Dual roles of α1,4-galactosyltransferase 1 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the development of Drosophila melanogaster; however, their role in spermatogenesis is still unclear. In this study, we found that α1,4-galactosyltransferase 1 (α4GT1) was expressed at a significantly higher level in the testis than in the ovary of Drosophila. Importantly, the hatching rate was significantly decreased when α4GT1 RNA interference (RNAi) males were crossed with w1118 females, with only a few mature sperm being present in the seminal vesicle of α4GT1 RNAi flies. Immunofluorescence staining further revealed that the individualization complex (IC) in the testes from α4GT1 RNAi flies was scattered and did not move synchronically, compared with the clustered IC observed in the control flies. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay showed that apoptosis signals in the sperm bundles of α4GT1 RNAi flies were significantly increased. Moreover, the expression of several individualization-related genes, such as Shrub, Obp44a and Hanabi, was significantly decreased, whereas the expression of several apoptosis-related genes, including Dronc and Drice, was significantly increased in the testes of α4GT1 RNAi flies. Together, these results suggest that α4GT1 may play dual roles in Drosophila spermatogenesis by regulating the sperm individualization process and maintaining the survival of sperm bundles.

N6-methyladenosine modification of RNA controls dopamine synthesis to influence labour division in ants.

The N6-methyladenosine (m6A) modification of RNA has been reported to remodel gene expression in response to environmental conditions; however, the biological role of m6A in social insects remains largely unknown. In this study, we explored the role of m6A in the division of labour by worker ants (Solenopsis invicta). We first determined the presence of m6A in RNAs from the brains of worker ants and found that m6A methylation dynamics differed between foragers and nurses. Depletion of m6A methyltransferase or chemical suppression of m6A methylation in foragers resulted in a shift to 'nurse-like' behaviours. Specifically, mRNAs of dopamine receptor 1 (Dop1) and dopamine transporter (DAT) were modified by m6A, and their expression increased dopamine levels to promote the behavioural transition from foragers to nurses. The abundance of Dop1 and DAT mRNAs and their stability were reduced by the inhibition of m6A modification caused by the silencing of Mettl3, suggesting that m6A modification in worker ants modulates dopamine synthesis, which regulates labour division. Collectively, our results provide the first example of the epitranscriptomic regulation of labour division in social insects and implicate m6A regulatory mechanism as a potential novel target for controlling red imported fire ants.

Rigidify styryl-pyridinium dyes to benzo[h]coumarin-based bright two-photon fluorescent probes for cellular bioimaging.

Fluorescence imaging of organelles at the cellular level is important for studying biological processes. The development of a highly emissive fluorescent probe that operates under a suitable excitation light source is a key step in high-quality fluorescence imaging. For long-term, high-fidelity fluorescence imaging of mitochondria-related cellular processes using two-photon microscopy and stimulated emission depletion microscopy, we developed a new benzocoumarin-based cationic fluorescent probe (BS-CN) that is far-red emitting, water-soluble, photostable, and very bright in cells. BS-CN showed a remarkably high quantum yield of 0.35 and a large two-photon excited fluorescence action cross-section of 76 GM, enabling the long-term tracking of mitochondria in live cells. In addition, BS-CN exhibited a certain affinity for RNA and stained nucleoli in fixed cells. A comparative assessment of the photophysical properties and bioimaging performance of benzo[h]coumarin-pyridinium and the structurally similar styryl-pyridinium (BS-MN) clearly indicated the importance of structural rigidity for fluorescence efficiency.

Long-Chain Fluorescent Probe for Straightforward and Nondestructive Staining Mitochondria in Fixed Cells and Tissues.

Normally, small-molecule fluorescent probes dependent on the mitochondrial membrane potential (MMP) are invalid for fixed cells and tissues, which limits their clinical applications when the fixation of pathological specimens is imperative. Given that mitochondrial morphology is closely associated with disease, we developed a long-chain mitochondrial probe for fixed cells and tissues, DMPQ-12, by installing a C12-alkyl chain into the quinoline moiety. In fixed cells stained with DMPQ-12, filament mitochondria and folded cristae were observed with confocal and structural illumination microscopy, respectively. In titration test with three major phospholipids, DMPQ-12 exhibited a stronger binding force to mitochondria-exclusive cardiolipin, revealing its targeting mechanism. Moreover, mitochondrial morphological changes in the three lesion models were clearly visualized in fixed cells. Finally, by DMPQ-12, three kinds of mitochondria with different morphologies were observed in situ in fixed muscle tissues. This work breaks the conventional concept that organic fluorescent probes only stain mitochondria with normal membrane potentials and opens new avenues for comprehensive mitochondrial investigations in research and clinical settings.

Maintaining Toll signaling in Drosophila brain is required to sustain autophagy for dopamine neuron survival.

