Arabinosylation of cell wall extensin is required for the directional response to salinity in roots.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.

Optimizing Interplanar Spacing, Oxygen Vacancies and Micromorphology via Lithium-Ion Pre-Insertion into Ammonium Vanadate Nanosheets for Advanced Cathodes in Aqueous Zinc-Ion Batteries.

Ammonium vanadates, featuring an N─H···O hydrogen bond network structure between NH4 + and V─O layers, have become popular cathode materials for aqueous zinc-ion batteries (AZIBs). Their appeal lies in their multi-electron transfer, high specific capacity, and facile synthesis. However, a major drawback arises as Zn2+ ions tend to form bonds with electronegative oxygen atoms between V─O layers during cycling, leading to irreversible structural collapse. Herein, Li+ pre-insertion into the intermediate layer of NH4 V4 O10 is proposed to enhance the electrochemical activity of ammonium vanadate cathodes for AZIBs, which extends the interlayer distance of NH4 V4 O10 to 9.8 Å and offers large interlaminar channels for Zn2+ (de)intercalation. Moreover, Li+ intercalation weakens the crystallinity, transforms the micromorphology from non-nanostructured strips to ultrathin nanosheets, and increases the level of oxygen defects, thus exposing more active sites for ion and electron transport, facilitating electrolyte penetration, and improving electrochemical kinetics of electrode. In addition, the introduction of Li+ significantly reduces the bandgap by 0.18 eV, enhancing electron transfer in redox reactions. Leveraging these unique advantages, the Li+ pre-intercalated NH4 V4 O10 cathode exhibits a high reversible capacity of 486.1 mAh g-1 at 0.5 A g-1 and an impressive capacity retention rate of 72% after 5,000 cycles at 5 A g-1 .

METTL14 promotes doxorubicin-induced cardiomyocyte ferroptosis by regulating the KCNQ1OT1-miR-7-5p-TFRC axis.

Doxorubicin (DOX) has toxic effects on the heart, causing cardiomyopathy and heart injury, but the underlying mechanisms of these effects require further investigation. This study investigated the role of DOX in promoting ferroptosis to induce myocardial injury. AC16 cardiomyocyte and neonatal rat ventricle cardiomyocytes were used as an in vitro model to study the molecules involved in myocardial injury using gene silencing, ectopic expression, and RNA immunoprecipitation. Messenger RNA and protein level analyses showed that DOX treatment resulted in the upregulation of methyltransferase-like 14 (METTL14), which catalyzes the m6A modification of the long non-coding RNA KCNQ1OT1, a miR-7-5p sponge. The RNA-binding protein IGF2BP1 is associated with KCNQ1OT1 to increase its stability and robustly inhibit miR-7-5p activity. Furthermore, a lack of miR-7-5p expression led to increased levels of transferrin receptor, promoting the uptake of iron and production of lipid reactive oxygen species and demonstrating that DOX-induced ferroptosis occurs in AC16 cells. Additionally, we found that miR-7-5p targets METTL14 in AC16 cells. Meanwhile, the role of METTL14/KCNQ1OT1/miR-7-5p axis in regulating ferroptosis in neonatal rat ventricle cardiomyocytes was also confirmed. Our results indicate that selectively inhibiting ferroptosis mediated by a METTL14/KCNQ1OT1/miR-7-5p positive feedback loop in cardiomyocytes could provide a new therapeutic approach to control DOX-induced cardiac injury.

SCF/SCFR signaling plays an important role in the early morphogenesis and neurogenesis of human embryonic neural retina.

The stem cell factor receptor (SCFR) has been demonstrated to be expressed in the neural retina of mice, rat and human for decades. Previous reports indicated that the SCFR correlates with glia differentiation of late retinal progenitor cells (RPCs), retinal vasculogenesis and homeostasis of the blood-retinal barrier. However, the role of SCF/SCFR signaling in the growth and development of the neural retina (NR), especially in the early embryonic stage, remains poorly understood. Here, we show that SCF/SCFR signaling orchestrates invagination of the human embryonic stem cell (hESC)-derived NR via regulation of cell cycle progression, cytoskeleton dynamic and apical constriction of RPCs in the ciliary marginal zone (CMZ). Furthermore, activation of SCF/SCFR signaling promotes neurogenesis in the central-most NR via acceleration of the migration of immature ganglion cells and repressing apoptosis. Our study reveals an unreported role for SCF/SCFR signaling in controlling ciliary marginal cellular behaviors during early morphogenesis and neurogenesis of the human embryonic NR, providing a new potential therapeutic target for human congenital eye diseases such as anophthalmia, microphthalmia and congenital high myopia.

