Microstructure-Assisted Wafer-Scale Fabrication of Perovskite Microlaser Arrays.

All inorganic lead halide perovskites exhibit fascinating optical and optoelectronic characteristics for on-chip lasing, but the lack of precise control of wafer-scale fabrication for perovskite microstructure arrays restricts their potential applications in on-chip-integrated devices. In this work, a microstructure-template assisted crystallization method is demonstrated via a designed chemical vapor deposition process, achieving the controllable fabrication of homogeneous perovskite micro-hemispheroid (PeMH) arrays spanning the entire surface area of a 4-inch wafer. Benefiting from the low-loss whispering gallery resonance and plasmon-enhanced light-matter interactions in well-confined hybrid cavities, this CsPbX3/Ag (X = Cl, Br) plasmonic microlasers exhibit quite low thresholds below 10 µJ cm-2. Interestingly, these thresholds can be efficiently modulated through the manipulation of plasmonic resonance and electromagnetic field mode in PeMHs owning various diameters. This strategy not only provides a valuable methodology for the large-scale fabrication of perovskite microstructures but also endorses the potential of all-inorganic perovskite nanostructures as promising candidates for on-chip-integrated light sources.

Efficacy of damage control orthopedics strategy in the management of lower limb trauma.

Background: Little is known about the efficacy of damage control (DC) surgery in the management of lower limb trauma. Here we compared the clinical parameters and complication rates of such patients received either DC or emergency comprehensive (EC) surgery treatment. Methods: This study is a retrospective study on patients with lower limb trauma that received surgical treatment. Data of 120 patients were divided into DC and EC surgery groups. Clinical parameters obtained at hospital admission and complications during follow-up were analyzed. Injury Severity Score (ISS), Gustilo classification and Mangled Extremity Severity Score (MESS) were used to assess trauma severity, open fractures and viability of injured limb, respectively. Results: Age, sex, ISS, fracture type, injury site, MESS, operation time, blood loss, pulmonary and cranial injuries were compared. We found that patients in the DC group had more severe injury as reflected by the higher injury severity score (ISS) (28.1 ± 10.9 vs 21.3 ± 7.4, P < 0.001). ISS was also identified as a significant influencer for the treatment selection (P < 0.001). In addition, patients treated with DC surgery demonstrated less complications (7 cases vs 27 cases), which was supported by the propensity score logistic regression analysis (Odd ratio 4.667). Conclusions: DC surgery is more often selected to treat patients with more severe lower limb injuries, which leads to lower complication rates.

Interleukin 4-driven reversal of self-reactive B cell anergy contributes to the pathogenesis of systemic lupus erythematosus.

OBJECTIVES: Reactivation of anergic autoreactive B cells (BND cells) is a key aetiological process in systemic lupus erythematosus (SLE), yet the underlying mechanism remains largely elusive. This study aimed to investigate how BND cells participate in the pathogenesis of SLE and the underlying mechanism. METHODS: A combination of phenotypical, large-scale transcriptome and B cell receptor (BCR) repertoire profiling were employed at molecular and single cell level on samples from healthy donors and patients with SLE. Isolated naïve B cells from human periphery blood were treated with anti-CD79b mAb in vitro to induce anergy. IgM internalisation was tracked by confocal microscopy and was qualified by flow cytometer. RESULTS: We characterised the decrease and disruption of BND cells in SLE patients and demonstrated IL-4 as an important cytokine to drive such pathological changes. We then elucidated that IL-4 reversed B cell anergy by promoting BCR recycling to the cell surface via STAT6 signalling. CONCLUSIONS: We demonstrated the significance of IL-4 in reversing B cell anergy and established the scientific rationale to treat SLE via blocking IL-4 signalling, also providing diagnostic and prognostic biomarkers to identify patients who are most likely going to benefit from such treatments.

Circular RNA circTIE1 drives proliferation, migration, and invasion of glioma cells through regulating miR-1286/TEAD1 axis.

