente artículo es una invitación a pensar, desde la escritura, el proceso de realización de una performance, donde desarrollo algunas ideas en las que se entretejen los conceptos de mapas, territorios y archivos. Este tejido reúne, en un ir y venir por el terreno de los recuerdos, una serie de experiencias de mi producción artística realizada entre los años 2013 y 2019, vinculadas unas al arte de performance y otras al orden de lo familiar. El modo de lo arácnido y la idea de acontecimiento me permitieron desplegar, en un trazar, estas producciones. En cada performance monto dispositivos que denomino mapas, en los cuales manipulo objetos y materiales que son restos y souvenires de algo que aconteció en otro momento, y que conforman algo parecido a un archivo. Intento elaborar un entretejido entre la experiencia de la escritura y la de hacer la performance.

Abstract Background Synechocystis sp. PCC 6803 provides a well-established reference point to cyanobacterial metabolic engineering as part of basic photosynthesis research, as well as in the development of next-generation biotechnological production systems. This study focused on expanding the current knowledge on genomic integration of expression constructs in Synechocystis, targeting a range of novel sites in the chromosome and in the native plasmids, together with established loci used in literature. The key objective was to obtain quantitative information on site-specific expression in reference to replicon copy numbers, which has been speculated but never compared side by side in this host. Results An optimized sYFP2 expression cassette was successfully integrated in two novel sites in Synechocystis chromosome (slr0944; sll0058) and in all four endogenous megaplasmids (pSYSM/slr5037-slr5038; pSYSX/slr6037; pSYSA/slr7023; pSYSG/slr8030) that have not been previously evaluated for the purpose. Fluorescent analysis of the segregated strains revealed that the expression levels between the megaplasmids and chromosomal constructs were very similar, and reinforced the view that highest expression in Synechocystis can be obtained using RSF1010-derived replicative vectors or the native small plasmid pCA2.4 evaluated in comparison. Parallel replicon copy number analysis by RT-qPCR showed that the expression from the alternative loci is largely determined by the gene dosage in Synechocystis, thereby confirming the dependence formerly proposed based on literature. Conclusions This study brings together nine different integrative loci in the genome of Synechocystis to demonstrate quantitative differences between target sites in the chromosome, the native plasmids, and a RSF1010-based replicative expression vector. To date, this is the most comprehensive comparison of alternative integrative sites in Synechocystis, and provides the first direct reference between expression efficiency and replicon gene dosage in the context. In the light of existing literature, the findings support the view that the small native plasmids can be notably more difficult to target than the chromosome or the megaplasmids, and that the RSF1010-derived vectors may be surprisingly well maintained under non-selective culture conditions in this cyanobacterial host. Altogether, the work broadens our views on genomic integration and the rational use of different integrative loci versus replicative plasmids, when aiming at expressing heterologous genes in Synechocystis.

BackgroundSepsis is one of the most important causes of morbidity and mortality in the neonatal period, especially in preterms. Diagnosis is difficult because of specific signs and symptoms. The diagnostic gold standard is blood culture, but its sensibility is low. Much effort has been made to identify early, sensitive, and specific diagnostic markers; among these markers particular attention was paid to procalcitonin. However, reference ranges of serum procalcitonin (PCT) shortly after birth have not been sufficiently studied in healthy preterms, and literature is still contradictory.ObjectivesThe aim of the study is to define PCT age-specific reference ranges in the first 72 h of life in uninfected VLBW preterms.MethodsSerum levels of PCT were assessed for each newborn at birth and every 24 h until the 3rd day of life. The eligible patients were classified into two groups according to their sepsis status.ResultsApproximately 343 patients were enrolled; 28 were septic and 315 non-septic. In non-septic infants, 1,015 determinations of PCT values were performed. Our data showed a trend in average value of PCT to increase after birth up to a peak between 24 and 48 h of life and, subsequently, to fall. The average peak value was 15.12 ng/ml achieved at nearly 36 h of life.ConclusionOur study shows a PCT nomogram of healthy preterms, which is different from the one of term newborns. Data agree with what is reported in literature on the reference ranges and trends of PCT in non-septic preterms shortly after birth.

