Large-scale deep tissue voltage imaging with targeted-illumination confocal microscopy.

Voltage imaging with cellular specificity has been made possible by advances in genetically encoded voltage indicators. However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines the signal-to-noise ratio and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating while also maximizing signal detection efficiency. The resulting benefits in signal-to-noise ratio and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different genetically encoded voltage indicator classes.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state.

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/ß proteins both on fast ps/ns as well as slower µs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

Ecosystem CO2 release driven by wind occurs in drylands at global scale.

Subterranean ventilation is a non-diffusive transport process that provokes the abrupt transfer of CO2 -rich air (previously stored) through water-free soil pores and cracks from the vadose zone to the atmosphere, under high-turbulence conditions. In dryland ecosystems, whose biological carbon exchanges are poorly characterized, it can strongly determine eddy-covariance CO2 fluxes that are used to validate remote sensing products and constrain models of gross primary productivity. Although subterranean ventilation episodes (VE) may occur in arid and semi-arid regions, which are unsung players in the global carbon cycle, little research has focused on the role of VE CO2 emissions in land-atmosphere CO2 exchange. This study shows clear empirical evidence of globally occurring VE. To identify VE, we used in situ quality-controlled eddy-covariance open data of carbon fluxes and ancillary variables from 145 sites in different open land covers (grassland, cropland, shrubland, savanna, and barren) across the globe. We selected the analyzed database from the FLUXNET2015, AmeriFlux, OzFlux, and AsiaFlux networks. To standardize the analysis, we designed an algorithm to detect CO2 emissions produced by VE at all sites considered in this study. Its main requirement is the presence of considerable and non-spurious correlation between the friction velocity (i.e., turbulence) and CO2 emissions. Of the sites analyzed, 34% exhibited the occurrence of VE. This vented CO2 emerged mainly from arid ecosystems (84%) and sites with hot and dry periods. Despite some limitations in data availability, this research demonstrates that VE-driven CO2 emissions occur globally. Future research should seek a better understanding of its drivers and the improvement of partitioning models, to reduce uncertainties in estimated biological CO2 exchanges and infer their contribution to the global net ecosystem carbon balance.

PLGA-TiO2 as a Carrier System for Drug Release.

This paper reports the results of the PLGA-TiO2 nanocomposite regarding the green synthesis of titanium dioxide nanoparticles using a natural extract, its characterization, and encapsulation with poly(lactic-co-glycolic acid) (PLGA). UV-visible spectrometry was used for the identification of terpenes present in the extracts. The morphology of the nanoparticles was determined by scanning electron microscopy. Infrared spectroscopy was used for the determination of functional groups, while X-ray diffraction was used to determine the crystal structure. The analysis of the extended release of the encapsulated extract in the matrix of the nanomaterial resulted in a maximum visible UV absorbance at approximately 260 nm and confirmed the synthesis of titanium dioxide nanoparticles. Moreover, terpenes enhance synthesis and stabilize titanium dioxide nanoparticles. The synthesized structures are spherical and amorphous, 44 nm in size, and encapsulated at 65 nm.

Usefulness of an organ donation opinion survey as a tool to promote organ donation among the adolescent population.

Opinion surveys on health issues are considered health promotion tools. However, no studies have confirmed this in deceased organ donation for transplantation. This study aimed to analyse the impact of completing an opinion questionnaire about deceased organ donation on the attitude towards organ donation among the adolescent population. This longitudinal study with repeated measurements of attitude towards deceased organ donation was conducted with an adolescent population. The measurement instrument was a validated questionnaire of the attitude towards organ donation (PCID-DTO-Ríos). The study process involved the application of the questionnaire at an initial time, 1 month later, and 6 months later. A total of 1374 adolescents participated in this study. The favourable attitude towards donation was 43.1%, which fell to 41.4% at 1 month (P = 0.145), and to 39.7% at 6 months (P = 0.019). Changes in the attitude were observed in all groups, both 1 and 6 months after the questionnaire was completed. There was no objective relationship between the adolescent's socio-family environment and the effect of completing the questionnaire on their attitude towards deceased organ donation. In conclusion, the opinion questionnaire was not useful for promoting organ donation and did not have a positive effect on adolescents' attitudes towards organ donation in the medium or long term.

