Added value of EUS-B-FNA to bronchoscopy and EBUS-TBNA in diagnosing and staging of lung cancer.

Background: Bronchoscopy and EBUS are standard procedures in lung cancer work-up but have low diagnostic yield in lesions outside the central airways and hilar/mediastinal lymph nodes. Growing evidence on introducing the EBUS endoscope into the oesophagus (EUS-B) in the same session as bronchoscopy/EBUS gives access to new anatomical areas that can be safely biopsied. Objective: To summarize the current evidence of the added value of EUS-B-FNA to bronchoscopy and EBUS-TBNA in lung cancer work-up. Methods: A narrative review. Results: Few randomized trials or prospective studies are available. Prospective studies show that add-on EUS-B-FNA increases diagnostic yield when sampling abnormal mediastinal lymph nodes, para-oesophageal lung and left adrenal gland. A large retrospective series on EUS-B-FNA from retroperitoneal lymph nodes suggests high diagnostic yield without safety concerns, as do casuistic reports on EUS-B-FNA from mediastinal pleural thickening, pancreatic lesions, ascites fluid and pericardial effusions. No study has systematically assessed both diagnostic yield, safety, patient reported outcomes, adverse events and costs. Conclusion: The diagnostic value of add-on EUS-B to standard bronchoscopy and EBUS in lung cancer work-up appears very promising without safety concerns, giving the pulmonologist access to a variety of sites out of reach with other minimally invasive techniques. Little is known on patient-reported outcomes and costs. Future and prospective research should focus on effectiveness aspects to clarify whether overall benefits of add-on EUS-B sufficiently exceed overall downsides.

The criteria for chronic rhinosinusitis in children with cystic fibrosis are rarely fulfilled after initiation of CFTR modulator treatment.

The vast majority of people with cystic fibrosis (pwCF) have untreated secondary chronic rhinosinusitis (CRS). Whereas the introduction of the cystic fibrosis transmembrane conductance regulator modulator (CFTRm) treatment regime has improved the lung function of pwCF, few studies have been published examining the effect on sinonasal symptoms in children. Our aim was to explore the effect of double CFTRm treatment on CRS and olfaction in children with CF. pwCF were included in this non-randomized cross-sectional study, where an otolaryngologist performed a complete ENT examination before initiating treatment with elaxacaftor/tezacaftor/ivacaftor (ETI). Twenty-three pwCF aged 6-12 years were included. Eighteen of 23 patients were on a double CFTRm treatment, and 5 patients were CFTRm naive, respectively. Altogether, 19 had normal olfaction, 20 had none or mild CRS symptoms according to SNOT-22, and 14 had a normal endoscopy. None of the patients had symptoms of chronic rhinosinusitis lasting for more than 12 weeks, thus none of the patients fulfilled the criteria for CRS. Children with CF treated with double CFTRm have few to no symptoms of CRS and normal olfaction, which is an improvement compared with children following treatment modalities prior to CFTRm.

Fluorescence microscopy and correlative brightfield videos of mitochondria and vesicles in H9c2 cardiomyoblasts.

This paper presents data acquired to study the dynamics and interactions of mitochondria and subcellular vesicles in living cardiomyoblasts. The study was motivated by the importance of mitochondrial quality control and turnover in cardiovascular health. Although fluorescence microscopy is an invaluable tool, it presents several limitations. Correlative fluorescence and brightfield images (label-free) were therefore acquired with the purpose of achieving virtual labelling via machine learning. In comparison with the fluorescence images of mitochondria, the brightfield images show vesicles and subcellular components, providing additional insights about sub-cellular components. A large part of the data contains correlative fluorescence images of lysosomes and/or endosomes over a duration of up to 400 timepoints (>30 min). The data can be reused for biological inferences about mitochondrial and vesicular morphology, dynamics, and interactions. Furthermore, virtual labelling of mitochondria or subcellular vesicles can be achieved using these datasets. Finally, the data can inspire new imaging experiments for cellular investigations or computational developments. The data is available through two large, open datasets on DataverseNO.

Using the Endoscope for Endobronchial Ultrasound in the Esophagus.

