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Bing-Xin Lin, Chun-Sen Xu Department of Breast Surgery, Union Hospital of Fujian Medical University, Fuzhou, People’s Republic of ChinaCorrespondence: Chun-Sen XuDepartment of Breast Surgery, Union Hospital of Fujian Medical University, No. 29 of Xinquan Road, Gulou District, Fuzhou, 350001, People’s Republic of ChinaTel/Fax +86 591 83357896Email chunsenxuparis@163.comBackground: To analyze the risk factors of a peripherally inserted central catheter (PICC)-related venous thrombosis in patients with breast cancer undergoing chemotherapy and explore its preventive measures.Methods: Data of 780 patients with breast cancer who underwent PICC chemotherapy in our hospital from January 2014 to June 2015 were retrospectively analyzed. The incidence of catheter-related thrombosis was observed, and related factors of venous thrombosis were analyzed.Results: Among the 780 patients with breast cancer, 36 developed PICC-related venous thrombosis. The incidence of which was 4.62% (36/780). The PICC retention time ranged between 60 and 136 days, and the median time was 92 days. Thrombosis was found to occur within seven days after catheterization in three patients (8.33%), between 7 and 30 days in 18 patients (50%), between 31 and 92 days in 12 patients (33.3%), and ≥ 92 days in three patients (8.33%). Basilic vein puncture-induced thrombosis occurred in 25 patients (3.68%), and median cubital vein and cephalic vein puncture-induced thrombosis occurred in 11 patients (10.78%). The difference was statistically significant (P = 0.001). Thrombosis was not associated with age, punctured limb, platelet count, or chemotherapy drugs (P > 0.05).Conclusion: Blood vessel puncture was the main factor that affected PICC-related thrombosis in breast cancer chemotherapy. The basilic vein should be the primary choice for blood vessel puncture. Prolonged catheter retention does not increase the risk of thrombosis.Keywords: breast cancer, peripherally inserted central catheter, thrombosis

Rubia tibetica Hook. f is a traditional Tibetan medicinal plant, with its roots serving as the primary medicinal part. Two new compounds, Rubiaxylm C and E (1 and 3), and one new natural compound, Rubiaxylm D (2) were isolated from R. tibetica, along with five known compounds (4-8). The structures of the previously undescribed compounds were elucidated by HR-ESIMS, 1D/2D NMR spectroscopic data and electronic circular dichroism calculations. Compound 1 is a rare anthrone substituted with a carbonyl group at position 1. Furthermore, all isolated compounds were tested for their cytotoxicity against HepG2 cell lines., (© 2024 Wiley‐VCH GmbH.)

Primary cilia are sensory organelles present in many cell types, partaking in various signaling processes. Primary cilia of pancreatic beta cells play pivotal roles in paracrine signaling and their dysfunction is linked to diabetes. Yet, the structural basis for their functions is unclear. We present three-dimensional reconstructions of beta cell primary cilia by electron and expansion microscopy. These cilia are spatially confined within deep ciliary pockets or narrow spaces between cells, lack motility components and display an unstructured axoneme organization. Furthermore, we observe a plethora of beta cell cilia-cilia and cilia-cell interactions with other islet and non-islet cells. Most remarkably, we have identified and characterized axo-ciliary synapses between beta cell cilia and the cholinergic islet innervation. These findings highlight the beta cell cilia's role in islet connectivity, pointing at their function in integrating islet intrinsic and extrinsic signals and contribute to understanding their significance in health and diabetes., (© 2024. The Author(s).)

Telmisartan, an angiotensin II type 1 receptor (AT1R) blocker, exhibits broad anti-tumor activity. However, in vitro, anti-proliferative effects are shown at doses far beyond the therapeutic plasma concentration. Considering the role of tumor microenvironment in glioma progression, glioma-astrocyte co-cultures were employed to test the anti-tumor potential of low-dose telmisartan. When a high dose was required for a direct anti-proliferative effect on glioma cell lines, a low dose significantly inhibited glioma cell proliferation and migration in the co-culture system. Under co-culture conditions, upregulated IL-6 expression in astrocytes played a critical role in glioma progression. Silencing IL-6 in astrocytes or IL-6R in glioma cells reduced proliferation and migration. Telmisartan (5 μM) inhibited astrocytic IL-6 expression, and its anti-tumor effects were reversed by silencing IL-6 or IL-6R and inhibiting signal transducer and activator of transcription 3 (STAT3) activity in glioma cells. Moreover, the telmisartan-driven IL-6 downregulation was not imitated by losartan, an AT1R blocker with little capacity of peroxisome proliferator-activated receptor-gamma (PPARγ) activation, but was eliminated by a PPARγ antagonist, indicating that the anti-glioma effects of telmisartan rely on its PPARγ agonistic activity rather than AT1R blockade. This study highlights the importance of astrocytic IL-6-mediated paracrine signaling in glioma growth and the potential of telmisartan as an adjuvant therapy for patients with glioma, especially those with hypertension., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The ventral pallidum (VP) is critical for motivated behaviors. While contemporary work has begun to elucidate the functional diversity of VP neurons, the molecular heterogeneity underlying this functional diversity remains incompletely understood. We used snRNA-seq and in situ hybridization to define the transcriptional taxonomy of VP cell types in mice, macaques, and baboons. We found transcriptional conservation between all three species, within the broader neurochemical cell types. Unique dopaminoceptive and cholinergic subclusters were identified and conserved across both primate species but had no homolog in mice. This harmonized consensus VP cellular atlas will pave the way for understanding the structure and function of the VP and identified key neuropeptides, neurotransmitters, and neuro receptors that could be targeted within specific VP cell types for functional investigations., Teaser: Genetic identity of ventral pallidum cell types is conserved across rodents and primates at the transcriptional level.

