Dynamic monitoring of TGW6 by selective autophagy during grain development in rice.

Crop yield must increase to achieve food security in the face of a growing population and environmental deterioration. Grain size is a prime breeding target for improving grain yield and quality in crop. Here, we report that autophagy emerges as an important regulatory pathway contributing to grain size and quality in rice. Mutations of rice Autophagy-related 9b (OsATG9b) or OsATG13a causes smaller grains and increase of chalkiness, whereas overexpression of either promotes grain size and quality. We also demonstrate that THOUSAND-GRAIN WEIGHT 6 (TGW6), a superior allele that regulates grain size and quality in the rice variety Kasalath, interacts with OsATG8 via the canonical Atg8-interacting motif (AIM), and then is recruited to the autophagosome for selective degradation. In consistent, alteration of either OsATG9b or OsATG13a expression results in reciprocal modulation of TGW6 abundance during grain growth. Genetic analyses confirmed that knockout of TGW6 in either osatg9b or osatg13a mutants can partially rescue their grain size defects, indicating that TGW6 is one of the substrates for autophagy to regulate grain development. We therefore propose a potential framework for autophagy in contributing to grain size and quality in crops.

Internalization of the TAT-PBX1 fusion protein significantly enhances the proliferation of human hair follicle-derived mesenchymal stem cells and delays their senescence.

OBJECTIVES: To express a TAT-PBX1 fusion protein using a prokaryotic expression system and to explore potential effects of TAT-PBX1 in the proliferation and senescence of human hair follicle-derived mesenchymal stem cells. RESULTS: The TAT-PBX1 fusion was produced in inclusion bodies and heterogenously expressed in Rosetta (DE3) cells. Immunofluorescence staining showed that TAT-PBX1 fusion proteins were internalized by human hair follicle-derived mesenchymal stem cells. The growth rate of cells was increased after treatment with more than 5.0 µg/mL of TAT-PBX1. The rate of senescence-associated ß-galactosidase positive cells was reduced in the 10.0 µg/mL TAT-PBX1 group (28%) than the 0 µg/mL control group (60%). Cells treated with the TAT-PBX1 fusion protein showed higher expression of p-AKT (1.22-fold that of the control), which indicates that TAT-PBX1 activated AKT pathway after cellular uptake. CONCLUSIONS: The TAT-PBX1 fusion protein increased the proliferation of hair follicle mesenchymal stem cells and delayed their senescence by activating the AKT pathway following internalization by cells.

Neurotoxic ß-amyloid oligomers cause mitochondrial dysfunction-the trigger for PANoptosis in neurons.

As the global population ages, the incidence of elderly patients with dementia, represented by Alzheimer's disease (AD), will continue to increase. Previous studies have suggested that ß-amyloid protein (Aß) deposition is a key factor leading to AD. However, the clinical efficacy of treating AD with anti-Aß protein antibodies is not satisfactory, suggesting that Aß amyloidosis may be a pathological change rather than a key factor leading to AD. Identification of the causes of AD and development of corresponding prevention and treatment strategies is an important goal of current research. Following the discovery of soluble oligomeric forms of Aß (AßO) in 1998, scientists began to focus on the neurotoxicity of AßOs. As an endogenous neurotoxin, the active growth of AßOs can lead to neuronal death, which is believed to occur before plaque formation, suggesting that AßOs are the key factors leading to AD. PANoptosis, a newly proposed concept of cell death that includes known modes of pyroptosis, apoptosis, and necroptosis, is a form of cell death regulated by the PANoptosome complex. Neuronal survival depends on proper mitochondrial function. Under conditions of AßO interference, mitochondrial dysfunction occurs, releasing lethal contents as potential upstream effectors of the PANoptosome. Considering the critical role of neurons in cognitive function and the development of AD as well as the regulatory role of mitochondrial function in neuronal survival, investigation of the potential mechanisms leading to neuronal PANoptosis is crucial. This review describes the disruption of neuronal mitochondrial function by AßOs and elucidates how AßOs may activate neuronal PANoptosis by causing mitochondrial dysfunction during the development of AD, providing guidance for the development of targeted neuronal treatment strategies.

Segregation of the stemness program from the proliferation program in intestinal stem cells.

Stem cells can undergo continuous self-renewal and meanwhile retain the stemness capability to differentiate to mature functional cells. However, it is unclear whether the proliferation property can be segregated from the stemness in stem cells. The intestinal epithelium undergoes fast renewal, and the Lgr5+ intestinal stem cells (ISCs) are essential to maintain homeostasis. Here, we report that methyltransferase-like 3 (Mettl3), a critical enzyme for N6-methyladenosine (m6A) methylation, is required for ISCs maintenance as its deletion results in fast loss of stemness markers but has no effect on cell proliferation. We further identify four m6A-modified transcriptional factors, whose ectopic expression can restore stemness gene expression in Mettl3-/- organoids, while their silencing leads to stemness loss. In addition, transcriptomic profiling analysis discerns 23 genes that can be segregated from the genes responsible for cell proliferation. Together, these data reveal that m6A modification sustains ISC stemness, which can be uncoupled from cell proliferation.

