High-throughput volumetric mapping of synaptic transmission.

Volumetric imaging of synaptic transmission in vivo requires high spatial and high temporal resolution. Shaping the wavefront of two-photon fluorescence excitation light, we developed Bessel-droplet foci for high-contrast and high-resolution volumetric imaging of synapses. Applying our method to imaging glutamate release, we demonstrated high-throughput mapping of excitatory inputs at >1,000 synapses per volume and >500 dendritic spines per neuron in vivo and unveiled previously unseen features of functional synaptic organization in the mouse primary visual cortex.

Replication stress promotes cell elimination by extrusion.

Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals1-3. During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer4. Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer1,5. The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos3, we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress6,7 triggered ATR-CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals.

A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Ultrafast two-photon fluorescence imaging of cerebral blood circulation in the mouse brain in vivo.

Characterizing blood flow dynamics in vivo is critical to understanding the function of the vascular network under physiological and pathological conditions. Existing methods for hemodynamic imaging have insufficient spatial and temporal resolution to monitor blood flow at the cellular level in large blood vessels. By using an ultrafast line-scanning module based on free-space angular chirped enhanced delay, we achieved two-photon fluorescence imaging of cortical blood flow at 1,000 two-dimensional (2D) frames and 1,000,000 one-dimensional line scans per second in the awake mouse. This orders-of-magnitude increase in temporal resolution allowed us to measure cerebral blood flow at up to 49 mm/s and observe pulsatile blood flow at harmonics of heart rate. Directly visualizing red blood cell (RBC) flow through vessels down to >800 µm in depth, we characterized cortical layer­dependent flow velocity distributions of capillaries, obtained radial velocity profiles and kilohertz 2D velocity mapping of multifile blood flow, and performed RBC flux measurements from penetrating blood vessels.

Reducing Cholesterol Level in Live Macrophages Improves Delivery Performance by Enhancing Blood Shear Stress Adaptation.

In recent years, live-cell-based drug delivery systems have gained considerable attention. However, shear stress, which accompanies blood flow, may cause cell death and weaken the delivery performance. In this study, we found that reducing cholesterol in macrophage plasma membranes enhanced their tumor targeting ability by more than 2-fold. Our study demonstrates that the reduced cholesterol level deactivated the mammalian target of rapamycin (mTOR) and consequently promoted the nuclear translocation of transcription factor EB (TFEB), which in turn enhanced the expression of superoxide dismutase (SOD) to reduce reactive oxygen species (ROS) induced by shear stress. A proof-of-concept system using low cholesterol macrophages attached to MXene (e.g., l-RX) was fabricated. In a melanoma mouse model, l-RX and laser irradiation treatments eliminated tumors with no recurrences observed in mice. Therefore, cholesterol reduction is a simple and effective way to enhance the targeting performance of macrophage-based drug delivery systems.

An adaptive optics module for deep tissue multiphoton imaging in vivo.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.

Regulating Electron Metal-Support Interaction to Suppress N2O Formation in the Selective Catalytic Oxidation of Ammonia.

N2O is a common byproduct in the selective catalytic oxidation of ammonia, and its generation often needs to be inhibited due to its strong greenhouse effect. In this paper, using Ag/ZSO-Y as a model catalyst, the N2O selectivity was reduced by 30% through modulation of the electron metal-support interaction. The results demonstrate that the work function of the support can be regulated by the content of the doping element. As the Zr content increases in SnO2, the work function of the support decreases. Moreover, there is a positive correlation between the charge transfer amount and the work function of the support. A series of in situ DRIFTS and density functional theory calculations revealed that the -NO and -N reactions are the primary pathways for N2O formation. By adjustment of the work function of the support through varying the Zr doping level, the electronic structure of Ag NPs was further tuned, resulting in an increased reaction energy barrier for -NO and -N reactions, effectively suppressing N2O formation.

Strategies to engineer various nanocarrier-based hybrid catalysts for enhanced chemodynamic cancer therapy.

