Search

The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome.

Live-cell imaging of biological structures at high resolution poses challenges in the microscope throughput regarding area and speed. For this reason, different parallelisation strategies have been implemented in coordinate- and stochastic-targeted switching super-resolution microscopy techniques. In this line, the molecular nanoscale live imaging with sectioning ability (MoNaLISA), based on reversible saturable optical fluorescence transitions (RESOLFT), offers

To investigate the association of viral load (VL) with (i) tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein-10, C-reactive protein, and a combinatorial score (BV score), and (ii) clinical severity.In this prospective, multicentre cohort substudy, children with respiratory tract infection or fever without source were enrolled. VL for influenza virus, rhinovirus, respiratory syncytial virus, and adenovirus was measured from nasopharyngeal swabs. The reference standard diagnosis was established based on expert panel adjudication.Of 1140 recruited patients, 333 had a virus monodetection. VL for the aggregated data set correlated with TRAIL and IP-10 levels, with the length of oxygen therapy, and inversely with the BV score. At a single viral level, only the influenza VL yielded a correlation with TRAIL, IP-10 levels, and the BV score. Children with a viral reference standard diagnosis had significantly higher VL than those with bacterial infection (p = 0.0005). Low TRAIL (incidence rate ratio [IRR] 0.6, 95% confidence interval [CI] 0.39-0.91) and young age (IRR 0.62, 95% CI 0.49-0.79) were associated with a longer hospital stay, while young age (IRR 0.33, 95% CI 0.18-0.61), low TRAIL (IRR 0.25, 95% CI 0.08-0.76), and high VL (IRR 1.16, 95% CI 1.00-1.33) were predictive of longer oxygen therapy.These findings indicate that VL correlates with biomarkers and may serve as a complementary tool pertaining to disease severity.

Programmed Death-1 (PD-1) is a coinhibitory receptor expressed on activated T cells that suppresses T-cell signaling and effector functions. It has been previously shown that binding to its ligand PD-L1 induces a spatial reorganization of PD-1 receptors into microclusters on the cell membrane. However, the roles of the spatial organization of PD-L1 on PD-1 clustering and T-cell signaling have not been elucidated. Here, we used DNA origami flat sheets to display PD-L1 ligands at defined nanoscale distances and investigated their ability to inhibit T-cell activation in vitro. We found that DNA origami flat sheets modified with CD3 and CD28 activating antibodies (FS-α-CD3-CD28) induced robust T-cell activation. Co-treatment with flat sheets presenting PD-L1 ligands separated by ∼200 nm (FS-PD-L1-200), but not 13 nm (FS-PD-L1-13) or 40 nm (FS-PD-L1-40), caused an inhibition of T-cell signaling, which increased with increasing molar ratio of FS-PD-L1-200 to FS-α-CD3-CD28. Furthermore, FS-PD-L1-200 induced the formation of smaller PD-1 nanoclusters and caused a larger reduction in IL-2 expression compared to FS-PD-L1-13. Together, these findings suggest that the spatial organization of PD-L1 determines its ability to regulate T-cell signaling and may guide the development of future nanomedicine-based immunomodulatory therapies.

Monitoring the proteins and lipids that mediate all cellular processes requires imaging methods with increased spatial and temporal resolution. STED (stimulated emission depletion) nanoscopy enables fast imaging of nanoscale structures in living cells but is limited by photobleaching. Here, we present event-triggered STED, an automated multiscale method capable of rapidly initiating two-dimensional (2D) and 3D STED imaging after detecting cellular events such as protein recruitment, vesicle trafficking and second messengers activity using biosensors. STED is applied in the vicinity of detected events to maximize the temporal resolution. We imaged synaptic vesicle dynamics at up to 24 Hz, 40 ms after local calcium activity; endocytosis and exocytosis events at up to 11 Hz, 40 ms after local protein recruitment or pH changes; and the interaction between endosomal vesicles at up to 3 Hz, 70 ms after approaching one another. Event-triggered STED extends the capabilities of live nanoscale imaging, enabling novel biological observations in real time.