Recent studies have verified that circRNAs (circular RNAs) play a critical role in glioma occurrence and malignant progression. However, numerous circRNAs with unknown functions remain to be explored with further research. qPCR (quantitative real-time polymerase chain reaction) was employed to detect circTIE1 expression in glioma tissues, NHAs (normal human astrocytes), and glioma cellular lines (U87, U118, U251, T98G, LN229). Cell viability was evaluated by CCK-8 assay. Cellular proliferation was evaluated by a 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay. Cell migration and aggression were both evaluated by transwell and migration assays. The direct binding and regulation among circTIE1, miR-1286 and TEAD1 was identified by western blotting, qPCR, luciferase reporter assay, and RNA immunoprecipitation (RIP) assay. Xenografts were generated by injecting glioma cells orthotopically into the brains of nude mice. Immunohistochemistry staining was implemented to evaluate the expression of the proliferation markers ki67 and TEAD1. We found that circTIE1 (circBase ID: hsa_circ_0012012) was upregulated in glioma tissues and glioma cellular lines in contrast to NBT (normal brain tissues) and NHA. CircTIE1 knockdown inhibited glioma cell viability, proliferation, migration and aggression both in vitro and in vivo. Mechanistically, circTIE1 could upregulate TEAD1 expression via miR-1286 sponging, and TEAD1 is a well-known functional gene that could promote malignant advancement in glioma. This research found a novel circRNA, circTIE1, which is an essential marker of glioma progression and diagnosis and may be anticipated to become a crucial target for molecular targeted therapy of glioma.

Oxidized mitochondrial DNA induces gasdermin D oligomerization in systemic lupus erythematosus.

Although extracellular DNA is known to form immune complexes (ICs) with autoantibodies in systemic lupus erythematosus (SLE), the mechanisms leading to the release of DNA from cells remain poorly characterized. Here, we show that the pore-forming protein, gasdermin D (GSDMD), is required for nuclear DNA and mitochondrial DNA (mtDNA) release from neutrophils and lytic cell death following ex vivo stimulation with serum from patients with SLE and IFN-γ. Mechanistically, the activation of FcγR downregulated Serpinb1 following ex vivo stimulation with serum from patients with SLE, leading to spontaneous activation of both caspase-1/caspase-11 and cleavage of GSDMD into GSDMD-N. Furthermore, mtDNA oxidization promoted GSDMD-N oligomerization and cell death. In addition, GSDMD, but not peptidyl arginine deiminase 4 is necessary for extracellular mtDNA release from low-density granulocytes from SLE patients or healthy human neutrophils following incubation with ICs. Using the pristane-induced lupus model, we show that disease severity is significantly reduced in mice with neutrophil-specific Gsdmd deficiency or following treatment with the GSDMD inhibitor, disulfiram. Altogether, our study highlights an important role for oxidized mtDNA in inducing GSDMD oligomerization and pore formation. These findings also suggest that GSDMD might represent a possible therapeutic target in SLE.

EIF4A3 induced circGRIK2 promotes the malignancy of glioma by regulating the miR-1303/HOXA10 axis.

In recent years, the role of circular RNAs (circRNAs) in glioma has become increasingly important. However, there are still many newly discovered circRNAs with unknown functions that require further study. In this study, circRNA sequencing, qPCR, MTS, EdU, Transwell, and other assays were conducted to detect the expression and malignant effects of a novel circRNA molecule, circGRIK2, in glioma. qPCR, western blotting, RIP, and luciferase reporter gene experiments were used to investigate the downstream molecular mechanisms of circGRIK2. Our study found that circGRIK2 was highly expressed in glioma and promoted glioma cell viability, proliferation, invasion, and migration. Mechanistically, circGRIK2 acted as a competitive sponge for miR-1303, upregulating the expression of HOXA10 to exert its oncogenic effects. Additionally, the RNA-binding protein EIF4A3 could bind to and stabilize circGRIK2, leading to its high expression in glioblastoma. The discovery of circGRIK2 in this study not only contributes to a better understanding of the biological mechanisms of circGRIK2 in glioma but also provides a new target for molecular targeted therapy.

Distinct impact of IgG subclass on autoantibody pathogenicity in different IgG4-mediated diseases.

IgG4 is the least potent human IgG subclass for the FcγR-mediated antibody effector function. Paradoxically, IgG4 is also the dominant IgG subclass of pathogenic autoantibodies in IgG4-mediated diseases. Here, we show that the IgG subclass and Fc-FcγR interaction have a distinct impact on the pathogenic function of autoantibodies in different IgG4-mediated diseases in mouse models. While IgG4 and its weak Fc-FcγR interaction have an ameliorative role in the pathogenicity of anti-ADAMTS13 autoantibodies isolated from thrombotic thrombocytopenic purpura (TTP) patients, they have an unexpected exacerbating effect on anti-Dsg1 autoantibody pathogenicity in pemphigus foliaceus (PF) models. Strikingly, a non-pathogenic anti-Dsg1 antibody variant optimized for FcγR-mediated effector function can attenuate the skin lesions induced by pathogenic anti-Dsg1 antibodies by promoting the clearance of dead keratinocytes. These studies suggest that IgG effector function contributes to the clearance of autoantibody-Ag complexes, which is harmful in TTP, but beneficial in PF and may provide new therapeutic opportunity.

