TCR+/BCR+ dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes.

Ahmed and colleagues recently described a novel hybrid lymphocyte expressing both a B and T cell receptor, termed double expresser (DE) cells. DE cells in blood of type 1 diabetes (T1D) subjects were present at increased numbers and enriched for a public B cell clonotype. Here, we attempted to reproduce these findings. While we could identify DE cells by flow cytometry, we found no association between DE cell frequency and T1D status. We were unable to identify the reported public B cell clone, or any similar clone, in bulk B cells or sorted DE cells from T1D subjects or controls. We also did not observe increased usage of the public clone VH or DH genes in B cells or in sorted DE cells. Taken together, our findings suggest that DE cells and their alleged public clonotype are not enriched in T1D. This Matters Arising paper is in response to Ahmed et al. (2019), published in Cell. See also the response by Ahmed et al. (2021), published in this issue.

Toll-like receptors TLR2 and TLR4 block the replication of pancreatic ß cells in diet-induced obesity.

Consumption of a high-energy Western diet triggers mild adaptive ß cell proliferation to compensate for peripheral insulin resistance; however, the underlying molecular mechanism remains unclear. In the present study we show that the toll-like receptors TLR2 and TLR4 inhibited the diet-induced replication of ß cells in mice and humans. The combined, but not the individual, loss of TLR2 and TLR4 increased the replication of ß cells, but not that of α cells, leading to enlarged ß cell area and hyperinsulinemia in diet-induced obesity. Loss of TLR2 and TLR4 increased the nuclear abundance of the cell cycle regulators cyclin D2 and Cdk4 in a manner dependent on the signaling mediator Erk. These data reveal a regulatory mechanism controlling the proliferation of ß cells in diet-induced obesity and suggest that selective targeting of the TLR2/TLR4 pathways may reverse ß cell failure in patients with diabetes.

m6A Modification Prevents Formation of Endogenous Double-Stranded RNAs and Deleterious Innate Immune Responses during Hematopoietic Development.

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.

Checkpoint kinase 2 controls insulin secretion and glucose homeostasis.

After the discovery of insulin, a century ago, extensive work has been done to unravel the molecular network regulating insulin secretion. Here we performed a chemical screen and identified AZD7762, a compound that potentiates glucose-stimulated insulin secretion (GSIS) of a human ß cell line, healthy and type 2 diabetic (T2D) human islets and primary cynomolgus macaque islets. In vivo studies in diabetic mouse models and cynomolgus macaques demonstrated that AZD7762 enhances GSIS and improves glucose tolerance. Furthermore, genetic manipulation confirmed that ablation of CHEK2 in human ß cells results in increased insulin secretion. Consistently, high-fat-diet-fed Chk2-/- mice show elevated insulin secretion and improved glucose clearance. Finally, untargeted metabolic profiling demonstrated the key role of the CHEK2-PP2A-PLK1-G6PD-PPP pathway in insulin secretion. This study successfully identifies a previously unknown insulin secretion regulating pathway that is conserved across rodents, cynomolgus macaques and human ß cells in both healthy and T2D conditions.

NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing.

BACKGROUND: Current approaches to profile the single-cell transcriptomics of human pancreatic endocrine cells almost exclusively rely on freshly isolated islets. However, human islets are limited in availability. Furthermore, the extensive processing steps during islet isolation and subsequent single cell dissolution might alter gene expressions. In this work, we report the development of a single-nucleus RNA sequencing (snRNA-seq) approach with targeted islet cell enrichment for endocrine-population focused transcriptomic profiling using frozen archival pancreatic tissues without islet isolation. RESULTS: We cross-compared five nuclei isolation protocols and selected the citric acid method as the best strategy to isolate nuclei with high RNA integrity and low cytoplasmic contamination from frozen archival human pancreata. We innovated fluorescence-activated nuclei sorting based on the positive signal of NKX2-2 antibody to enrich nuclei of the endocrine population from the entire nuclei pool of the pancreas. Our sample preparation procedure generated high-quality single-nucleus gene-expression libraries while preserving the endocrine population diversity. In comparison with single-cell RNA sequencing (scRNA-seq) library generated with live cells from freshly isolated human islets, the snRNA-seq library displayed comparable endocrine cellular composition and cell type signature gene expression. However, between these two types of libraries, differential enrichments of transcripts belonging to different functional classes could be observed. CONCLUSIONS: Our work fills a technological gap and helps to unleash frozen archival pancreatic tissues for molecular profiling targeting the endocrine population. This study opens doors to retrospective mappings of endocrine cell dynamics in pancreatic tissues of complex histopathology. We expect that our protocol is applicable to enrich nuclei for transcriptomics studies from various populations in different types of frozen archival tissues.