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon termed electroporation is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrhythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study, we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. Additionally, by analysing poration kinetics with Bayesian survival analysis we show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

The plasma membrane of a biological cell is a complex assembly of lipids and membrane proteins, which tightly regulate transmembrane transport. When a cell is exposed to a strong electric field, the membrane integrity becomes transiently disrupted by formation of transmembrane pores. This phenomenon, termed electroporation, is already utilized in many rapidly developing applications in medicine including gene therapy, cancer treatment, and treatment of cardiac arrythmias. However, the molecular mechanisms of electroporation are not yet sufficiently well understood; in particular, it is unclear where exactly pores form in the complex organization of the plasma membrane. In this study we combine coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis to identify how formation of pores depends on the local lipid organization. We show that pores do not form homogeneously across the membrane, but colocalize with domains that have specific features, the most important being high density of polyunsaturated lipids. We further show that knowing the lipid organization is sufficient to reliably predict poration sites with machine learning. However, by analysing poration kinetics with Bayesian survival analysis we then show that poration does not depend solely on local lipid arrangement, but also on membrane mechanical properties and the polarity of the electric field. Finally, we discuss how the combination of atomistic and coarse-grained molecular dynamics simulations, machine learning methods, and Bayesian survival analysis can guide the design of future experiments and help us to develop an accurate description of plasma membrane electroporation on the whole-cell level. Achieving this will allow us to shift the optimization of electroporation applications from blind trial-and-error approaches to mechanistic-driven design.

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities., Monomeric near-infrared (NIR) fluorescent proteins (FPs) from bacterial phytochromes bring potential advantages, but their brightness in cells is lower than dimeric NIR FPs. Here the authors develop enhanced monomeric NIR FPs enabling imaging across different scales without the trade-off between brightness and monomeric state.

The diffraction limit is no longer a concept that stands as a true constant in imaging, with fluorescence switching–based methods having made the breakthrough to circumvent this limit. Multiple ingenious solutions have been presented over the last decades and continue to be explored. The techniques used today have undergone constant development both conceptually and technically, which has enabled an increased number of biological studies at the molecular scale. Here we review recent developments to stimulated emission depletion microscopy, reversible saturable optical fluorescence transitions microscopy, single-molecule localization microscopy, and MINFLUX and mention key applications of these methods. Finally, we present our view on what the future holds for super-resolution imaging, especially in terms of even more challenging live-cell imaging in different biological model systems.

Cerebrospinal fluid-contacting (CSF-c) neurons line the central canal of the spinal cord and a subtype of CSF-c neurons expressing somatostatin, forms a homeostatic pH regulating system. Despite their importance, their intricate spatial organization is poorly understood. The function of another subtype of CSF-c neurons expressing dopamine is also investigated. Imaging methods with a high spatial resolution (5–10 nm) are used to resolve the synaptic and ciliary compartments of each individual cell in the spinal cord of the lamprey to elucidate their signalling pathways and to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine- and somatostatin-expressing CSF-c neuronal subtypes. The density of somatostatin-containing dense-core vesicles, resolved by stimulated emission depletion microscopy, was shown to be markedly reduced upon each exposure to either alkaline or acidic pH and being part of a homeostatic response inhibiting movements. Their cilia symmetry was unravelled by stimulated emission depletion microscopy in expanded tissues as sensory with 9 + 0 microtubule duplets. The dopaminergic CSF-c neurons on the other hand have a motile cilium with the characteristic 9 + 2 duplets and are insensitive to pH changes. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell-type classification.