Chemodynamic therapy (CDT) is a newly developed cancer-therapeutic modality that kills cancer cells by the highly toxic hydroxyl radical (˙OH) generated from the in situ triggered Fenton/Fenton-like reactions in an acidic and H2O2-overproduced tumor microenvironment (TME). By taking the advantage of the TME-activated catalytic reaction, CDT enables a highly specific and minimally-invasive cancer treatment without external energy input, whose efficiency mainly depends on the reactant concentrations of both the catalytic ions and H2O2, and the reaction conditions (including pH, temperature, and amount of glutathione). Unfortunately, it suffers from unsatisfactory therapy efficiency for clinical application because of the limited activators (i.e., mild acid pH and insufficient H2O2 content) and overexpressed reducing substance in TME. Currently, various synergistic strategies have been elaborately developed to increase the CDT efficiency by regulating the TME, enhancing the catalytic efficiency of catalysts, or combining with other therapeutic modalities. To realize these strategies, the construction of diverse nanocarriers to deliver Fenton catalysts and cooperatively therapeutic agents to tumors is the key prerequisite, which is now being studied but has not been thoroughly summarized. In particular, nanocarriers that can not only serve as carriers but are also active themselves for therapy are recently attracting increasing attention because of their less risk of toxicity and metabolic burden compared to nanocarriers without therapeutic capabilities. These therapy-active nanocarriers well meet the requirements of an ideal therapy system with maximum multifunctionality but minimal components. From this new perspective, in this review, we comprehensively summarize the very recent research progress on nanocarrier-based systems for enhanced CDT and the strategies of how to integrate various Fenton agents into the nanocarriers, with particular focus on the studies of therapy-active nanocarriers for the construction of CDT catalysts, aiming to guide the design of nanosystems with less components and more functionalities for enhanced CDT. Finally, the challenges and prospects of such a burgeoning cancer-theranostic modality are outlooked to provide inspirations for the further development and clinical translation of CDT.

Robust Thiazole-Linked Covalent Organic Frameworks for Water Sensing with High Selectivity and Sensitivity.

The rational design of covalent organic frameworks (COFs) with hydrochromic properties is of significant value because of the facile and rapid detection of water in diverse fields. In this report, we present a thiazole-linked COF (TZ-COF-6) sensor with a large surface area, ultrahigh stability, and excellent crystallinity. The sensor was synthesized through a simple three-component reaction involving amine, aldehyde, and sulfur. The thiazole and methoxy groups confer strong basicity to TZ-COF-6 at the nitrogen sites, making them easily protonated reversibly by water. Therefore, TZ-COF-6 displayed color change visible to the naked eye from yellow to red when protonated, along with a red shift in absorption in the ultraviolet-visible diffuse reflectance spectra (UV-vis DRS) when exposed to water. Importantly, the water-sensing process was not affected by polar organic solvents, demonstrating greater selectivity and sensitivity compared to other COF sensors. Therefore, TZ-COF-6 was used to detect trace amounts of water in organic solvents. In strong polar solvents, such as N,N-dimethyl formamide (DMF) and ethanol (EtOH), the limit of detection (LOD) for water was as low as 0.06% and 0.53%, respectively. Even after 8 months of storage and 15 cycles, TZ-COF-6 retained its original crystallinity and detection efficiency, displaying high stability and excellent cycle performance.

Rapid mesoscale volumetric imaging of neural activity with synaptic resolution.

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over the mesoscale with synaptic resolution. We applied the technology to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.

Kilohertz two-photon fluorescence microscopy imaging of neural activity in vivo.

Understanding information processing in the brain requires monitoring neuronal activity at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence microscope empowered by all-optical laser scanning, we imaged neuronal activity in vivo at up to 3,000 frames per second and submicrometer spatial resolution. This imaging method enabled monitoring of both supra- and subthreshold electrical activity down to 345 µm below the brain surface in head-fixed awake mice.

Feature issue introduction: ultrafast optical imaging.

This feature issue of Optics Express collects 20 articles that report the most recent progress of ultrafast optical imaging. This review provides a summary of these articles that cover the spectrum of ultrafast optical imaging, from new technologies to applications.

Simultaneous Quantification of Biomarkers for Bis-(2-chloroethyl) Sulfide and 1,2-Bis(2-chloroethylthio) Ethane Exposure in Human Urine at Trace Exposure Levels by Gas Chromatography Tandem Mass Spectrometry via Simultaneous Incubation and Extraction.

