Metabolite profiling and identification of enzymes responsible for the metabolism of hirsutine, a major alkaloid from Uncaria rhynchophylla.

The in vitro metabolism of hirsutine was determined using liver microsomes and human recombinant cytochrome P450 enzymes. Under the current conditions, a total of 14 phase I metabolites were tentatively identified.Ketoconazole showed significant inhibitory effect on the metabolism of hirsutine. Human recombinant cytochrome P450 enzyme analysis revealed that metabolism of hirsutine was mainly catalysed by CYP3A4.Our data revealed that hirsutine was metabolised via mono-oxygenation, di-oxygenation, N-oxygenation, dehydrogenation, demethylation and hydrolysis.In glutathione (GSH)-supplemented liver microsomes, four GSH adducts were identified. Hirsutine underwent facile P450-mediated metabolic activation, forming reactive 3-methyleneindolenine and iminoquinone intermediates.This study provided valuable information on the metabolic fates of hirsutine in liver microsomes, which would aid in understanding the hepatotoxicity caused by hirsutine or hirsutine-containing herb preparation.

Volumetric reduction in large cystic jaw lesions postoperative enucleation: a longitudinal clinical study.

BACKGROUND: Enucleation, a surgical procedure, is commonly used to treat large jaw cysts, unicystic ameloblastomas and keratocysts. However, it remains unclear to what extent the jaw bone regenerates after enucleation. We aimed to evaluate the percentage and the survival analysis of jaw bone regeneration, in terms of cavity volume residual (CVR), in patients who underwent enucleation of large jaw cysts, unicystic ameloblastomas and keratocysts. METHODS: We collected data longitudinally from 75 patients who underwent jaw cystic lesions enucleation at the Stomatological Hospital of Xi'an Jiaotong University, between January 2015 and June 2021. All patients had both preoperative and postoperative cone-beam computed tomography (CBCT) imaging data. CBCT images were analyzed using Image J. Changes in the CVR were assessed at various follow-up time points, and the Kaplan-Meier method was utilized to evaluate the CVR over time. RESULTS: The patients had a mean age of 31.7 years (range: 5.5-72 years) with 58.66% of them being male. The postoperative CVR was 32.20% at three months, 21.10% at six months, 15.90% at 12 months, and 5.60% at 24 months. The percentage of CVR during follow-up periods for the initial size Quartile (Q)1 (212.54-1569.60 mm3) was substantially lower than those of Q2 and Q3 at and after seven months of follow-up and became statistically significant at the 12-month mark. CONCLUSION: This study demonstrates that spontaneous bone regeneration can occur after enucleation of large jaw cysts, unicystic ameloblastomas and keratocysts, even without the use of filler materials. The initial size of the lesion had a significant impact on the outcome of cystic lesion enucleation over time. To minimize the risks associated with radiation exposure and expenses, we recommend reducing the frequency of CT imaging follow-ups for patients with small initial cavity sizes (ranging from 212.54 to 1569.60 mm3).

Magneto-mechanical stimulation modulates osteocyte fate via the ECM-integrin-CSK axis and wnt pathway.

Osteocytes are the mechano-sensors of bones. Large gradient high-static magnetic fields (LG-HMFs) produce stable, high-precision, and non-attenuation mechanical forces. We discovered that magnetic forces opposite to gravity inhibited MLO-Y4 osteocyte proliferation and viability by inducing structural damage and apoptosis. In contrast, magnetic force loading in the same direction as that of gravity promoted the proliferation and inhibited apoptosis of MLO-Y4 osteocytes. Differentially expressed gene (DEG) analysis after magnetic force stimulation indicated that the ECM-integrin-CSK axis responded most significantly to mechanical signals. Wisp2 was the most significant DEG between the 12 T upward and downward groups, showing the highest correlation with the Wnt pathway according to the STRING protein interaction database. Explaining the cellular and molecular mechanisms by which mechanical stimuli influence bone remodeling is currently the focus of osteocyte-related research. Our findings provide insights into the effects of LG-HMFs on bone cells, which have further implications in clinical practice.