Efficacy of Electroacupuncture Compared With Transcutaneous Electrical Stimulation for Severe Chronic Constipation: A Randomized Controlled Pilot Trial.

BACKGROUND: To treat severe chronic constipation, both electroacupuncture (EA) therapy and transcutaneous electrical stimulation (TES) are safe and effective, but there is a lack of comparison between the 2 treatments. We sought to compare the therapeutic effect of EA relative to TES for the treatment of severe chronic constipation. METHODS: We conducted a pilot randomized controlled trial. Seventy participants with severe chronic constipation were randomly allocated to the EA group or TES group. The acupoints in both groups are bilateral Tianshu (stomach 25), Fujie (spleen 14), and Shangjuxu (stomach 37). Each participant received 24 sessions of EA or TES during the treatment period of 8 weeks. Outcome measures were the proportion of participants with an increase from baseline of 1 or more complete spontaneous bowel movements (CSBMs) at week 8 and changes in the number of CSBMs and spontaneous bowel movements, stool character, difficulty in defecation, Patient Assessment of Constipation Quality of Life (PAC-QOL); the proportion of participants using rescue medicine and adverse reactions. Participants were followed for 24 weeks after the treatment. RESULTS: Sixty of the 70 eligible participants completed the whole trial, 32 participants (91.43%) in the EA group and 28 participants (80.00%) in the TES group. There were 10 participants (14.29%) who dropped out during the whole study, 3 (8.57%) in the EA group, and 7 (20.00%) in the TES group. At weeks 20 and 32, 22 of 32 (68.75%) and 19 of 32 (59.38%) in the EA group met the clinical outcome of an increase of 1 or more CSBMs from the baseline, compared with 10 of 28 (35.71%) and 9 of 28 (32.14%) in the TES group ( P =0.019 and 0.042, respectively). At the same time, the proportions of participants having 3 or more CSBMs per week were 18 of 32 (56.25%) and 15 of 32 (46.88%) in the EA group, respectively, compared with 6 of 28 (21.43%) and 5 of 28 (17.86%) in the TES group ( P =0.008 and 0.027, respectively). Statistical differences between the 2 groups were maintained in the change from baseline in CSBMs during the follow-up period ( P =0.007 and 0.013, at week 20 and 32, respectively) and in the change from baseline in spontaneous bowel movements at week 20 ( P =0.007). Statistical significance was also observed in the decrease of PAC-QOL score after 8 weeks of treatment (mean±SD, EA: 19.06±14.41 vs. TES: 12.48±9.13, P =0.031). No significant difference was noted between the 2 groups in defecation difficulty, stool character, and the use of rescue medicine. CONCLUSIONS: For the treatment of severe chronic constipation, EA performed better than TES in long-term follow-up and improving the quality of life. TES could be a better choice for patients with severe chronic constipation who are afraid of acupuncture because it is noninvasive. Well-designed and more rigorous clinical trials with larger sample sizes are needed.

Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.

The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.

Magnolol suppressed cell migration and invasion and induced cell apoptosis via inhibition of the NF-κB signaling pathway by upregulating microRNA-129 in multiple myeloma.

Multiple myeloma (MM) is incurable cancer in the blood system. Magnolol is an effective component against various cancers. This study tried to investigate the effect and mechanism of magnolol on MM via regulating miR-129. Human normal plasma cells (nPCs) and MM cells U266 and LP1 were used in this study, accompanied by treatment of magnolol. The miR-129 inhibitor was transfected into U266 and LP1 cells during experiments. Cell viability was detected by Cell Counting Kit-8 assay. Cell migration and invasion were tested by wound healing assay and Transwell assay. And Annexin-V-FITC/PI assay was utilized to assess cell apoptosis. miR-129, miR-1271-5p, miR-342-3p, and miR-124-3p expressions were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and western blot was adopted to evaluate Cyclin D1, matrix metalloprotein (MMP)-7, MMP-9, phosphorylation (p)-IκBα, p-p65, and p65 protein levels. In U266 and LP1 cells, with magnolol concentration increasing, cell viability, migration, and invasion rates, Cyclin D1, MMP-7, and MMP-9 expressions decreased, while cell apoptosis rose. And magnolol increased the miR-129 expression in MM cells. Besides, miR-129 inhibitor antagonized the above-mentioned effect of magnolol and partly offset the magnolol-induced decrease of p-IκBα and p-p65 expression, as well as the ratio of p-p65 to p65 in U266 and LP1 cells. Magnolol suppressed cell migration and invasion and induced cell apoptosis via inhibiting NF-κB pathway activation, by upregulating miR-129 in MM.

Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors.

In lower-order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans-differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long-Evens rat Müller glia were infected with Adeno-Lin28b or Adeno-GFP. Over-expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let-7 family of microRNAs. Following sub-retinal space transplantation, Müller glia-derived retinal progenitors improved b-wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS-P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up-regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors.

Curcumin Delays Retinal Degeneration by Regulating Microglia Activation in the Retina of rd1 Mice.

BACKGROUND/AIMS: Retinitis pigmentosa (RP) is characterized by degeneration of photoreceptors, and there are currently no effective treatments for this disease. However, curcumin has shown neuroprotectant efficacy in a RP rat and swine model, and thus, may have neuroprotective effects in this disease. METHODS: Immunofluorescence staining, electroretinogram recordings, and behavioral tests were used to analyze the effects of curcumin and the underlying mechanism in retinal degeneration 1 (rd1) mice. RESULTS: The number of apoptotic cells in the retina of rd1 mice at postnatal day 14 significantly decreased with curcumin treatment and visual function was improved. The activation of microglia and secretion of chemokines and matrix metalloproteinases in the retina were inhibited by curcumin. These effects were also observed in a co-culture of BV2 microglial cells and retina-derived 661W cells. CONCLUSIONS: Curcumin delayed retinal degeneration by suppressing microglia activation in the retina of rd1 mice. Thus, it may be an effective treatment for neurodegenerative disorders such as RP.

Neural stem cells transplanted to the subretinal space of rd1 mice delay retinal degeneration by suppressing microglia activation.

BACKGROUND AIMS: Retinal degeneration (RD) is an inherited eye disease characterized by irreversible photoreceptor loss. Conventionally, the activation of the resident microglia is secondary to the disease. Stem cell-based therapy has recently made rapid progress in treating RD. Although it has been demonstrated that the effect of stem cell therapy may include immunomodulation, the specific mechanisms have not been clarified. METHODS: Immunocytochemistry, terminal deoxynucleotidyl transferase UTP nick end labelling (TUNEL) assay and Western blot were used to analyze the microglia activation and photoreceptor apoptosis in the retina of rd1 mice. GFP-C17.2 neural stem cells (NSCs) were transplanted into the subretinal space to study the immunomodulatory and neuroprotective effects. The transwell co-culture of BV2 cells with GFP-C17.2 was performed to study the proliferation, apoptosis and secretion levels of inflammatory factors. Real time-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were performed to explore the gene and protein level of factors secreted by NSCs and microglia. RESULTS: TUNEL-positive cells were primarily distributed in the inner nuclear layer (INL) of rd1 mice on P8d, appeared in the outer nuclear layer (ONL) on P10d and peaked on P14d. Meanwhile, microglia migrated to the ONL and reached the maximum level, accompanied by the changes in the levels of fractalkine and its unique receptor CX3CR1 protein. After transplantation of NSCs on P7d into the subretinal space of rd1 mice, the activated microglia were inhibited and the degeneration of ONL was delayed. In addition, microglia activation was suppressed by co-cultured NSCs in vitro. The gene and protein level of tissue inhibitor of metalloproteinase (TIMP1) in NSCs was elevated, whereas that of matrix metalloproteinase (MMP9) in BV2 microglia was markedly suppressed in this co-culture system. CONCLUSIONS: Transplanted NSCs in the retina exerted immunomodulatory effects on microglia, thus delaying the degeneration of photoreceptors.

