Cecal necroptosis triggers lethal cardiac dysfunction in TNF-induced severe SIRS.

Tumor necrosis factor (TNF) induces systemic inflammatory response syndrome (SIRS), and severe SIRS can serve as a model for studying animal death caused by organ failure. Through strategic cecectomy, we demonstrate that necroptosis in the cecum initiates the death process in TNF-treated mice, but it is not the direct cause of death. Instead, we show that it is the cardiac dysfunction downstream of cecum damage that ultimately leads to the death of TNF-treated mice. By in vivo and ex vivo physiological analyses, we reveal that TNF and the damage-associated molecular patterns (DAMPs) released from necroptotic cecal cells jointly target cardiac endothelial cells, triggering caspase-8 activation and subsequent cardiac endothelial damage. Cardiac endothelial damage is a primary cause of the deterioration of diastolic function in the heart of TNF-treated mice. Our research provides insights into the pathophysiological process of TNF-induced lethality.

Cdc42 deletion yielded enamel defects by disrupting mitochondria and producing reactive oxygen species in dental epithelium.

Developmental defects of enamel are common due to genetic and environmental factors before and after birth. Cdc42, a Rho family small GTPase, regulates prenatal tooth development in mice. However, its role in postnatal tooth development, especially enamel formation, remains elusive. Here, we investigated Cdc42 functions in mouse enamel development and tooth repair after birth. Cdc42 showed highly dynamic temporospatial patterns in the developing incisors, with robust expression in ameloblast and odontoblast layers. Strikingly, epithelium-specific Cdc42 deletion resulted in enamel defects in incisors. Ameloblast differentiation was inhibited, and hypomineralization of enamel was observed upon epithelial Cdc42 deletion. Proteomic analysis showed that abnormal mitochondrial components, phosphotransferase activity, and ion channel regulator activity occurred in the Cdc42 mutant dental epithelium. Reactive oxygen species accumulation was detected in the mutant mice, suggesting that abnormal oxidative stress occurred after Cdc42 depletion. Moreover, Cdc42 mutant mice showed delayed tooth repair and generated less calcified enamel. Mitochondrial dysfunction and abnormal oxygen consumption were evidenced by reduced Apool and Timm8a1 expression, increased Atp5j2 levels, and reactive oxygen species overproduction in the mutant repair epithelium. Epithelium-specific Cdc42 deletion attenuated ERK1/2 signaling in the labial cervical loop. Aberrant Sox2 expression in the mutant labial cervical loop after clipping might lead to delayed tooth repair. These findings suggested that mitochondrial dysfunction, up-regulated oxidative stress, and abnormal ion channel activity may be among multiple factors responsible for the observed enamel defects in Cdc42 mutant incisors. Overall, Cdc42 exerts multidimensional and pivotal roles in enamel development and is particularly required for ameloblast differentiation and enamel matrix formation.

Mechanical force-activated CD109 on periodontal ligament stem cells governs osteogenesis and osteoclast to promote alveolar bone remodeling.

Mechanical force-mediated bone remodeling is crucial for various physiological and pathological processes involving multiple factors, including stem cells and the immune response. However, it remains unclear how stem cells respond to mechanical stimuli to modulate the immune microenvironment and subsequent bone remodeling. Here, we found that mechanical force induced increased expression of CD109 on periodontal ligament stem cells (PDLSCs) in vitro and in periodontal tissues from the force-induced tooth movement rat model in vivo, accompanied by activated alveolar bone remodeling. Under mechanical force stimulation, CD109 suppressed the osteogenesis capacity of PDLSCs through the JAK/STAT3 signaling pathway, whereas it promoted PDLSC-induced osteoclast formation and M1 macrophage polarization through paracrine. Moreover, inhibition of CD109 in vivo by lentivirus-shRNA injection increased the osteogenic activity and bone density in periodontal tissues. On the contrary, it led to decreased osteoclast numbers and pro-inflammatory factor secretion in periodontal tissues and reduced tooth movement. Mechanistically, mechanical force-enhanced CD109 expression via the repression of miR-340-5p. Our findings uncover a CD109-mediated mechanical force response machinery on PDLSCs, which contributes to regulating the immune microenvironment and alveolar bone remodeling during tooth movement.

