PAX5-miR-142 feedback loop promotes breast cancer proliferation by regulating DNMT1 and ZEB1.

BACKGROUND: Breast cancer is one of the most common malignancies occurred in female around the globe. Recent studies have revealed the crucial characters of miRNA and genes, as well as the essential roles of epigenetic regulation in breast cancer initiation and progression. In our previous study, miR-142-3p was identified as a tumor suppressor and led to G2/M arrest through targeting CDC25C. However, the specific mechanism is still uncertain. METHODS: We identified PAX5 as the upstream regulator of miR-142-5p/3p through ALGGEN website and verified by series of assays in vitro and in vivo. The expression of PAX5 in breast cancer was detected by qRT-PCR and western blot. Besides, bioinformatics analysis and BSP sequencing were performed to analyze the methylation of PAX5 promoter region. Finally, the binding sites of miR-142 on DNMT1 and ZEB1 were predicted by JASPAR, and proved by luciferase reporter assay, ChIP analysis and co-IP. RESULTS: PAX5 functioned as a tumor suppressor by positive regulation of miR-142-5p/3p both in vitro and in vivo. The expression of PAX5 was regulated by the methylation of its promoter region induced by DNMT1 and ZEB1. In addition, miR-142-5p/3p could regulate the expression of DNMT1 and ZEB1 through binding with their 3'UTR region, respectively. CONCLUSION: In summary, PAX5-miR-142-DNMT1/ZEB1 constructed a negative feedback loop to regulate the progression of breast cancer, which provided emerging strategies for breast cancer therapy.

Analysis of 4 imaging features in patients with COVID-19.

BACKGROUND: The aim of this was to analyze 4 chest CT imaging features of patients with coronavirus disease 2019 (COVID-19) in Shenzhen, China so as to improve the diagnosis of COVID-19. METHODS: Chest CT of 34 patients with COVID-19 confirmed by the nucleic acid test (NAT) were retrospectively analyzed. Analyses were performed to investigate the pathological basis of four imaging features("feather sign","dandelion sign","pomegranate sign", and "rime sign") and to summarize the follow-up results. RESULTS: There were 22 patients (65.2%) with typical "feather sign"and 18 (52.9%) with "dandelion sign", while few patients had "pomegranate sign" and "rime sign". The "feather sign" and "dandelion sign" were composed of stripe or round ground-glass opacity (GGO), thickened blood vessels, and small-thickened interlobular septa. The "pomegranate sign" was characterized as follows: the increased range of GGO, the significant thickening of the interlobular septum, complicated with a small amount of punctate alveolar hemorrhage. The "rime sign" was characterized by numerous alveolar edemas. Microscopically, the wall thickening, small vascular proliferation, luminal stenosis, and occlusion, accompanied by interstitial infiltration of inflammatory cells, as well as numerous pulmonary interstitial fibrosis and partial hyaline degeneration were observed. Repeated chest CT revealed the mediastinal lymphadenectasis in one patient. Re-examination of the NAT showed another positive anal swab in two patients. CONCLUSION: "Feather sign" and "dandelion sign" were typical chest CT features in patients withCOVID-19; "pomegranate sign" was an atypical feature, and "rime sign" was a severe feature. In clinical work, accurate identification of various chest CT signs can help to improve the diagnostic accuracy of COVID-19 and reduce the misdiagnosis or missed diagnosis rate.

Structural Basis of Reversible Phosphorylation by Maize Pyruvate Orthophosphate Dikinase Regulatory Protein.