In a newborn with very precocious liver failure, cholestatic jaundice, and low γ-glutamyl transpeptidase, progressive hepatosplenomegaly induced a progressively worsening respiratory distress, that was successfully treated with steroids. Laboratory and genetic tests did not find any disease usually associated with neonatal cholestasis. However, the patient was positive for a homozygous mutation of the HFE gene, which is associated with hereditary hemochromatosis, a disease with typical onset in adulthood. Although no firm conclusions can be drawn from a single clinical case, this experience suggests that hereditary hemochromatosis could have played a role in the induction of this serious cholestasis, probably already arisen in the uterus. We suggest that hereditary hemochromatosis ought to be included in the panel of the possible causes of neonatal cholestasis and that steroids ought to be added to the pharmacological armamentarium for treating specific conditions which cause cholestasis in newborns.

INTRODUCTION: In tauopathies, altered tau processing correlates with impairments in synaptic density and function. Changes in hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels contribute to disease‐associated abnormalities in multiple neurodegenerative diseases. METHODS: To investigate the link between tau and HCN channels, we performed histological, biochemical, ultrastructural, and functional analyses of hippocampal tissues from Alzheimer's disease (AD), age‐matched controls, Tau35 mice, and/or Tau35 primary hippocampal neurons. RESULTS: Expression of specific HCN channels is elevated in post mortem AD hippocampus. Tau35 mice develop progressive abnormalities including increased phosphorylated tau, enhanced HCN channel expression, decreased dendritic branching, reduced synapse density, and vesicle clustering defects. Tau35 primary neurons show increased HCN channel expression enhanced hyperpolarization‐induced membrane voltage "sag" and changes in the frequency and kinetics of spontaneous excitatory postsynaptic currents. DISCUSSION: Our findings are consistent with a model in which pathological changes in tauopathies impact HCN channels to drive network‐wide structural and functional synaptic deficits. Highlights: Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels are functionally linked to the development of tauopathy.Expression of specific HCN channels is elevated in the hippocampus in Alzheimer's disease and the Tau35 mouse model of tauopathy.Increased expression of HCN channels in Tau35 mice is accompanied by hyperpolarization‐induced membrane voltage "sag" demonstrating a detrimental effect of tau abnormalities on HCN channel function.Tau35 expression alters synaptic organization, causing a loosened vesicle clustering phenotype in Tau35 mice. [ABSTRACT FROM AUTHOR]

Exploiting light to drive redox reactions is currently a hot topic since light is considered as an environmentally friendly source of energy. Consequently, cyanobacteria, which can use light e.g., for generating NADPH, are in the focus of research. Previously, it has been shown that various heterologous redox enzymes could be expressed in these microorganisms. Here we demonstrated the successful inducer-free expression of α-keto-acid dehydrogenases (L-HicDH and D-HicDH) from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008 in Synechocystis sp. PCC 6803 ΔhoxYH mutant using replicative plasmids. While the L-HicDH showed poor activity limited by the amount of expressed enzyme, the D-HicDH was applied both in vivo and in vitro, transforming the selected α-keto acids to the corresponding optically pure (R)-α-hydroxy acids (ee >99%) in up to 53% and 90% conversion, respectively.

Acute disseminated encephalomyelitis (ADEM) is an inflammatory emyelinating disease of the central nervous system that is usually considered a monophasic disease Post-vaccination ADEM has been associated with several vaccines, however, there is scarce information related to SARS-CoV-2 vaccines. We present the case of a 26- year-old female who suffered from ADEM four weeks after Gam-COVID-Vac administration.