Diverging regulation of Bach2 protein and RNA expression determine cell fate in early B cell response.

During the early phase of primary humoral responses, activated B cells can differentiate into different types of effector cells, dependent on B cell receptor affinity for antigen. However, the pivotal transcription factors governing these processes remain to be elucidated. Here, we show that transcription factor Bach2 protein in activated B cells is transiently induced by affinity-related signals and mechanistic target of rapamycin complex 1 (mTORC1)-dependent translation to restrain their expansion and differentiation into plasma cells while promoting memory and germinal center (GC) B cell fates. Affinity-related signals also downregulate Bach2 mRNA expression in activated B cells and their descendant memory B cells. Sustained and higher concentrations of Bach2 antagonize the GC fate. Repression of Bach2 in memory B cells predisposes their cell-fate choices upon memory recall. Our study reveals that differential dynamics of Bach2 protein and transcripts in activated B cells control their cell-fate outcomes and imprint the fates of their descendant effector cells.

Promotion of a damage control concept in repairing orthopedic lower limb trauma.

OBJECTIVE: To explore the application value of the concept of damage control orthopaedics (DCO) in clinical treatment of lower limb fractures. METHODS: In this retrospective analysis, 157 patients with lower extremity fracture, who received surgery in Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine (Cangzhou Orthopaedic Hospital) during March 2019 and August 2020, were chosen as research subjects. Among them, 73 patients admitted from March to December 2019 were included in the control group, and the other 84 patients admitted from January to August 2020 were included in the observation group. The control group received conventional fracture treatment scheme, and the observation group was treated under the DCO concept. The operation time, postoperative hospital stay, fracture reduction quality, incidence of complications, and bone metabolism and bone healing pre- and post-treatment were compared between the groups. RESULTS: The operation time, time to achieve stable vital signs and hospital stay of the observation group were apparently shorter than those of the control group (P<0.05). Visual analog score (VAS) of patients in both groups on postoperative day 3 and 7 were substantially lower than those on postoperative day 1 (P<0.05), and VAS scores of patients in the observation group were markedly lower than those in the control group (P<0.05). Serum Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the two groups were significantly reduced on 14th day after surgery compared to those before surgery (P<0.05), and the indexes of observation group were lower than those of control group (P<0.05). Serum Osteocalcin (BGP), carboxyterminal of type I procollagen (PICP), alkaline phosphatase (ALP), Soluble intercellular adhesion molecule-1 (sICAM-1) and Insulin like growth factor-1 (IGF-1) in the two groups postoperative day 14 were obviously higher than those before operation (P<0.05), and the indicators in the observation group were higher than those of the control group (P<0.05). The incidence of complications in the control group was significantly higher than that in the observation group (P<0.05). CONCLUSION: The application of the DCO concept in the clinical treatment of lower extremity trauma can effectively promote the rehabilitation of patients with lower extremity trauma, minimize their complications, improve the bone metabolism and bone healing degree, and reduce the degree of pain.

The phosphatase PTEN links platelets with immune regulatory functions of mouse T follicular helper cells.

Beyond a function in hemostasis and thrombosis, platelets can regulate innate and adaptive immune responses. Hyperactive platelets are frequently associated with multiple human autoimmune diseases, yet their pathogenic functions in these diseases have not been fully established. Emerging studies show an essential function of the phosphatase and tensin homolog (PTEN) in maintenance of immune homeostasis. Here, we show that mice with platelet-specific deletion of Pten, develop age-related lymphoproliferative diseases and humoral autoimmunity not seen in wildtype animals. Platelet-specific Pten-deficient mice have aberrant T cell activation, excessive T follicular helper (Tfh) cell responses and accumulation of platelet aggregates in lymph nodes. Transferred Pten-deficient platelets are able to infiltrate into the peripheral lymphoid tissues and form more aggregates. Moreover, Pten-deficient platelets are hyperactive and overproduce multiple Tfh-promoting cytokines via activation of the PDK1/mTORC2-AKT-SNAP23 pathway. Pten-deficient platelets show enhanced interaction with CD4+ T cells and promote conversion of CD4+ T cells into Tfh cells. Our results implicate PTEN in platelet-mediated immune homeostasis, and provide evidence that hyperactive platelets function as an important mediator in autoimmune diseases using mouse models.

High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics.

