Rab5c-mediated endocytic trafficking regulates hematopoietic stem and progenitor cell development via Notch and AKT signaling.

It is well known that various developmental signals play diverse roles in hematopoietic stem and progenitor cell (HSPC) production; however, how these signaling pathways are orchestrated remains incompletely understood. Here, we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium (HE) specification by endocytic trafficking of Notch ligands and receptor. We further show that the interaction between Rab5c and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKT signaling. Interestingly, Rab5c overactivation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.

COM1, a factor of alternative non-homologous end joining, lagging behind the classic non-homologous end joining pathway in rice somatic cells.

The role of rice (Oryza sativa) COM1 in meiotic homologous recombination (HR) is well understood, but its part in somatic double-stranded break (DSB) repair remains unclear. Here, we show that for rice plants COM1 conferred tolerance against DNA damage caused by the chemicals bleomycin and mitomycin C, while the COM1 mutation did not compromise HR efficiencies and HR factor (RAD51 and RAD51 paralogues) localization to irradiation-induced DSBs. Similar retarded growth at the post-germination stage was observed in the com1-2 mre11 double mutant and the mre11 single mutant, while combined mutations in COM1 with the HR pathway gene (RAD51C) or classic non-homologous end joining (NHEJ) pathway genes (KU70, KU80, and LIG4) caused more phenotypic defects. In response to γ-irradiation, COM1 was loaded normally onto DSBs in the ku70 mutant, but could not be properly loaded in the MRE11RNAi plant and in the wortmannin-treated wild-type plant. Under non-irradiated conditions, more DSB sites were occupied by factors (MRE11, COM1, and LIG4) than RAD51 paralogues (RAD51B, RAD51C, and XRCC3) in the nucleus of wild-type; protein loading of COM1 and XRCC3 was increased in the ku70 mutant. Therefore, quite differently to its role for HR in meiocytes, rice COM1 specifically acts in an alternative NHEJ pathway in somatic cells, based on the Mre11-Rad50-Nbs1 (MRN) complex and facilitated by PI3K-like kinases. NHEJ factors, not HR factors, preferentially load onto endogenous DSBs, with KU70 restricting DSB localization of COM1 and XRCC3 in plant somatic cells.

OsATM Safeguards Accurate Repair of Meiotic Double-Strand Breaks in Rice.

ATAXIA TELANGIECTASIA-MUTATED (ATM) protein has been well studied for its roles in the DNA damage response. However, its role in meiosis has not been fully explored. Here, we characterized the functions of the rice (Oryza sativa) ATM homolog during meiosis. Aberrant chromosome associations and DNA fragmentations were observed after the completion of homologous pairing and synapsis in Osatm pollen mother cells (PMCs). Aberrant chromosome associations disappeared in Osspo11-1 Osatm-1 double mutants and more severe defects were observed in Osdmc1 Osatm, suggesting that OsATM functions downstream of OsSPO11-1-catalyzed double-strand break formation and in parallel with OsDMC1-mediated homologous recombination. We further demonstrated that phosphorylation of H2AX in PMCs did not depend on OsATM, in contrast to the situation in somatic cells. Moreover, the removal of OsDMC1 from chromosomes in Osatm PMCs was delayed and the number of HEI10 foci (markers of interference-sensitive crossover intermediates) decreased. Together, these findings suggest that OsATM plays important roles in the accurate repair of meiotic double-strand breaks in rice.

Recent advance in the discovery of tyrosinase inhibitors from natural sources via separation methods.

Tyrosinase (TYR) inhibitors are in great demand in the food, cosmetic and medical industrials due to their important roles. Therefore, the discovery of high-quality TYR inhibitors is always pursued. Natural products as one of the most important sources of bioactive compounds discovery have been increasingly used for TYR inhibitors screening. However, due to their complex compositions, it is still a great challenge to rapid screening and identification of biologically active components from them. In recent years, with the help of separation technologies and the affinity and intrinsic activity of target enzymes, two advanced approaches including affinity screening and inhibition profiling showed great promises for a successful screening of bioactive compounds from natural sources. This review summarises the recent progress of separation-based methods for TYR inhibitors screening, with an emphasis on the principle, application, advantage, and drawback of each method along with perspectives in the future development of these screening techniques and screened hit compounds.

OsPINOID Regulates Stigma and Ovule Initiation through Maintenance of the Floral Meristem by Auxin Signaling.