Sulfur mustard [HD; bis-(2-chloroethyl) sulfide] and other analogues are a kind of highly toxic vesicant and have been prohibited by the Organization for the Prohibition of Chemical Weapons (OPCW) since 1997. Exposures to HD could generate several adducts in the plasma and hydrolysis products in the urine, which are widely applied as biomarkers to identify HD exposure in forensic analysis. Several methods have been developed for the detection of related biomarkers. However, most methods are based on complex derivatization, and not enough attention is paid to HD analogues. A modified and convenient analytical method reported herein includes simultaneous incubation and organic solvent extraction. The biomarkers such as thiodiglycol and 1,2-bis (2-hydroxyethylthio) are transferred to HD and 1,2-bis(2-chloroethylthio) ethane via hydrochloric acid at the appropriate temperature. The analytes are analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) with 2-chloroethyl ethyl sulfide (2-CEES) applied as the internal standard. The interday and intraday study according to FDA rules has been achieved to evaluate the accuracy and precision of the method. The two targets are detected with a good linearity (R2 > 0.99) in the concentration ranges from 5 to 1000 ng/mL and 10 to 1000 ng/mL, with small relative standard deviations (RSD ≤6.62% and RSD ≤6.93%) and favorable recoveries between 90.3 and 107.3% and between 89.4 and 108.7%, respectively. The established method can be used for retrospective detection of sulfur mustards in biological samples and successfully applied in the biomedical proficiency testing organized by the OPCW.

Aponeurotic expansion of the supraspinatus tendon and concomitant shoulder pathologies.

OBJECTIVES: We investigated the correlation of aponeurotic expansion of the supraspinatus tendon (AESST) with shoulder pathologies such as long head of biceps tendon (LHB), supraspinatus tendon (SST), and subscapularis tendon (SSc). METHODS: We retrospectively evaluated 47 healthy patients and 163 patients with shoulder symptoms from August 2014 to March 2021. First, the presence of AESST was evaluated based on Moser et al.'s classification. Second, the presence of abnormal findings of including LHB tendinitis, LHB subluxation, SST tendinitis, SST tear, SSc tendinitis, and SSc tendon tear was evaluated. We analyzed the prevalence and type of AESST between the two study groups and the relationship between abnormal findings and the presence of AESST. RESULTS: The prevalence of AESST for readers 1 and 2 was 26.1% and 30.4% in the asymptomatic group, respectively, and 22.8% and 31.3% in the symptomatic group. Type 1 was most common (17.3-23.9%) followed by types 2a and 2b. There were no significant differences in the distribution of aponeurosis type between the two groups. In the AESST-positive groups, 45.9% and 47.1% had SST tears on examination by readers 1 and 2, respectively, whereas only 26.4% and 27.9% had SST tears in the AESST-negative group suggesting AESST is associated with SST tear. The odds ratio for SST tear in the presence of AESST was 2.370 and 2.294 (readers 1 and 2). CONCLUSIONS: There is an association between SST tears and the presence of AESST. KEY POINTS: • We evaluated the prevalence of aponeurotic expansion of the supraspinatus tendon (AESST) on MR imaging by type in both symptomatic and asymptomatic groups. • We investigated the correlation of AESST with shoulder pathologies such as biceps tendon and supraspinatus tendon tears. • There is an association between SST tears and the presence of AESST. • Radiologists should be aware of the risk of rotator cuff pathology if AESST is detected.

Semaphorin 3A inhibits tumor progression via the downregulation of Lin28B in ovarian cancer.

