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Acral burning pain triggered by fever, thermal hyposensitivity and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide. To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via gene editing. We subjected induced pluripotent stem cells to targeted peripheral neuronal differentiation and observed intra-lysosomal globotriaosylceramide accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in isogenic control neurons compared with healthy control neurons ( P < 0.001). Moreover, we demonstrate a drastic increase in Fabry sensory neuron calcium levels at 39°C mimicking clinical fever ( P < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons differentiated from induced pluripotent stem cells derived from Fabry patients compared with healthy control cells ( P < 0.001). Linear-nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed mitochondrial aggregation at sphingolipid accumulations within Fabry sensory neurites utilizing a click chemistry approach together with mitochondrial dysmorphism compared with healthy control cells. We pioneer pilot insights into the cellular mechanisms contributing to pain, thermal hyposensitivity and denervation in Fabry small fibre neuropathy and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches., Competing Interests: The authors report no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain.)

Considering the profound transformation affecting pathology practice, we aimed to develop a scalable artificial intelligence (AI) system to diagnose colorectal cancer from whole-slide images (WSI). For this, we propose a deep learning (DL) system that learns from weak labels, a sampling strategy that reduces the number of training samples by a factor of six without compromising performance, an approach to leverage a small subset of fully annotated samples, and a prototype with explainable predictions, active learning features and parallelisation. Noting some problems in the literature, this study is conducted with one of the largest WSI colorectal samples dataset with approximately 10,500 WSIs. Of these samples, 900 are testing samples. Furthermore, the robustness of the proposed method is assessed with two additional external datasets (TCGA and PAIP) and a dataset of samples collected directly from the proposed prototype. Our proposed method predicts, for the patch-based tiles, a class based on the severity of the dysplasia and uses that information to classify the whole slide. It is trained with an interpretable mixed-supervision scheme to leverage the domain knowledge introduced by pathologists through spatial annotations. The mixed-supervision scheme allowed for an intelligent sampling strategy effectively evaluated in several different scenarios without compromising the performance. On the internal dataset, the method shows an accuracy of 93.44% and a sensitivity between positive (low-grade and high-grade dysplasia) and non-neoplastic samples of 0.996. On the external test samples varied with TCGA being the most challenging dataset with an overall accuracy of 84.91% and a sensitivity of 0.996., (© 2024. The Author(s).)

High fidelity synaptic neurotransmission in the millisecond range is provided by a defined structural arrangement of synaptic proteins. At the presynapse multi-epitope scaffolding proteins are organized spatially at release sites to guarantee optimal binding of neurotransmitters at receptor clusters. The organization of pre- and postsynaptic proteins in trans-synaptic nanocolumns would thus intuitively support efficient information transfer at the synapse. Visualization of these protein-dense regions as well as the minute size of protein-packed synaptic clefts remains, however, challenging. To enable efficient labeling of these protein complexes, we developed post-gelation immunolabeling expansion microscopy combined with Airyscan super-resolution microscopy. Using ~8-fold expanded samples, Airyscan enables multicolor fluorescence imaging with 20-40 nm spatial resolution. Post-immunolabeling of decrowded (expanded) samples provides increased labeling efficiency and allows the visualization of trans-synaptic nanocolumns. Our approach is ideally suited to investigate the pathological impact on nanocolumn arrangement e.g., in limbic encephalitis with autoantibodies targeting trans-synaptic leucine-rich glioma inactivated 1 protein (LGI1)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sachs, Reinhard, Eilts, Sauer and Werner.)