Stigma and ovule initiation is essential for sexual reproduction in flowering plants. However, the mechanism underlying the initiation of stigma and ovule primordia remains elusive. We identified a stigma-less mutant of rice (Oryza sativa) and revealed that it was caused by the mutation in the PINOID (OsPID) gene. Unlike the pid mutant that shows typical pin-like inflorescences in maize (Zea mays) and Arabidopsis (Arabidopsis thaliana), the ospid mutant does not display any defects in inflorescence development and flower initiation, and fails to develop normal ovules in most spikelets. The auxin activity in the young pistil of ospid was lower than that in the wild-type pistil. Furthermore, the expression of most auxin response factor genes was down-regulated, and OsETTIN1, OsETTIN2, and OsMONOPTEROS lost their rearrangements of expression patterns during pistil and stamen primordia development in ospid Moreover, the transcription of the floral meristem marker gene, OSH1, was down-regulated and FLORAL ORGAN NUMBER4, the putative ortholog of Arabidopsis CLAVATA3, was up-regulated in the pistil primordium of ospid These results suggested that the meristem proliferation in the pistil primordium might be arrested prematurely in ospid Based on these results, we propose that the OsPID-mediated auxin signaling pathway plays a crucial role in the regulation of rice stigma and ovule initiation by maintaining the floral meristem.

On-line pre-column FRAP-based antioxidant reaction coupled with HPLC-DAD-TOF/MS for rapid screening of natural antioxidants from different parts of Polygonum viviparum.

Polygonum viviparum L. (PV) is a widely used resource plant with high medicinal, feeding and ecological values. Our studies show that PV has strong antioxidant activity. However, up to date, the antioxidant activity and components in other parts were not fully elucidated. In the present study, a new online pre-column ferric ion reducing antioxidant power (FRAP)-based antioxidant reaction coupled with high performance liquid chromatography-diode array detector-quadrupole-time-of-flight mass spectrometry (HPLC-DAD-TOF/MS) was developed for rapid and high-throughput screening of natural antioxidants from three different parts of PV including stems and leaves, fruits and rhizomes. In this procedure, it was assumed that the peak areas of compounds with potential antioxidant activity in HPLC chromatograms would be greatly diminished or vanish after incubating with the FRAP. The online incubation conditions including mixed ratios of sample and FRAP solution and reaction times were firstly optimized with six standards. Then, the repeatability of the screening system was evaluated by analysis of the samples of stems and leaves of PV. As a result, a total of 21 compounds mainly including flavonoids and phenolic acids were screened from the three parts of PV. In conclusion, the present study provided a simple and effective strategy to rapidly screen antioxidants in natural products.

The Influence of Different Extraction Techniques on the Chemical Profile and Biological Properties of Oroxylum indicum: Multifunctional Aspects for Potential Pharmaceutical Applications.

Oroxylum indicum (L.) Kurz (Bignoniaceae), a traditional Chinese herbal medicine, possesses various biological activities including antioxidant, anti-inflammatory, antibacterial, and anticancer. In order to guide the practical application of O. indicum in the pharmaceutical, food, and cosmetic industries, we evaluated the effects of five different extraction techniques (maceration extraction (ME), oxhlet extraction (SOXE), ultrasound-assisted extraction (UAE), tissue-smashing extraction (TSE), and accelerated-solvent extraction (ASE)) with 70% ethanol as the solvent on the phytochemical properties and biological potential. The UHPLC-DAD Orbitrap Elite MS technique was applied to characterize the main flavonoids in the extracts. Simultaneously, the antioxidant and enzyme inhibitory activities of the tested extracts were analyzed. SOXE extract showed the highest total phenolic content (TPC, 50.99 ± 1.78 mg GAE/g extract), while ASE extract displayed the highest total flavonoid content (TFC, 34.92 ± 0.38 mg RE/g extract), which displayed significant correlation with antioxidant activity. The extract obtained using UAE was the most potent inhibitor of tyrosinase (IC50: 16.57 ± 0.53 mg·mL-1), while SOXE extract showed the highest activity against α-glucosidase (IC50: 1.23 ± 0.09 mg·mL-1), succeeded by UAE, ME, ASE, and TSE extract. In addition, multivariate analysis suggested that different extraction techniques could significantly affect the phytochemical properties and biological activities of O. indicum. To sum up, O. indicum displayed expected biological potential and the data collected in this study could provide an experimental basis for further investigation in practical applications.

Rapid screening of natural-origin tyrosinase regulators from Vernonia anthelmintica (L.) Willd. by offline two-dimensional liquid chromatography coupled with mass spectrometry.

