Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

Enhancement of ß-catenin activity by BIG1 plus BIG2 via Arf activation and cAMP signals.

Multifunctional ß-catenin, with critical roles in both cell-cell adhesion and Wnt-signaling pathways, was among HeLa cell proteins coimmunoprecipitated by antibodies against brefeldin A-inhibited guanine nucleotide-exchange factors 1 and 2 (BIG1 or BIG2) that activate ADP-ribosylation factors (Arfs) by accelerating the replacement of bound GDP with GTP. BIG proteins also contain A-kinase anchoring protein (AKAP) sequences that can act as scaffolds for multimolecular assemblies that facilitate and limit cAMP signaling temporally and spatially. Direct interaction of BIG1 N-terminal sequence with ß-catenin was confirmed using yeast two-hybrid assays and in vitro synthesized proteins. Depletion of BIG1 and/or BIG2 or overexpression of guanine nucleotide-exchange factor inactive mutant, but not wild-type, proteins interfered with ß-catenin trafficking, leading to accumulation at perinuclear Golgi structures. Both phospholipase D activity and vesicular trafficking were required for effects of BIG1 and BIG2 on ß-catenin activation. Levels of PKA-phosphorylated ß-catenin S675 and ß-catenin association with PKA, BIG1, and BIG2 were also diminished after BIG1/BIG2 depletion. Inferring a requirement for BIG1 and/or BIG2 AKAP sequence in PKA modification of ß-catenin and its effect on transcription activation, we confirmed dependence of S675 phosphorylation and transcription coactivator function on BIG2 AKAP-C sequence.

STIM1 Knockout Enhances PDGF-Mediated Ca2+ Signaling through Upregulation of the PDGFR⁻PLCγ⁻STIM2 Cascade.

Platelet-derived growth factor (PDGF) has mitogenic and chemotactic effects on fibroblasts. An increase in intracellular Ca2+ is one of the first events that occurs following the stimulation of PDGF receptors (PDGFRs). PDGF activates Ca2+ elevation by activating the phospholipase C gamma (PLCγ)-signaling pathway, resulting in ER Ca2+ release. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx following depletion of ER Ca2+ stores and stromal interaction molecule 1 (STIM1) is a key molecule in the regulation of SOCE. In this study, wild-type and STIM1 knockout mouse embryonic fibroblasts (MEF) cells were used to investigate the role of STIM1 in PDGF-induced Ca2+ oscillation and its functions in MEF cells. The unexpected findings suggest that STIM1 knockout enhances PDGFR⁻PLCγ­STIM2 signaling, which in turn increases PDGF-BB-induced Ca2+ elevation. Enhanced expressions of PDGFRs and PLCγ in STIM1 knockout cells induce Ca2+ release from the ER store through PLCγ­IP3 signaling. Moreover, STIM2 replaces STIM1 to act as the major ER Ca2+ sensor in activating SOCE. However, activation of PDGFRs also activate Akt, ERK, and JNK to regulate cellular functions, such as cell migration. These results suggest that alternative switchable pathways can be observed in cells, which act downstream of the growth factors that regulate Ca2+ signaling.

Revealing the three dimensional architecture of focal adhesion components to explain Ca2+-mediated turnover of focal adhesions.

BACKGROUND: Focal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca2+/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca2+ influx induces cascades of FA turnover in living cells. METHODS: Images obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca2+ ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover. RESULTS: Vinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA. CONCLUSIONS: These results suggest that Ca2+-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca2+-mediated FA turnover. GENERAL SIGNIFICANCE: This study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca2+.

Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex.

Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells.

Colchicine derivative as a potential anti-glioma compound.