Objective: Characterize key factors and training needs of U.S. cancer centers in implementing family caregiver support services., Methods: Sequential explanatory mixed methods design consisting of: (1) a national survey of clinicians and administrators from Commission-on-Cancer-accredited cancer centers (N = 238) on factors and training needed for establishing new caregiver programs and (2) qualitative interviews with a subsample of survey respondents (N = 30) to elicit feedback on survey findings and the outline of an implementation strategy to facilitate implementation of evidence-based family caregiver support (the Caregiver Support Accelerator). Survey data was tabulated using descriptive statistics and transcribed interviews were analyzed using thematic analysis., Results: Top factors for developing new caregiver programs were that the program be: consistent with the cancer center's mission and strategic plan (87%), supported by clinic leadership (86.5%) and providers and staff (85.7%), and low cost or cost effective (84.9%). Top training needs were how to: train staff to implement programs (72.3%), obtain program materials (63.0%), and evaluate program outcomes (62.6%). Only 3.8% reported that no training was needed. Qualitative interviews yielded four main themes: (1) gaining leadership, clinician, and staff buy-in and support is essential; (2) cost and clinician burden are major factors to program implementation; (3) training should help with adapting and marketing programs to local context and culture; and (4) the Accelerator strategy is comprehensive and would benefit from key organizational partnerships and policy standards., Conclusion: Findings will be used to inform and refine the Accelerator implementation strategy to facilitate the adoption and growth of evidence-based cancer caregiver support in U.S. cancer centers., (© 2023 John Wiley & Sons Ltd.)

Tumor cell fraction (TCF) estimation is a common clinical task with well-established large interobserver variability. It thus provides an ideal test bed to evaluate potential impacts of employing a tumor cell fraction computer-aided diagnostic (TCFCAD) tool to support pathologists' evaluation. During a National Slide Seminar event, pathologists (n = 69) were asked to visually estimate TCF in 10 regions of interest (ROIs) from hematoxylin and eosin colorectal cancer images intentionally curated for diverse tissue compositions, cellularity, and stain intensities. Next, they re-evaluated the same ROIs while being provided a TCFCAD-created overlay highlighting predicted tumor vs nontumor cells, together with the corresponding TCF percentage. Participants also reported confidence levels in their assessments using a 5-tier scale, indicating no confidence to high confidence, respectively. The TCF ground truth (GT) was defined by manual cell-counting by experts. When assisted, interobserver variability significantly decreased, showing estimates converging to the GT. This improvement remained even when TCFCAD predictions deviated slightly from the GT. The standard deviation (SD) of the estimated TCF to the GT across ROIs was 9.9% vs 5.8% with TCFCAD (P < .0001). The intraclass correlation coefficient increased from 0.8 to 0.93 (95% CI, 0.65-0.93 vs 0.86-0.98), and pathologists stated feeling more confident when aided (3.67 ± 0.81 vs 4.17 ± 0.82 with the computer-aided diagnostic [CAD] tool). TCFCAD estimation support demonstrated improved scoring accuracy, interpathologist agreement, and scoring confidence. Interestingly, pathologists also expressed more willingness to use such a CAD tool at the end of the survey, highlighting the importance of training/education to increase adoption of CAD systems., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Importance: Family caregivers provide the majority of health care to the 18 million patients with cancer in the US. Yet despite providing complex medical and nursing care, a large proportion of caregivers report no formal support or training. In recognition of this gap, many interventions to support cancer caregivers have been developed and tested over the past 2 decades. However, there are few system-level data on whether US cancer centers have adopted and implemented these interventions., Objective: To describe and characterize the availability of family caregiver support programs in US cancer centers., Design, Setting, and Participants: This cross-sectional national survey study was conducted between September 1, 2021, and April 30, 2023. Participants comprised clinical and administrative staff of Commission on Cancer-accredited US cancer centers. Data analysis was performed in May and June 2023., Main Outcomes and Measures: Survey questions about the availability of 11 types of family caregiver programs (eg, peer mentoring, education classes, and psychosocial programs) were developed after literature review, assessment of similar program evaluation surveys, and discussions among a 13-member national expert advisory committee. Family caregiver programs were defined as structured, planned, and coordinated groups of activities and procedures aimed at specifically supporting family caregivers as part of usual care. Survey responses were tabulated using standard descriptive statistics, including means, proportions, and frequencies., Results: Of the surveys sent to potential respondents at 971 adult cancer centers, 238 were completed (response rate, 24.5%). After nonresponse weight adjustment, most cancer centers (75.4%) had at least 1 family caregiver program; 24.6% had none. The most common program type was information and referral services (53.6%). Cancer centers with no programs were more likely to have smaller annual outpatient volumes (χ2 = 11.10; P = .011). Few centers had caregiver programs on training in medical and/or nursing tasks (21.7%), caregiver self-care (20.2%), caregiver-specific distress screening (19.3%), peer mentoring (18.9%), and children caregiving for parents (8.3%). Very few programs were developed from published evidence in a journal (8.1%). The top reason why cancer centers selected their programs was community members requesting the program (26.3%); only 12.3% of centers selected their programs based on scientific evidence. Most programs were funded by the cancer center or hospital (58.6%) or by philanthropy (42.4%)., Conclusions and Relevance: In this survey study, most cancer centers had family caregiver programs; however, a quarter had none. Furthermore, the scope of programming was limited and rarely evidence based, with few centers offering caregiving education and training. These findings suggest that implementation strategies are critically needed to foster uptake of evidence-based caregiver interventions.