Euphorbia dentata Michx. is an invasive plant species in China, known for its toxicity and potential to reduce crop yields, posing numerous threats. To gain a deeper understanding of this invasive plant, phytochemical methods were employed to isolate 13 terpenoids ( 1 - 11 , 19 , 20 ) and 7 sterols ( 12 - 18 ) from the ethanol extract of E. dentata , identifying one new compound and 19 known compounds. Within spectroscopic methods such as NMR, HR-ESI-MS, and ECD, the structures and absolute configurations of these compounds were established. Among them, dentatacid A ( 11 ) possesses an unprecedented 2, 3- seco -arbor-2, 3-dioic skeleton within the potential biosynthetic pathway proposed. Dentatacid A also exhibited excellent anti-proliferative activity against the HT-29 (human colorectal adenocarcinoma) cell line, with an IC 50 value of 2.64 ± 0.78 μM, which was further confirmed through network pharmacology and molecular docking. This study significantly expands the chemical diversity of E. dentata and offers new insights into the resource utilization and management of this invasive plant from the perspective of natural product discovery.

Purpose: This study aimed to assess the drug risk of drug-related keratitis and track the epidemiological characteristics of drug-related keratitis., Methods: This study analyzed data from the U.S. Food and Drug Administration (FDA) Adverse Event Reporting System (FAERS) database from January 2004 to December 2023. A disproportionality analysis was conducted to assess drug-related keratitis with positive signals, and drugs were classified and assessed with regard to their drug-induced timing and risk of drug-related keratitis., Results: A total of 1606 drugs were reported to pose a risk of drug-related keratitis in the FAERS database, and, after disproportionality analysis and screening, 17 drugs were found to significantly increase the risk of drug-related keratitis. Among them, seven were ophthalmic medications, including dorzolamide (reporting odds ratio [ROR] = 3695.82), travoprost (ROR = 2287.27), and brimonidine (ROR = 2118.52), and 10 were non-ophthalmic medications, including tralokinumab (ROR = 2609.12), trazodone (ROR = 2377.07), and belantamab mafodotin (ROR = 680.28). The top three drugs having the highest risk of drug-related keratitis were dorzolamide (Bayesian confidence propagation neural network [BCPNN] = 11.71), trazodone (BCPNN = 11.11), and tralokinumab (BCPNN = 11.08). The drug-induced times for non-ophthalmic medications were significantly shorter than those for ophthalmic medications (mean days, 141.02 vs. 321.96, respectively; P < 0.001). The incidence of drug-related keratitis reached its peak in 2023., Conclusions: Prevention of drug-related keratitis is more important than treatment. Identifying the specific risks and timing of drug-induced keratitis can support the development of preventive measures., Translational Relevance: Identifying the specific drugs related to medication-related keratitis is of significant importance for drug vigilance in the occurrence of drug-related keratitis.

Purpose and Design: This study aimed to evaluate the risk of drug-related dry eye using real-world data, underscoring the significance of tracing pharmacological etiology for distinct clinical types of dry eye., Methods: Analyzing adverse event reports in the Food and Drug Administration Adverse Event Reporting System (FAERS) from January 2004 to September 2023, we employed disproportionality analysis and the Bayesian confidence propagation neural network algorithm. The analysis involved categorizing drugs causing dry eye, assessing risk levels, and conducting segmental assessments based on the time of onset of drug-related dry eye adverse reactions., Results: In the FAERS database, adverse reactions related to dry eye were linked to 1160 drugs. Disproportionality analysis identified 33 drugs with significant risk, notably in ophthalmic (brimonidine, bimatoprost), oncology (tisotumab vedotin, erdafitinib), and other medications (isotretinoin, oxymetazoline). The top three drugs with the highest risk of drug-related dry eye are isotretinoin (Bayesian confidence propagation neural network (BCPNN) = 6.88), tisotumab vedotin (BCPNN = 6.88), and brimonidine (BCPNN = 6.77). Among different categories of drugs, respiratory medications have the shortest mean onset time for drug-related dry eye, averaging 50.99 days. The prevalence skewed towards females (69.9 %), particularly in menopausal and elderly individuals (45-70 years old, mean age 54.7 ± 18.2). Reports of drug-related dry eye adverse reactions showed an annual increase., Conclusion: Informed clinical decision-making is crucial for preventing drug-related dry eye. Assessing the risk of dry eyes associated with both local and systemic medications helps optimize treatment and provide necessary cautionary information., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pacinian corpuscles are rapidly adapting mechanoreceptor end-organs that detect transient touch and high-frequency vibration. In the prevailing model, these properties are determined by the outer core, which acts as a mechanical filter limiting static and low-frequency stimuli from reaching the afferent terminal-the sole site of touch detection in corpuscles. Here, we determine the detailed 3D architecture of corpuscular components and reveal their contribution to touch detection. We show that the outer core is dispensable for rapid adaptation and frequency tuning. Instead, these properties arise from the inner core, composed of gap junction-coupled lamellar Schwann cells (LSCs) surrounding the afferent terminal. By acting as additional touch sensing structures, LSCs potentiate mechanosensitivity of the terminal, which detects touch via fast-inactivating ion channels. We propose a model in which Pacinian corpuscle function is mediated by an interplay between mechanosensitive LSCs and the afferent terminal in the inner core., Competing Interests: Competing Interests. CSX is an inventor of a US patent assigned to Howard Hughes Medical Institute for the enhanced FIB-SEM systems used in this work: Xu, C.S., Hayworth K.J., Hess H.F. (2020) Enhanced FIB-SEM systems for large-volume 3D imaging. US Patent 10,600,615, 24 Mar 2020. The authors declare no other competing interests.