Subretinal transplantation of retinal pigment epithelium overexpressing fibulin-5 inhibits laser-induced choroidal neovascularization in rats.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Choroidal neovascularization (CNV) is the abnormal angiogenesis that causes severe visual loss in AMD. Fibulin-5 (Fbln5), which functions as an angiogenesis inhibitor, plays an important role in the pathogenesis of AMD. Here, we investigated whether subretinal transplantation of Fbln5-overexpressing retinal pigment epithelial (RPE) cells can inhibit CNV in vivo. Adult Long-Evans rats were used in this study. CNV was induced by laser photocoagulation. One week after laser-induced CNV, RPE cells expressing pZlen-Fbln5-IRES-GFP or the control pZlen-IRES-GFP vectors were transplanted into the subretinal space of the right and left eyes, respectively. CNV was evaluated using fundus photography, fundus fluorescein angiography (FFA), and hematoxylin and eosin staining. We found that CNV occurred at 1 week after photocoagulation, reaching peak activity at 3 weeks and remaining at a high level at 4-5 weeks after photocoagulation. Transplanted RPE cells survived for at least 4 weeks and migrated toward the retina. Subretinal transplantation of Fbln5-overexpressing RPE cells resulted in a significant reduction in the total area of leakage and the number of leakage spots compared with transplantation of RPE cells expressing only green fluorescent protein. Our findings suggest that subretinal transplantation of Fbln5-overexpressing RPE cells inhibits laser-induced CNV in rats and thus represents a promising therapy for the treatment of AMD.

Small extracellular vesicles of organoid-derived human retinal stem cells remodel Müller cell fate via miRNA: A novel remedy for retinal degeneration.

Remodeling retinal Müller glial fate, including gliosis inhibition and pro-reprogramming, represents a crucial avenue for treating degenerative retinal diseases. Stem cell transplantation exerts effects on modulating retinal Müller glial fate. However, the optimized stem cell products and the underlying therapeutic mechanisms need to be investigated. In the present study, we found that retinal progenitor cells from human embryonic stem cell-derived retinal organoids (hERO-RPCs) transferred extracellular vesicles (EVs) into Müller cells following subretinal transplantation into RCS rats. Small EVs from hERO-RPCs (hERO-RPC-sEVs) were collected and were found to delay photoreceptor degeneration and protect retinal function in RCS rats. hERO-RPC-sEVs were taken up by Müller cells both in vivo and in vitro, and inhibited gliosis while promoting early dedifferentiation of Müller cells. We further explored the miRNA profiles of hERO-RPC-sEVs, which suggested a functional signature associated with neuroprotection and development, as well as the regulation of stem cell and glial fate. Mechanistically, hERO-RPC-sEVs might regulate the fate of Müller cells by miRNA-mediated nuclear factor I transcription factors B (NFIB) downregulation. Collectively, our findings offer novel mechanistic insights into stem cell therapy and promote the development of EV-centered therapeutic strategies.

Extracellular vesicles from organoid-derived human retinal progenitor cells prevent lipid overload-induced retinal pigment epithelium injury by regulating fatty acid metabolism.

Retinal degeneration (RD), a group of diseases leading to irreversible vision loss, is characterised by retinal pigment epithelium (RPE) or retinal neuron damage and loss. With fewer risks of immune rejection and tumorigenesis, stem cell-secreted extracellular vesicles (EVs) offer a new cell-free therapeutic paradigm for RD, which remains to be investigated. Human retinal organoid-derived retinal progenitor cells (hERO-RPCs) are an easily accessible and advanced cell source for RD treatment. However, hERO-RPCs-derived EVs require further characterisation. Here, we compared the characteristics of EVs from hERO-RPCs (hRPC-EVs) with those of human embryonic stem cell (hESC)-derived EVs (hESC-EVs) as controls. Based on in-depth proteomic analysis, we revealed remarkable differences between hRPC-EVs and hESC-EVs. A comparison between EVs and their respective cells of origin demonstrated that the protein loading of hRPC-EVs was more selective than that of hESC-EVs. In particular, hESC-EVs were enriched with proteins related to angiogenesis and cell cycle, whereas hRPC-EVs were enriched with proteins associated with immune modulation and retinal development. More importantly, compared with that of hESC-EVs, hRPC-EVs exhibited a lower correlation with cell proliferation and a unique capacity to regulate lipid metabolism. It was further confirmed that hRPC-EVs potentially eliminated lipid deposits, inhibited lipotoxicity and oxidative stress, and enhanced phagocytosis and survival of oleic acid-treated ARPE-19 cells. Mechanistically, hRPC-EVs are integrated into the mitochondrial network of oleic acid-treated ARPE-19 cells, and increased the level of mitochondrial fatty acid ß-oxidation-related proteins. Thus, organoid-derived hRPC-EVs represent a promising source of cell-free therapy for RD, especially for blinding diseases related to abnormal lipid metabolism in RPE cells.

Identifying Hmga2 preserving visual function by promoting a shift of Müller glia cell fate in mice with acute retinal injury.