Colchicine, an anti-microtubule and antimitotic drug, is a common therapeutically agent for gout, which is thought to have potential anti-tumor effects. Owing to concerns of colchicines poisoning, the development of derivatives with low dose efficacy and less side effects is of obvious interest. In this study, we characterized the inhibitory effects of a colchicine derivative named AD1 on the cell proliferation of human malignant glioblastoma (MG) cell lines, U87MG and U373MG. We found that 50 % of U87MG and U373MG cells were reduced in the cultures after exposure to AD1 for 24 h at 10 and 50 nM, respectively. Moreover, α-tubulin immunostaining indicated that AD1 induced the disruption of the microtubule polymerization in glioma cells with apoptotic features including membrane budding/blebbing or fragmented nuclei. Increased levels of reactive oxygen species (ROS) were also detected in AD1-treated U87MG and U373MG cells compared to that observed in the control culture. Moreover, examination of microtubule-associated protein 1A/1B-light chain 3 (LC3I)/LC3II conversion and acridine orange staining for autophagic vesicles, combined with flow cytometry, showed that treatment with AD1 induced the autophagic pathway in U87MG and U373MG cells. Furthermore, we found that the intermittent intravenous administration of AD1 suppressed glioma growth in rat brain receiving intracerebral injection with rat C6 glioma cells. Taken together, our findings reveal that treatment with AD1 at nanomolar scales can reduce glioma cell viability effectively, with the occurrence of a rise in ROS and cellular autophagy. In conjunction with the observations from in vivo study, the colchicine derivative AD1 has chemotherapeutic potential to suppress glioma progression.

Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin ß1 cycling and actin remodeling.

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-ribosylation factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-ribosylation factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin ß1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses. After a 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in the effects of integrin ß1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin ß1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cofilin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. Taken together, these data reveal a mechanism(s) through which BIG2 may coordinate actin cytoskeleton mechanics and membrane traffic in cell migration via integrin ß1 action and actin functions.

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing.

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.

Population growth of Tetranychus truncatus (Acari: Tetranychidae) on different drought-tolerant potato cultivars.

Tetranychus truncatus Ehara (Acari: Tetranychidae) has become one of the major phytophagous pests in China in recent years, and is found on a wide range of host plants. However, little information is available on the population performance of this arthropod pest on potatoes. In this study, we explored the population growth of T. truncatus on two drought-tolerant potato (Solanum tuberosum L.) cultivars under laboratory conditions using the age-stage, two-sex life table. Tetranychus truncatus completed its entire life history on both potato cultivars tested, Holland 15 and Longshu 10. There was no significant difference between two potato cultivars in developmental duration. Tetranychus truncatus had shorter adult longevity (20.61 days), adult female longevity (20.41 days), and total female longevity (33.66 days) on Longshu 10 than Holland 15 (21.16 days, 21.19 days, and 34.38 days, respectively). However, it exhibited a higher preadult survival rate, higher fecundity (F = 88.32 eggs per female), and relatively higher population parameters when reared on Longshu 10 than on Holland 15 (F = 75.70 eggs per female). Growth projection also showed that the population size of T. truncatus on Longshu 10 (expand 750-fold) was larger than that on Holland 15 (expand 273-fold) after 60 days. Our results demonstrate that the drought-sensitive potato variety, Holland 15, is relatively resistant to T. truncatus compared with the drought-tolerant variety, Longshu 10, and suggest that T. truncatus exhibited a trade-off between longevity and reproduction on both potato cultivars. Our findings provide information on population prediction, which may aid the management of this pest mite species of potatoes.

Conformally Large-Area Single-Crystal Piezocomposites with High Performance for Acoustic Transducers.

Large-area and conformal piezoelectric elements are highly desired for acoustic transducers to possess a large power source level and wide detecting range. To date, single-crystal piezocomposites attract much attention on enhancing the power source level and bandwidth for next-generation acoustic transducers, owing to their higher piezoelectric and electromechanical coupling properties compared to traditional piezocomposites. Unfortunately, it is still challenging to achieve large-area and conformal single-crystal piezocomposites because of the fragile nature, large anisotropy, and the limited grown size of piezoelectric single crystals. Here, we successfully fabricate the conformally large-area single-crystal piezocomposite with an area of 160 × 50 mm2 and a bending angle of 162° by a modified 3D-printing-assisted inserting method. The single-crystal piezocomposite exhibits a high thickness electromechanical coupling factor kt of 85% and a large piezoelectric coefficient d33 of 1150 pC/N, surpassing those of the reported large-area piezocomposites. The influence of the volume fraction and curvature radius of single-crystal PCs and acoustic transducers was investigated. Furthermore, we designed an acoustic transducer based on the conformal single-crystal piezocomposite. Benefiting from the excellent piezoelectric and electromechanical properties of the single-crystal piezocomposite, the transducer indicates a high maximum transmitting voltage response of 171.8 dB. Especially, its bandwidth (-3 dB) achieves 60 kHz with a resonant frequency of 292 kHz, which is about 1.8 times superior to the conformal acoustic transducer based on the ceramic piezocomposite with a similar resonant frequency. This work may benefit the future design and fabrication of high-performance and complex-shape piezoelectric composites as key materials for next-generation transducers.