Potentiometric Study of Carbon Nanotube/Surfactant Interactions by Ion-Selective Electrodes. Driving Forces in the Adsorption and Dispersion Processes.

The interaction (adsorption process) of commercial ionic surfactants with non-functionalized and functionalized carbon nanotubes (CNTs) has been studied by potentiometric measurements based on the use of ion-selective electrodes. The goal of this work was to investigate the role of the CNTs' charge and structure in the CNT/surfactant interactions. Non-functionalized single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), and amine functionalized SWCNT were used. The influence of the surfactant architecture on the CNT/surfactant interactions was also studied. Surfactants with different charge and hydrophobic tail length (sodium dodecyl sulfate (SDS), octyltrimethyl ammonium bromide (OTAB), dodecyltrimethyl ammonium bromide (DoTAB) and hexadecyltrimethyl ammonium bromide (CTAB)) were studied. According to the results, the adsorption process shows a cooperative character, with the hydrophobic interaction contribution playing a key role. This is made evident by the correlation between the free surfactant concentration (at a fixed [CNT]) and the critical micellar concentration, cmc, found for all the CNTs and surfactants investigated. The electrostatic interactions mainly determine the CNT dispersion, although hydrophobic interactions also contribute to this process.

Chemometric comparison of almond oxidation rates using kinetic parameters obtained by infrared spectroscopy.

BACKGROUND: The study of almond fat stability is essential from a quality control perspective meanly because, in most of the cases, almonds are sold skinned and thermally treated. In this work an alternative method to Rancimat test based on attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectrometry was adapted for checking the induced degradation at 75 °C of seven almond oil cultivars, three of the top Californian producing varieties, and, four traditional cultivars harvested in Spain. RESULTS: The thermal oil degradation evolution was followed by measuring the changes in the absorbance of the selected FTIR spectra bands (3470, 3006, 1730, 1630, 988 and 970 cm-1 ). A first-order kinetic behaviour was observed, after an induction time in all bands. CONCLUSIONS: Kinetic coefficients and induction times were easily obtained as the absorbance values (from difference spectra) fitted to pseudo-first-order kinetics after the induction time. Principal component analysis was applied to the kinetic parameters to visualize which variables could be useful to classify the almond cultivars based on their resistance to thermal oxidation processes. It was found that selecting only the induction times corresponding to the bands 3470, 3006, 1630 and 970 cm-1 a separate classification of the Californian cultivars from the Spanish ones was possible. Finally, a linear discriminant analysis was assayed using only the four induction times previously selected. Validated classification and correct in 100% of the cases was obtained for all the samples based on their Spanish or Californian origin. © 2020 Society of Chemical Industry.

Septins restrict inflammation and protect zebrafish larvae from Shigella infection.

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation.

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore-forming toxins.

During infection, plasma membrane (PM) blebs protect host cells against bacterial pore-forming toxins (PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT-induced blebbing. Gp96 interacts with non-muscle myosin heavy chain IIA (NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA-Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod-like structures from polarized migrating cells, a process that also promotes NMHCIIA-Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O (LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO-dependent killing.

Clinical results after the implementation of cell-free fetal DNA detection in maternal plasma.

