DataSheet1_Exploring the characteristics and driving forces of orchard expansion in ecological fragile region: A case study of three typical counties in the Loess Plateau.docx

The Loess Plateau exemplifies the type of ecologically fragile region that faces severe poverty challenges in China. Orchards have expanded rapidly over the past few decades and now constitute a considerable part of local economy. Not only do the characteristics of orchard expansion affect local economic development, but also exert additional pressure on the ecological environment. Therefore, it is essential for sustainable development on the Loess Plateau to investigate the characteristics and driving forces of orchard expansion. The Fuxian, Luochuan, Huangling, three typical orchard planting counties were chosen as the study area. Firstly, the orchard was extracted from the land use/cover classification from 1990–2020. It broadens the research approach to the identification of expansion cash crops by using the combination of linear spectral mixture analysis (LSMA) and decision tree. Secondly, the spatiotemporal dynamics of orchard expansion were quantitatively investigated based on spatial geometry center shift, physical geographical features, landscape pattern and orchard planting suitability. Then, we constructed an evaluation indicators system to detect the feature importance and partial dependence of different factors by random forest regression. It is more innovative to employ the machine learning method to investigate driving forces. Finally, the linkages between planting suitability and orchard expansion were further discussed, and subsequent policies were proposed. Findings demonstrated the orchard had continuously expanded over the past 30 years, with the fastest expansion rate during 1990–2005. Increased cohesion was accompanied by a shift in the orchard’s spatial distribution to the north central region and highly suitable planting regions. Slope turned out to be the primary factor affecting the orchard expansion. In the future, regions with aging orchard but high planting suitability should be the preferred choice for orchard expansion. Additionally, the transportation connectivity and governmental assistance are crucial considerations for the future planning of the orchard.</p

Salt-Induced Stability and Serum-Resistance of Polyglutamate Polyelectrolyte Brushes/Nuclear Factor-κB p65 siRNA Polyplex Enhance the Apoptosis and Efficacy of Doxorubicin

Short interfering RNAs (siRNAs) as chemotherapeutic RNAi agents hold great promise for a significant improvement in cancer therapy. Despite the promise, effective transport of siRNA with minimal side effects remains a challenge. The common problem associated with the low delivery efficiencies of current polycation-based gene delivery systems is their low stability in the presence of salt and serum. In the present study we developed the polyglutamate derivatives (PGS) polyelectrolyte brushes for NF-κB p65 siRNA delivery. The PGS polyelectrolyte brushes/siRNA polyplex was colloidally stable (150 nm diameter) in physiological saline (150 mM NaCl), likely due to the osmotic brushes of PGS. The size-controlled siRNA/PGS polyplex also showed the serum resistance resulting in their efficient cellular uptake was not negatively influenced by the presence of serum. The endothermic profile of ITC, their low values of Gibbs free energy and binding constants <i>K</i>_b under salt conditions provided the direct evidence that PGS polyelectrolyte brushes had a much lower binding affinity for serum proteins, compared with PEI 25KDa. PGS polyelectrolyte brushes delivering NF-κB p65 siRNA achieved efficient down-regulation of NF-κB p65 protein in HeLa cells. The NF-κB p65 down-regulation mediated by PGS polyelectrolyte brushes was more significant than PEI 25KDa and comparable to Lipofectamine 2000. Furthermore, the combination treatment with PGS polyelectrolyte brushes/NF-κB p65 siRNA polyplex and doxorubicin demonstrated synergistic apoptotic and cytotoxic effects on HeLa cancer cells. The high stability in physiological saline and salt-induced serum resistance of PGS polyelectrolyte brushes/siRNA polyplex has potential applications together with standard chemotherapies such as doxorubicin to be a viable method to improve the clinical outcomes in cancer therapies

MiR-125a Is a critical modulator for neutrophil development

<div><p>MicroRNAs are universal post-transcriptional regulators in genomes. They have the ability of buffering gene expressional programs, contributing to robustness of biological systems and playing important roles in development, physiology and diseases. Here, we identified a microRNA, miR-125a, as a positive regulator of granulopoiesis. <i>MiR125a</i> knockout mice show reduced infiltration of neutrophils in the lung and alleviated tissue destruction after endotoxin challenge as a consequence of decreased neutrophil numbers. Furthermore, we demonstrated that this significant reduction of neutrophils was due to impaired development of granulocyte precursors to mature neutrophils in an intrinsic manner. We showed that <i>Socs3</i>, a critical repressor for granulopoiesis, was a target of miR-125a. Overall, our study revealed a new microRNA regulating granulocyte development and supported a model in which miR-125a acted as a fine-tuner of granulopoiesis.</p></div

MiR-125a regulates maturation of neutrophils by targeting <i>Socs3 in vivo</i>.