Phosphorylation of caspase-8 by RSKs via organ-constrained effects controls the sensitivity to TNF-induced death.

Caspase-8 (Casp8) serves as an initiator of apoptosis or a suppressor of necroptosis in context-dependent manner. Members of the p90 RSK family can phosphorylate caspase-8 at threonine-265 (T265), which can inactivate caspase-8 for bypassing caspase-8-mediated blockade of necroptosis and can also decrease caspase-8 level by promoting its degradation. Mutating T265 in caspase-8 to alanine (A) in mice blocked TNF-induced necroptotic cecum damage but resulted in unexpectedly massive injury in the small intestine. Here, we show RSK1, RSK2, and RSK3 redundantly function in caspase-8 phosphorylation, and the duodenum is the most severely affected part of the small intestine when T265 phosphorylation of caspase-8 was prevented. Eliminating caspase-8 phosphorylation resulted in a duodenum-specific increase in basal caspase-8 protein level, which shall be responsible for the increased sensitivity to TNF-induced damage. Apoptosis of intestinal epithelial cells (IECs) was predominant in the duodenum of TNF-treated Rsk1-/-Rsk2-/-Rsk3-/- and Casp8T265A/T265A mice, though necroptosis was also observed. The heightened duodenal injury amplified systemic inflammatory responses, as evidenced by the contribution of hematopoietic cells to the sensitization of TNF-induced animal death. Further analysis revealed that hematopoietic and non-hematopoietic cells contributed differentially to cytokine production in response to the increased cell death. Collectively, RSKs emerges as a previously overlooked regulator that, via tissue/organ-constrained inactivating caspase-8 and/or downregulating caspase-8 protein level, controls the sensitivity to TNF-induced organ injury and animal death.

Clinical Features, Diagnosis, and Treatment of Congenital and Neonatal Tuberculosis: A Retrospective Study.

INTRODUCTION: Limited studies have explored the clinical features, treatment, and prognosis of neonatal tuberculosis (TB). Here, we attempted to delineate the clinical characteristics of neonatal TB, which may help clinicians further understand this disease. METHODS: A retrospective analysis of neonates diagnosed with congenital and/or neonatal TB disease from January 2010 to December 2020 was performed. Information on the demographic and epidemiological features, clinical symptoms, laboratory and imaging examinations, therapeutic regimens, and outcomes was collected. Kaplan-Meier analysis was used to present the time to disease onset, time to diagnosis, etc. Results: Forty-eight cases of neonatal TB were classified into congenital (n = 33) and postnatal (n = 15). The median time to disease onset in postnatal group was significantly longer than that in congenital group. Positive results for gastric fluid acid-fast bacilli, TB culture, Xpert MTB/RIF, interferon-γ release assay (IGRA), and tuberculin skin test were detected in 26/48 (54.2%), 14/34 (41.2%), 11/18 (61.1%), 19/29 (65.5%), and 8/24 (33.3%) patients, respectively. For lymphadenopathy, computed tomography (CT) scans showed a higher detection rate than did X-ray (80.0% vs. 0). Of the 48 infants, 44/48 (91.7%) received anti-TB therapy, and 33/44 (75%) were clinically improved or cured after 22.1 months (interquartile range: 12.4-27.7) of follow-up. Drug-induced liver injury occurred in 14/44 (31.8%) patients. DISCUSSION/CONCLUSION: IGRA and Xpert MTB/RIF showed good positive rate in neonatal TB infection/disease. In cases where TB is presumed but etiological evidence is lacking, low-dose CT could be considered. Prompt treatment under careful surveillance is important for preventing mortality and avoiding severe adverse effects.

Anthrax lethal toxin and tumor necrosis factor-α synergize on intestinal epithelia to induce mouse death.