AIM: Detection of cell-free fetal DNA in maternal blood is a type of noninvasive prenatal diagnosis test (NIPT), which has already been known for some time but has not yet been introduced in most of public hospitals in Spain. How the implementation of cell-free fetal DNA (cffDNA) in a contingent protocol has influenced the aneuploidy screening in our hospital is described. METHODS: Two cohorts of patients with positive combined screening were compared: the first one (years 2012-2013, 5747 patients) from a period of time in which the protocol valid until March 2016 - that included the use of invasive procedures - was applied; and the second one in which the current protocol - that included NIPT versus invasive procedures - was applied (first 7 months after protocol implementation, 898 patients). RESULTS: Comparison of both periods resulted in a 60.5% reduction of invasive procedures (P < 0,001) preserving the same chromosomopathy detection rate. The ratio of positive invasive procedures-indicated invasive procedures was improved by 15% in the first period to 50% in the second period (P = 0.01). CONCLUSION: NIPT introduction has caused a significant reduction of 60.5% of IP in high chromosomopathy risk patients after combined screening without modifying detection rate.

Man Thou Art Dust: Rites of Passage in Austere Times.

In response to recent calls for further cross-disciplinary research on austerity and a deeper sociological understanding of the impact and aftermath of the economic crisis on individuals and societies, this article builds on extant austerity literature through an exploration of its effects on European men. Informed by theories of liminality and rites of passage, this qualitative investigation examines the experience of austerity from the perspective of 11 men through the three liminal stages of separation, transition and reaggregation and investigates its impact on their identity, responsibilities and expectations. Our findings reveal the negative experiences of alienation and outsiderhood alongside positive experiences of communitas, solidarity and comradeship. The study provides a nuanced understanding of modern male Europeans and their 'rites of passage' through austere times.

Synthesis, Spectral Characterization of Several Novel Pyrene-Derived Aminophosphonates and Their Ecotoxicological Evaluation Using Heterocypris incongruens and Vibrio fisheri Tests.

Four diphenyl pyrene-derived aminophosphonates were synthesized. Attempts were made to synthesize diphenyl N-(R)-α-methylbenzylamino(pyren-1-yl)methylphosphonate (3e) in order to obtain the chiral aminophosphonate bearing a pyrene moiety. Because these attempts failed, dimethyl and dibenzyl N-(R)-α-methylbenzyl substituted aminophosphonates 4 and 5 were synthesized and the predominant diastereoisomer of dimethyl aminophosphonate 4 was isolated. The resolution of the diastereomeric mixture of 5 failed. Aminophosphonates 3a-d and the predominant diastereoisomer of 4 were investigated in terms of their ecotoxicity using tests performed on the ostracode Heterocypris incongruens and the fluorescent bacterium Vibrio fisheri. The tests confirmed the moderate-to-high ecotoxicity of aminophosphonates 3a-d and 4, but no evident correlation between the structure and toxicity has been found.

Synthesis, fluorescence properties and the promising cytotoxicity of pyrene-derived aminophosphonates.

A large series of variously substituted amino(pyren-1-yl)methylphosphonic acid derivatives was synthesized using a modified aza-Pudovik reaction in 20-97% yields. The fluorescence properties of the obtained compounds were investigated revealing that N-alkylamino(pyren-1-yl)methylphosphonic derivatives are stronger emissive compounds than the corresponding N-aryl derivatives. N-Benzylamino(pyren-1-yl)methylphosphonic acid displayed strong fluorescence (ΦF = 0.68) in phosphate-buffered saline (PBS). The influence of a series of derivatives on two colon cancer cell lines HT29 and HCT116 was also investigated. The most promising results were obtained for N-(4-methoxyphenyl)amino(pyren-1-yl)methylphosphonate, which was found to be cytotoxic for the HCT116 cancer cell line (IC50 = 20.8 µM), simultaneously showing weak toxicity towards normal lymphocytes (IC50 = 230.8 µM).

The zebrafish as a new model for the in vivo study of Shigella flexneri interaction with phagocytes and bacterial autophagy.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.

Results of transanal endorrectal descent in Hirschprung's disease.