The current standard treatment for ovarian cancer consists of surgery to reduce the size of the tumor, followed by treatment with chemotherapeutic drugs, which have major side effects. Therefore, finding a new natural product drug with fewer side effects is a strategy. Delphinium brunonianum (D. brunonianum) is a traditional Tibetan medicine, mainly from southern Tibet, China, whereas the chemical constituents in this plant remain elusive. The major metabolites in the dichloromethane fraction of D. brunonianum were analyzed and purified by HPLC and various column chromatography techniques. Nine diterpenoid alkaloids (1-9) and one amide alkaloid (10) were isolated from D. brunonianum, including three novel C19-type diterpenoid alkaloids (Brunonianines D-F) (1-3). Their structures were elucidated by 1D/2D NMR, HR-ESI-MS and single-crystal X-ray diffraction analyses. All compounds were evaluated for toxicity in four tumor cell lines. Most of the compounds exhibited potent inhibitory effects on Skov-3 cell lines, with IC 50 values ranging from 2.57 to 8.05 μM. The western blotting experiment was used to further analyze the expression levels of molecules in the Bax/Bcl-2/Caspase-3 signaling pathway for compound 1. Molecular docking was performed to predict the binding modes of Brunonianine D with target proteins. In vivo experiments were also performed and evaluated in real time by monitoring the size of the Skov-3 tumor. Additionally, tumor H&E staining and the TUNEL assay used to evaluate anti-tumor effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Hepatic ischemia-reperfusion (IR) injury significantly impacts liver transplantation success, yet current treatments remain inadequate. This study explores the role of Proto-oncogene serine/threonine-protein kinase (Pim-1) in liver IR, an area previously unexplored. Utilizing a mouse liver IR in vivo model and a MIHA cell hypoxia-reoxygenation in vitro model, we observed that Pim-1 expression increases following IR, inversely correlating with serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Increased Pim-1 expression stabilizes mitochondrial membranes by modifying Drp1 phosphorylation, reducing mitochondrial fission and apoptosis, thereby mitigating liver damage. Additionally, we discovered that elevated Pim-1 expression is dependent on the trimethylation of histone H3 lysine 9 during liver IR. These findings underscore the importance and potential clinical application of targeting Pim-1 in treating hepatic IR, presenting a novel therapeutic avenue., Competing Interests: The authors have declared that no conflict of interest exists., (© 2024 The Author(s).)

Single-particle tracking demonstrates that individual filaments in bundles of vimentin intermediate filaments are transported in the cytoplasm by motor proteins along microtubules. Furthermore, using 3D FIB-SEM the authors showed that vimentin filament bundles are loosely packed and coaligned with microtubules. Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a ~50 μm 3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time., Competing Interests: Statements and Declarations All authors declare no conflict of interest.

Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca 2+  is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress., Competing Interests: Declaration of interests Portions of the technology described here are covered by US Patent 10,600,615 titled “Enhanced FIB-SEM systems for large-volume 3D imaging,” which was issued to C.S.X., K.J.H., and H.F.H. and assigned to Howard Hughes Medical Institute on 24 March 2020., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vision provides animals with detailed information about their surroundings, conveying diverse features such as color, form, and movement across the visual scene. Computing these parallel spatial features requires a large and diverse network of neurons, such that in animals as distant as flies and humans, visual regions comprise half the brain's volume. These visual brain regions often reveal remarkable structure-function relationships, with neurons organized along spatial maps with shapes that directly relate to their roles in visual processing. To unravel the stunning diversity of a complex visual system, a careful mapping of the neural architecture matched to tools for targeted exploration of that circuitry is essential. Here, we report a new connectome of the right optic lobe from a male Drosophila central nervous system FIB-SEM volume and a comprehensive inventory of the fly's visual neurons. We developed a computational framework to quantify the anatomy of visual neurons, establishing a basis for interpreting how their shapes relate to spatial vision. By integrating this analysis with connectivity information, neurotransmitter identity, and expert curation, we classified the ~53,000 neurons into 727 types, about half of which are systematically described and named for the first time. Finally, we share an extensive collection of split-GAL4 lines matched to our neuron type catalog. Together, this comprehensive set of tools and data unlock new possibilities for systematic investigations of vision in Drosophila , a foundation for a deeper understanding of sensory processing.