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol—∆sps, ∆ggpS, and ∆sps∆ggpS—were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis’ native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

The work is focused on the formulation of a thermodynamically--based constitutive theory for granular, cemented geomaterials, often characterized by a open structure with high porosity and voids of large diameter. Upon mechanical degradation processes such as bond rupture and grain crushing, these material undergo large volumetric and shear strains, and in some cases the deformations are so large that the usual assumption of linearized kinematics may be not applicable. In the first part of this work, the theory of hyperplasticity is extended to the finite deformation regime by adopting a multiplicative split of the deformation gradient into an elastic and a plastic part, under the assumption of material isotropy. Grain breakage and bond damage processes are accounted for through two micromechanically--inspired internal variables. A specific constitutive model for carbonatic cemented sands and calcarenites is proposed as a relevant example of application. In the second part, an implicit stress--point algorithm has been developed which is amenable to closed form linearization, for the implementation of the model into standard FE platforms. A series of numerical tests have demonstrated the accuracy and efficiency of the proposed algorithm. The simulation of plane strain biaxial tests, modeled as boundary--value problems, has highlighted the role played by geometric non--linearity in determining the evolution of the specimen deformation upon reaching a bifurcation condition.

Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories.

[This corrects the article DOI: 10.1371/journal.pone.0226949.].

Baeocytous cyanobacteria (Pleurocapsales/Subsection II) can thrive in a wide range of habitats on Earth but, compared to other cyanobacterial lineages, they remain poorly studied at genomic level. In this study, we sequenced the first genome from a member of the Hyella genus – H. patelloides LEGE 07179, a recently described species isolated from the Portuguese foreshore. This genome is the largest of the thirteen baeocyte-forming cyanobacterial genomes sequenced so far, and diverges from the most closely related strains. Comparative analysis revealed strain-specific genes and horizontal gene transfer events between H. patelloides and its closest relatives. Moreover, H. patelloides genome is distinctive by the number and diversity of natural product biosynthetic gene clusters (BGCs). The majority of these clusters are strain-specific BGCs with a high probability of synthesizing novel natural products. One BGC was identified as being putatively involved in the production of terminal olefin. Our results showed that, H. patelloides produces hydrocarbon with C15 chain length, and synthesizes C14, C16, and C18 fatty acids exceeding 4% of the dry cell weight. Overall, our data contributed to increase the information on baeocytous cyanobacteria, and shed light on H. patelloides evolution, phylogeny and natural product biosynthetic potential.

Summary:. An autologous homologous skin construct (AHSC) has been developed for the repair and replacement of skin. It is created from a small, full-thickness harvest of healthy skin, which contains endogenous regenerative populations involved in native skin repair. A multicenter retrospective review of 15 wounds in 15 patients treated with AHSC was performed to evaluate the hypothesis that a single application could result in wound closure in a variety of wound types and that the resulting tissue would resemble native skin. Patients and wounds were selected and managed per provider’s discretion with no predefined inclusion, exclusion, or follow-up criteria. Dressings were changed weekly. Graft take and wound closure were documented during follow-up visits and imaged with a digital camera. Wound etiologies included 5 acute and chronic burn, 4 acute traumatic, and 6 chronic wounds. All wounds were closed with a single application of AHSC manufactured from a single tissue harvest. Median wound, harvest, and defect-to-harvest size ratio were 120 cm2 (range, 27–4800 cm2), 14 cm2 (range, 3–20 cm2), and 11:1 (range, 2:1–343:1), respectively. No adverse reactions with the full-thickness harvest site or the AHSC treatment site were reported. Average follow-up was 4 ± 3 months. An AHSC-treated area was biopsied, and a micrograph of the area was developed using immunofluorescent confocal microscopy, which demonstrated mature, full-thickness skin with nascent hair follicles and glands. This early clinical experience with ASHC suggests that it can close different wound types; however, additional studies are needed to verify this statement.

Cyanobacteria are a group of photosynthetic prokaryotes that contribute to primary production on a global scale. These microorganisms release vesicles to the extracellular environment, spherical nanosized structures, derived essentially from the outer membrane. Even though earlier works in model Gram-negative bacteria have hypothesized that outer membrane stability is crucial in vesicle formation, the mechanisms determining vesicle biogenesis in cyanobacteria remain unknown. Here, we report on the identification of six candidate genes encoding outer membrane proteins harboring SLH/OprB-domains in the genome of the model cyanobacterium Synechocystis sp. PCC 6803. Using a genetics-based approach, one gene was found to encode an essential protein (Slr1841), while the remaining five are not essential for growth under standard conditions. Vesicle production was monitored, and it was found that a mutant in the gene encoding the second most abundant SLH/OprB protein in Synechocystis sp. PCC 6803 outer membrane (Slr1908) produces more vesicles than any of the other tested strains. Moreover, the Slr1908-protein was also found to be important for iron uptake. Altogether, our results suggest that proteins containing the SLH/OprB-domains may have dual biological role, related to micronutrient uptake and to outer membrane stability, which, together or alone, seem to be involved in cyanobacterial vesicle biogenesis.