Oviposition Preference and Age-Stage, Two-Sex Life Table Analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) on Different Maize Varieties.

Host plants play an important role in the growth, development, and reproduction of insects. However, only a few studies have reported the effects of maize varieties on the growth and reproduction of S. frugiperda. In this study, a free-choice test was used to evaluate the oviposition preferences of female adults on ten common maize varieties and ten special maize varieties. The population fitness of S. frugiperda on six different maize varieties was also examined using the age-stage, two-sex life table method. The results showed that S. frugiperda oviposited and completed its life cycle across all maize cultivars. Moreover, the S. frugiperda females exhibited a significantly higher oviposition preference on the special maize varieties than on the common maize varieties. The highest number of eggs and egg masses occurred on Baitiannuo and the lowest on Zhengdan 958. The egg + larval stage, preadult, pupal stage, adult, APOP, TPOP, and total longevity of S. frugiperda were significantly shorter on the special maize varieties than on the common maize varieties. The fecundity, oviposition days, pupal weight, and hatching rate of S. frugiperda were significantly higher on the special maize varieties than on the common maize varieties. Specifically, S. frugiperda had the highest fecundity, female, and male pupal weight on Baitiannuo. Moreover, the net reproductive rate (R0), intrinsic rate of increase (r), and finite rate of increase (λ) of S. frugiperda were the greatest on Baitiannuo, whereas the shortest mean generation time (T) occurred on Zaocuiwang. The lowest R0, r, and λ, and longest T occurred on Zhengdan 958, suggesting that Zhengdan 958 is a non-preferred host plant compared to the other tested maize varieties. The findings of this study can provide a reference for the rational planting of maize and provide basic scientific information for the management of S. frugiperda.

Interaction of phosphodiesterase 3A with brefeldin A-inhibited guanine nucleotide-exchange proteins BIG1 and BIG2 and effect on ARF1 activity.

ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.

Responses of Fungi Maggot (Bradysia impatiens Johannsen) to Allyl Isothiocyanate and High CO2.

Botanical pesticide is highly recommended for integrated pest management (IPM), due to its merits such as environmental friendliness, safe to non-target organisms, operators, animals, and food consumers. The experiment was conducted to determine the lethal and sub-lethal effects of allyl isothiocyanate (AITC) on eggs, third instar larvae, pupae, and females and males of Bradysia impatiens Johannsen (B. impatiens). Different concentrations of AITC under ambient CO2 by the conical flask sealed fumigation method were used for the experiment. The results showed that there was a significant linear relationship between different concentrations of AITC and the toxicity regression equation of B. impatiens. The sub-lethal concentrations of AITC had significant effects on the larval stage, pupal stage, pupation rate, pupal weight, adult emergence rate, and oviposition. The pupation rate, pupal weight, and adult emergency rate were significantly (p < 0.05) affected by AITC fumigation. The pupation rate was the lowest after fumigation treatment of AITC at LC50 (36.67%), followed by LC25 (41.94%), compared with the CK (81.39%). Female longevity was significantly (p < 0.05) shortened by fumigation at LC25 (1.75 d) and LC50 (1.64 d), compared with that of CK (2.94 d). Male longevity was shorter at LC25 (1.56 d) than at LC50 (1.25 d) and had no significant difference between these two treatments. The fumigation efficiency of AITC was significantly increased under high CO2 condition. Furthermore, detoxification enzyme activities and antioxidant enzyme activities were accumulated under high CO2 condition. The fumigation method in the application of AITC can be useful in areas where B. impatiens is a major concern.