Significance: Understanding the organization of biomolecules into complexes and their dynamics is crucial for comprehending cellular functions and dysfunctions, particularly in neuronal networks connected by synapses. In the last two decades, various powerful super-resolution (SR) microscopy techniques have been developed that produced stunning images of synapses and their molecular organization. However, current SR microscopy methods do not permit multicolor fluorescence imaging with 20 to 30 nm spatial resolution., Aim: We developed a method that enables 4-color fluorescence imaging of synaptic proteins in neurons with 20 to 30 nm lateral resolution., Approach: We used post-expansion immunolabeling of eightfold expanded hippocampal neurons in combination with Airyscan and structured illumination microscopy (SIM)., Results: We demonstrate that post-expansion immunolabeling of approximately eightfold expanded hippocampal neurons enables efficient labeling of synaptic proteins in crowded compartments with minimal linkage error and enables in combination with Airyscan and SIM four-color three-dimensional fluorescence imaging with 20 to 30 nm lateral resolution. Using immunolabeling of Synaptobrevin 2 as an efficient marker of the vesicle pool allowed us to identify individual synaptic vesicles colocalized with Rab3-interacting molecule 1 and 2 (RIM1/2), a marker of pre-synaptic fusion sites., Conclusions: Our optimized expansion microscopy approach improves the visualization and location of pre- and post-synaptic proteins and can thus provide invaluable insights into the spatial organization of proteins at synapses., (© 2023 The Authors.)

The diffraction limit of light microscopy poses a problem that is frequently faced in structural analyses of social insect brains. With the introduction of expansion microscopy (ExM), a tool became available to overcome this limitation by isotropic physical expansion of preserved specimens. Our analyses focus on synaptic microcircuits (microglomeruli, MG) in the mushroom body (MB) of social insects, high-order brain centers for sensory integration, learning, and memory. MG undergo significant structural reorganizations with age, sensory experience, and during long-term memory formation. However, the changes in subcellular architecture involved in this plasticity have only partially been accessed yet. Using the western honeybee Apis mellifera as an experimental model, we established ExM for the first time in a social insect species and applied it to investigate plasticity in synaptic microcircuits within MG of the MB calyces. Using combinations of antibody staining and neuronal tracing, we demonstrate that this technique enables quantitative and qualitative analyses of structural neuronal plasticity at high resolution in a social insect brain., (© 2023. The Author(s).)