EUS-B is a procedure using the echoendobronchoscope in the esophagus and stomach. The procedure is a minimally invasive, safe, and feasible approach that pulmonologists can use to visualize and biopsy structures adjacent to the esophagus and stomach. EUS-B gives access to many structures of which some may also be reached by EBUS (mediastinal lymph nodes, lung or pleural tumors, pericardial fluid) while others cannot be reached such as retroperitoneal lymph nodes, ascites, and lesions in the liver, pancreas or left adrenal gland. The procedure is a pulmonologist- and patient- friendly version of the gastroenterologists' EUS using the thin EBUS endoscope that the pulmonologist already masters. Thus EUS-B training should be easy and a natural continuation of EBUS. With the patient under conscious sedation and in the supine position, the echoendoscope is introduced either through the nostril or mouth into the oropharynx. Then the patient is encouraged to swallow while the endoscope is slowly bent posteriorly and introduced into the esophagus and stomach. Using the ultrasonic image, the operator identifies the six landmarks by EUS-B and EUS: the left liver lobe, abdominal aorta (with the celiac trunk and superior mesenteric artery), left adrenal gland, and mediastinal lymph node stations 7, 4L, and 4R. Biopsies can be taken from suspected lesions under real-time ultrasonographic guidance- fine needle aspiration (EUS-B-FNA) using a technique similar to that used with EBUS-TBNA. The biopsy order is M1b-M1a-N3-N2-N1-T (M = metastasis, N = lymph node, T = tumor) to avoid iatrogenic upstaging. Pre- and post-procedural observation is similar to that of bronchoscopy. EUS-B is safe and feasible in the hands of experienced interventional pulmonologists and provides a significant expansion of the diagnostic possibilities in providing safe, fast, and thorough diagnosis and staging of lung cancer.

High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.

Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations: ATG: autophagy related; ATG7: autophagy related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OXPHOS: oxidative phosphorylation; PepA: pepstatin A; PLA: proximity ligation assay; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; RAB9A: RAB9A, member RAS oncogene family; ROS: reactive oxygen species; SIM: structured illumination microscopy; siRNA: short interfering RNA; SYNJ2BP: synaptojanin 2 binding protein; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.

Label-free superior contrast with c-band ultra-violet extinction microscopy.

In 1934, Frits Zernike demonstrated that it is possible to exploit the sample's refractive index to obtain superior contrast images of biological cells. The refractive index contrast of a cell surrounded by media yields a change in the phase and intensity of the transmitted light wave. This change can be due to either scattering or absorption caused by the sample. Most cells are transparent at visible wavelengths, which means the imaginary component of their complex refractive index, also known as extinction coefficient k, is close to zero. Here, we explore the use of c-band ultra-violet (UVC) light for high-contrast high-resolution label-free microscopy, as k is naturally substantially higher in the UVC than at visible wavelengths. Using differential phase contrast illumination and associated processing, we achieve a 7- to 300-fold improvement in contrast compared to visible-wavelength and UVA differential interference contrast microscopy or holotomography, and quantify the extinction coefficient distribution within liver sinusoidal endothelial cells. With a resolution down to 215 nm, we are, for the first time in a far-field label-free method, able to image individual fenestrations within their sieve plates which normally requires electron or fluorescence superresolution microscopy. UVC illumination also matches the excitation peak of intrinsically fluorescent proteins and amino acids and thus allows us to utilize autofluorescence as an independent imaging modality on the same setup.

Preparation of a fluorinated dental resin system and its anti-adhesive properties against S. mutans.

OBJECTIVES: The purpose of this study was to characterize physicochemical properties and investigate anti-bacterial adhesion effect of dental resins containing fluorinated monomers. METHOD: Fluorinated dimethacrylate FDMA was mixed with commonly used reactive diluent triethylene- glycol dimethacrylate (TEGDMA) and fluorinated diluent 1 H,1 H-heptafluorobutyl methacrylate (FBMA) separately at a mass ratio of 60 wt./40 wt. to prepare fluorinated resin systems. Double bond conversion (DC), flexural strength (FS) and modulus (FM), water sorption (WS) and solubility (SL), contact angle and surface free energy, surface element concentration, and anti-adhesion effect against Streptococcus mutans (S. mutans) were investigated according to standard or referenced methods. 2,2-bis[4-(2-hydroxy-3-methacryloy-loxypropyl)-phenyl]propane (Bis-GMA)/TEGDMA (60/40, wt./wt.) was used as control. RESULTS: Both fluorinated resin systems had higher DC than Bis-GMA based resin (p < 0.05); compared with Bis-GMA based resin (FS, FDMA/TEGDMA resin system had higher FS (p < 0.05) and comparable FM (p > 0.05), while FDMA/FBMA resins system had lower FS and FM (p < 0.05). Both fluorinated resin systems had lower WS and SL than Bis-GMA based resin (p < 0.05), and FDMA/TEGDMA resin system had the lowest WS (p < 0.05) in all experimental resin systems. Only FDMA/FBMA resin system showed lower surface free energy than Bis-GMA based resin (p < 0.05). When the surface was smooth, FDMA/FBMA resin system had lower amount of adherent S. mutans than Bis-GMA based resin (p < 0.05), while after the surface became roughness, FDMA/FBMA resin system had comparable amount of adherent S. mutans as Bis-GMA based resin (p > 0.05). SIGNIFICANCE: Resin system prepared exclusively with fluorinated methacrylate monomers reduced the S. mutans adhesion due to their increased hydrophobicity and decreased surface energy., while flexural properties of it should be improved.