The hepatocytes within the liver present an immense capacity to adapt to changes in nutrient availability. Here, by using high resolution volume electron microscopy, we map how hepatic subcellular spatial organization is regulated during nutritional fluctuations and as a function of liver zonation. We identify that fasting leads to remodeling of endoplasmic reticulum (ER) architecture in hepatocytes, characterized by the induction of single rough ER sheet around the mitochondria, which becomes larger and flatter. These alterations are enriched in periportal and mid-lobular hepatocytes but not in pericentral hepatocytes. Gain- and loss-of-function in vivo models demonstrate that the Ribosome receptor binding protein1 (RRBP1) is required to enable fasting-induced ER sheet-mitochondria interactions and to regulate hepatic fatty acid oxidation. Endogenous RRBP1 is enriched around periportal and mid-lobular regions of the liver. In obesity, ER-mitochondria interactions are distinct and fasting fails to induce rough ER sheet-mitochondrion interactions. These findings illustrate the importance of a regulated molecular architecture for hepatocyte metabolic flexibility., (© 2024. The Author(s).)

To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites 1,2 . Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites 3,4 . ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle 5,6 . However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation 7,8 , a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis., (© 2024. The Author(s).)

Adult vaccination is an important component of the life-course immunization for all. Strengthening adult vaccination in China contributes to shrinking immunization gaps between regions and groups, enhancing the overall immunity of our population, and promoting health equity and social prosperity. Chinese adults bear the heavy burden of vaccine preventable diseases such as influenza, pneumococcal diseases and shingles, and have low coverage of vaccines against those diseases, so it is necessary to make efforts to improve adult vaccination development. This article focuses on elaborating the values of adult vaccination, introducing the current status of adult vaccination abroad, and analyzing the challenges and existing foundations for China to provide adult vaccination, and makes suggestions for the building and development of adult vaccination.

Objective: To understand the current situation of vaccination services for adults in China, explore how to establish a stable and efficient vaccination service system for adults, and provide reference for formulating corresponding policies. Methods: The vaccination information systems of nine provinces in China were used to obtain information on urban and rural vaccination of influenza vaccine, 23-valent pneumococcal polysaccharide vaccine (PPV23), and human papillomavirus vaccine (HPV) from 2019 to 2021. The indicator, vaccination rate/full vaccination rate, was used for statistical description. Results: The vaccination rate/full vaccination rate of the three vaccines in eastern China was generally higher than that in central and western China. The vaccination rate/full vaccination rate in urban areas was generally higher than that in rural areas. From 2019 to 2021, the vaccination rates of influenza vaccine among people aged 60 years and above in urban and rural areas were 2.96%, 6.29%, 6.14% and 1.29%, 2.58%, 2.94%, respectively. The vaccination rates of the PPV23 among people aged 60 years and above in urban and rural areas increased year by year, with rates of 0.38%, 1.05%, 1.15% and 0.14%, 0.49%, 0.59%, respectively. From 2019 to 2021, the HPV coverage of female adults aged 27-45 years in urban and rural areas increased year by year, with rates of 0.46%, 0.93%, 1.88% and 0.17%, 0.40%, 1.08%, respectively. Conclusion: The vaccination rates of influenza vaccine,PPV23 vaccine and HPV vaccine for adults in China are relatively low, with higher rates in the eastern region than in the central and western regions, and higher rates in urban areas than in rural areas. It is recommended to formulate corresponding health and economic policies and explore a suitable vaccination service system for adults in China to improve vaccination rates.