The spatial location of cerebrospinal fluid contacting (CSF-c) neurons enables important regulatory homeostatic functions regarding pH and motion control. Their intricate organization, facing the central canal and extending across the spinal cord, in relation to specific subtypes is poorly understood. This calls for imaging methods with a high spatial resolution (5-10 nm) to resolve the synaptic and ciliary compartments of each individual cell to elucidate their signalling pathways and enough throughput to dissect the cellular organization. Here, light-sheet and expansion microscopy resolved the persistent ventral and lateral organization of dopamine and somatostatin CSF-c neuronal types.The number of somatostatin-containing dense core vesicles, resolved by STED microscopy, was shown to be markedly reduced upon each exposure to alkaline or acidic pH inhibiting any movement as part of a homeostatic response. Their cilia symmetry was unravelled by ExSTED as sensory in contrast with the motile one found in the dopaminergic ph insensitive neurons. This novel experimental workflow elucidates the functional role of CSF-c neuron subtypes in situ paving the way for further spatial and functional cell type classification.

The classic view of organelle cell biology is undergoing a constant revision fueled by the new insights unraveled by fluorescence nanoscopy, which enable sensitive, faster and gentler observation of specific proteins in situ. The endoplasmic reticulum (ER) is one of the most challenging structure to capture due the rapid and constant restructuring of fine sheets and tubules across the full 3D cell volume. Here we apply STED and parallelized 2D and 3D RESOLFT live imaging to uncover the tubular ER organization in the fine processes of neuronal cells with focus on mitochondria-ER contacts, which recently gained medical attention due to their role in neurodegeneration. Multi-color STED nanoscopy enables the simultaneous visualization of small transversal ER tubules crossing and constricting mitochondria all along axons and dendrites. Parallelized RESOLFT allows for dynamic studies of multiple contact sites within seconds and minutes with prolonged time-lapse imaging at ~50 nm spatial resolution. When operated in 3D super resolution mode it enables a new isotropic visualization of such contacts extending our understanding of the three-dimensional architecture of these packed structures in axons and dendrites.

The development of parallelized STED/RESOLFT super resolution microscopy techniques have shown impressive results combining diffraction unlimited lateral spatial resolution (< 70 nm) with increasingly fast acquisition speeds (~ 1-30Hz) and optical sectioning. However, the diffraction-unlimited spatial resolution in these systems are often limited to the lateral dimension. Here we present a novel design of illumination patterns that allows for 3-dimensional isotropic diffraction unlimited resolution while maintaining the rapid recording speed of a parallelized imaging system. Much like in the original RESOLFT-MoNaLISA set-up [1] the system is based on controlling the spatial distribution of ON-OFF states in a sample labelled with reversibly switchable fluorescent proteins (rsFPs) [2] . Using rsFPs as labels allows for high resolution imaging at minimal light doses and is thus suitable for live cell imaging. Designing illumination patterns that are modulated in all three spatial dimensions and combining these with the complete OFF-switching of rsFP allow to confine the emission spots in all the three spatial dimensions to sub-diffraction sized. The extended frequency content in the emission pattern manifests itself as isotropic diffraction unlimited resolution in the final images [3] .

Objectives Identifying infection aetiology is essential for appropriate antibiotic use. Previous studies showed a host-protein signature consisting of TNF-related apoptosis-induced ligand (TRAIL), interferon gamma-induced protein-10 (IP-10), and C-reactive protein (CRP) can accurately differentiate bacterial from viral infections. Methods This prospective, multicentre cohort study titled “AutoPilot-Dx”, aimed to validate signature performance and estimate its potential impact on antibiotic use across a broad paediatric population (>90 days to 18 years) with respiratory tract infections, or fever without source, at emergency departments and wards in Italy and Germany. Infection aetiology was adjudicated by experts based on clinical and laboratory investigations, including multiplex PCR and follow-up data. Results In total, 1140 patients were recruited (2/2017–12/2018), of which 1008 met eligibility criteria (mean age 3.5 years, 41.9% female). Viral and bacterial infections were adjudicated for 628 (85.8%) and 104 (14.2%) children, respectively; 276 patients were assigned an indeterminate reference standard outcome. For the 732 children with reference standard aetiology, the signature discriminated bacterial from viral infections with sensitivity of 93.7% (95% CI, 88.7–98.7), specificity of 94.2% (92.2–96.1), positive predictive value of 73.0% (65.0–81.0), negative predictive value of 98.9% (98.0–99.8), and 9.8% equivocal test results. The signature performed consistently across different patient subgroups and detected bacterial immune response in viral PCR positive patients. Conclusions The findings validate high diagnostic performance of the TRAIL/IP-10/CRP signature in a broad paediatric cohort and support its potential to reduce antibiotic overuse in children with viral infections. Trial registration Clinicaltrials.gov identifier: NCT03052088.