A static magnetic field improves bone quality and balances the function of bone cells with regulation on iron metabolism and redox status in type 1 diabetes.

Osteoporosis is one of the chronic complications of type 1 diabetes with high risk of fracture. The prevention of diabetic osteoporosis is of particular importance. Static magnetic fields (SMFs) exhibit advantages on improvement of diabetic complications. The biological effects and mechanism of SMFs on bone health of type 1 diabetic mice and functions of bone cells under high glucose have not been clearly clarified. In animal experiment, six-week-old male C57BL/6J mice were induced to type 1 diabetes and exposed to SMF of 0.4-0.7 T for 4 h/day lasting for 6 weeks. Bone mass, biomechanical strength, microarchitecture and metabolism were determined by DXA, three-point bending assay, micro-CT, histochemical and biochemical methods. Exposure to SMF increased BMD and BMC of femur, improved biomechanical strength with higher ultimate stress, stiffness and elastic modulus, and ameliorated the impaired bone microarchitecture in type 1 diabetic mice by decreasing Tb.Pf, Ct.Po and increasing Ct.Th. SMF enhanced bone turnover by increasing the level of markers for bone formation (OCN and Collagen I) as well as bone resorption (CTSK and NFAT2). In cellular experiment, MC3T3-E1 cells or primary osteoblasts and RAW264.7 cells were cultured in 25 mM high glucose-stimulated diabetic marrow microenvironment under differentiation induction and exposed to SMF. SMF promoted osteogenesis with higher ALP level and mineralization deposition in osteoblasts, and it also enhanced osteoclastogenesis with higher TRAP activity and bone resorption in osteoclasts under high glucose condition. Further, SMF increased iron content with higher FTH1 expression and regulated the redox level through activating HO-1/Nrf2 in tibial tissues, and lowered hepatic iron accumulation by BMP6-mediated regulation of hepcidin and lipid peroxidation in mice with type 1 diabetes. Thus, SMF may act as a potential therapy for improving bone health in type 1 diabetes with regulation on iron homeostasis metabolism and redox status.

Phenethyl isothiocyanate induces oxidative cell death in osteosarcoma cells with regulation on mitochondrial network, function and metabolism.

Phenethyl isothiocyanate (PEITC), a kind of isothiocyanate available in cruciferous vegetables, exhibits inhibitory effects on cancers. PEITC has been extensively recorded for its effect on regulation of redox status in cancer cells. Our previous studies revealed that PEITC induced ROS-dependent cell death in osteosarcoma. Mitochondria are the main sites for ROS generation and play significant role in deciding cell fate. To dissect the mechanism of PEITC's action on osteosarcoma cells, we detected the changes on mitochondrial network, function and metabolism in K7M2 and 143B cells. Here, PEITC induced cytosolic, lipid and mitochondrial ROS production in osteosarcoma cells. It changed mitochondrial morphology from elongated to punctate network and decreased mitochondrial mass. Meantime, PEITC increased mitochondrial transmembrane potential in short time, decreased it with time prolonged, and later collapsed it in K7M2 cells, and reduced it in 143B cells. PEITC inhibited proliferation potential of osteosarcoma cells with damage on mitochondrial respiratory chain complexes. Further, PEITC-treated osteosarcoma cells experienced a sudden increase in ATP level, and later its content was decreased. Moreover, PEITC downregulated the expressions of mitochondrial respiratory chain complexes including COX IV, UQCR, SDHA and NDUFA9 in 143B cells and COX IV in K7M2 cells. At last, by using ρ0 cells derived from K7M2 and 143B cells, we found that osteosarcoma cells that depleted mtDNA were less sensitive to PEITC-induced changes on cellular morphology, cytoskeleton filament, mitochondrial transmembrane potential and ROS generation. In conclusion, our study demonstrated that mitochondria may play important role in PEITC-induced oxidative cell death in osteosarcoma cells.