<p>(A) Flow cytometry analysis of GFP⁺ bone marrow neutrophils after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Bar graphs indicated numbers of GFP⁺ neutrophils per femur and tibia. (B) Flow cytometry analysis of GFP⁺ myeloid precursor cell populations after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Plots shown here were previously gated on Lin^-Sca-1^-c-Kit⁺ cells. Bar graphs indicated numbers of GFP⁺ GMPs (upper) or CMPs (lower) per femur and tibia. (C) Flow cytometry analysis of neutrophils incorporating BrdU in bone marrow GFP⁺ GMPs (upper) or CMPs (lower). Bar graphs indicate the mean fluorescence intencities of BrdU-incorporating GMPs (upper) or CMPs (lower). ns, none significant difference, *<i>P</i><0.05(Student’s <i>t</i>-test).</p

Lower mortality and neutrophil infiltration in LPS-induced lethal septic shock in <i>MiR125a</i>^<i>-/-</i> mice.

<p>(A) Flow cytometry analysis of infiltrating neutrophils from lungs of <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice challenged with 25 mg/kg LPS after 24 hours. Single cell suspensions of lung cells were previously gated with CD45. Neutrophils were stained with CD11b-Percp cy5.5 and Ly6G-APC. Bar graph shows the average percentage of infiltrating neutrophils (mean±s.d.,n = 3 mice of each genotype). (B) Hematoxylinand-eosin staining of lung sections from WT and KO mice 24 hours after 25 mg/kg LPS injection. Bar graph is the histopathological severity score of lung sections. Histopathological severity of randomly selected fields from the lung sections were graded as 0 (no findings or normal), 1 (mild), 2 (moderate) or 3 (severe) for each of the four parameters(congestion, edema, hemorrhage and inflammation). Theses results were confirmed by a blinded independent researcher. (C) Serum concentrations of aspartate aminotransferase (ALT), blood urea nitrogen (BUN), creatine kinase (CK) and creatinine (CREA) in <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice 24 h after injection of 25 mg/kg LPS (mean±s.d.,n = 5 mice of each genotype,). (D) Survival of <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice (n = 10 each genotype) intraperitoneally challenged with 45 mg/kg LPS. Data are presented as a Kaplan-Meier plot. P<0.05 (log-rank test). (E) TNF-α and IL-6 concentrations in serum of <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> mice 2h after intraperitoneal injection of 45 mg/kg LPS (mean±s.d., n = 5 mice of each genotype). ns, no significant difference (Student’s <i>t</i>-test). (F) Wild-type mice were first depleted of endogenous macrophages by pre-treatment with clodronate liposomes and then were transplanted with 1x10⁷ <i>MiR125a</i>^<i>+/+</i>and <i>MiR125a</i>^<i>-/-</i> bone marrow derived macrophages 6 hours before intraperitoneal injection with 45 mg/kg LPS. Survival percentage of these mice are presented as a Kaplan-Meier plot (n = 7 mice of each genotype;p = 0.7114, log-rank test).*<i>P</i><0.05,**<i>P</i><0.01, ***<i>P</i><0.001.</p

Hematological Parameters of <i>MiR125a</i>^<i>-/-</i> mice.

<p>Hematological Parameters of <i>MiR125a</i>^<i>-/-</i> mice.</p

<i>Socs3</i> ia a target of miR-125a.