For most model organisms in neuroscience, research into visual processing in the brain is difficult because of a lack of high-resolution maps that capture complex neuronal circuitry. The microinsect Megaphragma viggianii, because of its small size and non-trivial behavior, provides a unique opportunity for tractable whole-organism connectomics. We image its whole head using serial electron microscopy. We reconstruct its compound eye and analyze the optical properties of the ommatidia as well as the connectome of the first visual neuropil-the lamina. Compared with the fruit fly and the honeybee, Megaphragma visual system is highly simplified: it has 29 ommatidia per eye and 6 lamina neuron types. We report features that are both stereotypical among most ommatidia and specialized to some. By identifying the "barebones" circuits critical for flying insects, our results will facilitate constructing computational models of visual processing in insects., Competing Interests: Declaration of interests Portions of the technology described here are covered by U.S. Patent 10,600,615 titled “Enhanced FIB-SEM systems for large-volume 3D imaging,” which was issued to C.S.X. and H.F.H. (as well as Kenneth J. Hayworth) and assigned to the Howard Hughes Medical Institute on March 24, 2020., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: To explore the altered functional connectivity (FC) of the nucleus tractus solitarii (NTS) in patients with chronic cough after lung surgery using resting-state functional magnetic resonance imaging (rs-fMRI), and the association between abnormal FC and clinical scale scores., Methods: A total of 22 patients with chronic cough after lung surgery and 22 healthy controls were included. Visual analog scale (VAS), Mandarin Chinese version of the Leicester Cough Questionnaire (LCQ-MC), and Hamilton anxiety rating scale (HAMA) scores were assessed, and rs-fMRI data were collected. The FC analysis was performed using the NTS as the seed point, and FC values with all voxels in the whole brain were calculated. A two-sample t-test was used to compare FC differences between the two groups. The FC values of brain regions with differences were extracted and correlated with clinical scale scores., Results: In comparison to healthy controls, FC values in the NTS and anterior cingulate cortex(ACC) were reduced in patients with chronic cough after lung surgery (GRF correction, p-voxel < 0.005, p-cluster < 0.05) which were positively correlated with LCQ-MC scores (r = 0.534, p = 0.011), but with VAS (r = -0.500, p = 0.018), HAMA (r = -0.713, p < 0.001) scores were negatively correlated., Conclusions: Reduced FC of the NTS with ACC may be associated with cough hypersensitivity and may contribute to anxiety in patients with chronic cough after lung surgery., (© 2023 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Across mammalian skin, structurally complex and diverse mechanosensory end organs respond to mechanical stimuli and enable our perception of dynamic, light touch. How forces act on morphologically dissimilar mechanosensory end organs of the skin to gate the requisite mechanotransduction channel Piezo2 and excite mechanosensory neurons is not understood. Here, we report high-resolution reconstructions of the hair follicle lanceolate complex, Meissner corpuscle, and Pacinian corpuscle and the subcellular distribution of Piezo2 within them. Across all three end organs, Piezo2 is restricted to the sensory axon membrane, including axon protrusions that extend from the axon body. These protrusions, which are numerous and elaborate extensively within the end organs, tether the axon to resident non-neuronal cells via adherens junctions. These findings support a unified model for dynamic touch in which mechanical stimuli stretch hundreds to thousands of axon protrusions across an end organ, opening proximal, axonal Piezo2 channels and exciting the neuron., Competing Interests: Declaration of interests C.S.X. is the inventor of a US patent assigned to HHMI for the enhanced FIB-SEM systems used in this work: Xu, C.S., Hayworth, K.J., and Hess, H.F. (2020) Enhanced FIB-SEM systems for large-volume 3D imaging. US Patent 10,600,615, 24 Mar. 2020., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Astrocytes are crucially involved in neuroinflammation. Activated astrocytes exhibit at least two phenotypes, A1 (neurotoxic) and A2 (neuroprotective). The A1 phenotype is the major reactive astrocyte phenotype involved in aging and neurodegenerative diseases. Telmisartan, which is an antihypertensive agent, is a promising neuroprotective agent. This study aimed to investigate the effects of telmisartan on the phenotype of reactive astrocytes. Astrocytes were activated by culturing with the conditioned medium derived from lipopolysaccharide-stimulated microglia. This conditioned medium induced early, transient A2 astrocyte conversion (within 24 h) and late, sustained A1 conversion (beginning at 24 h and lasting up to 7 days), with a concomitant increase in the production of pro-inflammatory cytokines (interleukin [IL]-1β, tumor necrosis factor [TNF]α, and IL-6) and phosphorylation of nuclear factor-κB (NF-κB)/p65. Telmisartan treatment promoted and inhibited A2 and A1 conversion, respectively. Telmisartan reduced total and phosphorylated p65 protein levels. Losartan, a specific angiotensin II type-1 receptor (AT1R) blocker, did not influence the reactive state of astrocytes. Additionally, AT1R activation by angiotensin II did not induce the expression of pro-inflammatory cytokines and A1/A2 markers, indicating that the AT1R signaling pathway is not involved in the astrocyte-mediated inflammatory response. A peroxisome proliferator-activated receptor γ (PPARγ) antagonist reversed the effects of telmisartan. Moreover, telmisartan-induced p65 downregulation was reversed by the proteasome inhibitor MG132. These results indicate that telmisartan suppresses activated microglia-induced neurotoxic A1 astrocyte conversion through p65 degradation. Our findings contribute towards the elucidation of the anti-inflammatory activity of telmisartan in brain disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Clinician moral distress has been documented over the past several decades as occurring within numerous healthcare disciplines, often in relation to clinicians' involvement in patients' end-of-life decision-making. The resulting harms impact clinician well-being, patient well-being, and healthcare system functioning. Given Covid-19's catastrophic death toll and associated demands on end-of-life decision-making processes, the pandemic represents a particularly important context within which to understand clinician moral distress. Thus, we conducted a convergent mixed methods study to examine its prevalence, associations with clinicians' demographic and professional characteristics, and contributing circumstances among Veterans Health Administration (VA) clinicians. The study, conducted in April 2021, consisted of a cross-sectional on-line survey of VA clinicians at 20 VA Medical Centers with professional jurisdiction to place life-sustaining treatment orders working who were from a number of select specialties. The survey collected quantitative data on respondents' demographics, clinical practice characteristics, attitudes and behaviors related to goals of care conversations, intensity of moral distress during "peak-Covid," and qualitative data via an open-ended item asking for respondents to describe contributing circumstances if they had indicated any moral distress. To understand factors associated with heightened moral distress, we analyzed quantitative data using bivariate and multivariable regression analyses and qualitative data using a hybrid deductive/inductive thematic approach. Mixed methods analysis followed, whereby we compared the quantitative and qualitative datasets and integrated findings at the analytic level. Out of 3,396 eligible VA clinicians, 323 responded to the survey (9.5% adjusted response rate). Most respondents (81%) reported at least some moral distress during peak-Covid. In a multivariable logistic regression, female gender (OR 3.35; 95% CI 1.53-7.37) was associated with greater odds of moral distress, and practicing in geriatrics/palliative care (OR 0.40; 95% CI 0.18-0.87) and internal medicine/family medicine/primary care (OR 0.46; 95% CI 0.22-0.98) were associated with reduced odds of moral distress compared to medical subspecialties. From the 191 respondents who completed the open-ended item, five qualitative themes emerged as moral distress contributors: 1) patient visitation restrictions, 2) anticipatory actions, 3) clinical uncertainty related to Covid, 4) resource shortages, and 5) personal risk of contracting Covid. Mixed methods analysis found that quantitative results were consistent with these last two qualitative themes. In sum, clinician moral distress was prevalent early in the pandemic. This moral distress was associated with individual-, system-, and situation-level contributors. These identified contributors represent leverage points for future intervention to mitigate clinician moral distress and its negative outcomes during future healthcare crises and even during everyday clinical care., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Retinoid X receptor (RXR), particularly RXRα, has been implicated in cardiovascular diseases. However, the functional role of RXR activation in myocardial infarction (MI) remains unclear. This study aimed to determine the effects of RXR agonists on MI and to dissect the underlying mechanisms. Sprague-Dawley (SD) rats were subjected to MI and then treated (once daily for 4 weeks) with either RXR agonist bexarotene (10 or 30 mg/kg body weight) or vehicle. Heart function was determined using echocardiography and cardiac hemodynamic measurements. Four weeks post MI, myocardial tissues were collected to evaluate cardiac remodeling. Primary cardiac fibroblasts (CFs) were treated with or without RXR ligand 9-cis-RA followed by stimulation with TGF-β1. Immunoblot, immunofluorescence, and co-immunoprecipitation were performed to elucidate the regulatory role of RXR agonists in TGF-β1/Smad signaling. In vivo treatment with Bexarotene moderately affects systemic inflammation and apoptosis and ameliorated left ventricular dysfunction after MI in rat model. In contrast, bexarotene significantly inhibited post-MI myocardial fibrosis. Immunoblot analysis of heart tissue homogenates from MI rats revealed that bexarotene regulated the activation of the TGF-β1/Smad signaling pathway. In vitro, 9-cis-RA inhibited the TGF-β1-induced proliferation and collagen production of CFs. Importantly, upon activation by 9-cis-RA, RXRα interacted with p-Smad2 in cytoplasm, inhibiting the TGF-β1-induced nuclear translocation of p-Smad2, thereby negatively regulating TGF-β1/Smad signaling and attenuating the fibrotic response of CFs. These findings suggest that RXR agonists ameliorate post-infarction myocardial fibrosis, maladaptive remodeling, and heart dysfunction via attenuation of fibrotic response in CFs through inhibition of the TGF-β1/Smad pathway activation., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanosensory corpuscles detect transient touch and vibration in the skin of vertebrates, enabling precise sensation of the physical environment. The corpuscle contains a mechanoreceptor afferent surrounded by lamellar cells (LCs), but corpuscular ultrastructure and the role of LCs in touch detection are unknown. We report the three-dimensional architecture of the avian Meissner (Grandry) corpuscle acquired using enhanced focused ion beam scanning electron microscopy and machine learning-based segmentation. The corpuscle comprises a stack of LCs interdigitated with terminal endings from two afferents. Simultaneous electrophysiological recordings from both cell types revealed that mechanosensitive LCs use calcium influx to trigger action potentials in the afferent and thus serve as physiological touch sensors in the skin. The elaborate architecture and bicellular sensory mechanism in the corpuscles, which comprises the afferents and LCs, create the capacity for nuanced encoding of the submodalities of touch.