Currently, high-throughput approaches are lacking in the isolation of antibodies with functional readouts beyond simple binding. This situation has impeded the next generation of cancer immunotherapeutics, such as bispecific T cell engager (BiTE) antibodies or agonist antibodies against costimulatory receptors, from reaching their full potential. Here, we developed a highly efficient droplet-based microfluidic platform combining a lentivirus transduction system that enables functional screening of millions of antibodies to identify potential hits with desired functionalities. To showcase the capacity of this system, functional antibodies for CD40 agonism with low frequency (<0.02%) were identified with two rounds of screening. Furthermore, the versatility of the system was demonstrated by combining an anti-Her2 × anti-CD3 BiTE antibody library with functional screening, which enabled efficient identification of active anti-Her2 × anti-CD3 BiTE antibodies. The platform could revolutionize next-generation cancer immunotherapy drug development and advance medical research.

Bach2 attenuates IL-2R signaling to control Treg homeostasis and Tfr development.

Differentiation and homeostasis of Foxp3+ regulatory T cells (Tregs) are tightly controlled by the interleukin-2 receptor (IL-2R) signaling, yet the mechanisms governing these processes are incompletely understood. Here, we report that transcription factor Bach2 attenuates IL-2R signaling to coordinate Treg differentiation and homeostasis. Bach2 is required for the quiescence, survival, and maintenance of resting Treg cells (rTregs). Unexpectedly, Bach2 directly represses CD25 (IL-2Rα) and subsequently attenuates IL-2R signaling in Tregs. Upregulated CD25/IL-2R signaling in Bach2-deficient rTregs acts as a parallel pathway to partially counteract their poor survival and maintenance. Furthermore, Bach2 suppresses CD25/IL-2R signaling in T follicular regulatory (Tfr) cells. Bach2 deficiency in Tregs prevents the formation of highly differentiated Tfr cells, associated with aberrant GC response. Finally, a mild and late onset of autoimmune disease is observed in mice with Bach2-deficient Tregs. Thus, Bach2 balances IL-2R signaling to orchestrate development and homeostasis of various Treg subsets.

A general Fc engineering platform for the next generation of antibody therapeutics.

Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform. Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice. Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models.Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

In vivo Screen Identifies Zdhhc2 as a Critical Regulator of Germinal Center B Cell Differentiation.

Germinal center (GC) B cell differentiation is critical for the production of affinity-matured pathogen-specific antibodies, the dysregulation of which may lead to humoral immunodeficiency or autoimmunity. The development of an in vivo screening system for factors regulating GC B cell differentiation has been a challenge. Here we describe a small-scale in vivo screening system with NP-specific B1-8hi cells and a retroviral shRNA library targeting 78 candidate genes to search for B cell-intrinsic factors that specifically regulate GC B cell differentiation. Zdhhc2, a gene encoding palmitoyltransferase ZDHHC2 and highly expressed in GC B cells, is identified as a strong positive regulator of GC B cell differentiation. B1-8hi cells transduced with Zdhhc2-shRNA are severely compromised in differentiating into GC B cells. A further analysis of in vitro differentiated B cells transduced with Zdhhc2-shRNA shows that Zdhhc2 is critical for the proliferation and the survival of B cells stimulated by CD40L, BAFF, and IL-21 and consequently impacts on their differentiation into GC B cells and post-GC B cells. These studies not only identify Zdhhc2 as a novel regulator of GC B cell differentiation but also represent a proof of concept of in vivo screen for regulators of GC B cell differentiation.

Human immunoglobulin G hinge regulates agonistic anti-CD40 immunostimulatory and antitumour activities through biophysical flexibility.

Human immunoglobulin G (IgG) agonistic antibodies targeting costimulatory immunoreceptors represent promising cancer immunotherapies yet to be developed. Whether, and how, human IgG hinge and Fc impact on their agonistic functions have been disputed. Here, we show that different natural human IgGs confer divergent agonistic anti-CD40 immunostimulatory and antitumour activities in FcγR-humanized mice, including inactive IgG3 and superior IgG2. This divergence is primarily due to their CH1-hinges despite all human IgGs requiring Fc-FcγR binding for optimal agonistic activities. Unexpectedly, biophysical flexibility of these CH1-hinges inversely correlates with, and can modulate, their agonistic potency. Furthermore, IgG Fcs optimized for selective FcγR binding synergize with and still require IgG hinge, selected for rigidity, to confer improved anti-CD40 immunostimulatory and antitumour activities. These findings highlight the importance of both hinge rigidity and selective FcγR binding in antibody agonistic function, and the need for newer strategies to modulate antibody agonism for improved clinical application.

Excessive CD11c+Tbet+ B cells promote aberrant TFH differentiation and affinity-based germinal center selection in lupus.