OBJECTIVE: To present our experience of patients with Hirschsprung disease (HD) operated by transanal endorrectal descent (TED). METHODS: A retrospective study performed of TEDs. We correlate fundamentally the aganglionic segment length with: Complications, need for colostomy and surgical approach. RESULTS: Between 2003 and 2012 we performed 73 TED (57 men), 78.6% diagnosed in the neonatal period. The aganglionic segment length evidenced in the enema was correlated with surgical findings in 68.9% of cases, being higher in the short forms than in the long segments (80.9 vs. 44%, P<.001). Four patients required preoperative colostomy. The mean age of surgery was 6.3 months (4.5 to 33.7). Short forms represented 64.4% of cases. The abdominal approach was necessary in 10 patients, 9 of these were long/total colonic aganglionic segment. Postoperative enterocolitis presented in 10,9% (9 patients), with no relation with the length of aganglionic or the existence of residual dysplasia. Obstructions occurred in 19.2% of the long form or pancolonic forms. Currently, of the evaluable patients, 5 present constipationand 12 present occasional leakage. CONCLUSIONS: TED is the surgery of choice for patients with HD. The majority can be handled by preoperative «nursing¼ without colostomy. The diagnostic tests of choice are manometry with suction biopsy. No significant correlation was found between enterocolitis and length of aganglionic segment, although we observed a drastic reduction of postoperative enterocolitis, since the introduction of technical changes, such as the use of postoperative rectal tubes and the posterior section of the cuff muscles.

Population-based integrated care funding values and guiding principles: An empirical qualitative study.

There is wide agreement on the benefits of integrated care; yet funding barriers persist. We suggest that funding models could currently hinder quality of care and that identifying values is necessary to designing adequate funding models. Yet it is currently unclear what are these values that ought to shape healthcare policy decisions. To fill in this gap, we conducted semi-structure interviews with fourteen health policy officials, managers, and researchers to elicit and explore how they conceptualize the values and guiding principles underlying these funding policies. Our findings suggest that values guide population-based integrated funding models, namely: accountability & integrity, transparency, equity, and innovation. Overall, funding mechanisms could incentivize integrated population-based care when the following conditions are met: a) there is transparent governance, with a whole-system approach, political will, and engagement and collaboration across health system partners, organizations and institutions, b) regulatory and evaluative frameworks support accountability including in decision-making, in outcomes and quality of care, as well as financial accountability; c) funding is equitable with a fair distribution of resources and supports accessibility to services; and d) funding mechanisms design and implementation include innovation enabling change, which are continuously evaluated. These values and guiding principles could be used in the development of funding models and future studies need to evaluate the effect of these values on decisions made by policy makers with respect to funding allocations and investments.

Exploring uterine contractility frequency in infertile population: A comparative study among different control groups with and without a C-section defect.

OBJECTIVE: Women undergoing IVF who have had a previous c-section (CS) have a lower live birth rate than those with a previous vaginal delivery. However, the precise underlying mechanisms need clarification. Does a previous CS affect the pattern of uterine contractility?. METHODS: Prospective evaluation in patients undergoing frozen blastocyst embryo transfer in medicated endometrial preparation cycles. Twenty patients were included in groups: A/nulliparous. B/previous vaginal delivery. C/ previous CS without a niche, whereas fifteen patients were recruited in group D (CS and a niche). Patients employed estradiol compounds and 800 mg vaginal progesterone. A 3D-scan was performed the transfer-day where uterine contractility/minute was recorded. RESULTS: Baseline characteristics (age, BMI, smoking, endometrial thickness) were similar. Mean frequency of uterine contractions/minute was similar between groups (1.15, 1.01, 0.92, and 1.21 for groups A, B, C, and D, respectively). There was a slight increase in the number of contractions in patients with a sonographic niche versus controls, not reaching statistical significance (p=0.48). No differences were observed when comparing patients with a previous C-section (regardless of the presence of a niche) to those without a C-section, either nulliparous (p=0.78) or with a previous vaginal delivery (p=0.80). The frequency of uterine contractions was similar between patients who achieved a clinical pregnancy and those who did not (1.19 vs. 1.02 UC/min, p=0.219, respectively). CONCLUSIONS: Our study found no significant difference in the frequency of uterine contractility between patients with or without a previous C-section or sonographic diagnosed niche. Further investigation is necessary to understand the physiological mechanisms affecting implantation in patients with isthmocele.