Homozygous deletion (HD) of the CDKN2A/B locus has emerged as an unfavourable prognostic marker in diffuse gliomas, both IDH-mutant and IDH-wild-type. Testing for CDKN2A/B deletions can be performed by a variety of approaches, including copy number variation (CNV) analysis based on gene array analysis, next generation sequencing (NGS) or fluorescence in situ hybridisation (FISH), but questions remain regarding the accuracy of testing modalities. In this study, we assessed: (1) the utility of S-methyl-5'-thioadenosine phosphorylase (MTAP) and cellular tumour suppressor protein pl61NK4a (p16) immunostainings as surrogate markers for CDKN2A/B HD in gliomas, and (2) the prognostic value of MTAP, across different histological tumour grades and IDH mutation status. One hundred consecutive cases of diffuse and circumscribed gliomas (Cohort 1) were collected, in order to correlate MTAP and p16 expression with the CDKN2A/B status in the CNV plot of each tumour. IDH1 R132H, ATRX and MTAP immunohistochemistry was performed on next generation tissue microarrays (ngTMAs) of 251 diffuse gliomas (Cohort 2) for implementing survival analysis. Complete loss of MTAP and p16 by immunohistochemistry was 100% and 90% sensitive as well as 97% and 89% specific for CDKN2A/B HD, respectively, as identified on CNV plot. Only two cases (2/100) with MTAP and p16 loss of expression did not demonstrate CDKN2A/B HD in CNV plot; however, FISH analysis confirmed the HD for CDKN2A/B. Moreover, MTAP deficiency was associated with shortened survival in IDH-mutant astrocytomas (n=75; median survival 61 vs 137 months; p<0.0001), IDH-mutant oligodendrogliomas (n=59; median survival 41 vs 147 months; p<0.0001) and IDH-wild-type gliomas (n=117; median survival 13 vs 16 months; p=0.011). In conclusion, MTAP immunostaining is an important complement for diagnostic work-up of gliomas, because of its excellent correlation with CDKN2A/B status, robustness, rapid turnaround time and low costs, and provides significant prognostic value in IDH-mutant astrocytomas and oligodendrogliomas, while p16 should be used cautiously., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: COVID-19 has had enormous consequences on global health-care and has resulted in millions of fatalities. The exact mechanism and site of SARS-CoV-2 entry into the body remains insufficiently understood. Recently, novel virus receptors were identified, and alveolar macrophages were suggested as a potential viral entry cell type and vector for intra-alveolar virus transmission. Here, we investigated the protein expression of 10 well-known and novel virus entry molecules along potential entry sites in humans using immunohistochemistry., Methods and Results: Samples of different anatomical sites from up to 93 patients were incorporated into tissue microarrays. Protein expression of ACE2, TMPRSS2, furin, CD147, C-type lectin receptors (CD169, CD209, CD299), neuropilin-1, ASGR1 and KREMEN1 were analysed. In lung tissues, at least one of the three receptors ACE2, ASGR1 or KREMEN1 was expressed in the majority of cases. Moreover, all the investigated molecules were found to be expressed in alveolar macrophages, and co-localisation with SARS-CoV-2 N-protein was demonstrated using dual immunohistochemistry in lung tissue from a COVID-19 autopsy. While CD169 and CD209 showed consistent protein expression in sinonasal, conjunctival and bronchiolar tissues, neuropilin-1 and ASGR1 were mostly absent, suggesting a minor relevance of these two molecules at these specific sites., Conclusion: Our results extend recent discoveries indicating a role for these molecules in virus entry at different anatomical sites. Moreover, they support the notion of alveolar macrophages being a potential entry cell for SARS-CoV-2., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: For the improvement of outcome after renal transplantation it is important to predict future risk of major adverse cardiac events as well as all-cause mortality. We aimed to determine the relationship of pre-transplant NT-proBNP with major adverse cardiac events and all-cause mortality after transplant in patients on the waiting-list with preserved left ventricular ejection fraction., Patients and Methods: We included 176 patients with end-stage renal disease and preserved left ventricular ejection fraction who received a kidney transplant. MACE was defined as myocardial infarction (ST-segment elevation [STEMI] or non-ST-segment elevation [NSTEMI]), stroke or transient ischemic attack), coronary artery disease requiring intervention or bypass or death from cardiovascular causes., Results: MACE occurred in 28/176 patients. Patients with NT-proBNP levels above 4350 pg/ml had 1- and 5-year survival rates of 90.67% and 68.20%, whereas patients with NT-proBNP levels below 4350 pg/ml had 1- and 5-year survival rates of 100% and 90.48% (p < 0.01). 1- and 5-year MACE-free survival rates were calculated as 78.82% and 74.68% for patients with NT-proBNP > 4350 pg/ml and 93.33% and 91.21% for patients with NT-proBNP < 4350 pg/ml (p < 0.01)., Conclusions: Pre-transplant NT-proBNP might identify renal transplant candidates at risk for MACE after transplant., (© 2023. The Author(s).)