The deep-sea constitutes a true unexplored frontier and a potential source of innovative drug scaffolds. Here, we present the genome sequence of two novel marine actinobacterial strains, MA3_2.13 and S07_1.15, isolated from deep-sea samples (sediments and sponge) and collected at Madeira archipelago (NE Atlantic Ocean; Portugal). The de novo assembly of both genomes was achieved using a hybrid strategy that combines short-reads (Illumina) and long-reads (PacBio) sequencing data. Phylogenetic analyses showed that strain MA3_2.13 is a new species of the Streptomyces genus, whereas strain S07_1.15 is closely related to the type strain of Streptomyces xinghaiensis. In silico analysis revealed that the total length of predicted biosynthetic gene clusters (BGCs) accounted for a high percentage of the MA3_2.13 genome, with several potential new metabolites identified. Strain S07_1.15 had, with a few exceptions, a predicted metabolic profile similar to S. xinghaiensis. In this work, we implemented a straightforward approach for generating high-quality genomes of new bacterial isolates and analyse in silico their potential to produce novel NPs. The inclusion of these in silico dereplication steps allows to minimize the rediscovery rates of traditional natural products screening methodologies and expedite the drug discovery process.

The use of the versatile cyanobacterial extracellular polymeric substances (EPS) for biotechnological/biomedical applications implies an extensive knowledge of their biosynthetic pathways to improve/control polymer production yields and characteristics. The multiple copies of EPS-related genes, scattered throughout cyanobacterial genomes, adds another layer of complexity, making these studies challenging and time-consuming. Usually, this issue would be tackled by generating deletion mutants, a process that in cyanobacteria is also hindered by the polyploidy. Thus, the use of the CRISPRi multiplex system constitutes an efficient approach to addressing this redundancy. Here, three putative Synechocystis sp. PCC 6803 kpsM homologues (slr0977, slr2107, and sll0574) were repressed using this methodology. The characterization of the 3-sgRNA mutant in terms of fitness/growth and total carbohydrates, released and capsular polysaccharides, and its comparison with previously generated single knockout mutants pointed towards Slr0977 being the key KpsM player in Synechocystis EPS production. This work validates CRISPRi as a powerful tool to unravel cyanobacterial complex EPS biosynthetic pathways expediting this type of studies.

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.

El artículo propone la articulación de la noción de fuente abierta, proveniente del desarrollo de software, con el funcionamiento y las transformaciones de un lugar y una comunidad de trabajadoras culturales como Casa 13. Dicha articulación toma como punto de partida la noción de “habitar” una casa, un espacio o incluso un concepto, conflicto que atraviesa a Casa 13 desde, por ejemplo, su programa de residencia. Luego se hace foco en la conformación del Taller de Herramientas Libres, para lo cual se reconstruye la trayectoria de quienes promovieron la iniciativa en la casa y la red de relaciones que dio lugar a la convergencia entre este grupo y la comunidad organizadora del Festival Latinoamericano de Instalación de Software Libre en Córdoba. Resultado de este cruce fue la constitución del colectivo Libre Base en Casa 13, experiencia que da pie a las autoras para analizar algunos desbordes en los modos de producción casatrecina (tanto de manera retrospectiva como prospectiva) que invitan a pensar varias preguntas: ¿qué implicaría copiar, estudiar, modificar y redistribuir (las cuatro libertades básicas del copyleft) Casa 13? ¿Cuáles son las herramientas de ese espacio y cómo transformarlas en herramientas libres? ¿Cuáles son las consecuencias políticas de estos contagios entre ámbitos disciplinares?