Elementary school comprehensive intervention and myopia development: the Wenzhou Epidemiology of Refraction Error Study.

AIM: To investigate the effects of school-based comprehensive intervention on myopia development in elementary school children. METHODS: As a part of the Wenzhou Epidemiology of Refraction Error Study, there were 1524 participating elementary students (730 girls, 47.9%) in grades 1 to 3 from three campuses of one school, aged 7.3±0.9y, who were examined twice every year for a 2.5y follow up period. Comprehensive intervention and other reminders were given at school every semester for the intervention group. The control group did not receive comprehensive intervention and did not have reminders of it. RESULTS: There were 651 students in the intervention group [mean age 7.3±0.9y; 294 (45.2%) girls] and 737 students in the control group [mean age 7.2±0.9y; 346 (46.9%) girls]. Overall mean myopia progression during the 2.5y follow-up was -0.49±1.04 diopters (D) in the intervention group and -0.65±1.08 D in the control group (P=0.004). The majority that not get myopia at baseline spherical equivalent (SE≤-1.0 D). Their mean myopia progression during the 2.5y follow-up was -0.37±0.89 D in the intervention group and -0.51±0.93 D in the control group (27.5% reduction, P=0.009); Overall, mean axial length elongation was less in the intervention group (0.56±0.32 mm) than in the control group (0.61±0.38 mm, 10.5% reduction, P=0.009). The percentage of close reading distance (<30 cm) in the intervention group was less than in the control group (73.4% vs 76.2%, P<0.001), the percentage of everyday perform eye exercises in the intervention group was more than in the control group (27.8% vs 20.7%, P<0.001) 30mo later. CONCLUSION: The comprehensive intervention program at elementary school has a significant alleviating effect on myopia progression for children during the 2.5y follow-up, especially for those non-myopia at baseline.

Effects of baffle and intraocular pressure on aerosols generated in the noncontact tonometer measurement during COVID-19.

AIM: To investigate the effects of baffle and intraocular pressure (IOP) on the aerosols generated in the noncontact tonometer (NCT) measurement and provide recommendations for the standardized use of the NCT during coronavirus disease 2019 (COVID-19). METHODS: This clinical trial included 252 subjects (312 eyes) in The Eye Hospital, Wenzhou Medical University from March 7, 2020, to March 28, 2020. Sixty subjects (120 eyes) with normal IOP were divided into two groups. One group used an NCT without a baffle, another group used an NCT with a baffle. Another 192 subjects (192 eyes) were divided into four groups: Group A1 (without a baffle+normal IOP), Group A2 (without a baffle+high IOP), Group B1 (with a baffle+normal IOP) and Group B2 (with a baffle+high IOP). Particulate matter (PM) 2.5 and PM10 generated by all subjects were quantified during the NCT measurement. The IOP values were recorded simultaneously. Effects of baffle and IOP on aerosols generated during the NCT measurement were analyzed. RESULTS: In the normal eye group with a baffle, the aerosol density decreased in a wave-like shape near the NCT with the increase in the number of people measured for IOP, demonstrating no cumulative effect. However, in the normal eye group without a baffle, there was a cumulative effect. PM2.5 and PM10 in Group A2 were higher than Group A1 (both P<0.001). The PM2.5 and PM10 in Group B2 were higher than Group B1 (P<0.01, P<0.001 respectively). The PM10 of Group B1 was lower than Group A1 (P<0.01). PM2.5 in Group B2 were lower than Group A2 (P<0.01). The median of per capita PM2.5 and PM10 in the combined Group A1+A2 were 0.80 and 1.10 µg/m3 respectively, which were higher than 0.20 and 0.60 µg/m3 in the combined Group B1+B2 (both P<0.01). The median of per capita PM2.5 and PM10 in the combined Group A1+B1 were 0.10 and 0.20 µg/m3 respectively, which were lower than 1.30 and 1.70 µg/m3 in the combined Group A2+B2 (both P<0.001). CONCLUSION: More aerosols could be generated in patients with high IOP. After the NCT is equipped with a baffle, per capita aerosol density generated decreased significantly near the NCT; And with the increase in the number of people measured for IOP, the aerosols gradually dissipated near the NCT, demonstrating no cumulative effect. Therefore, it is suggested that the NCT should be equipped with a baffle, especially for patients with high IOP.

