Mutations in an AP2 Transcription Factor-Like Gene Affect Internode Length and Leaf Shape in Maize

Background Plant height is an important agronomic trait that affects yield and tolerance to certain abiotic stresses. Understanding the genetic control of plant height is important for elucidating the regulation of maize development and has practical implications for trait improvement in plant breeding. Methodology/Principal Findings In this study, two independent, semi-dwarf maize EMS mutants, referred to as dwarf & irregular leaf (dil1), were isolated and confirmed to be allelic. In comparison to wild type plants, the mutant plants have shorter internodes, shorter, wider and wrinkled leaves, as well as smaller leaf angles. Cytological analysis indicated that the leaf epidermal cells and internode parenchyma cells are irregular in shape and are arranged in a more random fashion, and the mutants have disrupted leaf epidermal patterning. In addition, parenchyma cells in the dil1 mutants are significantly smaller than those in wild-type plants. The dil1 mutation was mapped on the long arm of chromosome 6 and a candidate gene, annotated as an AP2 transcription factor-like, was identified through positional cloning. Point mutations near exon-intron junctions were identified in both dil1 alleles, resulting in mis-spliced variants. Conclusion An AP2 transcription factor-like gene involved in stalk and leaf development in maize has been identified. Mutations near exon-intron junctions of the AP2 gene give mis-spliced transcript variants, which result in shorter internodes and wrinkled leaves

Cdc42 Regulates Extracellular Matrix Remodeling in Three Dimensions*

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions

Involvement of Rho Family GTPases in p19Arf- and p53-Mediated Proliferation of Primary Mouse Embryonic Fibroblasts

The Rho family GTPases Rac1, RhoA, and Cdc42 function as molecular switches that transduce intracellular signals regulating gene expression and cell proliferation as well as cell migration. p19(Arf) and p53, on the other hand, are tumor suppressors that act both independently and sequentially to regulate cell proliferation. To investigate the functional interaction and cooperativeness of Rho GTPases with the p19(Arf)-p53 pathway, we examined the contribution of Rho GTPases to the gene transcription and cell proliferation unleashed by deletion of p19Arf or p53 in primary mouse embryo fibroblasts. We found that (i) p19(Arf) or p53 deficiency led to a significant increase in PI 3-kinase activity, which in turn upregulated RhoA and Rac1 activities; (ii) deletion of p19Arf or p53 led to an increase in cell growth rate that was in part dependent on RhoA, Rac1, and Cdc42 activities; (iii) p19(Arf) or p53 deficiency caused an enhancement of the growth-related transcription factor NF-κB and cyclin D1 activities that are partly dependent on RhoA or Cdc42 but not on Rac1; (iv) forced expression of the activating mutants of Rac1, RhoA, or Cdc42 caused a hyperproliferative phenotype of the p19Arf(−/−) and p53(−/−) cells and promoted transformation of both cells; (v) RhoA appeared to contribute to p53-regulated cell proliferation by modulating cell cycle machinery, while hyperactivation of RhoA further suppressed a p53-independent apoptotic signal; and (vi) multiple pathways regulated by RhoA, including that of Rho-kinase, were required for RhoA to fully promote the transformation of p53(−/−) cells. Taken together, these results provide strong evidence indicating that signals through the Rho family GTPases can both contribute to cell growth regulation by p19Arf and p53 and cooperate with p19Arf or p53 deficiency to promote primary cell transformation

RhoA of the Rho Family Small GTPases Is Essential for B Lymphocyte Development

RhoA is a member of the Rho family small GTPases that are implicated in various cell functions including proliferation and survival. However, the physiological role of RhoA in vivo remains largely unknown. Here, we deleted RhoA in the B cell and hematopoietic stem cell (HSC) populations in RhoA flox/flox mice with CD19 and Mx promoter-driven Cre expression, respectively. Deletion of RhoA by CD19 Cre/+ significantly blocked B cell development in spleen, leading to a marked reduction in the number of transitional, marginal zone, and follicular B cells. Surprisingly, neither B cell proliferation in response to either LPS or B cell receptor (BCR) engagement nor B cell survival rate in vivo was affected by RhoA deletion. Furthermore, RhoA 2/2 B cells, like control cells, were rescued from apoptosis by BCR crosslinking in vitro. In contrast, RhoA deficiency led to a defect in B cell activating factor (BAFF)-mediated B cell survival that was associated with a dampened expression of BAFF receptor and a loss of BAFF-mediated Akt activation. Finally, HSC deletion of RhoA by Mx-Cre severely reduced proB/preB and immature B cell populations in bone marrow while common lymphoid progenitors were increased, indicating that RhoA is also required for B cell progenitor/precursor differentiation. Taken together, our results uncover an important role for RhoA at multiple stages of B cell development

Mechanism of inert inflammation in an immune checkpoint blockade-resistant tumor subtype bearing transcription elongation defects

Summary: The clinical response to immune checkpoint blockade (ICB) correlates with tumor-infiltrating cytolytic T lymphocytes (CTLs) prior to treatment. However, many of these inflamed tumors resist ICB through unknown mechanisms. We show that tumors with transcription elongation deficiencies (TEdef+), which we previously reported as being resistant to ICB in mouse models and the clinic, have high baseline CTLs. We show that high baseline CTLs in TEdef+ tumors result from aberrant activation of the nucleic acid sensing-TBK1-CCL5/CXCL9 signaling cascade, which results in an immunosuppressive microenvironment with elevated regulatory T cells and exhausted CTLs. ICB therapy of TEdef+ tumors fail to increase CTL infiltration and suppress tumor growth in both experimental and clinical settings, suggesting that TEdef+, along with surrogate markers of tumor immunogenicity such as tumor mutational burden and CTLs, should be considered in the decision process for patient immunotherapy indication