BACKGROUND: Unlike in lower vertebrates, Müller glia (MG) in adult mammalian retinas lack the ability to reprogram into neurons after retinal injury or degeneration and exhibit reactive gliosis instead. Whether a transition in MG cell fate from gliosis to reprogramming would help preserve photoreceptors is still under exploration. METHODS: A mouse model of retinitis pigmentosa (RP) was established using MG cell lineage tracing mice by intraperitoneal injection of sodium iodate (SI). The critical time point for the fate determination of MG gliosis was determined through immunohistochemical staining methods. Then, bulk-RNA and single-cell RNA seq techniques were used to elucidate the changes in RNA transcription of the retina and MG at that time point, and new genes that may determine the fate transition of MG were screened. Finally, the selected gene was specifically overexpressed in MG cells through adeno-associated viruses (AAV) in the mouse RP model. Bulk-RNA seq technique, immunohistochemical staining methods, and visual function testing were used to elucidate and validate the mechanism of new genes function on MG cell fate transition and retinal function. RESULTS: Here, we found the critical time point for MG gliosis fate determination was 3 days post SI injection. Hmga2 was screened out as a candidate regulator for the cell fate transition of MG. After retinal injury caused by SI, the Hmga2 protein is temporarily and lowly expressed in MG cells. Overexpression of Hmga2 in MG down-regulated glial cell related genes and up-regulated photoreceptor related genes. Besides, overexpressing Hmga2 exclusively to MG reduced MG gliosis, made MG obtain cone's marker, and retained visual function in mice with acute retinal injury. CONCLUSION: Our results suggested the unique reprogramming properties of Hmga2 in regulating the fate transition of MG and neuroprotective effects on the retina with acute injury. This work uncovers the reprogramming ability of epigenetic factors in MG.

Carcinogenic health outcomes associated with endocrine disrupting chemicals exposure in humans: A wide-scope analysis.

Endocrine-disrupting chemicals (EDCs) are persistent and pervasive compounds that pose serious risks. Numerous studies have explored the effects of EDCs on human health, among which tumors have been the primary focus. However, because of study design flaws, lack of effective exposure levels of EDCs, and inconsistent population data and findings, it is challenging to draw clear conclusions on the effect of these compounds on tumor-related outcomes. Our study is the first to systematically integrate observational studies and randomized controlled trials from over 20 years and summarize over 300 subgroup associations. We found that most EDCs promote tumor development, and that exposure to residential environmental pollutants may be a major source of pesticide exposure. Furthermore, we found that phytoestrogens exhibit antitumor effects. The findings of this study can aid in the development of global EDCs regulatory health policies and alleviate the severe risks associated with EDCs exposure.

Human bone marrow stromal cells can differentiate to a retinal pigment epithelial phenotype when co-cultured with pig retinal pigment epithelium using a transwell system.

BACKGROUND: There is an increasing interest in generating retinal pigment epithelial (RPE) cells from stem cells for therapy against degenerative eye diseases. Human bone marrow stromal cells (hBMSCs) can be induced to express retinal neuron-specific markers when co-cultured with retinal neurons, however, whether hBMSCs can differentiate into RPE-like cells in a co-culture system has not been clarified. METHODS: The induction of hBMSCs into RPE-like cells was performed by combining hBMSCs and pig RPE cells in a transwell system. The biomarkers of hBMSCs-derived RPE cells were determined by quantitative RT-PCR and immunofluorescence. The function of induced cells was assayed by ELISA for secretion of neurotrophic factors. RESULTS: Intracellular pigment granules and many RPE markers existed in hBMSCs-derived RPE cells after co-culturing with pig RPE cells for 14 days. Typical RPE functions, such as phagocytosis of photoreceptor outer segments and secretion of the trophic factors, brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF), were observed in these induced cells. CONCLUSION: hBMSCs can be induced toward functional RPE cells simply by transwell-based co-culture with RPE cells.

Characterization of five pigmentation genes as transgenic markers in Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm, Spodoptera frugiperda (J. E. Smith), has become one of the most damaging pests worldwide since its invasion of Africa, Asia and Oceania from 2016, threatening plants in 76 families including important crops. Genetics-based methods have proved to be an efficient way to control pests, especially invasive species, but many difficulties must be overcome to develop a transgenic insect strain, especially for a non-model species. Here we thus sought to identify a visible marker that would facilitate the distinction between genetically modified (GM) and non-transgenic insects, thereby simplifying mutation identification and facilitating the broader application of genome editing tools in non-model insects. Five genes (sfyellow-y, sfebony, sflaccase2, sfscarlet, and sfok) that are orthologs of well-studied genes in pigment metabolism were knocked out using the CRISPR/Cas9 system to identify candidate gene markers. Two genes, Sfebony and Sfscarlet, were identified responsible for body and compound eye coloration, respectively, in S. frugiperda, and could be potential visual markers for genetics-based pest management strategies.