Semaphorin 3A (Sema3A) has recently been proven to play an essential role in tumorigenesis. Here, the role of Sema3A in ovarian cancer is explored. The prognostic value of Sema3A was evaluated using the Kaplan-Meier plotter database, and stable expression cells were established by the delivery of lentivirus harboring SEMA3A cDNA or shRNA into OVCA433 and SKOV3 cells, respectively. Then CCK-8 assay, colony-formation assay, wound-healing assay, and Transwell assay were utilized to verify the effect of Sema3A on tumorigenesis. Co-cultures of ovarian cancer cells (OVCA433 and SKOV3) with a conditional medium collected from the established cells were further utilized to confirm the function of Sema3A. Then, the RNA-seq assay was adopted to explore the underlying mechanism. The results demonstrated that low expression of Sema3A was predictive of poor overall survival in patients with ovarian cancer. Functional experiments revealed that Sema3A inhibited proliferation, migration, and invasion in ovarian cancer cells. Secreted Sema3A in a conditioned culture medium also exhibited an anti-tumor effect in ovarian cancer cells. RNA-seq assay suggested that focal adhesion and Lin28B were involved in regulating Sema3A. Rescue assays further verified that Lin28B/ROCK1 axis was vital in the regulation of Sema3A and Lin28B significantly upregulated ROCK1 through let-7g microRNA. The presented data indicate that Sema3A inhibits proliferation and metastasis via the downregulation of Lin28B/ROCK1 in ovarian cancer.

Ultrasonographic and strain elastographic features of epidermal cyst according to body location.

BACKGROUND: There are not many studies on the differences of ultrasound (US) findings between epidermal cysts (ECs) located in the trunk and those in the extremities. PURPOSE: To compare the sonographic findings of ECs according to location in the body (trunk vs. extremity) and evaluated the feasibility of strain elastography (SE). MATERIAL AND METHODS: This is a retrospective study of 76 patients with surgically confirmed non-ruptured EC who underwent US including SE. The US analyses included size, shape, ratio of depth to length (DLR), involvement of more than half the dermis, "submarine sign," and SE characters of each lesion. SE findings were assigned into four grades based on elasticity. RESULTS: The submarine sign was more significantly observed in ECs located in the trunk versus extremities (P value = 0.004 and 0.035, respectively). Truncal lesions were significantly more likely to possess an ovoid shape (P < 0.05) and exhibited higher DLR (P < 0.05). There were more cases with low elasticity according to SE (grade 3 or 4) compared to high elasticity (grade 1 or 2). However, we did not observe significant differences between the two locations (P > 0.05). More-than-half signs also did not exhibit a significant difference (P > 0.05). CONCLUSION: The submarine sign, ovoid shape, and tall lesions (higher DLR) are common in the trunk. However, the degree of elasticity and number of more-than-half signs did not differ between the two groups.

Intra- and extra-capsular ganglia at the gastrocnemius origin and association with meniscal tears and severity of osteoarthritis of the knee joint.

BACKGROUND: The association between size of ganglia or type of ganglia (intra-articular or extra-articular) and meniscal tears or severity of the osteoarthritis (OA) is not evaluated. PURPOSE: To evaluate the prevalence, size, and location of intra- and extra-capsular ganglia at the gastrocnemius origin and to assess their associations with meniscal injury and grades of OA. MATERIAL AND METHODS: This study included 301 consecutive patients who had knee pain and had undergone magnetic resonance imaging (MRI) of the knee. We evaluated presence of ganglia at the gastrocnemius muscle origin site and diagnosed whether it was an intra-capsular located or mixed-capsular located (intra-capsular and extra-capsular) and then measured the diameter of each ganglion. After two weeks, we evaluated whether articular cartilage injury existed. The presence of a meniscal tear was also recorded. RESULTS: A total of 186 patients (93%) had intra- and extra-capsular ganglia. Intra-capsular ganglia were found in 183 cases (91%) and mixed-capsular ganglia were found in 16 cases (8%). In cases with intra- and extra-capsular ganglia, more meniscal tears were found (P = 0.029). Intra-capsular ganglia showed more meniscal tears (P = 0.021). Intra-capsular ganglia were more likely to have high-grade OA (P = 0.043). Patients who had a meniscal tear displayed larger-sized ganglia, especially of the intra-capsular type (P = 0.044). CONCLUSION: Patients with intra- and extra-capsular ganglia, especially of the intra-capsular type, are more likely to have meniscal injury and more severe OA. Patients with a meniscal tear or OA are more likely to have larger intra- and extra-capsular ganglia, especially of the intra-capsular type.

Cyclops lesions associated with both bundles and selective bundle repair of the anterior cruciate ligament.