Genotype-specific reference interval of haptoglobin tests in a Chinese population on the BN II System

Abstract The distribution of Haptoglobin (HP) subtypes differs according to race and geography. It was also confirmed that the serum HP concentration was substantially affected by the HP subtypes. This study aimed to investigate the HP subtypes in northern Chinese and to establish reference intervals for the major HP subtypes using the BN II system. 1195 individuals were included in the study, grouped by haptoglobin subtype, and tested for concentrations by BN II System. Analysis of reference range was performed according to the EP28-A3c guideline. The need to establish reference ranges for subtype, gender, and age groupings was confirmed by the Z-test. The 2.5th and 97.5th percentiles were used as the upper and lower limits of the reference interval, respectively. In the population we investigated, the HP2-2 subtype had the highest proportion, accounting for 49.3%, followed by HP2-1 (38.0%), HP1-1 (7.2%). In addition, about 5.5% of individuals had HP del -related subtypes. The concentrations of the major subtypes (HP1-1, HP2-1, HP2-2) were significantly different, and it was necessary to establish reference ranges by grouping according to the results of the Z-test. The reference intervals were as follows: HP1-1, 0.37–2.19 g/L; HP2-1, 0.38–2.12 g/L; HP2-2, 0.12–1.51 g/L. Significant differences in HP concentrations between genders and ages were found, however, it was not necessary to establish separate reference interval since the results of the Z-test was negative. We have established reference ranges of serum haptoglobin concentrations based on subtypes, which are necessary for the clinical application of haptoglobin

RhoA is necessary for BAFF-mediated B cell survival but not proliferation.

<p>(A) Splenic B220⁺ B cells from <i>CD19^Cre/+; RhoA^+/+</i> (control) and <i>CD19^Cre/+; RhoA^flox/flox</i> (<i>RhoA^−/−</i>) mice were cultured for 48 hours on 96-well plates (4×10⁵ cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')₂ antibody or LPS. Cell growth rate was analyzed using the CellTiter 96® AQ_ueous Non-Radioactive Cell Proliferation Assay (MTS) kit. Data are expressed as absorbance OD₄₉₀. n = 5. (B) Splenocytes from control and <i>RhoA^−/−</i> mice were stained with anti-B220, -CD21, and -CD23 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (C) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were cultured for 72 hours on 96-well plates (2×10⁵ cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')₂ antibody or indicated concentrations of BAFF (left). Alternatively, control B cells were incubated with or without (−) BAFF and/or Y27632 (10 µM) (right). The cells were then stained with Annexin V and analyzed by flow cytometry. n = 5. (D) Splenocytes from control and <i>RhoA^−/−</i> mice were stained with antibodies against B220, CD21, CD23 and BAFFR, and then analyzed by flow cytometry. The numbers above bracketed lines indicate the percentage of BAFFR⁺ cells in each B cell subset and the numbers below the bracketed lines indicate mean fluorescence intensity (MFI) of BAFFR in each B cell subset (left). The percentage of BAFFR⁺ cells and MFI of BAFFR were averaged from 5 mice for each genotype (right). (E) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were subjected to Western blot for BAFFR and IgM (left). β-actin serves as loading control (left). The protein expression was quantified and normalized to β-actin and the data are expressed as fold of expression (right). n = 3. (F) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were analyzed for BAFFR mRNA levels by quantitative RT-PCR. The expression of GAPDH was used to normalize samples and the relative fold of expression is shown. n = 4. (G) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were stimulated with or without BAFF(100 ng/mL) for 30 min and then subjected to Western blot (left). Phospho (p)-Akt (S473) is quantified and normalized to total Akt and the data are expressed as relative fold of p-Akt (right). n = 3. Error bars represent mean ± SD. **p<0.01. *p<0.05. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.</p

B cell-specific deletion of RhoA impairs splenic B cell development.

<p>(A) Generation of <i>RhoA^−/−</i> B cells. Left, the loxP/Cre-mediated gene targeting strategy to generate the <i>RhoA</i> knockout allele (<i>RhoA⁻</i>) in B cells. Right, Western blot showing RhoA expression in B220⁺ B cells purified from bone marrow and spleen of <i>CD19^Cre/+; RhoA^+/+</i> (control) and <i>CD19^Cre/+; RhoA^flox/flox</i> (<i>RhoA^−/−</i>) mice. (B) Bone marrow cells from control and <i>RhoA^−/−</i> mice were stained with antibodies against B220 and IgM and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of bone marrow cells by the percentage of each subset of cells (right). n = 5. (C) Splenocytes from control and <i>RhoA^−/−</i> mice were stained with antibodies against B220, CD21 and CD23 and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of splenocytes by the percentage of each subset of cells (right). T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (D) Spleen sections from control and <i>RhoA^−/−</i> mice, stained with hematoxylin and eosin. Data are representative of 3 mice. Error bars represent mean ± SD. **p<0.01. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.</p