Galectin-3 impairs calcium transients and ß-cell function.

In diabetes, macrophages and inflammation are increased in the islets, along with ß-cell dysfunction. Here, we demonstrate that galectin-3 (Gal3), mainly produced and secreted by macrophages, is elevated in islets from both high-fat diet (HFD)-fed and diabetic db/db mice. Gal3 acutely reduces glucose-stimulated insulin secretion (GSIS) in ß-cell lines and primary islets in mice and humans. Importantly, Gal3 binds to calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) and inhibits calcium influx via the cytomembrane and subsequent GSIS. ß-Cell CACNG1 deficiency phenocopies Gal3 treatment. Inhibition of Gal3 through either genetic or pharmacologic loss of function improves GSIS and glucose homeostasis in both HFD-fed and db/db mice. All animal findings are applicable to male mice. Here we show a role of Gal3 in pancreatic ß-cell dysfunction, and Gal3 could be a therapeutic target for the treatment of type 2 diabetes.

Trimethylamine N-oxide impairs ß-cell function and glucose tolerance.

ß-Cell dysfunction and ß-cell loss are hallmarks of type 2 diabetes (T2D). Here, we found that trimethylamine N-oxide (TMAO) at a similar concentration to that found in diabetes could directly decrease glucose-stimulated insulin secretion (GSIS) in MIN6 cells and primary islets from mice or humans. Elevation of TMAO levels impairs GSIS, ß-cell proportion, and glucose tolerance in male C57BL/6 J mice. TMAO inhibits calcium transients through NLRP3 inflammasome-related cytokines and induced Serca2 loss, and a Serca2 agonist reversed the effect of TMAO on ß-cell function in vitro and in vivo. Additionally, long-term TMAO exposure promotes ß-cell ER stress, dedifferentiation, and apoptosis and inhibits ß-cell transcriptional identity. Inhibition of TMAO production improves ß-cell GSIS, ß-cell proportion, and glucose tolerance in both male db/db and choline diet-fed mice. These observations identify a role for TMAO in ß-cell dysfunction and maintenance, and inhibition of TMAO could be an approach for the treatment of T2D.

Tracking endogenous proteins based on RNA editing-mediated genetic code expansion.

Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

Bright and stable monomeric green fluorescent protein derived from StayGold.

The high brightness and photostability of the green fluorescent protein StayGold make it a particularly attractive probe for long-term live-cell imaging; however, its dimeric nature precludes its application as a fluorescent tag for some proteins. Here, we report the development and crystal structures of a monomeric variant of StayGold, named mBaoJin, which preserves the beneficial properties of its precursor, while serving as a tag for structural proteins and membranes. Systematic benchmarking of mBaoJin against popular green fluorescent proteins and other recently introduced monomeric and pseudomonomeric derivatives of StayGold established mBaoJin as a bright and photostable fluorescent protein, exhibiting rapid maturation and high pH/chemical stability. mBaoJin was also demonstrated for super-resolution, long-term live-cell imaging and expansion microscopy. We further showed the applicability of mBaoJin for neuronal labeling in model organisms, including Caenorhabditis elegans and mice.

Readily releasable ß cells with tight Ca2+-exocytosis coupling dictate biphasic glucose-stimulated insulin secretion.

Biphasic glucose-stimulated insulin secretion (GSIS) is essential for blood glucose regulation, but a mechanistic model incorporating the recently identified islet ß cell heterogeneity remains elusive. Here, we show that insulin secretion is spatially and dynamically heterogeneous across the islet. Using a zinc-based fluorophore with spinning-disc confocal microscopy, we reveal that approximately 40% of islet cells, which we call readily releasable ß cells (RRßs), are responsible for 80% of insulin exocytosis events. Although glucose up to 18.2 mM fully mobilized RRßs to release insulin synchronously (first phase), even higher glucose concentrations enhanced the sustained secretion from these cells (second phase). Release-incompetent ß cells show similarities to RRßs in glucose-evoked Ca2+ transients but exhibit Ca2+-exocytosis coupling deficiency. A decreased number of RRßs and their altered secretory ability are associated with impaired GSIS progression in ob/ob mice. Our data reveal functional heterogeneity at the level of exocytosis among ß cells and identify RRßs as a subpopulation of ß cells that make a disproportionally large contribution to biphasic GSIS from mouse islets.