Exposure to a static magnetic field attenuates hepatic damage and function abnormality in obese and diabetic mice.

Static magnetic fields (SMFs) exhibit significant effect on health care. However, the effect of SMF on hepatic metabolism and function in obesity and diabetes are still unknown. Liver is not only the main site for glucolipid metabolism but also the core part for iron metabolism regulation. Dysregulations of iron metabolism and redox status are risk factors for the development of hepatic injury and affect glucolipid metabolism in obesity and diabetes. Mice of HFD-induced obesity and HFD/streptozocin-induced diabetes were exposed to a moderate-intensity SMF (0.4-0.7 T, direction: upward, 4 h/day, 8 weeks). Results showed that SMF attenuated hepatic damage by decreasing inflammation and fibrosis in obese and diabetic mice. SMF had no effects on improving glucose/insulin tolerance but regulated proteins (GLUT1 and GLUT4) and genes (G6pc, Pdk4, Gys2 and Pkl) participating in glucose metabolism with phosphorylation of Akt/AMPK/GSK3ß. SMF also reduced lipid droplets accumulation through decreasing Plin2 and Plin5 and regulated lipid metabolism with elevated hepatic expressions of PPARγ and C/EBPα in obese mice. In addition, SMF decreased hepatic iron deposition with lower FTH1 expression and modulated systematic iron homeostasis via BMP6-mediated regulation of hepcidin. Moreover, SMF balanced hepatic redox status with regulation on mitochondrial function and MAPKs/Nrf2/HO-1 pathway. Finally, we found that SMF activated hepatic autophagy and enhanced lipophagy by upregulating PNPLA2 expression in obese and diabetic mice. Our results demonstrated that SMF significantly ameliorated the development of hepatic injury in obese and diabetic mice by inhibiting inflammatory level, improving glycolipid metabolism, regulating iron metabolism, balancing redox level and activating autophagy.

Biophysical mechanisms underlying the effects of static magnetic fields on biological systems.

With the widespread use of static magnetic fields (SMFs) in medicine, it is imperative to explore the biological effects of SMFs and the mechanisms underlying their effects on biological systems. The presence of magnetic materials within cells and organisms could affect various biological metabolism and processes, including stress responses, proliferation, and structural alignment. SMFs were generally found to be safe at the organ and organism levels. However. human subjects exposed to strong SMFs have reported side effects. In this review, we combined the magnetic properties of biological samples to illustrate the mechanism of action of SMFs on biological systems from a biophysical point of view. We suggest that the mechanisms of action of SMFs on biological systems mainly include the induction of electric fields and currents, generation of magnetic effects, and influence of electron spins. An electrolyte flowing in a static magnetic field generates an induced current and an electric field. Magnetomechanical effects include orientation effects upon subjecting biological samples to SMFs and movement of biological samples in strong field gradients. SMFs are thought to affect biochemical reaction rates and yields by influencing electron spin. This paper helps people how can harness the favorable biological effects of SMFs.

Serum Total Bile Acids in Relation to Gastrointestinal Cancer Risk: A Retrospective Study.