Deadly and venomous Lonomia caterpillars are more than the two usual suspects.

Caterpillars of the Neotropical genus Lonomia (Lepidoptera: Saturniidae) are responsible for some fatal envenomation of humans in South America inducing hemostatic disturbances in patients upon skin contact with the caterpillars' spines. Currently, only two species have been reported to cause hemorrhagic syndromes in humans: Lonomia achelous and Lonomia obliqua. However, species identifications have remained largely unchallenged despite improved knowledge of venom diversity and growing evidence that the taxonomy used over past decades misrepresents and underestimates species diversity. Here, we revisit the taxonomic diversity and distribution of Lonomia species using the most extensive dataset assembled to date, combining DNA barcodes, morphological comparisons, and geographical information. Considering new evidence for seven undescribed species as well as three newly proposed nomenclatural changes, our integrative approach leads to the recognition of 60 species, of which seven are known or strongly suspected to cause severe envenomation in humans. From a newly compiled synthesis of epidemiological data, we also examine the consequences of our results for understanding Lonomia envenomation risks and call for further investigations of other species' venom activities. This is required and necessary to improve alertness in areas at risk, and to define adequate treatment strategies for envenomed patients, including performing species identification and assessing the efficacy of anti-Lonomia serums against a broader diversity of species.

Biological characterization of the Bothrops brazili snake venom and its neutralization by Brazilian Bothrops antivenom produced by the Butantan Institute.

We evaluated the ability of the Bothrops antivenom produced by the Butantan Institute to neutralize the lethal, hemorrhagic, myotoxic and phospholipase A2 activities induced by B. brazili venom from Rondônia state, Brazil, and verified its cross-reactivity against this venom. This antivenom neutralized the cited biological activities. It also showed cross-reactivity with this venom, and preferentially recognized components with a relative mass above 66 kDa. Our results suggest that Brazilian Bothrops antivenom can be used in B. brazili envenomation in this region.

Biological characterization of bristle extract of Lonomia descimoni caterpillar (Lepidoptera, Saturniidae) and effectiveness of Lonomia antivenom to neutralize experimental envenomation in rats.

Contact with Lonomia caterpillars can cause severe envenomation with hemorrhagic syndrome, consumptive coagulopathy, acute renal failure, and death. In Brazil, an antivenom was produced using extracts from L. obliqua caterpillar bristles as antigen and has been used in other countries in South America to treat envenomation caused by distinct species of Lonomia. This study aimed to characterize the activities of toxins from Lonomia descimoni caterpillars found in Colombia and the neutralization of these toxins by the Brazilian Lonomia antivenom. The protein composition and coagulant, phospholipase A2, hyaluronidase, and defibrinogenating activities were evaluated and compared with the same parameters of the L. obliqua bristle extract. Immune recognition and the neutralizing ability of Lonomia antivenom were also determined. The results showed that the L. descimoni bristle extract presented marked differences in electrophoretic and mass spectrometry profiles and had coagulant, phospholipase A2, and hyaluronidase activities significantly less intense than those of the L. obliqua extract. In rats, L. descimoni extract induced coagulopathy and hemoglobinuria when injected by intravenous or intraperitoneal routes. The Lonomia antivenom recognized the toxins in the extract of L. descimoni and reversed the experimental envenomation in rats. Our results indicate that L. descimoni caterpillars possess toxins with weaker activities than those of L. obliqua but with the potential to cause envenomation. Moreover, the Lonomia antivenom recognized and neutralized the toxins in the L. descimoni bristle extract.

Virtual labeling of mitochondria in living cells using correlative imaging and physics-guided deep learning.

Mitochondria play a crucial role in cellular metabolism. This paper presents a novel method to visualize mitochondria in living cells without the use of fluorescent markers. We propose a physics-guided deep learning approach for obtaining virtually labeled micrographs of mitochondria from bright-field images. We integrate a microscope's point spread function in the learning of an adversarial neural network for improving virtual labeling. We show results (average Pearson correlation 0.86) significantly better than what was achieved by state-of-the-art (0.71) for virtual labeling of mitochondria. We also provide new insights into the virtual labeling problem and suggest additional metrics for quality assessment. The results show that our virtual labeling approach is a powerful way of segmenting and tracking individual mitochondria in bright-field images, results previously achievable only for fluorescently labeled mitochondria.