Physical Contacts Between Mitochondria and WPBs Participate in WPB Maturation.

BACKGROUND: Weibel-Palade bodies (WPBs) are endothelial cell-specific cigar-shaped secretory organelles containing various biologically active molecules. WPBs play crucial roles in thrombosis, hemostasis, angiogenesis, and inflammation. The main content of WPBs is the procoagulant protein vWF (von Willebrand factor). Physical contacts and functional cross talk between mitochondria and other organelles have been demonstrated. Whether an interorganellar connection exists between mitochondria and WPBs is unknown. METHODS: We observed physical contacts between mitochondria and WPBs in human umbilical vein endothelial cells by electron microscopy and living cell confocal microscopy. We developed an artificial intelligence-assisted method to quantify the duration and length of organelle contact sites in live cells. RESULTS: We found there existed physical contacts between mitochondria and WPBs. Disruption of mitochondrial function affected the morphology of WPBs. Furthermore, we found that Rab3b, a small GTPase on the WPBs, was enriched at the mitochondrion-WPB contact sites. Rab3b deficiency reduced interaction between the two organelles and impaired the maturation of WPBs and vWF multimer secretion. CONCLUSIONS: Our results reveal that Rab3b plays a crucial role in mediating the mitochondrion-WPB contacts, and that mitochondrion-WPB coupling is critical for the maturation of WPBs in vascular endothelial cells.

Quantitatively mapping local quality of super-resolution microscopy by rolling Fourier ring correlation.

In fluorescence microscopy, computational algorithms have been developed to suppress noise, enhance contrast, and even enable super-resolution (SR). However, the local quality of the images may vary on multiple scales, and these differences can lead to misconceptions. Current mapping methods fail to finely estimate the local quality, challenging to associate the SR scale content. Here, we develop a rolling Fourier ring correlation (rFRC) method to evaluate the reconstruction uncertainties down to SR scale. To visually pinpoint regions with low reliability, a filtered rFRC is combined with a modified resolution-scaled error map (RSM), offering a comprehensive and concise map for further examination. We demonstrate their performances on various SR imaging modalities, and the resulting quantitative maps enable better SR images integrated from different reconstructions. Overall, we expect that our framework can become a routinely used tool for biologists in assessing their image datasets in general and inspire further advances in the rapidly developing field of computational imaging.

Outcomes of surgery and subsequent therapy for central nervous system oligoprogression in EGFR-mutated NSCLC patients.

BACKGROUND: Oligoprogression is an emerging issue in patients with epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC). However, the surgical treatment for central nervous system (CNS) oligoprogression is not widely discussed. We investigated the outcomes of craniotomy with adjuvant whole-brain radiotherapy (WBRT) and subsequent therapies for CNS oligoprogression in patients with EGFR-mutated NSCLC. METHODS: NSCLC patients with CNS oligoprogression were identified from a tertiary medical center. The outcomes of surgery with adjuvant WBRT or WBRT alone were analyzed, along with other variables. Overall survival and progression-free survival were analyzed using the log-rank test as the primary and secondary endpoints. A COX regression model was used to identify the possible prognostic factors. RESULTS: Thirty-seven patients with CNS oligoprogression who underwent surgery or WBRT were included in the study after reviewing 728 patients. Twenty-one patients underwent surgery with adjuvant WBRT, and 16 received WBRT alone. The median overall survival for surgery and WBRT alone groups was 43 (95% CI 17-69) and 22 (95% CI 15-29) months, respectively. Female sex was a positive prognostic factor for overall survival (OR 0.19, 95% CI 0.06-0.57). Patients who continued previous tyrosine kinase inhibitors (OR 3.48, 95% CI 1.06-11.4) and induced oligoprogression (OR 3.35, 95% CI 1.18-9.52) were associated with worse overall survival. Smoking history (OR 4.27, 95% CI 1.54-11.8) and induced oligoprogression (OR 5.53, 95% CI 2.1-14.7) were associated with worse progression-free survival. CONCLUSIONS: Surgery combined with adjuvant WBRT is a feasible treatment modality for CNS oligoprogression in patients with EGFR-mutated NSCLC. Changing the systemic-targeted therapy after local treatments may be associated with improved overall survival.