Macroautophagy/autophagy is a conserved process in eukaryotic cells to degrade and recycle damaged intracellular components. Higher level of autophagy in the brain has been observed, and autophagy dysfunction has an impact on neuronal health, but the molecular mechanism is unclear. In this study, we showed that overexpression of Toll-1 and Toll-7 receptors, as well as active Spätzle proteins in Drosophila S2 cells enhanced autophagy, and Toll-1/Toll-7 activated autophagy was dependent on Tube-Pelle-PP2A. Interestingly, Toll-1 but not Toll-7 mediated autophagy was dMyd88 dependent. Importantly, we observed that loss of functions in Toll-1 and Toll-7 receptors and PP2A activity in flies decreased autophagy level, resulting in the loss of dopamine (DA) neurons and reduced fly motion. Our results indicated that proper activation of Toll-1 and Toll-7 pathways and PP2A activity in the brain are necessary to sustain autophagy level for DA neuron survival.

Direct synthesis of phenanthrenyl triflates from 1-biphenylyl-2-diazo-2-aryl ketones and triflic anhydride.

A strategy for direct synthesis of phenanthrenyl triflates from 1-biphenylyl-2-diazo-2-aryl ketones and triflic anhydride is described. The reaction of 1-biphenylyl-2-diazo-2-aryl ketones with triflic anhydride proceeded smoothly in the presence of 2,6-di-tert-butylpyridine under mild conditions to produce phenanthrenyl triflates in high to excellent yields. The phenanthrenyl triflate products were demonstrated to be utilized as coupling partners in various coupling reactions. The proposed mechanism involves an intramolecular Friedel-Crafts reaction of a vinyl cation intermediate formed in situ.

Visible-Light-Driven Iron-Catalyzed Intermolecular Benzylic C(sp3)-H Amination with 1,2,3,4-Tetrazoles.

A visible-light-driven iron-catalyzed C(sp3)-H amination of diphenylmethane derivatives with 1,2,3,4-tetrazoles under mild conditions has been developed. The reaction proceeds with photosensitizer-free conditions and features satisfactory to good yields. Mechanistic studies revealed that the reaction proceeded via an iron-nitrene intermediate, and H atom abstraction was the rate-determining step. Computational studies showed that the denitrogenation of 1,2,3,4-tetrazole depends on the conversion of the sextet ground state of 1,2,3,4-tetrazole-bounding iron species to the quartet spin state under visible-light irradiation.

Immediate breast reconstruction with laparoscopically harvested omental flap: A retrospective analysis with a maximum 12-year follow-up.

PURPOSE: To evaluate the clinical efficacy of immediate breast reconstruction with free or pedicled laparoscopically harvested omental flaps (LHOFs). METHODS: Between March 2011 and 2021, 82 patients who underwent immediate breast reconstruction with free or pediculated omental flaps were enrolled. Breast total or partial mastectomy, laparoscopic greater omentum harvest, and breast reconstruction were carried out in an orderly manner. Postoperative operative results, cosmetic outcomes, and complications were investigated. RESULTS: Seventeen cases of free LHOF and 65 cases of pedicled LHOF were performed. Cosmetic results were mostly satisfactory (61% excellent, 35% good), with a soft breast that was natural in appearance. Satisfaction investigation showed that 96.2% of patients were satisfied with the reconstructed breast. Uneventful follow-up showed no abdominal complications at the donor site, and the surface skin displayed no swelling. No major complications were found, except for three cases of necrosis. One patient developed slight hematoma. Two patients were found to have local recurrence, and one had distant metastasis. Twenty-four patients accepted radiotherapy, but no size reduction was noted after radiotherapy. We followed the patients to determine their survival status. All patients were alive, except for 1 in the free LHOF group who died 31.2 months after surgery. CONCLUSION: Immediate breast reconstruction with LHOF provides a soft reconstructed breast with relatively little donor-site deformity and is useful for breast tumor-specific immediate reconstruction.

Identification and functional analysis of CG3526 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2 H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.

Expression profiles of lncRNAs, miRNAs, and mRNAs and interaction analysis indicate their potential involvement during testicular fusion in Spodoptera litura.

Testicular fusion of Spodoptera litura occures during metamorphosis, which benefits sperms development. Previous research identified involvement of ECM-integrin interaction pathways, MMPs in testicular fusion, but the regulatory mechanism remains unclear. RNA-seq was performed to analyze long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in testes, aiming to uncover potential regulatory mechanisms of testicular fusion. 2150 lncRNAs, 2742 targeted mRNAs, and 347 miRNAs were identified in testes at three different developmental stages. Up-regulated DElncRNAs and DEmRNAs, as well as down-regulated DEmiRNAs, were observed during testicular fusion, while the opposite expression pattern was observed after fusion. Enrichment analysis of DEmRNAs revealed that cAMP signal pathway, ECM remodeling enzymes, ECM-integrin interaction pathways, and cell adhesion molecules were potentially associated with testicular fusion. The identified DElncRNA-DEmiRNA-DEmRNA regulatory network related to cAMP signal pathway, ECM remodeling enzymes suggests their roles during testicular fusion. Our research will provide new targets for studying the mechanism of testicular fusion.