Responses of Six Wheat Cultivars (Triticum aestivum) to Wheat Aphid (Sitobion avenae) Infestation.

Resistant variety screening is widely recommended for the management of Sitobion avenae. The purpose of this study was to assess responses of six wheat varieties (lines) to S. avenae. The aphid quantity ratio (AQR) was used to assess S. avenae resistance. Pearson's correlation coefficient was used to perform a correlation analysis between AQR, biological parameters, and the accumulation of total phenolic and flavonoid content. When compared to the other cultivars, the results showed that two cultivars, Yongliang No.15 and Ganchun No.18, had high resistance against S. avenae. The correlation analysis revealed a positive relationship between total phenol and flavonoid content accumulation and developmental duration (DD), and a negative relationship between accumulation and weight gain (WG) and mean relative growth rate (MRGR). The correlation between flavonoid and biological parameters was statistically stronger than the correlation between total phenol and biological parameters. This research provides critical cues for screening and improving aphid-resistant wheat varieties in the field and will aid in our understanding of the resistance mechanism of wheat varieties against S. avenae.

ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling.

ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.

Arl4D-EB1 interaction promotes centrosomal recruitment of EB1 and microtubule growth.

ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. End-binding 1 (EB1) is a microtubule (MT) plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing MTs and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates MT nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EB homology region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal MT nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased MT nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and the EB1, which is important for MT stabilization. Together, our results indicate that Arl4D modulates MT nucleation through regulation of the EB1-p150 association at the centrosome.

Cloning and promoter analysis of palladin 90-kDa, 140-kDa, and 200-kDa isoforms involved in skeletal muscle cell maturation.

OBJECTIVE: Palladin is a ubiquitous phosphoprotein expressed in vertebrate cells that works as a scaffolding protein. Several isoforms deriving from alternative splicing are originated from the palladin gene and involved in mesenchymal and muscle cells formation, maturation, migration, and contraction. Recent studies have linked palladin to the invasive spread of cancer and myogenesis. However, since its discovery, the promoter region of the palladin gene has never been studied. The objective of this study was to predict, identify, and measure the activity of the promoter regions of palladin gene. RESULTS: By using promoter prediction programs, we successfully identified the transcription start sites for the Palld isoforms and revealed the presence of a variety of transcriptional regulatory elements including TATA box, GATA, MyoD, myogenin, MEF, Nkx2-5, and Tcf3 upstream promoter regions. The transcriptome profiling approach confirmed the active role of predicted transcription factors in the mouse genome. This study complements the missing piece in the characterization of palladin gene and certainly contributes to understanding the complexity and enrollment of palladin regulatory factors in gene transcription.

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To optimize the effects of nitrate (NO3--N) to ammonium (NH4+-N) ratios on water consumption characteristics, grain yield and water use efficiency (WUE) of maize under full film mulching on double ridges, a field experiment was conducted at semi-arid Loess Plateau of Gansu Province, China during 2015 to 2017 cropping seasons. The treatments with different ratios of NO3--N to NH4+-N included: N1 (1:0), N2 (1:1), N3 (1:3) and N4 (3:1). The results showed that different NO3--N/NH4+-N ratios had significant impacts on soil water storage in 0-200 cm soil layer. Treatment N3 had the lowest soil water storage. Treatment N4 significantly increased total water consumption by 2.9%, 1.9% and 0.9% in 2015, and 2.3%, 1.4%, and 2.2% in 2017, compared with N1, N2 and N3 treatments, respectively. Compared with the other treatments, treatment N4 increased grain yield by 3.3%-9.9%, 3.5%-24.2% and 8.3%-36.1% and improved WUE by 1.6%-6.8%, 4.9%-21.8%, and 6.6%-32.9% in 2015, 2016 and 2017, respectively. Treatment N4 had the highest partial productivity of nitrogen fertilizer, followed by N2, N3 and N1, respectively. We recommended treatment N4 as the best nitrate and ammonium ratio to improve water use efficiency, N partial productivity, and grain yield of maize in arid and semi-arid Loess Plateau.