Development of PEG-mediated genetic transformation and gene editing system of Bryum argenteum as an abiotic stress tolerance model plant.

KEY MESSAGE: To establish a sterile culture system and protoplast regeneration system for Bryum argenteum, and to establish and apply CRISPR/Cas9 system in Bryum argenteum. Bryum argenteum is a fascinating, cosmopolitan, and versatile moss species that thrives in various disturbed environments. Because of its comprehensive tolerance to the desiccation, high UV and extreme temperatures, it is emerging as a model moss for studying the molecular mechanisms underlying plant responses to abiotic stresses. However, the lack of basic tools such as gene transformation and targeted genome modification has hindered the understanding of the molecular mechanisms underlying the survival of B. argenteum in different environments. Here, we reported the protonema of B. argenteum can survive up to 95.4% water loss. In addition, the genome size of B. argenteum is approximately 313 Mb by kmer analysis, which is smaller than the previously reported 700 Mb. We also developed a simple method for protonema induction and an efficient protoplast isolation and regeneration protocol for B. argenteum. Furthermore, we established a PEG-mediated protoplast transient transfection and stable transformation system for B. argenteum. Two homologues of ABI3(ABA-INSENSITIVE 3) gene were successfully cloned from B. argenteum. To further investigate the function of the ABI3 gene in B. argenteum, we used the CRISPR/Cas9 genetic editing system to target the BaABI3A and BaABI3B gene in B. argenteum protoplasts. This resulted in mutagenesis at the target in about 2-5% of the regenerated plants. The isolated abi3a and abi3b mutants exhibited increased sensitivity to desiccation, suggesting that BaABI3A and BaABI3B play redundant roles in desiccation stress. Overall, our results provide a rapid and simple approach for molecular genetics in B. argenteum. This study contributes to a better understanding of the molecular mechanisms of plant adaptation to extreme environmental.

A fetal wound healing program after intrauterine bile duct injury may contribute to biliary atresia.

BACKGROUND & AIMS: Biliary atresia (BA) is an obstructive cholangiopathy that initially affects the extrahepatic bile ducts (EHBDs) of neonates. The etiology is uncertain, but evidence points to a prenatal cause. Fetal tissues have increased levels of hyaluronic acid (HA), which plays an integral role in fetal wound healing. The objective of this study was to determine whether a program of fetal wound healing is part of the response to fetal EHBD injury. METHODS: Mouse, rat, sheep, and human EHBD samples were studied at different developmental time points. Models included a fetal sheep model of prenatal hypoxia, human BA EHBD remnants and liver samples taken at the time of the Kasai procedure, EHBDs isolated from neonatal rats and mice, and spheroids and other models generated from primary neonatal mouse cholangiocytes. RESULTS: A wide layer of high molecular weight HA encircling the lumen was characteristic of the normal perinatal but not adult EHBD. This layer, which was surrounded by collagen, expanded in injured ducts in parallel with extensive peribiliary gland hyperplasia, increased mucus production and elevated serum bilirubin levels. BA EHBD remnants similarly showed increased HA centered around ductular structures compared with age-appropriate controls. High molecular weight HA typical of the fetal/neonatal ducts caused increased cholangiocyte spheroid growth, whereas low molecular weight HA induced abnormal epithelial morphology; low molecular weight HA caused matrix swelling in a bile duct-on-a-chip device. CONCLUSION: The fetal/neonatal EHBD, including in human EHBD remnants from Kasai surgeries, demonstrated an injury response with prolonged high levels of HA typical of fetal wound healing. The expanded peri-luminal HA layer may swell and lead to elevated bilirubin levels and obstruction of the EHBD. IMPACT AND IMPLICATIONS: Biliary atresia is a pediatric cholangiopathy associated with high morbidity and mortality rates; although multiple etiologies have been proposed, the fetal response to bile duct damage is largely unknown. This study explores the fetal pathogenesis after extrahepatic bile duct damage, thereby opening a completely new avenue to study therapeutic targets in the context of biliary atresia.

Biological cell trapping and manipulation of a photonic nanojet by a specific microcone-shaped optical fiber tip.