<p>(A) Protein expression of SOCS3 in bone marrow neutrophils from <i>MiR125a</i>^<i>+/+</i> and <i>MiR125a</i>^<i>-/-</i> mice. Cell lysates were analyzed by immunoblot using SOCS3 antibody. (B) Schematic presentation of a potential miR-125a binding sites in the 3’UTR regions of Socs3. Sequences below indicate the mutant form of this site. (C) Luciferase reporter gene assay performed on 293T cells transfected with plasmids on which the luciferase reporter gene fused to the fragment of wild-type or mutant 3’UTRs of Socs3. Values were normalized to a firefly gene’s activity on the same construct (mean±s.d., n = 3). (D) The mRNA expression of Socs3 in sorted GFP⁺ GMPs which were transduced with retrovirus of Socs3 shRNA or a control(Ctrl) shRNA. (E-G) 1000 GMPs were sorted from <i>MiR125a</i>^<i>-/-</i> bone marrow lin^- cells which were transduced with retrovirus of Socs3 shRNA or a Ctrl shRNA and then cultivated in G-CSF containing methylcellulose media. Photographed CFUs (E), colony numbers (F) and cell number per CFUs (G) were shown. Representative data were from three independent experiments. <i>**P</i><0.01 (Student’s <i>t</i>-test).</p

Impaired differentiation from granulocyte progenitors to mature neutrophils in <i>MiR125a</i>^<i>-/-</i> mice.

<p>(A) Flow cytometry analysis of myeloid precursor cell populations of 8-week-old mice. Plots shown here were previously gated on Lin^-Sca-1^-c-Kit⁺ cells. The right panel shows the overall number of precursors per bone marrow sample isolated from femurs and tibiae (mean±s.d.,n = 6 mice of each genotype). (B-C) Colony numbers of bone marrow cells in methylcellulose colony assays. Myeloid precursors were analyzed in complete methylcellulose medium containing SCF, IL-3, IL-6, and EPO (B) or varying concentrations of G-CSF (C). Values were represented as mean±s.d., n = 3 mice of each genotype. (D-G) 1000 GMPs were sorted from <i>MiR125a</i>^<i>+/+</i> or <i>MiR125a</i>^<i>-/-</i> mice and cultivate in G-CSF containing methylcellulose media. Colony numbers (D), photographed CFUs (E), cell number per CFUs (F) and total cell number of 1000 CFUs (g) were shown. Values were represented as mean±s.d., n = 3 mice of each genotype. ns, none significant difference,*<i>P</i><0.05, ***<i>P</i><0.001(Student’s <i>t</i>-test).</p

Impaired G-CSF signaling in <i>MiR125a</i>^-/- neutrophils.

<p>(A) Proliferation of bone marrow neutrophils in respond to various concentrations of G-CSF. 2x10⁴ bone marrow neutrophils were cultured with G-CSF in various concentrations for 24 hours. Cell number were then counted. (B) Flow cytometry analysis of BrdU incorporating bone marrow neutrophils in response to G-CSF. Bar graphs show the average percentage of BrdU-incorporating neutrophils. (C) Apoptosis of bone marrow neutrophils in response to G-CSF for 48 hours. The bar graph shows the percentage of Annexin V + neutrophils. (D) Activation of STAT1, ERK and STAT3 in response to G-CSF. Bone marrow neutrophils were stimulated with 10 ng/ml G-CSF for 15 min, 30 min, 60 min and 120 min. Cell lysates were analyzed by immunoblot using antibodies specific for phosphorylated and total STATs, ERK and GAPDH. Representative data are from three independent experiments. (E) The ratio of phosphorylated STAT1, ERK and STAT3 vs. total STAT1, ERK and STAT3. Image J was used to quantitatively analyze the western blots results in (D). All values were represented as mean±s.d., n = 3 mice of each genotype. Ns, none significant difference, ** <i>P</i><0.01, *<i>P</i> <0.05 (Student’s <i>t</i>-test).</p

MiR-125a regulates maturation of neutrophils by targeting <i>Socs3 in vivo</i>.

<p>(A) Flow cytometry analysis of GFP⁺ bone marrow neutrophils after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Bar graphs indicated numbers of GFP⁺ neutrophils per femur and tibia. (B) Flow cytometry analysis of GFP⁺ myeloid precursor cell populations after bone marrow transplantation of miR-125a^-/- ST-HSCs which are transduced with lentivirus of Socs3 shRNA or a control(Ctrl) shRNA. Plots shown here were previously gated on Lin^-Sca-1^-c-Kit⁺ cells. Bar graphs indicated numbers of GFP⁺ GMPs (upper) or CMPs (lower) per femur and tibia. (C) Flow cytometry analysis of neutrophils incorporating BrdU in bone marrow GFP⁺ GMPs (upper) or CMPs (lower). Bar graphs indicate the mean fluorescence intencities of BrdU-incorporating GMPs (upper) or CMPs (lower). ns, none significant difference, *<i>P</i><0.05(Student’s <i>t</i>-test).</p