Fusobacterium nucleatum exacerbates the progression of pulpitis by regulating the STING-dependent pathway.

Bacterial infection is the main cause of pulpitis. However, whether a dominant bacteria can promote the progression of pulpitis and its underlying mechanism remains unclear. We provided a comprehensive assessment of the microbiota alteration in pulpitis using 16S rRNA sequencing. Fusobacterium nucleatum was the most enriched in pulpitis and played a pathogenic role accelerating pulpitis progression in rat pulpitis model. After odontoblast-like cells cocultured with F. nucleatum, the stimulator of interferon genes (STING) pathway and autophagy were activation. There was a float of STING expression during F. nucleatum stimulation. STING was degraded by autophagy at the early stage. At the late stage, F. nucleatum stimulated mitochondrial Reactive Oxygen Species (ROS) production, mitochondrial dysfunction and then mtDNA escape into cytosol. mtDNA, which escaped into cytosol, caused more cytosolic mtDNA binds to cyclic GMP-AMP synthase (cGAS). The release of IFN-ß was dramatically reduced when mtDNA-cGAS-STING pathway inhibited. STING-/- mice showed milder periapical bone loss and lower serum IFN-ß levels compared with wildtype mice after 28 days F. nucleatum-infected pulpitis model establishment. Our data demonstrated that F. nucleatum exacerbated the progression of pulpitis, which was mediated by the STING-dependent pathway.

STING inhibition alleviates bone resorption in apical periodontitis.

AIM: The goal of this study was to investigate the potential effects of an immunotherapeutic drug targeting STING to suppress the overreactive innate immune response and relieve the bone defect in apical periodontitis. METHODOLOGY: We established an apical periodontitis mouse model in Sting-/- and WT mice in vivo. The progression of apical periodontitis was analysed by micro-CT analysis and H&E staining. The expression level and localization of STING in F4/80+ cells were identified by IHC and immunofluorescence staining. RANKL in periapical tissues was tested by IHC staining. TRAP staining was used to detect osteoclasts. To clarify the effect of STING inhibitor C-176 as an immunotherapeutic drug, mice with apical periodontitis were treated with C-176 and the bone loss was identified by H&E, TRAP, RANKL staining and micro-CT. Bone marrow-derived macrophages (BMMs) were isolated from Sting-/- and WT mice and induced to osteoclasts in a lipopolysaccharide (LPS)-induced inflammatory environment in vitro. Moreover, WT BMMs were treated with C-176 to determine the effect on osteoclast differentiation by TRAP staining. The expression levels of osteoclast-related genes were tested using qRT-PCR. RESULTS: Compared to WT mice, the bone resorption and inflammatory cell infiltration were reduced in exposed Sting-/- mice. In the exposed WT group, STING was activated mainly in F4/80+ macrophages. Histological staining revealed the less osteoclasts and lower expression of osteoclast-related factor RANKL in Sting-/- mice. The treatment of the STING inhibitor C-176 in an apical periodontitis mice model alleviated inflammation progression and bone loss, similar to the effect observed in Sting-/- mice. Expression of RANKL and osteoclast number in periapical tissues were also decreased after C-176 administration. In vitro, TRAP staining showed fewer positive cells and qRT-PCR reflected decreased expression of osteoclastic marker, Src and Acp5 were detected during osteoclastic differentiation in Sting-/- and C-176 treated BMMs. CONCLUSIONS: STING was activated and was proven to be a positive factor in bone loss and osteoclastogenesis in apical periodontitis. The STING inhibitor C-176 administration could alleviate the bone loss via modulating local immune response, which provided immunotherapy to the treatment of apical periodontitis.

Extensional Stress-Induced Ductility of Poly(l-lactide) Films: Role of the Entangled Network in Amorphous Regions.