Trapping and manipulating mesoscopic biological cells with high precision and flexibility are very important for numerous biomedical applications. In particular, a photonic nanojet based on a non-resonance focusing phenomenon can serve as a powerful tool for manipulating red blood cells and tumor cells in blood. In this study, we demonstrate an approach to trap and drive cells using a high-quality photonic nanojet which is produced by a specific microcone-shaped optical-fiber tip. The dynamic chemical etching method is used to fabricate optical-fiber probes with a microcone-shaped tip. Optical forces and potentials exerted on a red blood cell by a microcone-shaped fiber tips are analyzed based on finite-difference time-domain calculations. Optical trapping and driving experiments are done using breast cancer cells and red blood cells. Furthermore, a cell chain is formed by adjusting the magnitude of the optical force. The real-time backscattering intensities of multiple cells are detected, and highly sensitive trapping is achieved. This microcone-shaped optical fiber probe is potentially a powerful device for dynamic cell assembly, optical sorting, and the precise diagnosis of vascular diseases.

In-plane subwavelength optical capsule for lab-on-a-chip nano-tweezers.

In this Letter, we propose a new, to the best of our knowledge, proof-of-concept of optical nano-tweezers based on a pair of dielectric rectangular structures that are capable of generating a finite-volume in-plane optical capsule. Finite-difference time-domain simulations of light spatial distributions and optical trapping forces of a gold nanoparticle immersed in water demonstrate the physical concept of an in-plane subwavelength optical capsule integrated with a microfluidic mesoscale device. It is shown that the refractive index of and the distance between the two dielectric rectangular structures can effectively control the shape and axial position of the optical capsule. Such an in-plane mesoscale structure provides a new path for manipulating absorbing nano-particles or bio-particles in a compact planar architecture, and should thus lead to promising perspectives in lab-on-a-chip domains.

Simulation and experimental observations of axial position control of a photonic nanojet by a dielectric cube with a metal screen.

In this Letter, we report on a numerical study, fabrication, and experimental observations of photonic nanojet (PNJ) shaping by control of a tangential electric field component. Here the PNJs are generated by a single mesoscale micro-cube that is fabricated from polydimethylsiloxane, deposited on a silicon substrate and placed on thick metal screen at illuminating wavelengths of 405, 532, and 671 nm. It is shown that the length, focal length, and width of the PNJ can be significantly reduced in the presence of the metal masks along the side faces of the micro-cube. Experimental measurements of the PNJ imaging are performed by a scanning optical microscope with laser sources. Our experimental results are in reasonable agreement with simulation predictions of the finite-difference time-domain method. Due to the appearance of the metal masks, the PNJ focal length decreases 1.5 times, the PNJ decay length decreases 1.7 times, and the PNJ resolution increases 1.2 times. Such PNJs possess great potential in complex manipulation, including integrated plasmonic circuits, biosensing, and optical tweezers.

Experimental demonstration of a tunable photonic hook by a partially illuminated dielectric microcylinder.

In this Letter, we report the experimental observations of a tunable curved photonic nanojet (photonic hook) generated by a 5 µm polydimethylsiloxane microcylinder deposited on a silicon substrate and illuminated by 405 nm laser beam. A moveable opaque aluminum-mask is mounted in front of the microcylinder implementing partial illumination and imparting spatial curvature to the photonic nanojet. Experimental results of main parameters (tilt angle, width, and intensity) of emerging photonic hooks exhibit close agreement with numerical predictions of the near-field optical structures. The experimentally measured full widths at half-maximum of photonic hooks are 0.48λ, 0.56λ, and 0.76λ for tilt angles of θ=0∘, 5.7°, and 20.1°, respectively. Photonic hooks possess great potential in complex manipulation such as super-resolution imaging, surface fabrication, and optomechanical manipulation along curved trajectories.

Experimental verification of twin photonic nanojets from a dielectric microcylinder.

In this Letter, the direct generation of twin photonic nanojets (PNJs) through two coherent illuminations of a microcylinder is investigated theoretically and experimentally. The dielectric microcylinder (polydimethylsiloxane) with 5 µm diameter and 6 µm height is employed to generate symmetric twin PNJs. The finite-difference time-domain calculation is used to simulate the electric field distributions inside and outside the microcylinder. The scanning optical microscope system is performed for experimental verification of twin photonic nanojets. In both theory and in practice, the intensity null of electric field creates two separate PNJs. Compared to a single PNJ, twin PNJs have a smaller subwavelength waist and more complex intensity distribution. The focal distance, interval, and full width at half-maximum of twin PNJs are a function of the offset angle. The twin PNJs will provide novel applications in nanolithography, optical trapping, biophotonic sensing, and therapy.