Visible-Light-Driven Iron-Catalyzed Decarboxylative C-N Coupling Reaction of Alkyl Carboxylic Acids with NaNO2.

An efficient visible-light-driven iron-catalyzed decarboxylative C-N coupling reaction of alkyl carboxylic acids with NaNO2 under mild conditions was developed. The reaction proceeds under photosensitizer-free conditions and features good to excellent yields, broad functional group tolerance, and an easy operation procedure. Preliminary mechanistic investigations showed that visible-light-driven iron catalysis not only achieved oxidative decarboxylation of alkyl carboxylic acids to alkyl radicals but also promoted the reduction of NO2- to NO, thus leading to the C-N radical coupling reaction.

Visible-light-induced iron-catalyzed reduction of nitroarenes to anilines.

An efficient visible-light-induced iron-catalyzed reduction of nitroarenes to anilines by using N-ethylmorpholine (NEM) as a reductant under mild conditions has been developed. The reaction proceeds with photosensitizer-free conditions and features good to excellent yields and broad functional group tolerance. Preliminary mechanistic investigations showed that this reaction was conducted via ligand-to-metal (NEM to Fe3+) charge transfer and nitro triplet biradical-induced hydrogen atom transfer processes.

RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation.

BACKGROUND AND AIMS: The pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. METHODS: For in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. RESULTS: PDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αß (PDGFRαß) rather than PDGFRαα or PDGFRßß in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. CONCLUSIONS: The results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

The Impact of Bamboo Consumption on the Spread of Antibiotic Resistance Genes in Giant Pandas.

The spread of antibiotic resistance genes (ARGs) in the environment exacerbates the contamination of these genes; therefore, the role plants play in the transmission of resistance genes in the food chain requires further research. Giant pandas consume different bamboo parts at different times, which provides the possibility of investigating how a single food source can affect the variation in the spread of ARGs. In this study, metagenomic analysis and the Comprehensive Antibiotic Resistance Database (CARD) database were used to annotate ARGs and the differences in gut microbiota ARGs during the consumption of bamboo shoots, leaves, and culms by captive giant pandas. These ARGs were then compared to investigate the impact of bamboo part consumption on the spread of ARGs. The results showed that the number of ARGs in the gut microbiota of the subjects was highest during the consumption of bamboo leaves, while the variety of ARGs was highest during the consumption of shoots. Escherichia coli, which poses a higher risk of ARG dissemination, was significantly higher in the leaf group, while Klebsiella, Enterobacter, and Raoultella were significantly higher in the shoot group. The ARG risk brought by bamboo shoots and leaves may originate from soil and environmental pollution. It is recommended to handle the feces of giant pandas properly and regularly monitor the antimicrobial and virulence genes in their gut microbiota to mitigate the threat of antibiotic resistance.

Visible-Light-Driven α-Substituted Amines Enabled by In Situ Formation of Amine Substrate Aggregates.

A visible-light-driven, photocatalyst-free, air-promoted, α-substituted reaction of amines with varying nucleophiles is described. The amine substrate aggregates formed in situ through physical π-π stacking by H2O regulation in organic solvent can absorb visible light and then generate iminium ion intermediates, which undergo nucleophilic substitution reactions with varying nucleophiles to afford α-substituted amines. This reaction features catalyst-free, good functional group tolerance, simple operation procedure, and green reaction conditions.

One-pot synthesis of multisubstituted propenylbenzenes from benzyl chlorides through relay catalysis of palladium.

A strategy for the synthesis of multisubstituted propenylbenzenes using benzyl chlorides as starting materials is described. The palladium-catalyzed allylative dearomatization and the subsequent Wagner-Meerwein rearrangement as well as the olefin isomerization proceeded smoothly under mild conditions to produce propenylation products in good yields with high regioselectivity. Control experiments and cyclic voltammetry analysis suggest that Bu3SnCl, a by-product generated in the first step of allylative dearomatization, plays an essential role in the third step of olefin isomerization in the presence of a Brønsted acid.

Identification of 5 hub genes for diagnosis of coronary artery disease.