Background: Bile acids (BAs) have been proposed to promote gastrointestinal cells carcinogenesis. However, studies on serum total bile acid (TBA) levels and gastrointestinal cancers (GICs) risk are rare. Methods: We conducted a retrospective case-control study from 2015 to 2019 at the First Affiliated Hospital of Air Force Military Medical University, in which 4,256 GICs cases and 1,333 controls were recruited. Patients' demographic, clinical and laboratory data were collected. The odds ratios (ORs) with 95% confidence intervals (CIs) were estimated using binary logistic regression models. Results: Positive associations were observed between serum TBA levels and risks of esophageal cancer (EC), gastric cancer (GC) and colorectal cancer (CRC). Overall, ORs of EC, GC and CRC risk rose with the TBA levels increasing. After adjustment for potential confounders, the OR of TBA-positive for EC risk was 4.89 (95% CI: 3.20-7.49), followed by GC (OR: 3.92, 95% CI: 2.53-6.08), and CRC (OR: 3.32, 95% CI: 2.04-5.11). Patients aged 60 years or older have a higher risk of GICs, especially for EC patients. Males are associated with a higher risk of GC, while females are associated with a higher risk of CRC. Preoperative serum TBA positive and negative was significantly different in the presence or absence of hematogenous metastasis among EC patients (P=0.014), and lymph node metastasis among GC patients (P=0.018). Conclusions: This retrospective study showed positive associations between serum TBA level and GICs risk, and a higher serum TBA level constitutes a risk factor for GICs.

Iron plays a role in sulfasalazine-induced ferroptosis with autophagic flux blockage in K7M2 osteosarcoma cells.

Osteosarcoma is the most common primary bone malignancy in children and young adults, with a very poor prognosis. It is of great importance to develop targeted therapeutic strategies for osteosarcoma. Sulfasalazine (SAS) is an FDA-approved drug for the treatment of Crohn's disease, rheumatoid arthritis, and inflammatory bowel disease. It acts as an inhibitor of cystine/glutamate system, which is important for cellular glutathione synthesis and maintenance of GPx4 activity. Nowadays, SAS has been repurposed as an antitumor drug for inducing ferroptosis in cancers. This study aimed to uncover the role of iron in SAS-induced ferroptotic cell death in K7M2 osteosarcoma cells. Herein, SAS led to an iron-dependent cell death mode in K7M2 cells, accompanied with decreased antioxidant defense and increased production of cytosolic and lipid reactive oxygen species. Results also showed that iron supplement with ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS) exacerbated the declined cell viability of SAS-treated K7M2 cells, while in the case of iron depletion, it weakened such suppression. Furthermore, iron promoted SAS-induced alterations on cell cycle, cytoskeleton, mitochondria morphology and function, and redox system. Iron also induced the dysfunction of autophagic activity in SAS-treated K7M2 cells. In conclusion, our study uncovered the essential role of iron in SAS's effects on K7M2 cells and provided the potential combined therapy of inhibition on antioxidant defense and an increase in oxidative potential, which further disturbed the redox status in tumor cells.

Evaluating the biological safety on mice at 16 T static magnetic field with 700 MHz radio-frequency electromagnetic field.

OBJECTIVES: This study evaluated the associated biological effects of radio-frequency (RF) exposure at 16 T magnetic resonance imaging (MRI) on mice health. MATERIAL AND METHODS: A total of 48 healthy 8-week-old male C57BL/6 mice were investigated. A 16 T high static magnetic field (HiSMF) was generated by a superconducting magnet, and a radiofrequency (RF) electromagnetic field for hydrogen resonance at 16 T (700 MHz) was transmitted via a homemade RF system. The mice were exposed inside the 16 T HiSMF with the 700 MHz RF field for 60 min, and the body weight, organ coefficients, histomorphology of major organs, and blood indices were analyzed for the basal state of the mice on day 0 and day 14. The Heat Shock Protein 70 (HSP70), cyclooxygenase 2 (COX2), and interleukin- 6 (IL-6) were used to evaluate the thermal effects on the brain. Locomotor activity, the open field test, tail suspension test, forced swimming test, and grip strength test were used to assess the behavioral characteristics of the mice. RESULTS: The 16 T HiSMF with 700 MHz RF electromagnetic field exposure had no significant effects on body weight, organ coefficients, or histomorphology of major organs in the mice. On day 0, the expressions of HSP70 and COX2 in the brain were increased by 16 T HiSMF with 700 MHz RF electromagnetic field exposure. However, the expression of HSP70, COX2, and IL-6 had no significant difference compared with the sham group on day 14. Compared with the sham groups, the meancorpuscularvolume (MCV) on day 0 and the total protein (TP) on day 14 were increased significantly, whereas the other blood indices did not change significantly. The 16 T HiSMF with 700 MHz RF electromagnetic field exposure caused the mice to briefly circle tightly but had no effect on other behavioral indicators. CONCLUSIONS: In summary, 16 T HiSMF with 700 MHz RF electromagnetic field exposure for 60 min did not have severe effects on mice.