PBX1-SIRT1 Positive Feedback Loop Attenuates ROS-Mediated HF-MSC Senescence and Apoptosis.

Stem cell senescence and depletion are major causes of aging and aging-related diseases. The NAD (Nicotinamide adenine dinucleotide) - SIRT1 (Silent Information Regulator 1) - PARP1 (Poly (ADP-ribose) polymerase-1) axis has gained interest owing to its significant role in regulating stem cell senescence and organismal aging. A recent study from our lab showed that pre-B-cell leukemia transcription factor1 (PBX1) overexpression attenuates hair follicle-derived mesenchymal stem cells (HF-MSCs) senescence and apoptosis by regulating ROS-mediated DNA damage via PARP1 downregulation; thus, suggesting that PARP1 downregulation is a common manifestation of the roles of both PBX1 and SIRT1 in HF-MSCs senescence attenuation, and implying a potential link between PBX1 and SIRT1. To this end, HF-MSCs overexpressing PBX1, overexpressing both PBX1 and PARP1, downregulating SIRT1, and overexpressing PBX1 as well as downregulating SIRT1 were generated, and senescence, apoptosis, DNA damage, and repair biomarkers were analyzed. Our results showed that (1) PBX1 overexpression alleviated HF-MSCs senescence and apoptosis accompanied by SIRT1 upregulation, PARP1 downregulation, and increased intracellular NAD and ATP levels. (2) SIRT1 knockdown enhanced cellular senescence and apoptosis, accompanied by increased ROS accumulation, DNA damage aggravation, and decreased intracellular NAD and ATP levels. (3) PBX1 overexpression rescued HF-MSCs senescence and apoptosis induced by SIRT1 knockdown. (4) PBX1 rescued PARP1 overexpression-mediated ATP and NAD depletion, accompanied by increased SIRT1 expression. Collectively, our results revealed that a positive interaction feedback loop exists between PBX1 and SIRT1. To the best of our knowledge we are the first to report that there is a PBX1-SIRT1-PARP1 axis that plays a critical role in alleviating HF-MSCs senescence and apoptosis. We provide a new perspective on the mechanisms underlying stem cell senescence as well as age-related disease prevention and treatment.

L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells.

Methylmercury (MeHg) is a toxin that causes severe neuronal oxidative damage. As vitamin C is an antioxidant well-known to protect neurons from oxidative damage, our goal was to elucidate its protective mechanism against MeHg-induced oxidative stress in human neuroblastomas (SHSY5Y). We treated cells with MeHg, L-ascorbic acid 2-phosphate (AA2P), or both, and used MTT, flow cytometry, and Western blot analyses to assess cell damage. We found that MeHg significantly decreased the survival rate of SH-SY5Y cells in a time- and dose-dependent manner, increased apoptosis, downregulated PAR and PARP1 expression, and upregulated AIF, Cyto C, and cleaved Caspase-3 expression. A time course study showed that MeHg increased reactive oxygen species (ROS) accumulation; enhanced apoptosis; increased DNA damage; upregulated expression ofγH2A.X, KU70, 67 and 57 kDa AIF, CytoC, and cleaved Caspase-3; and downregulated expression of 116 kDa PARP1, PAR, BRAC1, and Rad51. Supplementation with AA2P significantly increased cell viability and decreased intrinsic ROS accumulation. It also reduced ROS accumulation in cells treated with MeHg and decreased MeHg-induced apoptosis. Furthermore, AA2P conversely regulated gene expression compared to MeHg. Collectively, we demonstrate that AA2P attenuates MeHg-induced apoptosis by alleviating ROS-mediated DNA damage and is a potential treatment for MeHg neurotoxicity.

Intrinsic ROS Drive Hair Follicle Cycle Progression by Modulating DNA Damage and Repair and Subsequently Hair Follicle Apoptosis and Macrophage Polarization.

