Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis.

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

TNFSF15 inhibits progression of diabetic retinopathy by blocking pyroptosis via interacting with GSDME.

Diabetic retinopathy is a common microvascular complication of diabetes and a leading cause of blindness. Pyroptosis has emerged as a mechanism of cell death involved in diabetic retinopathy pathology. This study explored the role of GSDME-mediated pyroptosis and its regulation by TNFSF15 in diabetic retinopathy. We found GSDME was upregulated in the progression of diabetic retinopathy. High glucose promoted GSDME-induced pyroptosis in retinal endothelial cells and retinal pigment epithelial cells, attributed to the activation of caspase-3 which cleaves GSDME to generate the pyroptosis-executing N-terminal fragment. TNFSF15 was identified as a binding partner and inhibitor of GSDME-mediated pyroptosis. TNFSF15 expression was increased by high glucose but suppressed by the caspase-3 activator Raptinal. Moreover, TNFSF15 protein inhibited high glucose- and Raptinal-induced pyroptosis by interacting with GSDME in retinal cells. Collectively, our results demonstrate TNFSF15 inhibits diabetic retinopathy progression by blocking GSDME-dependent pyroptosis of retinal cells, suggesting the TNFSF15-GSDME interaction as a promising therapeutic target for diabetic retinopathy.

Heme oxygenase-1 increases intracellular iron storage and suppresses inflammatory response of macrophages by inhibiting M1 polarization.

Heme oxygenase-1 (HO-1) catalyzes the first and rate-limiting enzymatic step of heme degradation, producing carbon monoxide, biliverdin, and free iron. Most iron is derived from aged erythrocytes by the decomposition of heme, which happened mainly in macrophages. However, the role of HO-1 on iron metabolism and function of macrophage is unclear. The present study investigated the effect of HO-1 on iron metabolism in macrophages, and explored the role of HO-1 on inflammatory response, polarization, and migration of macrophages. HO-1 inducer Hemin or HO-1 inhibitor zinc protoporphyrin was intravenously injected to C57BL/6 J mice every 4 d for 28 d. We found that HO-1 was mainly located in the cytoplasm of splenic macrophages of mice. Activation of HO-1 by Hemin significantly increased iron deposition in the spleen, up-regulated the gene expression of ferritin and ferroportin, and down-regulated gene expression of divalent metal transporter 1 and hepcidin. Induced HO-1 by Hemin treatment increased intracellular iron levels of macrophages, slowed down the absorption of extracellular iron, and accelerated the excretion of intracellular iron. In addition, activation of HO-1 significantly decreased the expression of pro-inflammatory cytokines including interleukin (IL)-6, IL-1ß, and inducible nitric oxide synthase, but increased the expression of anti-inflammatory cytokines such as IL-10. Furthermore, activation of HO-1 inhibited macrophages to M1-type polarization, and increased the migration rate of macrophages. This study demonstrated that HO-1 was able to regulate iron metabolism, exert anti-inflammatory effects, and inhibit macrophages polarization to M1 type.

Efficacy of subthreshold micropulse laser photocoagulation therapy versus anti-vascular endothelial growth factor therapy for refractory macular edema secondary to non-ischemic branch retinal vein occlusion.

OBJECTIVE: To assess the efficacy of subthreshold micropulse laser photocoagulation (SMLP) therapy versus anti-vascular endothelial growth factor (anti-VEGF) therapy in patients with refractory macular edema (ME) secondary to non-ischemic branch retinal vein occlusion (BRVO). METHODS: This single-center, prospective, nonrandomized, case-control trial involved patients with refractory ME that responded poorly to three or more initial anti-VEGF injections. The patients were examined and divided into two groups according to their chosen treatment: the intravitreal ranibizumab (IVR) group and the SMLP group. Both groups were followed up monthly for 12 months. Therapeutic efficacy and safety were assessed throughout the follow-up period. RESULTS: The IVR group comprised 49 eyes, and the SMLP group comprised 45 eyes. The improvements in the optical coherence tomography findings and visual acuity were comparable between the two groups at the final follow-up. The total number of injections was significantly lower in the SMLP than IVR group. No serious adverse events occurred during the study period. CONCLUSIONS: SMLP therapy is better for patients with central macular thickness (CMT) of ≤400 µm. For patients with CMT of >400 µm, we advise continuation of anti-VEGF agents to reduce ME followed by application of SMLP therapy when CMT has decreased to ≤400 µm.

PI3K/AKT/mTOR signaling participates in insulin-mediated regulation of pathological myopia-related factors in retinal pigment epithelial cells.