Formation of high-quality photonic nanojets by decorating spider silk.

The photonic nanojet is a highly concentrated beam with low divergence on the shadow side of dielectric microparticles. In this Letter, we first theoretically and experimentally investigate the formation of high-quality photonic nanojets by decorating spider silk. The dragline silks are directly extracted from cellar spiders and capable of efficiently collecting ultraviolet cure adhesive. The liquid-collecting capacity of the captured silk is the result of a singular fiber structure with periodic spindle knots. Using a scanning-optical-microscope system, we show that high-quality photonic nanojets are generated by silk fiber with spindle knots. With the variation in spindle-knot dimensions, the properties of photonic nanojets, such as intensity distribution, focal length, and full width at half-maximum, are able to tune flexibly. By combining the unique biocompatibility, flexibility, and tensile strength, the silk filaments with spindle knots pave a potential way for original bio-photonic applications.

Islet transplantation improved penile tissue fibrosis in a rat model of type 1 diabetes.

BACKGROUND: Glycaemic control is one of the most effective strategies for the treatment of diabetes-related erectile dysfunction (DMED). Compared to conventional anti-diabetic drugs and insulin, islet transplantation is more effective in the treatment of diabetic complications. The aim of this study was to investigate the efficacy of islet transplantation for reversing advanced-stage DMED in rats and to observe its influence on corpus cavernosum fibrosis. METHODS: Wistar rats were intraperitoneally injected with streptozotocin to establish a diabetes model. After 12 weeks, the rats were divided into 4 groups: diabetic, insulin, islet transplantation, and normal control. Following supplementation, the changes in blood glucose and weight were determined sequentially. Penile erectile function was evaluated by apomorphine experiments in the fourth week, and the penile corpus cavernosum was also collected for assessment by Masson staining, immunohistochemistry and Western blot to observe the spongy tissue and the related cellular changes at the molecular level. RESULTS: Islet transplantation significantly ameliorated penile erectile function in advanced-stage diabetic rats. The ratio of corpus cavernosum smooth muscle cells to fibroblasts and the expression level of α-SMA in the islet transplantation group were significantly higher than those in the diabetic and insulin groups. In addition, the expression levels of TGF-ß1, p-Samd2, and connective tissue growth factor (CTGF) in the islet transplantation and insulin groups were much lower than those in the diabetic group, while those in the islet transplantation group were significantly lower than those in the insulin group. CONCLUSIONS: Our findings strongly suggest that islet transplantation can promote the regeneration of smooth muscle cells and ameliorate corpus cavernosum fibrosis to restore its normal structure in advanced-stage diabetic rats. The possible mechanism of ameliorating corpus cavernosum fibrosis by islet transplantation may be associated with improvement of the hyperglycaemic status in diabetic rats, thereby inhibiting the TGF-ß1/Samd2/CTGF pathway.

Menin and PRMT5 suppress GLP1 receptor transcript and PKA-mediated phosphorylation of FOXO1 and CREB.

Menin is a scaffold protein that interacts with several epigenetic mediators to regulate gene transcription, and suppresses pancreatic ß-cell proliferation. Tamoxifen-inducible deletion of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes the protein menin, increases ß-cell mass in multiple murine models of diabetes and ameliorates diabetes. Glucagon-like-peptide-1 (GLP1) is another key physiological modulator of ß-cell mass and glucose homeostasis. However, it is not clearly understood whether menin crosstalks with GLP1 signaling. Here, we show that menin and protein arginine methyltransferase 5 (PRMT5) suppress GLP1 receptor (GLP1R) transcript levels. Notably, a GLP1R agonist induces phosphorylation of forkhead box protein O1 (FOXO1) at S253, and the phosphorylation is mediated by PKA. Interestingly, menin suppresses GLP1-induced and PKA-mediated phosphorylation of both FOXO1 and cAMP response element binding protein (CREB), likely through a protein arginine methyltransferase. Menin-mediated suppression of FOXO1 and CREB phosphorylation increases FOXO1 levels and suppresses CREB target genes, respectively. A small-molecule menin inhibitor reverses menin-mediated suppression of both FOXO1 and CREB phosphorylation. In addition, ex vivo treatment of both mouse and human pancreatic islets with a menin inhibitor increases levels of proliferation marker Ki67. In conclusion, our results suggest that menin and PRMT5 suppress GLP1R transcript levels and PKA-mediated phosphorylation of FOXO1 and CREB, and a menin inhibitor may reverse this suppression to induce ß-cell proliferation.