Hair follicles (HFs) maintain homeostasis through the hair cycles; therefore, disrupting the hair cycle may lead to hair loss. Our previous study showed that apoptosis-inducing factor (AIF) nuclear translocation and poly [ADP-ribose] polymerase 1 (PARP1) upregulation induced apoptosis in mouse hair follicles during the hair cycle transition from anagen to catagen. However, the mechanism underlying this phenomenon remains unclear. In this study, we found that intrinsic ROS levels increased during the hair follicle cycle transition from anagen to catagen, followed by abrupt DNA breaks and activation of homologous recombinant and nonhomologous end joining DNA repair, along with the enhancement of apoptosis. Mice in different stages of the hair cycle were sacrificed, and the dorsal skins were collected. The results of western blot and histological staining indicated that AIF-PARP1 plays a key role in HF apoptosis, but their role in the regulation of the HF cycle is not clear. Mice were treated with inhibitors from anagen to catagen: treatment with BMN 673, a PARP1 inhibitor, increased DNA breaks and activated the cytochrome c/caspase-3-mediated apoptotic pathway, accelerating HF regression. Ac-DEVD-CHO (Ac), a caspase-3 inhibitor, attenuated HF degeneration by upregulating PARP1 expression, suggesting a seesaw relationship between cytochrome c-caspase-3- and AIF-PARP1-mediated apoptosis, wherein PARP1 may be the fulcrum. In addition, macrophages were involved in regulating the hair cycle, and the rate of M1 macrophages around HFs increased during catagen, while more M2 macrophages were found during anagen and telogen. Our results indicate that intrinsic ROS drive HF cycle progression through DNA damage and repair, followed by apoptosis. Intrinsic ROS drive hair follicle cycle progression by modulating DNA damage and repair, and consecutively, hair follicle apoptosis and macrophage polarization work together to promote the hair follicle cycle.

ATG8-Interacting Motif: Evolution and Function in Selective Autophagy of Targeting Biological Processes.

Autophagy is an evolutionarily conserved vacuolar process functioning in the degradation of cellular components for reuse. In plants, autophagy is generally activated upon stress and its regulation is executed by numbers of AuTophaGy-related genes (ATGs), of which the ATG8 plays a dual role in both biogenesis of autophagosomes and recruitment of ATG8-interacting motif (AIM) anchored selective autophagy receptors (SARs). Such motif is either termed as AIM or ubiquitin-interacting motif (UIM), corresponding to the LC3-interacting region (LIR)/AIM docking site (LDS) or the UIM docking site (UDS) of ATG8, respectively. To date, dozens of AIM or UIM containing SARs have been characterized. However, the knowledge of these motifs is still obscured. In this review, we intend to summarize the current understanding of SAR proteins and discuss the conservation and diversification of the AIMs/UIMs, expectantly providing new insights into the evolution of them in various biological processes in plants.

PBX1 Attenuates Hair Follicle-Derived Mesenchymal Stem Cell Senescence and Apoptosis by Alleviating Reactive Oxygen Species-Mediated DNA Damage Instead of Enhancing DNA Damage Repair.

Tissues and organs undergo structural deterioration and functional decline during aging. DNA damage is considered a major cause of stem cell senescence. Although stem cells develop sophisticated DNA repair systems, when the intrinsic and extrinsic insults exceed the DNA repair capacity, cellular senescence, and age-related diseases inevitably occur. Therefore, the prevention and alleviation of DNA damage is an alternative to DNA repair in attenuating stem cell senescence and preventing age-related diseases. Pre-B-cell leukaemia homeobox 1 (PBX1) participates in maintaining the pluripotency of human embryonic and haematopoietic stem cells. Our recent studies showed that PBX1 promotes hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation, decreases cellular senescence and apoptosis, and enhances induced pluripotent stem cell generation. Whether PBX1 attenuates HF-MSC senescence and apoptosis by alleviating DNA damage or by enhancing DNA repair remains unknown. In this study, we aimed to determine the effects of PBX1 on the intrinsic ROS or extrinsic H2O2-induced cellular senescence of HF-MSCs. To this end, we generated HF-MSCs overexpressing either PBX1, or poly (ADP-ribose) polymerase 1, or both. Our results showed that PBX1 overexpression attenuates HF-MSC senescence and apoptosis by alleviating reactive oxygen species (ROS)-mediated DNA damage instead of enhancing DNA repair. This is the first study to report that PBX1 attenuates stem cell senescence and apoptosis by alleviating DNA damage. It provides new insight into the mechanism of stem cell senescence and lays the foundation for the development of strategies for age-related disease prevention and treatment, and in particular, hair follicle repair and regeneration.

Melanocytes derived from mouse hair follicles: A novel study model to assess pigmentation disorders.

Melanocytes are the major cells responsible for skin and fair pigmentation in vertebrates. They localize to hair follicles(HFs) and the epidermis during embryonic development. A reduced number or dysfunction of melanocytes results in pigmentation disorders.Thus, methods for isolation, culture, and identification of melanocytes in mouse hair follicles provide an experimental basis for thestudy of of pigmentation disorders. In the current work, we harvested the melanocytes from the anagen phase dorsal skin of C57BL/6 mice.After its separation from the skin, the dermis was digested, and the HFs were released. HFs were then also digested, and the cells released from HFs were cultured in melanocyte growth medium. Immunofluorescence and immunohistochemistry staining were used to localize the distribution of melanocytes in HFs . Reverse transcription polymerase chain reaction was performed to detect the expression of specific melanocyte marker genes. Immunofluorescence, immunohistochemistry, flow cytometry, and western blot were carried out to detect the expression of marker proteins in cells. 3,4-Dihydroxy-L-phenylalanine (L-DOPA) staining was used to detect the pigmentation functionality of melaonocytes. Based on our results, we conclude that mature and functional melanocytes can be successfully obtained from theHFs, providing a cell model to study pigmentation disorders. The current findings provide novel insights for the treatment of pigmentation disorders by autologous cell transplantation. Further, we believe that issues related to skin damage, insufficient numbers of autologous cells, and autoimmune problems can be resolved in future though the use of functional melanocytes.