In the last 20 years, gut microbiota in patients with cystic fibrosis (CF) has become an object of interest. It was shown that these patients had gut dysbiosis and this could explain not only the intestinal manifestations of the disease but also part of those involving the respiratory tract. The acquisition of previously unknown information about the importance of some bacteria, i.e., those partially or totally disappeared in the gut of CF patients, in the regulation of the activity and function of the gut and the lung was the base to suggest the use of probiotics in CF patients. The main aim of this paper is to discuss the biological basis for probiotic administration to CF patients and which results could be expected. Literature analysis showed that CF intestinal dysbiosis depends on the same genetic mutations that condition the clinical picture of the diseases and is aggravated by a series of therapeutic interventions, such as dietary modifications, the use of antibiotics, and the administration of antacids. All this translates into a significant worsening of the structure and function of organs, including the lung and intestine, already deeply penalized by the genetic alterations of CF. Probiotics can intervene on dysbiosis, reducing the negative effects derived from it. However, the available data cannot be considered sufficient to indicate that these bacteria are essential elements of CF therapy. Further studies that take into account the still unsolved aspects on how to use probiotics are absolutely necessary.

Objective: Lombardy was the most affected Italian region by COVID-19. To limit the spread of infection, the government issued a national social lockdown. The obstetrical-gynaecological emergencies and essential services were guaranteed to protect pregnant women's health, and a return to a medicalised childbirth was necessary. This situation could had amplified risk factors on the psychological wellbeing of mothers-to-be. Indeed, the last trimester of pregnancy is a period of increased vulnerability itself.Method: For better support women who experience pregnancy during social lockdown, we explored the impact of COVID-19 on psychic wellbeing of two samples of pregnant women (40 living in Lombardy and 35 in Tuscany).Results: T-test and correlations analyses revealed that women living in the Lombardy had a higher perception of the centrality of COVID-19. Further, women that considered the pandemic as a significant event, experienced a higher perinatal depressive symptom. Those symptoms also arose in women who presented a higher number of intrusion and hyperarousal symptoms and a lower ability to plan.Conclusion: Pregnant women should be closely monitored and supported, especially those who live in high-risk areas, such as Lombardy Region. The target intervention could be focused on improving resilience to reduce depressive symptomatology.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress. Fil: Smoler, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Coceano, Giovanna. Royal Institute of Technology; Suecia Fil: Testa, Ilaria. Royal Institute of Technology; Suecia Fil: Bruno, Luciana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Calculo. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Calculo; Argentina Fil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

Elucidating the volumetric architecture of organelles and molecules inside cells requires microscopy methods with a sufficiently high spatial resolution in all three dimensions. Current methods are limited by insufficient resolving power along the optical axis, long recording times and photobleaching when applied to live cell imaging. Here, we present a 3D, parallelized, reversible, saturable/switchable optical fluorescence transition (3D pRESOLFT) microscope capable of delivering sub-80-nm 3D resolution in whole living cells. We achieved rapid (1-2 Hz) acquisition of large fields of view (~40 × 40 µm