Excessive self-reactive and inadequate affinity-matured antigen-specific antibody responses have been reported to coexist in lupus, with elusive cellular and molecular mechanisms. Here, we report that the antigen-specific germinal center (GC) response-a process critical for antibody affinity maturation-is compromised in murine lupus models. Importantly, this defect can be triggered by excessive autoimmunity-relevant CD11c+Tbet+ age-associated B cells (ABCs). In B cell-intrinsic Ship-deficient (ShipΔB) lupus mice, excessive CD11c+Tbet+ ABCs induce deregulated follicular T-helper (TFH) cell differentiation through their potent antigen-presenting function and consequently compromise affinity-based GC selection. Excessive CD11c+Tbet+ ABCs and deregulated TFH cell are also present in other lupus models and patients. Further, over-activated Toll-like receptor signaling in Ship-deficient B cells is critical for CD11c+Tbet+ ABC differentiation, and blocking CD11c+Tbet+ ABC differentiation in ShipΔB mice by ablating MyD88 normalizes TFH cell differentiation and rescues antigen-specific GC responses, as well as prevents autoantibody production. Our study suggests that excessive CD11c+Tbet+ ABCs not only contribute significantly to autoantibody production but also compromise antigen-specific GC B-cell responses and antibody-affinity maturation, providing a cellular link between the coexisting autoantibodies and inadequate affinity-matured antigen-specific antibodies in lupus models and a potential target for treating lupus.

Ilamycin E, a natural product of marine actinomycete, inhibits triple-negative breast cancer partially through ER stress-CHOP-Bcl-2.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among women in the worldwide. Triple-negative breast cancer (TNBC) has a poor clinical outcome. The antitumor efficacy of Ilamycins, natural products with anti-tuberculosis activity isolated from deep sea-derived Streptomyces atratus, in TNBC has not been investigated, and the mechanisms remain elusive. Here, we demonstrated that Ilamycin-E, but not -F, decreases cell viability, inhibits G1/S cell cycle progression, and promotes apoptosis in the TNBC cell lines HCC1937 and MDA-MB-468. Ilamycin E promotes apoptosis via activation of endoplasmic reticulum (ER) stress, increasing the expression of CHOP, and down-regulating the expression of anti-apoptotic protein Bcl-2. Depletion of CHOP or overexpression of Bcl2 significantly rescued Ilamycin E-induced apoptosis. These findings indicate that Ilamycin E has anti-cancer activity in TNBC.

Sin1/mTORC2 regulate B cell growth and metabolism by activating mTORC1 and Myc.

Proper control of B cell growth and metabolism is crucial for B-cell-mediated immunity, but the underlying molecular mechanisms remain incompletely understood. In this study, Sin1, a key component of mTOR complex 2 (mTORC2), specifically regulates B cell growth and metabolism. Genetic ablation of Sin1 in B cells reduces the cell size at either the transitional stage or upon antigen stimulation and severely impairs metabolism. Sin1 deficiency also severely impairs B-cell proliferation, antibody responses, and anti-viral immunity. At the molecular level, Sin1 controls the expression and stability of the c-Myc protein and maintains the activity of mTORC1 through the Akt-dependent inactivation of GSK3 and TSC1/2, respectively. Therefore, our study reveals a novel and specific role for Sin1 in coordinating the activation of mTORC2 and mTORC1 to control B cell growth and metabolism.

BCL6-Mediated Silencing of PD-1 Ligands in Germinal Center B Cells Maintains Follicular T Cell Population.

The programmed cell death protein 1 (PD-1) ligands PD-L1 and PD-L2 on germinal center (GC) B cells deliver coinhibitory signals to follicular T cells. The PD-L1/L2-PD-1 axis modulates the quality and quantity of follicular T cells and has been shown to influence the GC responses. However, the transcriptional control of PD-1 ligands on GC B cells remains largely unknown. In this study, we report that the transcription factor BCL6 is a key negative regulator of the PD-1 ligands PD-L1 and PD-L2 in GC B cells. Acute deletion of Bcl6 in mature GC B cells resulted in marked upregulation of mRNA and protein abundance of PD-1 ligands. Moreover, the expression levels of BCL6 and PD-1 ligands were inversely correlated during GC B cell development and in human GC-derived lymphoma specimens. Mechanically, BCL6 directly bound to the promoter region of PD-L1 and intron 2 of PD-L2 to suppress their transcription. In addition, BCL6 indirectly inhibited the transcription of PD-1 ligands by repressing the expression of STAT1/STAT3 and IRF1. Moreover, BCL6 exerted these effects via its BTB domain. Finally, PD-1 blockade promoted cell survival to sustain the follicular T cell pool in the presence of Bcl6-deficinet GC B cells. In summary, B cell-specific expression of BCL6 dampens the PD-L1/L2-PD-1 signaling to maintain the size of follicular T cells during GC development.