Bacillus anthracis lethal toxin (LT) is a determinant of lethal anthrax. Its function in myeloid cells is required for bacterial dissemination, and LT itself can directly trigger dysfunction of the cardiovascular system. The interplay between LT and the host responses is important in the pathogenesis, but our knowledge on this interplay remains limited. Tumor necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine induced by bacterial infections. Since LT accumulates and cytokines, predominantly TNF, amass during B. anthracis infection, co-treatment of TNF + LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts. Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells (IECs) rather than that of hematopoietic cells led to LT + TNF-induced lethality. Inhibition of p38α mitogen-activated protein kinase (MAPK) signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs, leading to intestinal damage and mouse death. Consistently, p38α inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells. As intestinal damage is one of the leading causes of lethality in anthrax patients, the IEC damage caused by LT + TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Detection of necroptosis by phospho-MLKL immunohistochemical labeling.

TNF-induced necroptosis is involved in many physiological and pathological processes. Phospho-MLKL is a hallmark of necroptosis. Cecum is a sensitive organ with extensive necroptosis responses to TNF in vivo. Here, taking advantage of commercially available mouse TNF and easily accessible reagents and materials, we systematically provide a detailed and highly versatile protocol of detecting necroptosis signaling in mouse cecum by immunohistochemical labeling, which can also be used in other tissues or antibodies in immunohistochemical staining. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020) and Chen et al. (2015).

FOXQ1 promotes the osteogenic differentiation of bone mesenchymal stem cells via Wnt/ß-catenin signaling by binding with ANXA2.

BACKGROUND: This study investigated the role of Forkhead box Q1 (FOXQ1) in the osteogenic differentiation of bone mesenchymal stem cells. METHODS: Mouse bone mesenchymal stem cells (mBMSCs) were transfected with lentivirus to generate Foxq1-overexpressing mBMSCs, Foxq1-suppressed mBMSCs, and mBMSC controls. The activity of osteogenic differentiation was evaluated with alizarin red staining, alkaline phosphatase activity assay, and RT-qPCR. Wnt/ß-catenin signaling activities were compared among groups by TOPFlash/FOPFlash assay, immunofluorescence staining, and western blot assay of beta-catenin (CTNNB1). Coimmunoprecipitation mass spectrometry was also carried out to identify proteins binding with FOXQ1. RESULTS: Our data showed that FOXQ1 expression was positively correlated with the osteogenic differentiation of the mBMSCs. FOXQ1 also promoted the nuclear translocation of CTNNB1 in the mBMSCs, enhancing Wnt/ß-catenin signaling, which was also shown to be essential for the osteogenic differentiation-promoting effect of FOXQ1 in the mBMSCs. Annexin A2 (ANXA2) was bound with FOXQ1, and its depletion reversed the promoting effect of FOXQ1 on Wnt/ß-catenin signaling. CONCLUSION: These results showed that FOXQ1 binds with ANXA2, promoting Wnt/ß-catenin signaling in bone mesenchymal stem cells, which subsequently promotes osteogenic differentiation.

A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

Osteoimmunomodulatory effects of biomaterial modification strategies on macrophage polarization and bone regeneration.

Biomaterials as bone substitutes are always considered as foreign bodies that can trigger host immune responses. Traditional designing principles have been always aimed at minimizing the immune reactions by fabricating inert biomaterials. However, clinical evidence revealed that those methods still have limitations and many of which were only feasible in the laboratory. Currently, osteoimmunology, the very pioneering concept is drawing more and more attention-it does not simply regard the immune response as an obstacle during bone healing but emphasizes the intimate relationship of the immune and skeletal system, which includes diverse cells, cytokines, and signaling pathways. Properties of biomaterials like topography, wettability, surface charge, the release of cytokines, mediators, ions and other bioactive molecules can impose effects on immune responses to interfere with the skeletal system. Based on the bone formation mechanisms, the designing methods of the biomaterials change from immune evasive to immune reprogramming. Here, we discuss the osteoimmunomodulatory effects of the new modification strategies-adjusting properties of bone biomaterials to induce a favorable osteoimmune environment. Such strategies showed potential to benefit the development of bone materials and lay a solid foundation for the future clinical application.