Methods of three-dimensional electron microscopy have been actively developed recently and open up great opportunities for morphological work. This approach is especially useful for studying microinsects, since it is possible to obtain complete series of high-resolution sections of a whole insect. Studies on the genus Megaphragma are especially important, since the unique phenomenon of lysis of most of the neuron nuclei was discovered in species of this genus. In this study we reveal the anatomical structure of the head of Megaphragma viggianii at all levels from organs to subcellular structures. Despite the miniature size of the body, most of the organ systems of M. viggianii retain the structural plan and complexity of organization at all levels. The set of muscles and the well-developed stomatogastric nervous system of this species correspond to those of larger insects, and there is also a well-developed tracheal system in the head of this species. Reconstructions of the head of M. viggianii at the cellular and subcellular levels were obtained, and of volumetric data were analyzed. A total of 689 nucleated cells of the head were reconstructed. The ultrastructure of M. viggianii is surprisingly complex, and the evolutionary benefits of such complexity are probably among the factors limiting the further miniaturization of parasitoid wasps., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Goals-of-care conversations (GoCCs) are essential for individualized end-of-life care. Shared decision-making (SDM) that elicits patients' goals and values to collaboratively make life sustaining treatment (LST) decisions is best practice. However, it is unknown how the COVID-19 pandemic onset and associated changes to care delivery, stress on providers, and clinical uncertainty affected SDM and recommendation-making during GoCCs. Aim: To assess providers' attitudes and behaviors related to GoCCs during the COVID-19 pandemic and identify factors associated with provision of LST recommendations. Design: Survey of United States Veterans Health Administration (VA) health care providers. Setting/Participants: Health care providers from 20 VA facilities with high COVID-19 caseloads early in the pandemic who had authority to place LST orders and practiced in select specialties ( n  = 3398). Results: We had 323 respondents (9.5% adjusted response rate). Most were age ≥50 years (51%), female (63%), non-Hispanic white (64%), and had ≥1 GoCC per week during peak-COVID-19 (78%). Compared with pre-COVID-19, providers believed it was less appropriate and felt less comfortable giving an LST recommendation during peak-COVID-19 ( p  < 0.001). One-third (32%) reported either "never" or "rarely" giving an LST recommendation during GoCCs at peak-COVID-19. In adjusted regression models, being a physician and discussing patients' goals and values were positively associated with giving an LST recommendation ( B  = 0.380, p  = 0.031 and B  = 0.400, p  < 0.001, respectively) at peak-COVID-19. Conclusion: Providers who discuss patients' preferences and values are more likely to report giving a recommendation; both behaviors are markers of SDM during GoCCs. Our findings suggest potential areas for training in conducting patient-centered GoCCs.