BACKGROUND: The remnant of a ruptured anterior cruciate ligament (ACL) can increase the risk of impingement or a cyclops lesion, which can increase challenges to proper tunnel placement. PURPOSE: To evaluate the prevalence of cyclops lesions after ACL reconstruction and to assess the difference in the incidence of cyclops lesions between single-bundle repair and selective bundle repair of the ACL. MATERIAL AND METHODS: This retrospective study included 151 patients who were diagnosed with an ACL tear after knee joint magnetic resonance imaging (MRI) who underwent ACL reconstruction surgery. MRI diagnosis of cyclops lesion formation was made if a soft-tissue mass was seen anteriorly in the intercondylar notch near the tibial insertion of the reconstructed ACL, based on sagittal T2-weighted (T2W) imaging. The size of the cyclops lesions was recorded as the largest diameter on the sagittal T2W imaging. RESULTS: A cyclops lesion was detected in 74 (38.5%) cases. Cyclops lesions were detected more frequently in cases with single-bundle repair of the ACL, but the results were not statistically significant (P = 0.609). Compared with selective bundle repair, cyclops lesions had a significantly higher prevalence in the posterolateral (PL) bundle repair than in the anteromedial (AM) bundle repair (P = 0.027) based on MR images at 6-12 months after surgery. CONCLUSION: The incidence of cyclops lesions did not differ significantly in single-bundle repair and selective bundle repair of ACL. However, selective PL bundle repair of the ACL showed a significantly increased incidence of cyclops lesions compared with selective AM bundle repair.

Anti-Warburg effect via generation of ROS and inhibition of PKM2/ß-catenin mediates apoptosis of lambertianic acid in prostate cancer cells.

To elucidate the underlying antitumor mechanism of lambertianic acid (LA) derived from Pinus koraiensis, the role of cancer metabolism related molecules was investigated in the apoptotic effect of LA in DU145 and PC3 prostate cancer cells. MTT assay for cytotoxicity, RNA interference, cell cycle analysis for sub G1 population, nuclear and cytoplasmic extraction, lactate, Glucose and ATP assay by ELISA, Measurement of reactive oxygen species (ROS) generation, Western blotting, and immunoprecipitation assay were conducted in DU145 and PC3 prostate cancer cells. Herein LA exerted cytotoxicity, increased sub G1 population and attenuated the expression of pro-Caspase3 and pro-poly (ADP-ribose) polymerase (pro-PARP) in DU145 and PC3 cells. Also, LA reduced the expression of lactate dehydrogenase A (LDHA), glycolytic enzymes such as hexokinase 2 and pyruvate kinase M2 (PKM2) with reduced production of lactate in DU145 and PC3 cells. Notably, LA decreased phosphorylation of PKM2 on Tyr105 and inhibited the expression of p-STAT3, cyclin D1, C-Myc, ß-catenin, and p-GSK3ß with the decrease of nuclear translocation of p-PKM2. Furthermore, LA disturbed the binding of p-PKM2 and ß-catenin in DU145 cells, which was supported by Spearman coefficient (0.0463) of cBioportal database. Furthermore, LA generated ROS in DU145 and PC3 cells, while ROS scavenger NAC (N-acetyl L-cysteine) blocked the ability of LA to reduce p-PKM2, PKM2, ß-catenin, LDHA, and pro-caspase3 in DU145 cells. Taken together, these findings provide evidence that LA induces apoptosis via ROS generation and inhibition of PKM2/ß-catenin signaling in prostate cancer cells.

Invited Session IV: Studies of the visual cortex with sub-millimeter resolution: Imaging the visual brain at high spatiotemporal resolution.

Since the discovery of orientation-selective neurons in the cat primary visual cortex (V1), how the mammalian nervous system computes the orientation of visual stimuli has been a flagship question in neuroscience. With the recent advances in in vivo imaging of neural activity using genetically encoded indicators and two-photon fluorescence microscopy, we can now achieve synapse-resolving functional imaging and kilohertz imaging of membrane voltage in the mouse V1. I will describe our recent advancements in imaging visual processing at high spatiotemporal resolution, as well as two studies on the orientation selectivity of neurons in the mouse visual pathway.