Primitive chain network simulations of probe rheology.

Probe rheology experiments, in which the dynamics of a small amount of probe chains dissolved in immobile matrix chains is discussed, have been performed for the development of molecular theories for entangled polymer dynamics. Although probe chain dynamics in probe rheology is considered hypothetically as single chain dynamics in fixed tube-shaped confinement, it has not been fully elucidated. For instance, the end-to-end relaxation of probe chains is slower than that for monodisperse melts, unlike the conventional molecular theories. In this study, the viscoelastic and dielectric relaxations of probe chains were calculated by primitive chain network simulations. The simulations semi-quantitatively reproduced the dielectric relaxation, which reflects the effect of constraint release on the end-to-end relaxation. Fair agreement was also obtained for the viscoelastic relaxation time. However, the viscoelastic relaxation intensity was underestimated, possibly due to some flaws in the model for the inter-chain cross-correlations between probe and matrix chains.

Direct imaging of optimal photonic nanojets from core-shell microcylinders.

We first experimentally evaluate the direct imaging of photonic nanojets from core-shell microcylinders. The optimal photonic nanojet with long length, a high intensity spot, and low divergence is observed at the designed gold-silver-coating microcylinder. A special microcylinder consists of multilayered metallic shells (gold, silver, and copper) and a dielectric core (polydimethylsiloxane) at a diameter of 5 µm and a height of 6 µm. The electromagnetic distributions inside and outside the core-shell microcylinders are calculated by using the finite-difference time-domain method. The direct-imaging measurements for photonic nanojets are performed with a scanning-optical-microscope system. Such core-shell microcylinders provide new pathways for high-resolution optical imaging, which are useful for biophotonics, plasmonics, and optical data storage.

Photonic jets produced by dielectric micro cuboids.

The formation of photonic jets produced by dielectric micro cuboids is reported. The spatial electromagnetic field has been numerically analyzed on the basis of the finite-difference time-domain calculation. The characteristics of photonic jets, such as propagation length and location, can be drastically changed by controlling the cuboid dimensions. Visually three morphological types of photonic jets are introduced for classification. Combining key parameters of photonic jets, the quality criterion is used to describe the jet quality. The super resolution imaging of the dielectric micro cuboid can be expected from the long focal length and small beam waist. The simulation results show that it can be of interest for several potential applications, such as subdiffraction resolution optical microlenses, ultradirectional optical antennae, and nanolithography based on the micro cuboid.

Regulation of glucagon secretion in normal and diabetic human islets by γ-hydroxybutyrate and glycine.

Paracrine signaling between pancreatic islet ß-cells and α-cells has been proposed to play a role in regulating glucagon responses to elevated glucose and hypoglycemia. To examine this possibility in human islets, we used a metabolomic approach to trace the responses of amino acids and other potential neurotransmitters to stimulation with [U-(13)C]glucose in both normal individuals and type 2 diabetics. Islets from type 2 diabetics uniformly showed decreased glucose stimulation of insulin secretion and respiratory rate but demonstrated two different patterns of glucagon responses to glucose: one group responded normally to suppression of glucagon by glucose, but the second group was non-responsive. The non-responsive group showed evidence of suppressed islet GABA levels and of GABA shunt activity. In further studies with normal human islets, we found that γ-hydroxybutyrate (GHB), a potent inhibitory neurotransmitter, is generated in ß-cells by an extension of the GABA shunt during glucose stimulation and interacts with α-cell GHB receptors, thus mediating the suppressive effect of glucose on glucagon release. We also identified glycine, acting via α-cell glycine receptors, as the predominant amino acid stimulator of glucagon release. The results suggest that glycine and GHB provide a counterbalancing receptor-based mechanism for controlling α-cell secretory responses to metabolic fuels.

B lymphocyte-directed immunotherapy promotes long-term islet allograft survival in nonhuman primates.

We found that an induction immunotherapy regimen consisting of rabbit anti-thymocyte globulin (Thymoglobulin) and the monoclonal antibody to CD20 rituximab (Rituxan) promoted long-term islet allograft survival in cynomolgus macaques maintained on rapamycin monotherapy. B lymphocyte reconstitution after rituximab-mediated depletion was characterized by a preponderance of immature and transitional cells, whose persistence was associated with long-term islet allograft survival. Development of donor-specific alloantibodies was abrogated only in the setting of continued rapamycin monotherapy.