Evolution has generated an enormous variety of morphological, physiological, and behavioral traits in animals. How do behaviors evolve in different directions in species equipped with similar neurons and molecular components? Here we adopted a comparative approach to investigate the similarities and differences of escape behaviors in response to noxious stimuli and their underlying neural circuits between closely related drosophilid species. Drosophilids show a wide range of escape behaviors in response to noxious cues, including escape crawling, stopping, head casting, and rolling. Here we find that D. santomea, compared with its close relative D. melanogaster, shows a higher probability of rolling in response to noxious stimulation. To assess whether this behavioral difference could be attributed to differences in neural circuitry, we generated focused ion beam-scanning electron microscope volumes of the ventral nerve cord of D. santomea to reconstruct the downstream partners of mdIV, a nociceptive sensory neuron in D. melanogaster. Along with partner interneurons of mdVI (including Basin-2, a multisensory integration neuron necessary for rolling) previously identified in D. melanogaster, we identified two additional partners of mdVI in D. santomea. Finally, we showed that joint activation of one of the partners (Basin-1) and a common partner (Basin-2) in D. melanogaster increased rolling probability, suggesting that the high rolling probability in D. santomea is mediated by the additional activation of Basin-1 by mdIV. These results provide a plausible mechanistic explanation for how closely related species exhibit quantitative differences in the likelihood of expressing the same behavior., Competing Interests: Declaration of interests Portions of the technology described here are covered by U.S. Patent 10,600,615 titled “Enhanced FIB-SEM systems for large-volume 3D imaging,” which was issued to C.S.X., Kenneth J. Hayworth, and H.F.H. and assigned to Howard Hughes Medical Institute on 24 March 2020., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The effectiveness and safety of marginal donor livers remain controversial. This study aimed to investigate the clinical efficacy of marginal donor livers in patients with liver transplantation (LT)., Methods: This study included 199 liver donors (including 16 split donors) and 206 liver recipients from January 1, 2018 to January 27, 2020, with case follow-up until July 31, 2021. Clinical data of donors and recipients were retrospectively analyzed and were divided into the marginal donor and standard donor groups according to the criteria of marginal donor livers. Indices of liver and kidney functions, complications, and survival curves of the two groups were compared., Results: Compared with the standard donor group, the blood creatinine levels were significantly higher in the marginal donor group in the first week after operation (P < 0.05); there were no significant differences in alanine aminotransferase, aspartate aminotransferase, and total bilirubin levels after LT (all P > 0.05); there was no significant difference in the incidence of complications after LT (P > 0.05); there was also no significant difference in the survival curve (P = 0.335)., Conclusions: There were no significant differences in liver and kidney function and survival curve between the standard donor and marginal donor groups. The marginal donor liver appears safe and reliable for LT and may be an important strategy to expand the donor pool and solve the shortage of organs., (Copyright © 2022 First Affiliated Hospital, Zhejiang University School of Medicine in China. Published by Elsevier B.V. All rights reserved.)

Objective: This study aims to construct and validate a predictable deep learning model associated with clinical data and multi-sequence magnetic resonance imaging (MRI) for short-term postoperative facial nerve function in patients with acoustic neuroma., Methods: A total of 110 patients with acoustic neuroma who underwent surgery through the retrosigmoid sinus approach were included. Clinical data and raw features from four MRI sequences (T1-weighted, T2-weighted, T1-weighted contrast enhancement, and T2-weighted-Flair images) were analyzed. Spearman correlation analysis along with least absolute shrinkage and selection operator regression were used to screen combined clinical and radiomic features. Nomogram, machine learning, and convolutional neural network (CNN) models were constructed to predict the prognosis of facial nerve function on the seventh day after surgery. Receiver operating characteristic (ROC) curve and decision curve analysis (DCA) were used to evaluate model performance. A total of 1050 radiomic parameters were extracted, from which 13 radiomic and 3 clinical features were selected., Results: The CNN model performed best among all prediction models in the test set with an area under the curve (AUC) of 0.89 (95% CI, 0.84-0.91)., Conclusion: CNN modeling that combines clinical and multi-sequence MRI radiomic features provides excellent performance for predicting short-term facial nerve function after surgery in patients with acoustic neuroma. As such, CNN modeling may serve as a potential decision-making tool for neurosurgery., (© 2023. Huazhong University of Science and Technology.)