Transcriptional profiling of zebrafish identifies host factors controlling susceptibility to Shigella flexneri.

Shigella flexneri is a human-adapted pathovar of Escherichia coli that can invade the intestinal epithelium, causing inflammation and bacillary dysentery. Although an important human pathogen, the host response to S. flexneri has not been fully described. Zebrafish larvae represent a valuable model for studying human infections in vivo. Here, we use a Shigella-zebrafish infection model to generate mRNA expression profiles of host response to Shigella infection at the whole-animal level. Immune response-related processes dominate the signature of early Shigella infection (6 h post-infection). Consistent with its clearance from the host, the signature of late Shigella infection (24 h post-infection) is significantly changed, and only a small set of immune-related genes remain differentially expressed, including acod1 and gpr84. Using mutant lines generated by ENU, CRISPR mutagenesis and F0 crispants, we show that acod1- and gpr84-deficient larvae are more susceptible to Shigella infection. Together, these results highlight the power of zebrafish to model infection by bacterial pathogens and reveal the mRNA expression of the early (acutely infected) and late (clearing) host response to Shigella infection.

Mapping inter-professional collaboration in oncogenetics: Results from a scoping review.

Inter-professional collaboration could improve timely access and quality of oncogenetic services. Here, we present the results of a scoping review conducted to systematically identify collaborative models available, unpack the nature and extent of collaboration proposed, synthesize evidence on their implementation and evaluation, and identify areas where additional research is needed. A comprehensive search was conducted in four journal indexing databases on June 13th, 2022, and complemented with searches of the grey literature and citations. Screening was conducted by two independent reviewers. Eligible documents included those describing either the theory of change, planning, implementation and/or evaluation of collaborative oncogenetic models. 165 publications were identified, describing 136 unique interventions/studies on oncogenetic models with somewhat overlapping collaborative features. Collaboration appears to be mostly inter-professional in nature, often taking place during risk assessment and pre-testing genetic counseling. Yet, most publications provide very limited information on their collaborative features, and only a few studies have set out to formally evaluate them. Better quality research is needed to comprehensively examine and make conclusions regarding the value of collaboration in this oncogenetics. We propose a definition, logic model, and typology of collaborative oncogenetic models to strengthen future planning, implementation, and evaluation in this field.

Synergistic antifungal activity against Candida albicans between voriconazole and cyclosporine a loaded in polymeric nanoparticles.

The goal of this work is to investigate if the synergistic antifungal activity between cyclosporine A, CsA, and voriconazole, VRZ, increases when both drugs are encapsulated in a nanocarrier as compared when they are free. The preparation and characterization of blank and VRZ and CsA loaded polymeric based PLGA nanoparticles (PLGA, PLGA-PEG, and PLGA+PEG) was a necessary previous step. Using the more suitable NPs, those of PLGA, the antifungal susceptibility tests performed with VRZ-loaded PLGA NPs, show no significant increase of the antifungal activity in comparison to that of free VRZ. However, the synergistic behavior found for the (VRZ+CsA)-loaded PLGA NPs was fourfold stronger than that observed for the two free drugs together. On the other hand, the investigation into the suppression of C. albicans biofilm formation showed that blank PLGA NPs inhibit the biofilm formation at high NPs concentrations. However, a minor effect or even a slight biofilm increase formation was observed at low and moderate NPs concentrations. Therefore, the enhancement of the biofilm inhibition found for the three tested treatments (CsA alone, VRZ alone, and VRZ+CsA) when comparing free and encapsulated drugs, within the therapeutic window, can be attributed to the drug encapsulation approach. Indeed, polymeric PLGA NPs loaded with CsA, VRZ, or VRZ+CsA are more effective at inhibiting the C. albicans biofilm growth than their free counterparts.