Significance Basement membrane is a particular kind of sheet-like extracellular matrix that separates tissue compartments. Invasion of cells through basement membrane barriers is a key aspect of many normal and pathological processes, including organ development and cancer cell metastasis. Invasive protrusions are rich in actin, a cytoskeletal biopolymer, the self-assembly of which can produce force and remodel extracellular matrix. However, in the invasive protrusion, actin has been attributed a function in scaffolding and trafficking, and its mechanical role has not been explored. Here we show that invading cells in C. elegans apply forces via actin assembly to break through basement membrane, and that force production is one of the key features of invasion., During invasion, cells breach basement membrane (BM) barriers with actin-rich protrusions. It remains unclear, however, whether actin polymerization applies pushing forces to help break through BM, or whether actin filaments play a passive role as scaffolding for targeting invasive machinery. Here, using the developmental event of anchor cell (AC) invasion in Caenorhabditis elegans, we observe that the AC deforms the BM and underlying tissue just before invasion, exerting forces in the tens of nanonewtons range. Deformation is driven by actin polymerization nucleated by the Arp2/3 complex and its activators, whereas formins and cross-linkers are dispensable. Delays in invasion upon actin regulator loss are not caused by defects in AC polarity, trafficking, or secretion, as appropriate markers are correctly localized in the AC even when actin is reduced and invasion is disrupted. Overall force production emerges from this study as one of the main tools that invading cells use to promote BM disruption in C. elegans.

Homologous recombination (HR) is essential for the accurate repair of double-stranded DNA breaks (DSBs); it begins when the RecBCD2 complex resects the ends of the DSB into 3' single-stranded DNA (ssDNA) on which a RecA filament assembles. HR depends on the ability of this RecA-ssDNA filament to locate the homologous repair template on the sister chromosome. The mechanism by which the homology is located among vast amounts of heterologous DNA is not yet understood, despite a long history of research. Here, we directly visualize the repair of DSBs in hundreds of individual cells, using high-throughput microfluidics and fluorescence microscopy. We find that in E. coli, DSB repair is completed in 15 minutes without fitness loss. We further show that the search takes less than 10 minutes and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose a model in which the architecture of the RecA filament effectively reduces search dimensionality to two dimensions. The model is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA, and also predicts a search time that is consistent with our measurement. Since the RecA family proteins are conserved in all organisms, our results also translate to other systems that rely on homologous recombination.

The performance of fluorescence microscopy and nanoscopy is often discussed by the effective point spread function and the optical transfer function. However, due to the complexity of the fluorophore properties such as photobleaching or other forms of photoswitching, which introduce a variance in photon emission, it is not trivial to choose optimal imaging parameters and to predict the spatial resolution. In this paper, we analytically derive a theoretical framework for estimating the achievable resolution of a microscope depending on parameters such as photoswitching, labeling densities, exposure time and sampling. We developed a numerical simulation software to analyze the impact of reversibly switchable probes in RESOLFT imaging.

The volumetric architecture of organelles and molecules inside cells can only be investigated with microscopes featuring sufficiently high resolving power in all three spatial dimensions. Current methods suffer from severe limitations when applied to live cell imaging such as long recording times and/or photobleaching. By introducing a novel optical scheme to switch reversibly switchable fluorescent molecules, we demonstrate volumetric nanoscopy of living cells with resolution below 100 nm in 3D, large field of view and minimal illumination intensities (W-kW/cm2).