The virus that causes COVID-19 (Corona Virus Disease 2019), SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), is causing a worldwide pandemic, posing a substantial threat to human health. Patients show signs of pneumonia, ARDS, shock, acute cardiac injury, acute kidney injury and other complications. The SARS-CoV-2 receptor is angiotensin converting enzyme 2 (ACE2), which is an important component of the renin-angiotensin system (RAS). In addition, TMPRSS2 or other cofactors are needed to allow the virus to enter the host. Clinical patients have exhibited varying degrees of genitourinary and endocrine system damage, and some studies have also reported potential risks to the genitourinary and endocrine systems. This article reviews the mechanism underlying SARS-CoV-2 infection and the current studies on the male genitourinary and endocrine systems and proposes that more attention should be directed towards human reproductive and endocrine health during the SARS-CoV-2 epidemic., (©The Author(s) 2023. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

The ability to view biomolecules in cells and measure changes in their structure, quantity, distribution, and interaction is fundamental to understanding biology. By coupling nano -scale resolution with meso and even macro scale volumes, the enhanced focused ion beam-scanning electron microscopy (FIB-SEM) pipeline has enabled numerous transformational discoveries in life science, many of which were major new landmarks in their fields. This pipeline consists of EM sample preparation, FIB-SEM sample preparation, FIB-SEM imaging, data alignment, and image analysis. While the EM sample preparation, data alignment, and image analysis are consistent with those from other volume Electron Microscopy (vEM) approaches, the enhanced FIB-SEM sample preparation and imaging are unique to the rest of comparable methods. We here illustrate the detailed methods of enhanced FIB-SEM sample preparation and image acquisition that have not been previously described. These methods can also be applied to the conventional FIB-SEM platforms for improved image acquisition quality and pipeline throughput., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Deriving the detailed synaptic connections of an entire nervous system is the unrealized goal of the nascent field of connectomics. For the fruit fly Drosophila , in particular, we need to dissect the brain, connectives, and ventral nerve cord as a single continuous unit, fix and stain it, and undertake automated segmentation of neuron membranes. To achieve this, we designed a protocol using progressive lowering of temperature dehydration (PLT), a technique routinely used to preserve cellular structure and antigenicity. We combined PLT with low temperature en bloc staining (LTS) and recover fixed neurons as round profiles with darkly stained synapses, suitable for machine segmentation and automatic synapse detection. Here we report three different PLT-LTS methods designed to meet the requirements for FIB-SEM imaging of the Drosophila brain. These requirements include: good preservation of ultrastructural detail, high level of en bloc staining, artifact-free microdissection, and smooth hot-knife cutting to reduce the brain to dimensions suited to FIB-SEM. In addition to PLT-LTS, we designed a jig to microdissect and pre-fix the fly's delicate brain and central nervous system. Collectively these methods optimize morphological preservation, allow us to image the brain usually at 8 nm per voxel, and simultaneously speed the formerly slow rate of FIB-SEM imaging., 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 © 2022 Lu, Xu, Hayworth, Pang, Shinomiya, Plaza, Scheffer, Rubin, Hess, Rivlin and Meinertzhagen.)

Insect antennae are astonishingly versatile and have multiple sensory modalities. Audition, detection of airflow, and graviception are combined in the antennal chordotonal organs. The miniaturization of these complex multisensory organs has never been investigated. Here we present a comprehensive study of the structure and scaling of the antennal chordotonal organs of the extremely miniaturized parasitoid wasp Megaphragma viggianii based on 3D electron microscopy. Johnston's organ of M. viggianii consists of 19 amphinematic scolopidia (95 cells); the central organ consists of five scolopidia (20 cells). Plesiomorphic composition includes one accessory cell per scolopidium, but in M. viggianii this ratio is only 0.3. Scolopale rods in Johnston's organ have a unique structure. Allometric analyses demonstrate the effects of scaling on the antennal chordotonal organs in insects. Our results not only shed light on the universal principles of miniaturization of sense organs, but also provide context for future interpretation of the M. viggianii connectome., (© 2022. The Author(s).)

In the context of the global pandemic of COVID-19, the epidemic intensity, epidemic characteristics and infection risk of influenza have presented new features. COVID-19 and influenza have simultaneously emerged in many regions of the world. COVID-19 and influenza are similar in terms of transmission mode, clinical symptoms and other aspects. There are also similarities in the mechanism of influenza virus and novel coronavirus on cells. At the same time, it is feasible and significant to do a good job in the prevention and control of COVID-19 and influenza. This paper discusses the relevant strategies and measures for the joint prevention and control of influenza and novel coronavirus from the aspects of influenza vaccination to prevent co-infection, simultaneous vaccination of influenza vaccine and novel coronavirus vaccine, etc., and puts forward corresponding thoughts and suggestions, in order to provide scientific support for the formulation of strategies on seasonal influenza vaccine and novel coronavirus vaccination.

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical G αq/11 -RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state., Competing Interests: Declaration of interests Portions of the technology described herein are covered by U.S. Patent 10,600,615 titled “enhanced FIB-SEM systems for large-volume 3D imaging,” which was issued to C.S.X. and H.F.H. and assigned to Howard Hughes Medical Institute., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A) and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, whereas its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, on the basis of reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.