Telemedicine for outpatient treatment of depressive disorders.

BACKGROUND: The newly developed app TellUs is a digital offering for psychiatric outpatient treatment that includes diagnostic and therapeutic tools. The aim of this study was to test the clinical efficiency and patient satisfaction of TellUs. METHODS: Sixty-four patients with depressive disorder took part in the study for 3 months. The intervention group was treated digitally with TellUs and the control group received visiting treatment (treatment as usual) during that time. RESULTS: In both groups, a significant decrease of depressive symptoms and general strain through psychological symptoms, along with an increase of quality of life in the psychological domain, was shown. Furthermore, both groups were highly satisfied with the treatment. CONCLUSIONS: TellUs was shown to be equivalent to treatment as usual in terms of clinical efficiency and patient satisfaction.

Multidisciplinary biophotonics, open science, and plug & pray deep learning?

Due to its great creative potential for innovation and scientific discovery, inter- and multidisciplinary collaboration is being increasingly encouraged by institutions and funding agencies. The increased opportunities in the multidisciplinary arena also come with significant challenges like the added experimental, analytical and logistical complexity, blended with a high likelihood of miscommunications. When is multidisciplinarity worth the effort, and how can we be better collaborators? With a focus on cross-disciplinary collaborative work to answer burning questions in biology and biomedicine, this paper discusses both large challenges and opportunities with multidisciplinary biophotonics, how we can better navigate the arena of big data and artificial intelligence combined with open, reproducible science and biological discoveries.

In-Depth Profiling of O-Glycan Isomers in Human Cells Using C18 Nanoliquid Chromatography-Mass Spectrometry and Glycogenomics.

O-Glycosylation is an omnipresent modification of the human proteome affecting many cellular functions, including protein cleavage, protein folding, and cellular signaling, interactions, and trafficking. The functions are governed by differentially regulated O-glycan types and terminal structures. It is therefore essential to develop analytical methods that facilitate the annotation of O-glycans in biological material. While various successful strategies for the in-depth profiling of released O-glycans have been reported, these methods are often limitedly accessible to the nonspecialist or challenged by the high abundance of O-glycan structural isomers. Here, we developed a high-throughput sample preparation approach for the nonreductive release and characterization of O-glycans from human cell material. Reducing-end labeling allowed efficient isomer separation and detection using C18 nanoliquid chromatography coupled to Orbitrap mass spectrometry. Using the method in combination with a library of genetically glycoengineered cells displaying defined O-glycan types and structures, we were able to annotate individual O-glycan structural isomers from a complex mixture. Applying the method in a model system of human keratinocytes, we found a wide variety of O-glycan structures, including O-fucose, O-glucose, O-GlcNAc, and O-GalNAc glycosylation, with the latter carrying both elongated core1 and core2 structures and varying numbers of fucoses and sialic acids. The method, including the now well-characterized standards, provides the opportunity to study glycomic changes in human tissue and disease models using rather mainstream analytical equipment.

Three-dimensional structured illumination microscopy data of mitochondria and lysosomes in cardiomyoblasts under normal and galactose-adapted conditions.

This three-dimensional structured illumination microscopy (3DSIM) dataset was generated to highlight the suitability of 3DSIM to investigate mitochondria-derived vesicles (MDVs) in H9c2 cardiomyoblasts in living or fixed cells. MDVs act as a mitochondria quality control mechanism. The cells were stably expressing the tandem-tag eGFP-mCherry-OMP25-TM (outer mitochondrial membrane) which can be used as a sensor for acidity. A part of the dataset is showing correlative imaging of lysosomes labeled using LysoTracker in fixed and living cells. The cells were cultivated in either normal or glucose-deprived medium containing galactose. The resulting 3DSIM data were of high quality and can be used to undertake a variety of studies. Interestingly, many dynamic tubules derived from mitochondria are visible in the 3DSIM videos under both glucose and galactose-adapted growth conditions. As the raw 3DSIM data, optical parameters, and reconstructed 3DSIM images are provided, the data is especially suitable for use in the development of SIM reconstruction algorithms, bioimage analysis methods, and for biological studies of mitochondria.

Psychosocial functioning as a personal resource promoting a milder course of schizophrenia.