The efficacy and safety of different bismuth agents in Helicobacter pylori first-line eradication: A multicenter, randomized, controlled clinical trial.

BACKGROUND: The aim of this study was to evaluate the efficacy and safety of bismuth pectin capsules and bismuth pectin granules in the first-line quadruple treatment of Helicobacter pylori (H. pylori). METHODS: This study was a multicenter, randomized, open-labelled controlled clinical trial. Patients with a H. pylori infection were randomized into 4 groups (1:1:1:1) and treated with a 14-day bismuth-containing quadruple therapy. The 4 groups received either bismuth potassium citrate capsules (220 mg), colloidal bismuth pectin capsules (200 mg), bismuth pectin granules (150 mg), or bismuth pectin granules (300 mg). The primary outcome was the eradication rate of H. pylori. The secondary outcomes included symptom improvement, patient compliance, and incidence of adverse events. This study was registered at ClinicalTrials.gov (NCT04209933). RESULTS: A total of 240 patients were included in this study, and 211 patients completed the follow-up. An intention-to-treat analysis showed that the H. pylori eradication rates of the 4 groups were 73.3%, 76.7%, 75.0%, and 71.7%, respectively. The per-protocol analysis showed that the H. pylori eradication rates of the 4 groups were 86.3%, 82.1%, 83.3%, and 86.0%. There was no significant difference among the 4 groups in the H. pylori eradication rate (P > .05). There were also no significant differences in the symptom improvement rate, overall adverse reaction rate, or patient compliance among the 4 groups. CONCLUSIONS: Bismuth pectin capsules and bismuth pectin granules had similar efficacy and safety for H. pylori eradication compared to bismuth potassium citrate. These data suggest that bismuth pectin can be an alternative to bismuth potassium citrate to eradicate H. pylori when using bismuth-containing quadruple therapy.

Development of a competitive ELISA for detecting antibodies against genotype 1 hepatitis E virus.

Hepatitis E, a significant global public health issue in China, is caused by sporadic infections with regional hepatitis E virus (HEV) genotypes 1, 3, and 4. To date, most immunoassays currently used to test human sera for the presence of anti-HEV antibodies cannot identify HEV at the genotype level. However, such information would be useful for identifying the source of infecting virus. Therefore, here we describe the development of a competitive enzyme-linked immunosorbent assay (ELISA) for detecting anti-genotype 1 HEV antibodies in human sera. Using recombinant genotype 1 HEV ORF3 protein as immunogen, traditional hybridoma technology was employed to generate seven monoclonal antibodies (mAbs), of which two mAbs specifically reacted with the immunogen. One of these two mAbs, 1D2, was labeled with horseradish peroxidase (HRP) for use in competitive ELISA (cELISA). After cELISA optimization using a checkerboard assay design, the amount of ORF3SAR-55 as coating antigen (100 ng/well), HRP-1D2 mAb concentration (1 µg/mL), and test serum dilution (1:10) were selected and a result ≥ 19.5 was used as the cutoff for a positive result. Importantly, cross-genotype cELISA results indicated that the cELISA could not detect anti-genotype 3 rabbit and 4 swine HEV antibodies. Moreover, human sera confirmed as negative for anti-HEV antibodies using the commercial ELISA kit were all negative via cELISA. However, because the commercial ELISA kit detects anti-all genotypes HEV antibodies and the cELISA only detects anti-genotype 1 HEV antibodies, the consistence rate of two assays detecting positive sera is low. In summary, here a cELISA for detecting anti-genotype 1 HEV antibodies was developed for use in epidemiological investigations of genotype 1 HEV infections in humans. KEY POINTS: • Seven mAbs were produced using genotype 1 HEV ORF3 protein as immunogen. • One mAb that specifically bound to genotype 1 HEV ORF3 protein was selected and labeled for use in a cELISA to detect anti-genotype 1 HEV antibodies. • The competitive ELISA developed here will aid clinical diagnosis of HEV infections and will be useful for large-scale serological testing of genotype 1 HEV infections in humans.