BACKGROUND: Insulin positively correlates with the length of the eye axis and is increased in the vitreous and serum of patients with pathological myopia (PM). How insulin influences the physiological process of retinal pigment epithelial (RPE) cells in PM remains unclear. This study aimed to explore the effect of insulin on the ultrastructure and function of RPE cells and the role of PI3K/AKT/mTOR signaling involved in the development of PM. METHODS: The ARPE-19 cells were treated with different concentrations of insulin to analyze the cell morphology, cell viability, the protein level of insulin receptor ß, and the mRNA and protein levels of and PM-related factors (TIMP-2, MMP-2, bFGF, and IGF-1). The ultrastructure of APRE-19 cells was also observed after insulin treatment. Besides, the PI3K/AKT/mTOR signaling was studied with or without the PI3K inhibitor LY294002 in ARPE-19 cells. RESULTS: Insulin enhanced the cell viability of ARPE-19 cells and caused the endoplasmic reticulum to expand and vesiculate, suggesting increased secretion of growth factors and degeneration in ARPE-19 cells. Furthermore, the insulin receptor ß was stimulated with insulin treatment, subsequently, the phosphorylation of AKT and mTOR was positively activated, which was adversely suppressed in the presence of LY294002. The secretion of TIMP-2 and bFGF was significantly decreased, and the secretion of MMP-2 and IGF-1 was highly elevated with insulin treatment depending on the concentration in ARPE-19 cells. Furthermore, the effect of insulin on PM-related proteins was restored with the addition of LY294002. CONCLUSIONS: Our results indicated that insulin regulated the secretion of PM-related factors via the PI3K/AKT/mTOR signaling pathway in retinal pigment epithelial cells, and thus probably promoted the development of PM through transducing regulation signals from retina to choroid and sclera.

[Polarized activation affects iron metabolism in macrophages].

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.

Accumulation and health implications of metals in topsoil of an urban riparian zone adjacent to different functional areas in a subtropical city.

The riparian zone is a river-land ecotone, and its environmental conditions have a significant effect on the river ecosystem and population health. In this study, As, Cu, Cr, Cd, Mn, Fe, Ni, Pb, and Zn in the topsoils of urban riparian zones in a subtropical city in southeast China were quantitatively estimated by inductively coupled plasma-optical emission spectrometry. The geoaccumulation index and health risk evaluation model were adopted to assess the accumulation characteristic and health risk of residents' exposure to metals. Principle component analysis was used to determine the source of metals. The results showed that the mean contents of metals (except Fe) were higher than the soil background value, but none of the metal contents exceeded the mass limit of environmental quality standards. The order of the geoaccumulation index was Cd > Mn > Cu > Cr > Pb > Zn > As > Ni > Fe. The contamination level of Cd was classified as slight, whereas the other metals did not contribute to pollution. The spatial distribution of metals in the riparian zone was compatible with the pattern of functional zones in the adjacent urban areas, where levels of Cr, Cd, Fe, Mn, and Ni were higher in commercial areas, as were Pb and Zn in under-construction land and As in residential and industrial areas. Carcinogenic risks of Cr, As, and Pb were acceptable. The hazard index indicated no significant noncarcinogenic risks from any metals. However, noncarcinogenic risks of metals other than Mn were higher for children than for adults, and the primary exposure route of metal into the human body was ingestion for children and inhalation for adults. Principle component analysis indicated that the primary sources of Cr, Ni, Mn, Cu, and Fe were pedogenic processes and mineral weathering, whereas Zn, As, Pb, and Cd mainly originated from anthropogenic sources, specifically, Zn from transportation emission, Pb from transportation emission and industry waste, As from coal combustion and residential waste, and Cd from pigments/paint used in commercial buildings, urban greening, consumer waste, and transportation emission.

Generation of nonhuman primate retinitis pigmentosa model by in situ knockout of RHO in rhesus macaque retina.

Retinitis pigmentosa (RP) is a form of inherited retinal degenerative diseases that ultimately involves the macula, which is present in primates but not in the rodents. Therefore, creating nonhuman primate (NHP) models of RP is of critical importance to study its mechanism of pathogenesis and to evaluate potential therapeutic options in the future. Here we applied adeno-associated virus (AAV)-delivered CRISPR/SaCas9 technology to knockout the RHO gene in the retinae of the adult rhesus macaque (Macaca mulatta) to investigate the hypothesis whether non-germline mutation of the RHO gene is sufficient to recapitulate RP. Through a series of studies, we were able to demonstrate successful somatic editing of the RHO gene and reduced RHO protein expression. More importantly, the mutant macaque retinae displayed clinical RP phenotypes, including photoreceptor degeneration, retinal thinning, abnormal rod subcellular structures, and reduced photoresponse. Therefore, we suggest somatic editing of the RHO gene is able to phenocopy RP, and the reduced time span in generating NHP mutant accelerates RP research and expands the utility of NHP model for human disease study.