Background: Beta Trace Protein (BTP) is a biomarker for residual kidney function which has been linked to cardiovascular and all-cause mortality in haemodialysis patients. Following renal transplantation, recipients remain at increased risk for cardiovascular events compared with the general population. We aimed to determine the relationship of pre-transplant BTP to major adverse cardiac events (MACE) in patients following kidney transplantation., Methods: We included 384 patients with end-stage renal disease who received a kidney transplant. MACE was defined as myocardial infarction (ST-segment elevation or non-ST-segment elevation, stroke or transient ischemic attack), coronary artery disease requiring intervention or bypass or death for cardiovascular reason. The association between pre-transplant serum BTP concentration and post-transplant MACE was evaluated by Kaplan-Meier and Cox regression analyses., Results: Post-transplant MACE occurred in 70/384 patients. Pre-transplant BTP was significantly higher in patients with post-transplant MACE (14.36 ± 5.73 mg/l vs. 11.26 ± 5.11 mg/l; p < .01). Next to smoking (HR 1.81), age > 56.38 years (HR 1.97) and pre-existing coronary heart disease (HR 8.23), BTP above the cut off value of 12.7 mg/l was confirmed as independent risk factor for MACE (HR 2.02, all p < .05). MACE-free survival inversely correlated with pre-transplant BTP levels., Conclusions: Pre-transplant serum BTP concentration may identify renal transplant recipients with higher risk of post-transplant MACE., (© 2022 The Authors. Nephrology published by John Wiley & Sons Australia, Ltd on behalf of Asian Pacific Society of Nephrology.)

Background: Localization-based super-resolution microscopy resolves macromolecular structures down to a few nanometers by computationally reconstructing fluorescent emitter coordinates from diffraction-limited spots. The most commonly used algorithms are based on fitting parametric models of the point spread function (PSF) to a measured photon distribution. These algorithms make assumptions about the symmetry of the PSF and thus, do not work well with irregular, non-linear PSFs that occur for example in confocal lifetime imaging, where a laser is scanned across the sample. An alternative method for reconstructing sparse emitter sets from noisy, diffraction-limited images is compressed sensing, but due to its high computational cost it has not yet been widely adopted. Deep neural network fitters have recently emerged as a new competitive method for localization microscopy. They can learn to fit arbitrary PSFs, but require extensive simulated training data and do not generalize well. A method to efficiently fit the irregular PSFs from confocal lifetime localization microscopy combining the advantages of deep learning and compressed sensing would greatly improve the acquisition speed and throughput of this method., Results: Here we introduce ReCSAI, a compressed sensing neural network to reconstruct localizations for confocal dSTORM, together with a simulation tool to generate training data. We implemented and compared different artificial network architectures, aiming to combine the advantages of compressed sensing and deep learning. We found that a U-Net with a recursive structure inspired by iterative compressed sensing showed the best results on realistic simulated datasets with noise, as well as on real experimentally measured confocal lifetime scanning data. Adding a trainable wavelet denoising layer as prior step further improved the reconstruction quality., Conclusions: Our deep learning approach can reach a similar reconstruction accuracy for confocal dSTORM as frame binning with traditional fitting without requiring the acquisition of multiple frames. In addition, our work offers generic insights on the reconstruction of sparse measurements from noisy experimental data by combining compressed sensing and deep learning. We provide the trained networks, the code for network training and inference as well as the simulation tool as python code and Jupyter notebooks for easy reproducibility., (© 2022. The Author(s).)

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor., (© 2022. The Author(s).)

SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2-RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor.