Iron Chelator Induces Apoptosis in Osteosarcoma Cells by Disrupting Intracellular Iron Homeostasis and Activating the MAPK Pathway.

Osteosarcoma is a common malignant bone tumor in clinical orthopedics. Iron chelators have inhibitory effects on many cancers, but their effects and mechanisms in osteosarcoma are still uncertain. Our in vitro results show that deferoxamine (DFO) and deferasirox (DFX), two iron chelators, significantly inhibited the proliferation of osteosarcoma cells (MG-63, MNNG/HOS and K7M2). The viability of osteosarcoma cells was decreased by DFO and DFX in a concentration-dependent manner. DFO and DFX generated reactive oxygen species (ROS), altered iron metabolism and triggered apoptosis in osteosarcoma cells. Iron chelator-induced apoptosis was due to the activation of the MAPK signaling pathway, with increased phosphorylation levels of JNK, p38 and ERK, and ROS generation; in this process, the expression of C-caspase-3 and C-PARP increased. In an orthotopic osteosarcoma transplantation model, iron chelators (20 mg/kg every day, Ip, for 14 days) significantly inhibited the growth of the tumor. Immunohistochemical analysis showed that iron metabolism was altered, apoptosis was promoted, and malignant proliferation was reduced with iron chelators in the tumor tissues. In conclusion, we observed that iron chelators induced apoptosis in osteosarcoma by activating the ROS-related MAPK signaling pathway. Because iron is crucial for cell proliferation, iron chelators may provide a novel therapeutic strategy for osteosarcoma.

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro.

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.

Static magnetic field of 0.2-0.4 T promotes the recovery of hindlimb unloading-induced bone loss in mice.

PURPOSE: Bone loss is one of the most serious medical problem associated with prolonged weightlessness in long-term spaceflight mission. Skeletal reloading after prolonged spaceflight have indicated incomplete recovery of lost bone, which may lead to an increased risk of fractures in astronauts when returning to Earth. Substantial studies have revealed the capacity of static magnetic fields (SMFs) on treating various bone disorders, whereas it is unknown whether SMFs have the potential regulatory effects on bone quality in unloaded mice during unloading. This study was conducted to investigate the potential effects of whole-body SMF exposure with 0.2-0.4 T on the recovery of unloading-induced bone loss. MATERIALS AND METHODS: Eight-week-old male C57BL/6J mice were subjected to hindlimb unloading (HLU) for 4 weeks, following the mice were reloaded for 4 weeks under geomagnetic field (GMF) and SMF of 0.2-0.4 T. Bone quality indexes, including bone mineral density (BMD) and bone mineral content (BMC), bone microarchitecture, and bone mechanical properties were examined by the measurement of dual energy X-ray absorptiometry (DEXA), micro-computed tomography (Micro-CT), and 3-point bending. Bone turnover was evaluated by bone histomorphometric and serum biochemical assay. RESULTS: We found that SMF exposure for 4 weeks significantly promoted the recovery in HLU-induced decrease of BMD and BMC, deterioration of bone microarchitecture, and reduction of bone strength. The results from bone turnover determination revealed that SMF exposure for 4 weeks induced lower osteoclast number of trabecular bone and serum TRAP-5b levels in reloaded mice, whereas SMF showed no significant alteration in skeletal osteoblast number and serum osteocalcin levels. CONCLUSIONS: Together, our findings suggest that SMF of 0.2-0.4 T facilitated the recovery of unloading-induced bone loss by inhibiting the increase of bone resorption in reloaded mice, and indicate that SMF might become a promising biophysical countermeasure for maintaining bone health in astronauts after landing.