Health Risk Assessment and Source Apportionment for Heavy Metals in a Southern Chinese Reservoir Impacted by Stone Mining Activities.

Metal contaminants in drinking water pose a potential threat to human health. Metal elements (Fe, Mn, Cu, Cr, Cd, As, and Pb) in Shanzi Reservoir, China, a drinking water source for nearby cities, were measured in 2013 and 2014. The distribution characteristics of metal elements in water were identified and a health risk assessment model was used to evaluate potential harm. Principal component analysis and cluster analysis were used to determine the main sources of metal pollutants. The results showed that Pb and As exceeded the standard at some sampling sites, whereas other metal elements met the drinking water standards. The spatial distribution of metal elements was extremely uneven and might be affected by either the geochemical environment or human activities in the study region. The total risk value of metals (5 × 10-5 a-1 ) was below the recommended value of the United States Environmental Protection Agency (USEPA), the total cancer risk was higher than the total noncancer risk, and both risks were higher for children than for adults. Arsenic was the priority control pollutant, and the priority control site was located upstream of the reservoir. Source analysis showed that Fe, Mn, and Cu were mainly from soil formation and stone mining and processing industries; Pb and As were mainly from agricultural activities, free dumping and burning of domestic garbage, and atmospheric deposition from transportation emissions; Cd was mainly from agricultural application of fertilizers and pesticides; and Cr was from the stone mining and processing industry and from the electroplating and metal manufacturing industries. Integr Environ Assess Manag 2020;16:342-352. © 2019 SETAC.

Plant begomoviruses subvert ubiquitination to suppress plant defenses against insect vectors.

Most plant viruses are vectored by insects and the interactions of virus-plant-vector have important ecological and evolutionary implications. Insect vectors often perform better on virus-infected plants. This indirect mutualism between plant viruses and insect vectors promotes the spread of virus and has significant agronomical effects. However, few studies have investigated how plant viruses manipulate plant defenses and promote vector performance. Begomoviruses are a prominent group of plant viruses in tropical and sub-tropical agro-ecosystems and are transmitted by whiteflies. Working with the whitefly Bemisia tabaci, begomoviruses and tobacco, we revealed that C2 protein of begomoviruses lacking DNA satellites was responsible for the suppression of plant defenses against whitefly vectors. We found that infection of plants by tomato yellow leaf curl virus (TYLCV), one of the most devastating begomoviruses worldwide, promoted the survival and reproduction of whitefly vectors. TYLCV C2 protein suppressed plant defenses by interacting with plant ubiquitin. This interaction compromised the degradation of JAZ1 protein, thus inhibiting jasmonic acid defense and the expression of MYC2-regulated terpene synthase genes. We further demonstrated that function of C2 protein among begomoviruses not associated with satellites is well conserved and ubiquitination is an evolutionarily conserved target of begomoviruses for the suppression of plant resistance to whitefly vectors. Taken together, these results demonstrate that ubiquitination inhibition by begomovirus C2 protein might be a general mechanism in begomovirus, whitefly and plant interactions.

Three-Dimensional Analysis of Chloroplast Structures Associated with Virus Infection.

Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.

NbPHAN, a MYB transcriptional factor, regulates leaf development and affects drought tolerance in Nicotiana benthamiana.

MYB transcriptional factors, characterized by the presence of conserved DNA-binding domains (BDs) (MYB domain), are involved in diverse processes including plant growth, development, metabolic and stress responses. In this study, a new R2R3-type MYB gene, NbPHAN (Nicotiana benthamiana PHANTASTICA), was identified in N. benthamiana. The NbPHAN encodes a protein of 362 amino acids and shares high sequence identities with the AS1-RS2-PHANs (ARPs) from other plant species. The NbPHAN protein targets to and forms homodimers in the nucleus. The MYB domain and C-terminal region of NbPHAN determine its subcellular localization and homodimerization, respectively. Using virus-induced gene silencing, we showed that the NbPHAN-silenced leaves exhibited severe downward curling and abnormal growth of blades along the main veins through suppressing the expression of the NTH20 gene. In addition, we found NbPHAN plays an important role in drought tolerance. The NbPHAN-silenced plants exhibited severe wilting and increased rate of water loss than that found in the non-silenced plants when growing under the water deficit condition. Although abscisic acid accumulation was not altered in the NbPHAN-silenced plants as compared with that in the non-silenced plants, several other stress-inducible genes were clearly repressed under the water deficit condition. Our results provide strong evidence that other than controlling leaf development, the ARP genes can also regulate plant tolerance to drought stress.

Protective effects of human umbilical cord blood stem cell intravitreal transplantation against optic nerve injury in rats.