Background: Coronary artery disease (CAD) is a main cause leading to increasing mortality of cardiovascular disease (CVD) worldwide. We aimed to discover marker genes and develop a diagnostic model for CAD. Methods: CAD-related target genes were searched from DisGeNET. Count expression data and clinical information were screened from the GSE202626 dataset. edgeR package identified differentially expressed genes (DEGs). Using online STRING tool and Cytoscape, protein-protein reactions (PPI) were predicted. WebGestaltR package was employed to functional enrichment analysis. We used Metascape to conduct module-based network analysis. VarElect algorithm provided genes-phenotype correlation analysis. Immune infiltration was assessed by ESTIMATE package and ssGSEA analysis. mRNAsi was determined by one class logistic regression (OCLR). A diagnostic model was constructed by SVM algorithm. Results: 162 target genes were screened by intersection 1,714 DEGs and 1,708 CAD related target genes. 137 target genes of the 162 target genes were obtained using PPI analysis, in which those targets were enriched in inflammatory cytokine pathways, such as chemokine signaling pathway, and IL-17 signaling pathway. From the above 137 target genes, four functional modules (MCODE1-4) were extracted. From the 162 potential targets, CAD phenotype were directly and indirectly associated with 161 genes and 22 genes, respectively. Finally, 5 hub genes (CCL2, PTGS2, NLRP3, VEGFA, LTA) were screened by intersections with the top 20, directly and indirectly, and genes in MCODE1. PTGS2, NLRP3 and VEGFA were positively, while LTA was negatively correlated with immune cells scores. PTGS2, NLRP3 and VEGFA were negatively, while LTA was positively correlated with mRNAsi. A diagnostic model was successfully established, evidenced by 92.59% sensitivity and AUC was 0.9230 in the GSE202625 dataset and 94.11% sensitivity and AUC was 0.9706 in GSE120774 dataset. Conclusion: In this work, we identified 5 hub genes, which may be associated with CAD development.

Eurycomanone (EN) Activates Transcription Factor FoxO by Inhibiting the Insulin Signaling Pathway to Suppress the Development of Spodoptera frugiperda.

The insulin-like signaling (IIS) pathway is essential for insect growth and development. In this study, we showed that eurycomanone (EN) is an active compound with growth inhibitory activity against Spodoptera frugiperda larvae. Experiments in cells and RNA-seq analysis in the midgut showed that EN targeted the IIS pathway in S. frugiperda to activate the transcription factor SfFoxO (S. frugiperda forkhead boxO) to regulate mRNA levels associated with nutrient catabolism. Additionally, mass spectrometry imaging revealed that EN was distributed in the larval gut and enriched in the inner membrane of the gut. Immunofluorescence, western blotting, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed that EN induced program cell death (PCD) in the larvae midgut. Thus, EN targeted the insulin receptor to inhibit the IIS signaling pathway, exerting inhibitory activity on the growth and development of S. frugiperda larvae. Our results suggest that EN has great potential as a botanical pesticide, and the IIS signaling pathway may be an effective target for botanical pesticides.

Visible-Light-Promoted Mn-Catalyzed C(sp3)-H Amidation with Dioxazolones.

A visible-light-driven Mn-catalyzed C(sp3)-H amidation of diphenylmethane derivatives with dioxazolones was described. These reactions occur with an external photosensitizer-free process and feature satisfactory to good yields (up to 81%) under mild conditions. Mechanistic investigations revealed that the reaction proceeded via a Mn-acyl nitrene intermediate and that H-atom abstraction was the rate-determining step. Computational studies showed that the decarboxylation of dioxazolone depends on the conversion of ground sextet state dioxazolone-bounding Mn species to quartet spin state via visible-light irradiation.

A fluorescent probe for detecting mitochondrial viscosity and its application in distinguishing human breast cancer cells from normal ones.

Mitochondrial viscosity is closely associated with intracellular physiological activities yet their abnormality will result in various diseases. In particular, viscosity in cancer cells is different from that in normal cells, which is thought to be an indicator for cancer diagnosis. However, there were few fluorescent probes able to distinguish homologous cancer and normal cells by detecting mitochondrial viscosity. Herein, we designed a viscosity-sensitive fluorescent probe (named NP) based on the twisting intramolecular charge transfer (TICT) mechanism. NP exhibited exquisite sensitivity to viscosity and selectivity to mitochondria and excellent photophysical properties, such as large Stokes shift and high molar extinction coefficient, which enables wash-free, high-fidelity and fast imaging mitochondria. Moreover, it was capable of detecting mitochondrial viscosity in living cells and tissue, as well as monitoring apoptosis process. Significantly, considering numerous breast cancer cases in every country of the world, NP was successfully applied to distinguish human breast cancer cells (MCF-7) from normal cells (MCF-10A) by difference in fluorescence intensity originated from abnormality in mitochondrial viscosity. All the results indicated that NP could serve as a robust tool for effectively detecting mitochondrial viscosity changes in-situ.