ZBP1 mediates interferon-induced necroptosis.

Interferons (IFNs) play an important role in immunomodulatory and antiviral functions. IFN-induced necroptosis has been reported in cells deficient in receptor-interacting protein kinase 1 (RIPK1), Fas-associated protein with death domain (FADD), or caspase-8, but the mechanism is largely unknown. Here, we report that the DNA-dependent activator of IFN regulatory factors (ZBP1, also known as DAI) is required for both type I (ß) and type II (γ) IFN-induced necroptosis. We show that L929 fibroblast cells became susceptible to IFN-induced necroptosis when RIPK1, FADD, or Caspase-8 was genetically deleted, confirming the antinecroptotic role of these proteins in IFN signaling. We found that the pronecroptotic signal from IFN stimulation depends on new protein synthesis and identified ZBP1, an IFN-stimulated gene (ISG) product, as the de novo synthesized protein that triggers necroptosis in IFN-stimulated cells. The N-terminal domain (ND) of ZBP1 is important for ZBP1-ZBP1 homointeraction, and its RHIM domain in the C-terminal region interacts with RIPK3 to initiate RIPK3-dependent necroptosis. The antinecroptotic function of RIPK1, FADD, and caspase-8 in IFN-treated cells is most likely executed by caspase-8-mediated cleavage of RIPK3, since the inhibitory effect on necroptosis was eliminated when the caspase-8 cleavage site in RIPK3 was mutated. ZBP1-mediated necroptosis in IFN-treated cells is likely physiologically relevant, as ZBP1 KO mice were significantly protected against acute systemic inflammatory response syndrome (SIRS) induced by TNF + IFN-γ.

Simultaneous Discrimination of Single-Base Mismatch and Full Match Using a Label-Free Single-Molecule Strategy.

Identification of single-base mismatches has found wide applications in disease diagnosis, pharmacogenetics, and population genetics. However, there is still a great challenge in the simultaneous discrimination of single-base mismatch and full match. Combined with a nanopore electrochemical sensor, a shared-stem structure of molecular beacon was designed that did not need the labeling, but achieved an enhanced signal-to-background ratio of ∼104, high thermodynamic stability to bind with target sequences, and a fast hybridization rate. Fully matched and single-base mismatched sequences were simultaneously discriminated at the single-molecule level, which is expected to have potential applications ranging from the quick detection, early clinical diagnostics to point-of-care research.

Insight into How Telomeric G-Quadruplexes Enhance the Peroxidase Activity of Cellular Hemin.

The toxic oxidative damage of G-quadruplexes (G4), linked to neurodegenerative diseases, may arise from their ability to bind and oxidatively activate cellular hemin. However, there have been no precise studies on how telomeric G4 enhances the low intrinsic peroxidase activity of hemin. Herein, a label-free and nanopore-based strategy was developed to explore the enhancement mechanism of peroxidase activity of hemin induced by telomeric G4 (d(TTAGGG)n ). The nanopore-based strategy demonstrated that there were simultaneously two different binding modes of telomere G4 to hemin. At the single-molecule level, it was found that the hybrid structural telomeric G4 directly binds to hemin (the affinity constant (Ka )≈106 m-1 ) to form a tight complex, and some of them underwent a topological change to a parallel structure with an enhancement of Ka to approximately 107 m-1 . Through detailed analysis of the topology and peroxidase activity and molecular modeling investigations, the parallel telomere G4/hemin DNAzyme structure was proven to be preferable for high peroxidase activity. Upon strong π-π stacking, the parallel structural telomere G4 supplied a key axial ligand to the hemin iron, which accelerated the intermediate compound formation with H2 O2 in the catalytic cycle. Our studies developed a label-free and single-molecule strategy to fundamentally understand the catalytic activity and mechanism of telomeric DNAzyme, which provides some support for utilizing the toxic, oxidative-damage property in cellular oxidative disease and anticancer therapeutics.