The relationship between the density of the entangled amorphous network and the ductility of oriented poly(l-lactide) (PLLA) films is explored based on the preferential hydrolysis of the amorphous regions in phosphate buffer solution (PBS). PLLA films with a balance of ductility and stiffness have been prepared by the "casting-annealing stretching" based on mechanical rejuvenation, and the structural evolution and mechanical properties at different hydrolysis durations have been identified. Various stages are found during the transition of ductility to brittleness for hydrolyzed PLLA films. First, the elongation at break for hydrolyzed PLLA films remains unchanged in the first stage of hydrolysis and then gradually decreases. Eventually, the films turn to be brittle in the third stage. The strain-hardening modulus (GR) of the hydrolyzed films is utilized to reflect the density of the entangled amorphous network, and a gradual decrease of GR with hydrolysis time indicates the decisive role of the amorphous entanglement network in the mechanical rejuvenation-induced ductility of PLLA. The quantitative relationship between the entangled amorphous network and the stress-induced ductility of PLLA films is revealed. The dependence of deformation behavior on entangled amorphous network density is closely correlated to activated primary structure during deformation. The intact chain network plays a crucial role in sufficiently activating the primary structure to yield and disentanglement during the subsequent necking. These findings could advance the understanding of the PLLA's ductility induced by mechanical rejuvenation and offer guidance for awakening the intrinsic toughness of PLLA.

Nanoselenium transformation and inhibition of cadmium accumulation by regulating the lignin biosynthetic pathway and plant hormone signal transduction in pepper plants.

Selenium (Se) can promote the growth and resistance of agricultural crops as fertilizers, while the role of nano-selenium (nano-Se) against Cd remains unclear in pepper plants (Capsicum annuum L.). Biofortification with nano-Se observably restored Cd stress by decreasing the level of Cd in plant tissues and boosting the accumulation in biomass. The Se compounds transformed by nano-Se were primarily in the form of SeMet and MeSeCys in pepper tissues. Differential metabolites and the genes of plant signal transduction and lignin biosynthesis were measured by employing transcriptomics and determining target metabolites. The number of lignin-related genes (PAL, CAD, 4CL, and COMT) and contents of metabolites (sinapyl alcohol, phenylalanine, p-coumaryl alcohol, caffeyl alcohol, and coniferaldehyde) were remarkably enhanced by treatment with Cd1Se0.2, thus, maintaining the integrity of cell walls in the roots. It also enhanced signal transduction by plant hormones and responsive resistance by inducing the biosynthesis of genes (BZR1, LOX3, and NCDE1) and metabolites (brassinolide, abscisic acid, and jasmonic acid) in the roots and leaves. In general, this study can enable a better understanding of the protective mechanism of nano-Se in improving the capacity of plants to resist environmental stress.

Genetic spectrum of MCM3AP and its relationship with phenotype of Charcot-Marie-Tooth disease.

Mutations in MCM3AP have recently been reported to cause autosomal recessive Charcot-Marie-Tooth disease (CMT). However, only nine CMT families with MCM3AP mutations have been reported and genotype-phenotype correlation remains unclear. This study aimed to investigate the genetic spectrum of MCM3AP and its relationship with phenotype of CMT. Whole-exome sequencing (WES) was performed in the family and variants were validated by Sanger sequencing. Reverse transcription-PCR (RT-PCR) were performed in splicing analysis. We reported a novel splicing variant (c.5634-1G>T) and a known missense variant (c.2633G>A, p.Arg878His). Functional studies showed that c.5634-1G>T led to splicing defect and aberrant transcript eliminated by nonsense-mediated mRNA decay. The symptom of the patient was less severe and slowly progressed with axonal peripheral neuropathy compared to the reported CMT patients. Genotype-phenotype correlation analysis indicated that affected individuals with null mutations presented with delayed independent walking. The percentage of intellectual disability and loss of ambulation in the null group tended to be greater, although this failed to reach statistical significance. Our findings expand the genetic spectrum of MCM3AP and suggest that genotype-phenotype correlation would help genetic counseling of MCM3AP in CMT patients.