Nanobody Nb6 fused with porcine IgG Fc as the delivering tag to inhibit porcine reproductive and respiratory syndrome virus replication in porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly contagious virus that has led to enormous economic loss worldwide because of ineffective prevention and treatment. In view of their minimized size, high target specificity and affinity, nanobodies have been extensively investigated as diagnostic tools and treatments of many diseases. Previously, a PRRSV Nsp9-specific nanobody (Nb6) was identified as a PRRSV replication inhibitor. When it was fused with cell-penetrating peptide (CPP) TAT, Nb6-TAT could enter the cells for PRRSV suppression. However, delivery of molecules by CPP lack cell specificity and have a short duration of action. PRRSV has a tropism for monocyte/macrophage lineage, which expresses high levels of Fcγ receptors. Herein, we designed a nanobody containing porcine IgG Fc (Fcγ) to inhibit PRRSV replication in PRRSV permissive cells. Fcγ fused Nb6 chimeric antibody (Nb6-pFc) was assembled into a dimer with interchain disulfide bonds and expressed in a Pichia pastoris system. The results show that Nb6-pFc exhibits a well-binding ability to recombinant Nsp9 or PRRSV-encoded Nsp9 and that FcγR-mediated endocytosis of Nb6-pFc into porcine alveolar macrophages (PAM) was in a dose-dependent manner. Nb6-pFc can inhibit PRRSV infection efficiently not only by binding with Nsp9 but also by upregulating proinflammatory cytokine production in PAM. Together, this study proposes the design of a porcine IgG Fc-fused nanobody that can enter PRRSV susceptible PAM via FcγR-mediated endocytosis and inhibit PRRSV replication. This research reveals that nanobody-Fcγ chimeric antibodies might be effective for the control and prevention of monocyte/macrophage lineage susceptible pathogeneses.

Magnetic fields as a potential therapy for diabetic wounds based on animal experiments and clinical trials.

Diabetes mellitus (DM) is a chronic metabolic disorder with various complications that poses a huge worldwide healthcare burden. Wounds in diabetes, especially diabetic foot ulcers (DFUs), are difficult to manage, often leading to prolonged wound repair and even amputation. Wound management in people with diabetes is an extremely clinical and social concern. Nowadays, physical interventions gain much attention and have been widely developed in the fields of tissue regeneration and wound healing. Magnetic fields (MFs)-based devices are translated into clinical practice for the treatment of bone diseases and neurodegenerative disorder. This review attempts to give insight into the mechanisms and applications of MFs in wound care, especially in improving the healing outcomes of diabetic wounds. First, we discuss the pathological conditions associated with chronic diabetic wounds. Next, the mechanisms involved in MFs' effects on wounds are explored. At last, studies and reports regarding the effects of MFs on diabetic wounds from both animal experiments and clinical trials are reviewed. MFs exhibit great potential in promoting wound healing and have been practised in the management of diabetic wounds. Further studies on the exact mechanism of MFs on diabetic wounds and the development of suitable MF-based devices could lead to their increased applications into clinical practice.

Anticancer mechanisms of metformin: A review of the current evidence.

Metformin, a US Food and Drug Administration-approved "star" drug used for diabetes mellitus type 2, has become a topic of increasing interest to researchers due to its anti-neoplastic effects. Growing evidence has demonstrated that metformin may be a promising chemotherapeutic agent, and several clinical trials of metformin use in cancer treatment are ongoing. However, the anti-neoplastic effects of metformin and its underlying mechanisms have not been fully elucidated. In this review, we present the newest findings on the anticancer activities of metformin, and highlight its diverse anticancer mechanisms. Several clinical trials, as well as the limitations of the current evidence are also demonstrated. This review explores the crucial roles of metformin and provides supporting evidence for the repurposing of metformin as a treatment of cancer.