The topic of lactation following cancer diagnosis will become increasingly more current. Although oncological research confirms that breastfeeding after cancer might be possible, there is a lack of guidelines and a good recommendation for oncological women. In the absence of specific recommendations, women with past cancer may be at higher risk for psychological distress related to breastfeeding. The objective of this article was to analyse the experience of breastfeeding in new mothers with a history of cancer compared to women without a cancer diagnosis. First, we explored the impact of the cancer diagnosis on the breastfeeding choice. Second, we evaluated the relationship between different feeding methods and the mother’s mood states in women with and without a history of cancer. The sample was composed of 74 mothers divided into two groups: 34 with a cancer history (clinical sample) and 40 without a cancer diagnosis (control group). Participants were requested to complete a questionnaire three months after childbirth which assessed: socio-demographic and clinical data, feeding modes (breastfeeding, formula and mixed feeding) and the profile of mood states (POMS). Results showed that women in the clinical group breastfeed significantly less and use formula more than those in the control group. Moreover, in the clinical group, women who breastfeed feel reported higher levels of confusion (according to POMS) than mothers who bottle-feed or use a mixed feeding method. On the contrary, in the control sample, women who breastfeed feel significantly more vigorous than puerperae who bottle-feed or use mixed methods according to POMS. Our findings suggest the need for a specific warm chain of support and the development of guidelines with clear and specific information for women with a cancer diagnosis in order to reduce their confusion around breastfeeding.

Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation, in order to deliver therapeutic molecules into the cell. One type of events that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study we used molecular dynamics (MD) simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields induce pores in the voltage-sensor domains (VSDs) of different VGICs, and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into so-called complex pores, which become stabilized by lipid head-groups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophyiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents following pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall our study reveals a new mechanism of electroporation through membranes containing voltage-gated ion channels.Statement of SignificancePulsed electric fields are often used for treatment of excitable cells, e.g., for gene delivery into skeletal muscles, ablation of the heart muscle or brain tumors. Voltage-gated ion channels (VGICs) underlie generation and propagation of action potentials in these cells, and consequently are essential for their proper function. Our study reveals the molecular mechanisms by which pulsed electric fields directly affect VGICs and addresses questions that have been previously opened by electrophysiologists. We analyze VGICs’ characteristics, which make them prone for electroporation, including hydration and electrostatic properties. This analysis is easily transferable to other membrane proteins thus opening directions for future investigations. Finally, we propose a mechanism for long-lived membrane permeability following pulse treatment, which to date remains poorly understood.

Pulsed electric fields are increasingly used in medicine to transiently increase the cell membrane permeability via electroporation to deliver therapeutic molecules into the cell. One type of event that contributes to this increase in membrane permeability is the formation of pores in the membrane lipid bilayer. However, electrophysiological measurements suggest that membrane proteins are affected as well, particularly voltage-gated ion channels (VGICs). The molecular mechanisms by which the electric field could affects these molecules remain unidentified. In this study, we used molecular dynamics simulations to unravel the molecular events that take place in different VGICs when exposing them to electric fields mimicking electroporation conditions. We show that electric fields can induce pores in the voltage-sensor domains (VSDs) of different VGICs and that these pores form more easily in some channels than in others. We demonstrate that poration is more likely in VSDs that are more hydrated and are electrostatically more favorable for the entry of ions. We further show that pores in VSDs can expand into so-called complex pores, which become stabilized by lipid headgroups. Our results suggest that such complex pores are considerably more stable than conventional lipid pores, and their formation can lead to severe unfolding of VSDs from the channel. We anticipate that such VSDs become dysfunctional and unable to respond to changes in transmembrane voltage, which is in agreement with previous electrophysiological measurements showing a decrease in the voltage-dependent transmembrane ionic currents after pulse treatment. Finally, we discuss the possibility of activation of VGICs by submicrosecond-duration pulses. Overall, our study reveals a new, to our knowledge, mechanism of electroporation through membranes containing VGICs.

Fluorescence microscopy methods have been developed to circumvent the diffraction limit by exploiting nonlinearities in the interactions between light and fluorophores. Initially, these methods were up to orders of magnitude slower than standard microscopies. In recent years, a wide array of technological advances have increased the throughput of super-resolution microscopies, through parallelization, smart scanning or data processing, and sample expansion. Here, we review re cent innovations for increased throughput, some applications that have benefitted from them, and how they could be improved in the future.