Background: Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could contribute to axonopathy and presynaptic dysfunction in SMA., Methods: Using super-resolution microscopy, proximity ligation assay (PLA) and live imaging of cultured motoneurons from a mouse model of SMA, we investigated the dynamics of the axonal ER and ribosome distribution and activation., Results: We observed that the dynamic remodeling of ER was impaired in axon terminals of Smn-deficient motoneurons. In addition, in axon terminals of Smn-deficient motoneurons, ribosomes failed to respond to the brain-derived neurotrophic factor stimulation, and did not undergo rapid association with the axonal ER in response to extracellular stimuli., Conclusions: These findings implicate impaired dynamic interplay between the ribosomes and ER in axon terminals of motoneurons as a contributor to the pathophysiology of SMA and possibly also other motoneuron diseases., (© 2022. The Author(s).)

The development of endosomal disruptive agents is a major challenge in the field of drug delivery and pharmaceutical chemistry. Current endosomal disruptive agents are composed of polymers, peptides, and nanoparticles and have had limited clinical impact. Alternatives to traditional endosomal disruptive agents are therefore greatly needed. In this report, we introduce a new class of low molecular weight endosomal disruptive agents, termed caged surfactants, that selectively disrupt endosomes via reversible PEGylation under acidic endosomal conditions. The caged surfactants have the potential to address several of the limitations hindering the development of current endosomal disruptive agents, such as high toxicity and low excretion, and are amenable to traditional medicinal chemistry approaches for optimization. In this report, we synthesized three generations of caged surfactants and demonstrated that they can enhance the ability of cationic lipids to deliver mRNA into primary cells. We also show that caged surfactants can deliver siRNA into cells when modified with the RNA-binding dye thiazole orange. We anticipate that the caged surfactants will have numerous applications in pharmaceutical chemistry and drug delivery given their versatility.

Background: The novel beta-coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), enters the human body via mucosal surfaces of the upper and/or lower respiratory tract. Viral entry into epithelial cells is mediated via angiotensin-converting enzyme 2 (ACE2) and auxiliary molecules, but the precise anatomic site of infection still remains unclear., Methods: Here, we systematically investigated the main SARS-CoV-2 receptor proteins ACE2 and transmembrane serine protease 2 (TMPRSS2), as well as 2 molecules potentially involved in viral entry, furin and CD147, in formalin-fixed, paraffin-embedded human tissues. Tissue microarrays incorporating a total of 879 tissue cores from conjunctival (n = 84), sinonasal (n = 95), and lung (bronchiolar/alveolar; n = 96) specimens were investigated for protein expression by immunohistochemistry., Results: ACE2 and TMPRSS2 were expressed in ciliated epithelial cells of the conjunctivae and sinonasal tissues, with highest expression levels observed in the apical cilia. In contrast, in the lung, the expression of those molecules in bronchiolar and alveolar epithelial cells was much rarer and only very focal when present. Furin and CD147 were more uniformly expressed in all tissues analyzed, including the lung. Interestingly, alveolar macrophages consistently expressed high levels of all 4 molecules investigated., Conclusions: Our study confirms and extends previous findings and contributes to a better understanding of potential SARS-CoV-2 infection sites along the human respiratory tract., (© 2022 S. Karger AG, Basel.)

In neurons, the endoplasmic reticulum (ER) forms a highly dynamic network that enters axons and presynaptic terminals and plays a central role in Ca2+ homeostasis and synapse maintenance; however, the underlying mechanisms involved in regulation of its dynamic remodeling as well as its function in axon development and presynaptic differentiation remain elusive. Here, we used high-resolution microscopy and live-cell imaging to investigate rapid movements of the ER and ribosomes in axons of cultured motoneurons after stimulation with brain-derived neurotrophic factor. Our results indicate that the ER extends into axonal growth cone filopodia, where its integrity and dynamic remodeling are regulated mainly by actin and the actin-based motor protein myosin VI (encoded by Myo6). Additionally, we found that in axonal growth cones, ribosomes assemble into 80S subunits within seconds and associate with the ER in response to extracellular stimuli, which describes a novel function of axonal ER in dynamic regulation of local translation. This article has an associated First Person interview with Chunchu Deng, joint first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)