ß-Phenethyl Isothiocyanate Induces Cell Death in Human Osteosarcoma through Altering Iron Metabolism, Disturbing the Redox Balance, and Activating the MAPK Signaling Pathway.

Osteosarcoma is the most common primary malignancy of the skeleton in children and adults. The outcomes of people with osteosarcomas are unsatisfied. ß-Phenethyl isothiocyanate (PEITC) exhibits chemoprevention and chemotherapeutic activities against many human cancers. The molecular mechanism underlying its action on osteosarcoma is still unknown. This study was aimed at investigating the effect of PEITC on human osteosarcoma both in vitro and in vivo. The results showed that PEITC reduced cell viability, inhibited proliferation, and caused G2/M cell cycle arrest in four human osteosarcoma cell lines (MNNG/HOS, U-2 OS, MG-63, and 143B). Then, we found that PEITC altered iron metabolism related to the processes of iron import, storage, and export, which resulted in increased labile iron. Expectedly, PEITC caused oxidative stress as a consequence of GSH depletion-inducing ROS generation and lipid peroxidation. Multiple cell death modalities, including ferroptosis, apoptosis, and autophagy, were triggered in human osteosarcoma cells. Three MAPKs (ERK, p38, and JNK) were all activated after PEITC treatment; however, they presented different responses among the four human osteosarcoma cell lines. ROS generation was proved to be the major cause of PEITC-induced decreased proliferative potential, altered iron metabolism, cell death, and activated MAPKs in human osteosarcoma cells. In addition, PEITC also significantly delayed tumor growth in a xenograft osteosarcoma mouse model with a 30 mg/kg administration dose. In conclusion, this study reveals that PEITC simultaneously triggers ferroptosis, apoptosis, and autophagy in human osteosarcoma cells by inducing oxidative stress.

PEITC triggers multiple forms of cell death by GSH-iron-ROS regulation in K7M2 murine osteosarcoma cells.

Phenethyl isothiocyanate (PEITC) is an isothiocyanate that largely exists in cruciferous vegetables and exhibits chemopreventive and chemotherapeutic potential against various cancers. However, it is little known about the molecular mechanisms of its antitumor action against osteosarcoma, which is the second highest cause of cancer-related death in children and adolescents. In this study, we investigated the effects of PEITC on K7M2 murine osteosarcoma both in vitro and in vivo. We found that treatment with PEITC dose-dependently inhibited the viability of K7M2 murine osteosarcoma cells with an IC50 value of 33.49 µM at 24 h. PEITC (1, 15, 30 µM) dose-dependently inhibited the cell proliferation, caused G2/M cell cycle arrest, depleted glutathione (GSH), generated reactive oxygen species (ROS), altered iron metabolism, and triggered multiple forms of cell death, namely ferroptosis, apoptosis, and autophagy in K7M2 cells. We further revealed that PEITC treatment activated MAPK signaling pathway, and ROS generation was a major cause of PEITC-induced cell death. In a syngeneic orthotopic osteosarcoma mouse model, administration of PEITC (30, 60 mg/kg every day, ig, for 24 days) significantly inhibited the tumor growth, but higher dose of PEITC (90 mg/kg every day) compromised its anti-osteosarcoma effect. Histological examination showed that multiple cell death processes were initiated, iron metabolism was altered and MAPK signaling pathway was activated in the tumor tissues. In conclusion, we demonstrate that PEITC induces ferroptosis, autophagy, and apoptosis in K7M2 osteosarcoma cells by activating the ROS-related MAPK signaling pathway. PEITC has promising anti-osteosarcoma activity. This study sheds light on the redox signaling-based chemotherapeutics for cancers.