Schizophrenia has been shown repeatedly to be associated with a low level of psychosocial functioning. It is assumable that psychosocial functioning is related not only to current, but also to future symptom severity. To test this assumption, a follow-up study with two measurement time points with an interval of 18 months was conducted. In total, 154 inpatients from five psychiatric hospitals with a diagnosis of a schizophrenic disorder took part at both visits. Psychosocial functioning was measured with the Personal and Social Performance Scale (PSP scale) at baseline, and schizophrenic symptoms were assessed with the Positive and Negative Syndrome Scale (PANSS) at baseline and at follow-up. Two PSP subscales, i.e. socially useful activities and control over disturbing and aggressive behavior, turned out to be significant predictors of symptom severity 18 months later. The findings reveal that personal resources in the occupational domain and in adequate interpersonal behavior can have a positive impact on the long-term course of schizophrenia.

Gender, age at onset, and duration of being ill as predictors for the long-term course and outcome of schizophrenia: an international multicenter study.

BACKGROUND: The aim of the current study was to explore the effect of gender, age at onset, and duration on the long-term course of schizophrenia. METHODS: Twenty-nine centers from 25 countries representing all continents participated in the study that included 2358 patients aged 37.21 ± 11.87 years with a DSM-IV or DSM-5 diagnosis of schizophrenia; the Positive and Negative Syndrome Scale as well as relevant clinicodemographic data were gathered. Analysis of variance and analysis of covariance were used, and the methodology corrected for the presence of potentially confounding effects. RESULTS: There was a 3-year later age at onset for females (P < .001) and lower rates of negative symptoms (P < .01) and higher depression/anxiety measures (P < .05) at some stages. The age at onset manifested a distribution with a single peak for both genders with a tendency of patients with younger onset having slower advancement through illness stages (P = .001). No significant effects were found concerning duration of illness. DISCUSSION: Our results confirmed a later onset and a possibly more benign course and outcome in females. Age at onset manifested a single peak in both genders, and surprisingly, earlier onset was related to a slower progression of the illness. No effect of duration has been detected. These results are partially in accord with the literature, but they also differ as a consequence of the different starting point of our methodology (a novel staging model), which in our opinion precluded the impact of confounding effects. Future research should focus on the therapeutic policy and implications of these results in more representative samples.

Mitochondrial dynamics and quantification of mitochondria-derived vesicles in cardiomyoblasts using structured illumination microscopy.

Mitochondria are essential energy-providing organelles of particular importance in energy-demanding tissue such as the heart. The production of mitochondria-derived vesicles (MDVs) is a cellular mechanism by which cells ensure a healthy pool of mitochondria. These vesicles are small and fast-moving objects not easily captured by imaging. In this work, we have tested the ability of the optical super-resolution technique 3DSIM to capture high-resolution images of MDVs. We optimized the imaging conditions both for high-speed video microscopy and fixed-cell imaging and analysis. From the 3DSIM videos, we observed an abundance of MDVs and many dynamic mitochondrial tubules. The density of MDVs in cells was compared for cells under normal growth conditions and cells during metabolic perturbation. Our results indicate a higher abundance of MDVs in H9c2 cells during glucose deprivation compared with cells under normal growth conditions. Furthermore, the results reveal a large untapped potential of 3DSIM in MDV research.

Fluorescence fluctuation-based super-resolution microscopy using multimodal waveguided illumination.

Photonic chip-based total internal reflection fluorescence microscopy (c-TIRFM) is an emerging technology enabling a large TIRF excitation area decoupled from the detection objective. Additionally, due to the inherent multimodal nature of wide waveguides, it is a convenient platform for introducing temporal fluctuations in the illumination pattern. The fluorescence fluctuation-based nanoscopy technique multiple signal classification algorithm (MUSICAL) does not assume stochastic independence of the emitter emission and can therefore exploit fluctuations arising from other sources, as such multimodal illumination patterns. In this work, we demonstrate and verify the utilization of fluctuations in the illumination for super-resolution imaging using MUSICAL on actin in salmon keratocytes. The resolution improvement was measured to be 2.2-3.6-fold compared to the corresponding conventional images.

Use of freeze-dried trivalent antivenom to neutralize the toxic activities of Bothrops atrox snake venoms from the Amazon.

In Brazil, antivenom for snakebite is currently formulated in liquid form and requires storage at 4 °C. Here, a new freeze-dried trivalent antivenom, which would enable cold-chain free storage, was determined to have efficacy in neutralizing the biological activities of Bothrops atrox venoms from Manaus (Brazil) and Leticia (Colombia), exhibiting an efficacy similar to those of currently available liquid Bothrops antivenoms. These results indicate that freeze-dried trivalent antivenom may be beneficial for applications in the Brazilian and Colombian Amazon regions.