Annihilation of Non-small Cell Lung Cancer by NKG2D CAR-T Cells Produced from T Cells from Peripheral Blood of Healthy Donors.

Some progress has been made in immunotherapy with chimeric antigen receptor (CAR)-T cells targeting NKG2D-NKG2DL with the purpose of eradicating solid tumors. Non-small cell lung cancer (NSCLC) has been shown to express NKG2DL. This study hence evaluated the therapeutic effect of NKG2D CAR-T cells on NSCLC. Accordingly, NKG2D CAR-T cells were obtained from diverse human autologous T cell sources. T cells from peripheral blood T lymphocytes of healthy volunteers (without NKG2D CAR insertion) were used as NT-T cells. Coculture of effector cells (CAR-T cells or NT-T cells) with target cells (NSCLC cells such as PC-9 or NCL-H460 cells) was performed at different ratios. The cytotoxicity of CAR-T cells was examined using lactate dehydrogenase assay kits. Murine xenograft assay was conducted to investigate the in vivo antitumor effect of CAR-T cells. Cytokines secreted from CAR-T cells were assessed by enzyme-linked immunosorbent assay. CAR-T cell infiltration into xenografts was observed through immunochemical assay. Based on the results, NKG2DL was highly expressed in NSCLC cells. Compared with NT-T cells, NKG2D CAR-T cells from different sources of T cells delivered stronger toxicity, and secreted more effector and memory function-related cytokines to NSCLC cells, and those from the peripheral blood of healthy donors (H-T cells) exhibited the strongest effect. Furthermore, compared with NT-T cells, H-T cells and NKG2D CAR-T cells from NSCLC patients' peripheral blood diminished tumor, improved survival, increased body weight and tumor-infiltrating capacity, and upregulated serum IFN-γ level in NOG mice. Collectively speaking, NKG2D CAR-T cells exhibit a robust effect on eradicating NSCLC in a NKG2DL-dependent manner, thus making themselves a promising therapeutic candidate for NSCLC patients.

Organoid-derived human retinal progenitor cells promote early dedifferentiation of Müller glia in Royal College of Surgeons rats.

AIM: To explore whether the subretinal transplantation of retinal progenitor cells from human embryonic stem cell-derived retinal organoid (hERO-RPCs) could promote Müller glia dedifferentiation and transdifferentiation, thus improving visual function and delaying retinal degenerative progression. METHODS: hERO-RPCs were subretinally transplanted into Royal College of Surgeons (RCS) rats. Electroretinography (ERG) recording was performed at 4 and 8wk postoperation to assess retinal function. Using immunofluorescence, the changes in outer nuclear layer (ONL) thickness and retinal Müller glia were explored at 2, 4, and 8wk postoperation. To verify the effect of hERO-RPCs on Müller glia in vitro, we cocultured hERO-RPCs with Müller glia with a Transwell system. After coculture, Ki67 staining and quantitative polymerase chain reaction (qPCR) were performed to measure the proliferation and mRNA levels of Müller glia respectively. Cell migration experiment was used to detect the effect of hERO-RPCs on Müller glial migration. Comparisons between two groups were performed by the unpaired Student's t-test, and comparisons among multiple groups were made with one-way ANOVA followed by Tukey's multiple comparison test. RESULTS: The visual function and ONL thickness of RCS rats were significantly improved by transplantation of hERO-RPCs at 4 and 8wk postoperation. In addition to inhibiting gliosis at 4 and 8wk postoperation, hERO-RPCs significantly increased the expression of dedifferentiation-associated transcriptional factor in Müller glia and promoted the migration at 2, 4 and 8wk postoperation, but not the transdifferentiation of these cells in RCS rats. In vitro, using the Transwell system, we found that hERO-RPCs promoted the proliferation and migration of primary rat Müller glia and induced their dedifferentiation at the mRNA level. CONCLUSION: These results show that hERO-RPCs might promote early dedifferentiation of Müller glia, which may provide novel insights into the mechanisms of stem cell therapy and Müller glial reprogramming, contributing to the development of novel therapies for retinal degeneration disorders.