A tool kit of highly selective and sensitive genetically encoded neuropeptide sensors.

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution. However, this has been hindered by the lack of direct, sensitive, and noninvasive tools. We developed a series of GRAB (G protein-coupled receptor activation‒based) sensors for detecting somatostatin (SST), corticotropin-releasing factor (CRF), cholecystokinin (CCK), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors, which enable detection of specific neuropeptide binding at nanomolar concentrations, establish a robust tool kit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

Two-Dimensional Infrared Spectroscopy of Isolated Molecular Ions.

Two-dimensional infrared (2D IR) spectroscopy of mass-selected, cryogenically cooled molecular ions is presented. Nonlinear response pathways, encoded in the time-domain photodissociation action response of weakly bound N2 messenger tags, were isolated using pulse shaping techniques following excitation with four collinear ultrafast IR pulses. 2D IR spectra of Re(CO)3(CH3CN)3+ ions capture off-diagonal cross-peak bleach signals between the asymmetric and symmetric carbonyl stretching transitions. These cross peaks display intensity variations as a function of pump-probe delay time due to coherent coupling between the vibrational modes. Well-resolved 2D IR features in the congested fingerprint region of protonated caffeine (C8H10N4O2H+) are also reported. Importantly, intense cross-peak signals were observed at 3 ps waiting time, indicating that tag-loss dynamics are not competing with the measured nonlinear signals. These demonstrations pave the way for more precise studies of molecular interactions and dynamics that are not easily obtainable with current condensed-phase methodologies.

RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

Ultrafast transient vibrational action spectroscopy of cryogenically cooled ions.

Ultrafast transient vibrational action spectra of cryogenically cooled Re(CO)3(CH3CN)3+ ions are presented. Nonlinear spectra were collected in the time domain by monitoring the photodissociation of a weakly bound N2 messenger tag as a function of delay times and phases between a set of three infrared pulses. Frequency-resolved spectra in the carbonyl stretch region show relatively strong bleaching signals that oscillate at the difference frequency between the two observed vibrational features as a function of the pump-probe waiting time. This observation is consistent with the presence of nonlinear pathways resulting from underlying cross-peak signals between the coupled symmetric-asymmetric C≡O stretch pair. The successful demonstration of frequency-resolved ultrafast transient vibrational action spectroscopy of dilute molecular ion ensembles provides an exciting, new framework for the study of molecular dynamics in isolated, complex molecular ion systems.

Blocking IL-6 signaling improves glucose tolerance via SLC39A5-mediated suppression of glucagon secretion.