DNA methylation profile is associated with the osteogenic potential of three distinct human odontogenic stem cells.

Among the various sources of human autologous stem cells, stem cells isolated from dental tissues exhibit excellent properties in tissue engineering and regenerative medicine. However, the distinct potential of these odontogenic cell lines remains unclear. In this study, we analyzed DNA methylation patterns to determine whether specific differences existed among three different odontogenic cell types. Using the HumanMethylation450 Beadchip, the whole genomes of human dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) were compared. Then, the osteogenic potential of these cells was evaluated both in vitro and in vivo, and the methylation levels of certain genes related to bone formation differed among the three cell lines. P values less than 0.05 were considered to indicate statistical significance. The three cell types showed highly similar DNA methylation patterns, although specific differences were identified. Gene ontology analysis revealed that one of the most significantly different gene categories was related to bone formation. Thus, expression of cell surface epitopes and osteogenic-related transcription factors as well as the bone formation capacity were compared. The results showed that compared with DFPCs and DPSCs, PDLSCs had higher transcription levels of osteogenic-related factors, a higher in vitro osteogenic potential, and an increased new bone formation capacity in vivo. In conclusion, the results of this study suggested that the differential DNA methylation profiles could be related to the osteogenic potential of these human odontogenic cell populations. Additionally, the increased osteogenic potential of PDLSCs might aid researchers or clinicians in making better choices regarding tissue regeneration and clinical therapies.

Magnetic Bead-Based Serum Peptidome Profiling in Patients with Gestational Diabetes Mellitus.

Gestational diabetes mellitus (GDM) is a frequent medical condition during pregnancy. Early diagnosis and treatment of GDM are crucial for both the mother and the baby. In the present study, we aimed to identify specific biomarkers to assist in the early detection of GDM and give some clues to the possible causes of GDM by comparing serum peptide profile differences between GDM patients and healthy controls. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used in combination with weak cation exchange magnetic bead (WCX-MB). Levels of four peptides (4418.9, 2219.7, 2211.5, and 1533.4 Da) were significantly different. Interestingly, three of them (4418.9, 2211.5, and 1533.4 Da) were identified when GDM patients with two degrees of glucose intolerance were compared. Additionally, peptides 2211.5 and 1533.4 Da showed a decreasing trend as glucose intolerance increased, while peptide 4418.9 Da exhibited the reverse tendency. In conclusion, our study provides novel insights into the altered serum peptide profile of GDM patients. The specific candidate biomarkers may contribute to the development of GDM.

[Analysis of the electromyographic characteristics on shoulder fatigue caused by lifting load].

OBJECTIVE: To analyze and evaluate the fatigue of shoulder skeletal muscle caused by different lifting loads with surface electromyography (sEMG). METHODS: According to the loading standard of1 Repetition Maximum (1RM), ten male volunteers performed 3 tasks of upper limb flexion, i.e. 10%, 50% and 90%-1RM. During action process, the signals of Upper Trapezius (UT), Lower Trapezius (LT), Serratus Anterior (SA) and Anterior Deltoid (AD) were recorded by sEMG. The Mean Amplitude (MA) served as an index to evaluate the changes in skeletal muscle fatigue. RESULTS: The scores of Borg were 15.6, 15.9 and 15.2 for 3 loads of 10%-1RM, 50%-1RM and 90%-1RM, respectively (P > 0.05). The mean amplitudes (MAs) of Upper Trapezius, Lower Trapezius, Anterior Deltoid and Serratus Anterior in shoulders increased obviously. Under the load intensity of 10%-1RM, the MAs of Upper Trapezius and Anterior Deltoid increased significantly (P < 0.05), which were 0.898 and 0.736, respectively. After the exhaustion, the contribution of mean amplitude in shoulder muscle did not change significantly (P > 0.05). CONCLUSION: The low-load action for long time can induce easily the fatigue of upper trapezius and anterior deltoid.