Finding and developing safe and effective tyrosinase (TYR) regulators is of great significance for the prevention and treatment of melanin-related skin diseases in the medical and cosmetic industries. In the current research, an approach based on offline two-dimensional liquid chromatography coupled with mass spectrometry (offline 2D LC-MS) was established to screen TYR modulators from Vernonia anthelmintica (L.) Willd. (VA) extract. Firstly, the reliability of the proposed method was evaluated by using kojic acid (inhibitor), psoralen (activator) and ranitidine as positive and negative control, respectively. Some significant parameters including incubation time, TYR concentrations, and reaction temperature were investigated. Then, the developed new method was successfully applied to rapidly discover the active compounds from VA extract. Seven TYR ligands were successfully screened by comparing the chromatographic profiles of VA extract incubated with active and denatured TYR, respectively. To verify the activity of the screened compounds, in vitro bioassay was carried out and the result showed two of them, isorhamnetin and luteolin, had good TYR inhibitory activity with IC50 value of 0.86 and 1.00 mg/mL, respectively, while the other five compounds including eriodictyol, butochalcone, chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C showed strong activation against TYR. Furthermore, molecular docking displayed that these compounds could bind to the amino acid residues in TYR catalytic pocket. The results demonstrate that the established technique can be efficiently used for rapid screening of TYR-active compounds from plant extracts.

Identification of Ferroptosis Biomarker in AHH-1 Lymphocytes Associated with Low Dose Radiation.

ABSTRACT: The impact of long-term low-dose radiation on human health has always been a concern. Long-term low-dose gamma radiation causes cells continuous injury and causes chromosomal mutations to greatly increase the chance of cancer. Because it is significant to identify biomarkers for long-term low-dose gamma radiation, we investigate the influence of low dose rate on the gene expressions in the AHH-1 lymphocytes cell line (AHH-1 cells) for long-term irradiation. Different dose rates (7, 14, 26, 34, and 43 µGy h-1) of irradiation from gamma radiation in uranium tailings powder were used to irradiate AHH-1 lymphocytes. We used flow cytometry to test the apoptosis of AHH-1 lymphocytes at different dose rates and irradiation times (7-84 d). It was found that 14 µGy h-1 is the most sensitive dose rate of AHH-1 lymphocyte irradiation. The 7-, 14-, and 21-d (2.4, 4.8, and 7.2 mGy) irradiation groups were sensitive, and the 84-d (28.8 mGy) irradiation group was insensitive to low dose gamma radiation. Microarray analysis was conducted on the significantly differentially expressed genes (p<0.05) in the 2.4, 4.8, 7.2, and 28.8 mGy irradiation groups. We found that TFRC1, SLC3A2, SLC39A8, FTH1, ACSL4, and GPX4 are significant genes with low-dose radiation and were constituents of the ferroptosis signaling pathway. In the range of 0-4.8 mGy radiation dose, the expressions of these genes were downregulated with increasing radiation dose, while in the range of 4.8-28.8 mGy, its expression increased with increasing radiation dose. RT-PCR and Western blot were used to detect the mRNA and protein expression of these genes. The results were consistent with those from microarray analysis. Our findings indicate that expression of the TFRC, SLC3A2, SLC39A, FTH1, ACSL4, and GPX4 genes is sensitive to low-dose radiation, and they are main members of the ferroptosis signaling pathway. Therefore, there is a very important connection between ferroptosis and low-dose radiation, which has become a hot topic in international research. These results can provide reference to the effect of ferroptosis on human health with low-dose radiation.

Ultra-rapid, enhanced and eco-friendly extraction of four main flavonoids from the seeds of Oroxylum indicum by deep eutectic solvents combined with tissue-smashing extraction.

Rapid, green and efficient extraction of active compounds followed by fast analysis is always pursued in the field of food analysis and/or industry. Herein, a green and highly efficient extraction of four active flavonoids from the seeds of Oroxylum indicum using a combination of natural deep eutectic solvents (DESs) and tissue-smashing extraction (TSE) technique was applied and a UPLC method was developed for their sensitive and selective quantification. RSM coupled with BBD procedure was used to optimize the extraction conditions based on single factors, such as liquid-solid ratios, extraction speed and extraction time. Compared with other conventional methods, the TSE greatly shortens extraction time, obviously raises the extraction production, and decreases energy consumption. By combination of the DES-based TSE and UPLC, the analysis of flavonoids was accomplished within only 6 min, providing an ultra-rapid, environmentally friendly and promising choice for extraction and analysis of active compounds in natural products.