Genetic spectrum and clinical profiles in a southeast Chinese cohort of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of inherited sensorimotor neuropathies. To clarify the genetic spectrum and clinical profiles in Chinese CMT patients, we enrolled 150 unrelated CMT patients from southeast China. We performed multiplex ligation-dependent probe amplification (MLPA) testing in all patients and next-generation sequencing (NGS) among those patients without PMP22 rearrangements. We identified PMP22 duplications in 40 patients and deletions in 12 patients. In addition, we found 19 novel variants and 36 known mutations in 57 patients. Among these 55 variants, 45 pathogenic or likely pathogenic variants were identified in 48 cases, and 10 variants with uncertain significance were identified in 9 cases. Therefore, we obtained a genetic diagnosis in 63.8% (88/138) of CMT patients and 66.7% (100/150) of all included patients. PMP22, GJB1, and MFN2 are the most common causative genes in CMT1 (demyelinated form), intermediate CMT, and CMT2 (axonal form), respectively. In this study, we identified a higher proportion of intermediate CMT, a relatively high frequency of NDRG1 mutations and clinical features of later onset age in CMT1A patients. Our results broaden the genetic and clinical spectrum of CMT patients, which can help optimize the genetic and clinical diagnosis.

Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening.

Hand, foot, and mouth disease (HFMD) is a common pediatric illness mainly caused by enteroviruses, which are important human pathogens. Currently, there are no available antiviral agents for the therapy of enterovirus infection. In this study, an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed. Using this screening system, we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, exhibits potential inhibitory effects against various enteroviruses that cause HFMD, such as EV-A71, CV-A10, CV-B3 and CV-A16. Further investigations revealed that FAN targets the early stage of the enterovirus life cycle. Through the selection of FAN-resistant EV-A71 viruses, we demonstrated that the VP1 protein could be a potential target of FAN, as two mutations in VP1 (E145G and V258I) resulted in viral resistance to FAN. Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.

Biallelic SORD pathogenic variants cause Chinese patients with distal hereditary motor neuropathy.

Sorbitol dehydrogenase gene (SORD) has been identified as a novel causative gene of recessive forms of hereditary neuropathy, including Charcot-Marie-Tooth disease type 2 and distal hereditary motor neuropathy (dHMN). Our findings reveal two novel variants (c.404 A > G and c.908 + 1 G > C) and one known variant (c.757delG) within SORD in four Chinese dHMN families. Ex vivo cDNA polymerase chain reaction confirmed that c.908 + 1 G > C variant was associated with impaired splicing of the SORD transcript. In vitro cell functional studies showed that c.404 A > G variant resulted in aggregate formation of SORD and low protein solubility, confirming the pathogenicity of SORD variants. We have provided more evidence to establish SORD as a causative gene for dHMN.

Identification and functional characterization of novel GDAP1 variants in Chinese patients with Charcot-Marie-Tooth disease.

OBJECTIVE: To identify and characterize the pathogenicity of novel variants in Chinese patients with Charcot-Marie-Tooth disease. METHODS: Multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES) were performed in 30 unrelated CMT patients. Minigene assay was used to verify the effect of a novel splicing variant (c.694+1G>A) on pre-mRNA. Primary fibroblast cell lines were established from skin biopsies to characterize the biological effects of the novel variants p.L26R and p.S169fs. The mitochondrial structure was observed by an electron microscope. The expression level of protein was analyzed by Western Blotting. Mitochondrial dynamics and mitochondrial membrane potential (MMP, Δψm) were analyzed via immunofluorescence study. Mitochondrial ATP levels were analyzed via bioluminescence assay. The rate of oxygen consumption was measured with a Seahorse Bioscience XF-96 extracellular flux analyzer. RESULTS: We identified 10 pathogenic variants in three known CMT related genes, including three novel variants (p.L26R, p.S169fs, c.694+1G>A) and one known pathogenic variant (p.R120W) in GDAP1. Further, we described the clinical features of patients carrying pathogenic variants in GDAP1 and found that almost all Chinese CMT patients with GDAP1 variants present axonal type. The effect of c.694+1G>A on pre-mRNA was verified via minigene splice assay. Cellular biological effects showed ultrastructure damage of mitochondrial, reduced protein levels, different patterns of mitochondrial dynamics, decreased mitochondrial membrane potential (Δψm), ATP content, and defects in respiratory capacity in the patient carrying p.L26R and p.S169fs in GDAP1. INTERPRETATION: Our results broaden the genetic spectrum of GDAP1 and provided functional evidence for mitochondrial pathways in the pathogenesis of GDAP1 variants.