BACKGROUND AND AIMS: Hyperinsulinemia, hyperglucagonemia, and low-grade inflammation are frequently presented in obesity and type 2 diabetes (T2D). The pathogenic regulation between hyperinsulinemia/insulin resistance (IR) and low-grade inflammation is well documented in the development of diabetes. However, the cross-talk of hyperglucagonemia with low-grade inflammation during diabetes progression is poorly understood. In this study, we investigated the regulatory role of proinflammatory cytokine interleukin-6 (IL-6) on glucagon secretion. METHODS: The correlations between inflammatory cytokines and glucagon or insulin were analyzed in rhesus monkeys and humans. IL-6 signaling was blocked by IL-6 receptor-neutralizing antibody tocilizumab in obese or T2D rhesus monkeys, glucose tolerance was evaluated by intravenous glucose tolerance test (IVGTT). Glucagon and insulin secretion were measured in isolated islets from wild-type mouse, primary pancreatic α-cells and non-α-cells sorted from GluCre-ROSA26EYFP (GYY) mice, in which the enhanced yellow fluorescent protein (EYFP) was expressed under the proglucagon promoter, by fluorescence-activated cell sorting (FACS). Particularly, glucagon secretion in α-TC1 cells treated with IL-6 was measured, and RNA sequencing was used to screen the mediator underlying IL-6-induced glucagon secretion. SLC39A5 was knocking-down or overexpressed in α-TC1 cells to determine its impact in glucagon secretion and cytosolic zinc density. Dual luciferase and chromatin Immunoprecipitation were applied to analyze the signal transducer and activator of transcription 3 (STAT3) in the regulation of SLC39A5 transcription. RESULTS: Plasma IL-6 correlate positively with plasma glucagon levels, but not insulin, in rhesus monkeys and humans. Tocilizumab treatment reduced plasma glucagon, blood glucose and HbA1c in spontaneously obese or T2D rhesus monkeys. Tocilizumab treatment also decreased glucagon levels during IVGTT, and improved glucose tolerance. Moreover, IL-6 significantly increased glucagon secretion in isolated islets, primary pancreatic α-cells and α-TC1 cells. Mechanistically, we found that IL-6-activated STAT3 downregulated the zinc transporter SLC39A5, which in turn reduced cytosolic zinc concentration and ATP-sensitive potassium channel activity and augmented glucagon secretion. CONCLUSIONS: This study demonstrates that IL-6 increases glucagon secretion via the downregulation of zinc transporter SLC39A5. This result revealed the molecular mechanism underlying the pathogenesis of hyperglucagonemia and a previously unidentified function of IL-6 in the pathophysiology of T2D, providing a potential new therapeutic strategy of targeting IL-6/glucagon to preventing or treating T2D.

A practical multicellular sample preparation pipeline broadens the application of in situ cryo-electron tomography.

The structural studies of macromolecules in their physiological context, particularly in tissue, is constrained by the bottleneck of sample preparation. In this study, we present a practical pipeline for preparing multicellular samples for cryo-electron tomography. The pipeline comprises sample isolation, vitrification, and lift-out-based lamella preparation using commercially available instruments. We demonstrate the efficacy of our pipeline by visualizing pancreatic ß cells from mouse islets at the molecular level. This pipeline enables the determination of the properties of insulin crystals in situ for the first time, using unperturbed samples.

Evaluation of the safety, radiographic and resident training results of thoracic pedicle screws placement using resection of the transverse process.

OBJECTIVE: This study aimed at the evaluation and assessment of a simple method, the transverse process resection (TPR) technique, for freehand thoracic pedicle screw placement and the learning curve for trainee surgeons. METHODS: In the TPR technique, the tip of the thoracic transverse process (TP) is removed to create an entry point in the cancellous bone of the TP, and the thoracic pedicle is cannulated from the TP. We retrospectively evaluated the safety and radiographic results of the TPR technique and compared with that of conventional pedicle screws. The training performance of seven neurosurgical residents with TPR techniques were evaluated. RESULTS: Among 46 patients, a total of 322 thoracic screws were analyzed, including 178 screws placed using the TPR technique and 144 screws using the conventional straight-forward (SF) technique. TPR screws had greater medial angulations in all levels from T2 to T12 compared to SF screws (p < 0.001). The incidence of pedicle breach was lower in the TPR screws compared to SF screws (6.2% vs. 21.5%, p < 0.001), especially for screws placed by residents (6.7% vs. 29.6%, p < 0.001). Residents had improved performance following a cadaveric training course on the TPR technique (p = 0.001). CONCLUSION: This study demonstrated the safety of the TPR technique for thoracic pedicle screw placement and its short learning curve for trainee surgeons.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation.

Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development. Initially identified as a lysosomal protein, the TMEM106B D252N mutant has recently been associated with hypomyelination. However, how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood. We used superresolution Hessian structured illumination microscopy (Hessian-SIM) and spinning disc-confocal structured illumination microscopy (SD-SIM) to find that the wild-type TMEM106B protein is targeted to the plasma membrane, filopodia, and lysosomes in human oligodendrocytes. The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress. Most importantly, we detected reductions in the length and number of filopodia in cells expressing the D252N mutant. PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B, and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells. Therefore, interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes, which may be sustained by the delivery of these proteins from lysosomes via exocytosis.