BACKGROUND: The majority of studies addressing traumatic optic neuropathy (TON) have focused on drugs, proteins, cytokines, and various surgical techniques. A recent study reported that transplantation of human umbilical cord blood stem cells (hUCBSCs) achieved therapeutic effects on TON, but the exact effects on optic nerve injury are still unknown, and the mechanisms underlying nerve protection remain poorly understood. METHODS: A total of 135 healthy Sprague-Dawley adult rats were randomly assigned to three groups: sham-surgery, model and transplantation, with 45 rats in each group. TON was induced in the model and transplantation groups via optic nerve crush injury. The crush injury was not performed in the sham-surgery group. Seven days after the injury, 10(6) hUCBSCs were injected into the rat vitreous cavity of transplantation group, and an equal volume of physiological saline was administered to the model and sham-surgery groups. Pathological observation of rat retina tissues was performed by hematoxylin-eosin (H&E) staining at days 3, 7, 14, 21 and 28 post-surgery. The number of retinal ganglion cells (RGCs) and mRNA expression levels of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were assessed by the Fluorogold (FG) retrograde labeling and reverse transcriptase-polymerase chain reaction (RT-PCR) methods, respectively. RESULTS: The number of labeled RGCs and the expression of BDNF and GDNF mRNA obviously increased, and pathological injury was significantly ameliorated in the transplantation group compared to the model group (P < 0.05). CONCLUSIONS: Via intravitreal transplantation, the hUCBSCs resulted in a significant increase in the survival of the RGCs, and improved pathological changes in the rat retina, following TON. The protective mechanism is correlated with the continuous secretion of BDNF and GDNF in vivo of retina in optic nerve injury rats by the transplanted hUCBSCs.

Two cases of primary intraocular lymphoma: fine needle aspiration diagnosis and intravitreal methotrexate treatment.

We described clinical process of two cases of intraocular lymphoma in aspects of early diagnosis by fine needle aspiration (FNA) and biopsy and treatment by intravitreal methotrexate (MTX). Two patients were suspected to have primary intraocular lymphoma (PIOL) with geographic yellow-white infiltrates and vitreous opacity. FNA confirmed malignant intraocular lymphoma in one patient and failed in the other patient due to complication of vitreous hemorrhage. Subsequent vitreous biopsy confirmed malignant intraocular lymphoma in the other patient. Both patients were treated by intravitreal methotrexate. In case 1 the tumor had complete remission and follow-up of 12 months had not found any signs of recurrence. In case 2 the patient died of brain metastasis 22 months after the ocular biopsy. Our findings demonstrate that although cytological examination of vitrectomy specimens remains the gold standard in diagnosis of PIOL, examination of FNA and biopsy increases the reliability of early diagnosing or excluding a PIOL. Individualized intravitreal methotrexate can be used to effectively treat PIOL. More effective integrated program treating primary central nervous system lymphoma/PIOL is worthy of looking forward to.

Expression of a begomoviral DNAbeta gene in transgenic Nicotiana plants induced abnormal cell division.

An increasing number of monopartite begomoviruses are being identified that a satellite molecule (DNAbeta) is required to induce typical symptoms in host plants. DNAbeta encodes a single gene (termed betaC1) encoded in the complementary-sense. We have produced transgenic Nicotiana benthamiana and N. tabacum plants expressing the betaC1 gene of a DNAbeta associated with Tomato yellow leaf curl China virus (TYLCCNV), under the control of the Cauliflower mosaic virus 35S promoter. Transgenic plants expressing betaC1 showed severe developmental abnormalities in both species. Microscopic analysis of sections of both transgenic and non-transgenic N. tabacum leaves showed abnormal outgrowths of transgenic N. tabacum to be due to disorganized cell division (hyperplasia) of spongy and palisade parenchyma. Immuno-gold labeling of sections with a polyclonal antibody against the betaC1 protein showed that the betaC1 protein accumulated in the nuclei of cells. The possible biological function of the betaC1 protein was discussed.

[Prokaryotic expression and immuno-localization of replication protein gene of Tobacco curly shoot virus].

The replication protein (Rep) gene of Tobacco curly shoot virus (TbCSV) Y35 isolate was obtained from the infected tobacco plants by PCR and cloned into expression vector pGEX-4T-1 to generate the recombinant plasmid pGEX-Y35Rep. The recombinant plasmid was introduced into Escherichia coli strain BL21(DE3) pLys S, and TbCSV Rep fusion protein was expressed with induction by IPTG and some products were soluble. The Rep fusion protein was purified with GST-Sepharose 4B affinity chromatography and its polyclonal antibody was produced in a rabbit. Studies on subcellular distribution of TbCSV Rep protein in infected tobacco leaves revealed that Rep protein mainly exists in the fractions containing the nucleus. Immuno-gold labeling with the antibody against Rep fusion protein also indicated that Rep protein localized in the nuclei of infected tobacco cells.