Human hair follicle-derived mesenchymal stem cells: Isolation, expansion, and differentiation.

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.

MSC-derived exosomes attenuate cell death through suppressing AIF nucleus translocation and enhance cutaneous wound healing.

BACKGROUND: Skin wounding is very common and may be slow to heal. Increasing evidence shows that exosomes derived from mesenchymal stem cells (MSCs) dramatically enhance skin wound healing in a paracrine manner. However, the mechanism underlying this phenomenon has not yet been elucidated. Thus, the objective of the present study was to identify the signaling pathways and paracrine factors by which MSC-derived exosomes promote de novo skin tissue regeneration in response to wound healing. METHODS: In vitro and in vivo skin wound healing models were created by treating immortalized human keratinocytes (HaCaT) with hydrogen peroxide (H2O2) and excising full-thickness mouse skin, respectively. Exosomes were extracted from human umbilical cord Wharton's jelly MSCs (hucMSC-Ex) by ultracentrifugation of cell culture supernatant. RESULTS: The hucMSC-Ex treatment significantly increased HaCaT cell proliferation and migration in a time- and dose-dependent manner, suppressed HaCaT apoptosis induced with H2O2 by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and upregulating poly ADP ribose polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). The animal experiments showed that relative to hucMSCs, hucMSC-Ex attenuated full-thickness skin wounding by enhancing epidermal re-epithelialization and dermal angiogenesis. CONCLUSIONS: These findings indicated that direct administration of hucMSC-Ex may effectively treat cutaneous wounding and could be of great value in clinical settings.

PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis.

BACKGROUND: PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3ß pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs. METHODS: A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT. RESULTS: Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of ß-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3ß pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency. CONCLUSIONS: PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3ß signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3ß pathway during the early stages of reprogramming.

Growth and Physiological Responses to Water Depths in Carex schmidtii Meinsh.

A greenhouse experiment was performed to investigate growth and physiological responses to water depth in completely submerged condition of a wetland plant Carex schmidtii Meinsh., one of the dominant species in the Longwan Crater Lake wetlands (China). Growth and physiological responses of C. schmidtii were investigated by growing under control (non-submerged) and three submerged conditions (5 cm, 15 cm and 25 cm water level). Total biomass was highest in control, intermediate in 5 cm treatment and lowest in the other two submerged treatments. Water depth prominently affected the first-order lateral root to main root mass ratio. Alcohol dehydrogenase (ADH) activity decreased but malondialdehyde (MDA) content increased as water depth increased. The starch contents showed no differences among the various treatments at the end of the experiment. However, soluble sugar contents were highest in control, intermediate in 5 cm and 15 cm treatments and lowest in 25 cm treatment. Our data suggest that submergence depth affected some aspects of growth and physiology of C. schmidtii, which can reduce anoxia damage not only through maintaining the non-elongation strategy in shoot part but also by adjusting biomass allocation to different root orders rather than adjusting root-shoot biomass allocation.

Fungal pretreatment of cornstalk with Phanerochaete chrysosporium for enhancing enzymatic saccharification and hydrogen production.

The feasibility of fungal pretreatment of cornstalk with Phanerochaete chrysosporium for enzymatic saccharification and H(2) production was investigated in this study. Firstly, cornstalk was pretreated with P. chrysosporium at 29 °C under static condition for 15 d, lignin reduction was up to 34.3% with holocellulose loss less than 10%. Microscopic structure observation combined FTIR analysis further demonstrated that the lignin and crystallinity were decreased. Subsequently, the fungal-pretreated cornstalk was subjected to enzymatic hydrolysis by the crude cellulase from Trichoderma viride to produce fermentable sugars which were then fermented to bio-H(2) using Thermoanaerobacterium thermosaccharolyticum W16. The maximum enzymatic saccharification was found to be 47.3% which was 20.3% higher than the control without pretreatment. Upon fermentation of enzymatic hydrolysate, the yield of H(2) was calculated to be 80.3 ml/g-pretreated cornstalk. The present results suggested the potential of using hydrogen-producing bacteria for high-yield conversion of cornstalk into bio-H(2) integrate with biological pretreatment and enzymatic saccharification.