Quantitative structured illumination microscopy via a physical model-based background filtering algorithm reveals actin dynamics.

Despite the prevalence of superresolution (SR) microscopy, quantitative live-cell SR imaging that maintains the completeness of delicate structures and the linearity of fluorescence signals remains an uncharted territory. Structured illumination microscopy (SIM) is the ideal tool for live-cell SR imaging. However, it suffers from an out-of-focus background that leads to reconstruction artifacts. Previous post hoc background suppression methods are prone to human bias, fail at densely labeled structures, and are nonlinear. Here, we propose a physical model-based Background Filtering method for living cell SR imaging combined with the 2D-SIM reconstruction procedure (BF-SIM). BF-SIM helps preserve intricate and weak structures down to sub-70 nm resolution while maintaining signal linearity, which allows for the discovery of dynamic actin structures that, to the best of our knowledge, have not been previously monitored.

Quantification of vessel separation using the carotid-jugular angle to predict the nerve origin of neck peripheral nerve sheath tumours: a pooled analysis of cases from the literature and a single-center cohort.

BACKGROUND: Postoperative nerve palsy is a major complication following resection of neck peripheral nerve sheath tumours (PNSTs). Accurate preoperative identification of the nerve origin (NO) can improve surgical outcomes and patient counselling. MATERIAL AND METHODS: This study was a retrospective cohort and quantitative analysis of the literature. The authors introduced a parameter, the carotid-jugular angle (CJA), to differentiate the NO. A literature review of neck PNST cases from 2010 to 2022 was conducted. The CJA was measured from eligible imaging data, and quantitative analysis was performed to evaluate the ability of the CJA to predict the NO. External validation was performed using a single-centre cohort from 2008 to 2021. RESULTS: In total, 17 patients from our single-centre cohort and 88 patients from the literature were analyzed. Among them, 53, 45, and 7 patients had sympathetic, vagus, and cervical nerve PNSTs, respectively. Vagus nerve tumours had the largest CJA, followed by sympathetic tumours, whereas cervical nerve tumours had the smallest CJA ( P <0.001). Multivariate logistic regression identified a larger CJA as a predictor of vagus NO ( P <0.001), and receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) of 0.907 (0.831-0.951) for the CJA to predict vagus NO ( P <0.001). External validation showed an AUC of 0.928 (0.727-0.988) ( P <0.001). Compared with the AUC of the previously proposed qualitative method (AUC=0.764, 0.673-0.839), that of the CJA was greater ( P =0.011). The cut-off value identified to predict vagus NO was greater than or equal to 100°. Receiver operating characteristic analysis showed an AUC of 0.909 (0.837-0.956) for the CJA to predict cervical NO ( P <0.001), with a cut-off value less than 38.5°. CONCLUSIONS: A CJA greater than or equal to 100° predicted a vagus NO and a CJA less than 100° predicted a non-vagus NO. Moreover, a CJA less than 38.5 was associated with an increased likelihood of cervical NO.

Mesenchymal Migration on Adhesive-Nonadhesive Alternate Surfaces in Macrophages.

Mesenchymal migration usually happens on adhesive substrates, while cells adopt amoeboid migration on low/nonadhesive surfaces. Protein-repelling reagents, e.g., poly(ethylene) glycol (PEG), are routinely employed to resist cell adhering and migrating. Contrary to these perceptions, this work discovers a unique locomotion of macrophages on adhesive-nonadhesive alternate substrates in vitro that they can overcome nonadhesive PEG gaps to reach adhesive regions in the mesenchymal mode. Adhering to extracellular matrix regions is a prerequisite for macrophages to perform further locomotion on the PEG regions. Podosomes are found highly enriched on the PEG region in macrophages and support their migration across the nonadhesive regions. Increasing podosome density through myosin IIA inhibition facilitates cell motility on adhesive-nonadhesive alternate substrates. Moreover, a developed cellular Potts model reproduces this mesenchymal migration. These findings together uncover a new migratory behavior on adhesive-nonadhesive alternate substrates in macrophages.