Pyruvate orthophosphate dikinase (PPDK) is one of the most important enzymes in C4 photosynthesis. PPDK regulatory protein (PDRP) regulates the inorganic phosphate-dependent activation and ADP-dependent inactivation of PPDK by reversible phosphorylation. PDRP shares no significant sequence similarity with other protein kinases or phosphatases. To investigate the molecular mechanism by which PDRP carries out its dual and competing activities, we determined the crystal structure of PDRP from maize (Zea mays). PDRP forms a compact homo-dimer in which each protomer contains two separate N-terminal (NTD) and C-terminal (CTD) domains. The CTD includes several key elements for performing both phosphorylation and dephosphorylation activities: the phosphate binding loop (P-loop) for binding the ADP and inorganic phosphate substrates, residues Lys-274 and Lys-299 for neutralizing the negative charge, and residue Asp-277 for protonating and deprotonating the target Thr residue of PPDK to promote nucleophilic attack. Surprisingly, the NTD shares the same protein fold as the CTD and also includes a putative P-loop with AMP bound but lacking enzymatic activities. Structural analysis indicated that this loop may participate in the interaction with and regulation of PPDK. The NTD has conserved intramolecular and intermolecular disulfide bonds for PDRP dimerization. Moreover, PDRP is the first structure of the domain of unknown function 299 enzyme family reported. This study provides a structural basis for understanding the catalytic mechanism of PDRP and offers a foundation for the development of selective activators or inhibitors that may regulate photosynthesis.

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity.

In C4 plants, pyruvate orthophosphate dikinase (PPDK) activity is tightly dark/light regulated by reversible phosphorylation of an active-site threonine (Thr) residue; this process is catalyzed by PPDK regulatory protein (PDRP). Phosphorylation and dephosphorylation of PPDK lead to its inactivation and activation, respectively. Here, we show that light intensity rather than the light/dark transition regulates PPDK activity by modulating the reversible phosphorylation at Thr-527 (previously termed Thr-456) of PPDK in maize (Zea mays). The amount of PPDK (unphosphorylated) involved in C4 photosynthesis is indeed strictly controlled by light intensity, despite the high levels of PPDK protein that accumulate in mesophyll chloroplasts. In addition, we identified a transit peptide cleavage site, uncovered partial amino-terminal acetylation, and detected phosphorylation at four serine (Ser)/Thr residues, two of which were previously unknown in maize. In vitro experiments indicated that Thr-527 and Ser-528, but not Thr-309 and Ser-506, are targets of PDRP. Modeling suggests that the two hydrogen bonds between the highly conserved residues Ser-528 and glycine-525 are required for PDRP-mediated phosphorylation of the active-site Thr-527 of PPDK. Taken together, our results suggest that the regulation of maize plastid PPDK isoform (C4PPDK) activity is much more complex than previously reported. These diverse regulatory pathways may work alone or in combination to fine-tune C4PPDK activity in response to changes in lighting.

Light-regulated phosphorylation of maize phosphoenolpyruvate carboxykinase plays a vital role in its activity.

Phosphoenolpyruvate carboxykinase (PEPCK)-the major decarboxylase in PEPCK-type C4 plants-is also present in appreciable amounts in the bundle sheath cells of NADP-malic enzyme-type C4 plants, such as maize (Zea mays), where it plays an apparent crucial role during photosynthesis (Wingler et al., in Plant Physiol 120(2):539-546, 1999; Furumoto et al., in Plant Mol Biol 41(3):301-311, 1999). Herein, we describe the use of mass spectrometry to demonstrate phosphorylation of maize PEPCK residues Ser55, Thr58, Thr59, and Thr120. Western blotting indicated that the extent of Ser55 phosphorylation dramatically increases in the leaves of maize seedlings when the seedlings are transferred from darkness to light, and decreases in the leaves of seedlings transferred from light to darkness. The effect of light on phosphorylation of this residue is opposite that of the effect of light on PEPCK activity, with the decarboxylase activity of PEPCK being less in illuminated leaves than in leaves left in the dark. This inverse relationship between PEPCK activity and the extent of phosphorylation suggests that the suppressive effect of light on PEPCK decarboxylation activity might be mediated by reversible phosphorylation of Ser55.

A systematic proteomic analysis of NaCl-stressed germinating maize seeds.

Salt (NaCl) is a common physiological stressor of plants. To better understand how germinating seeds respond to salt stress, we examined the changes that occurred in the proteome of maize seeds during NaCl-treated germination. Phenotypically, salt concentrations less than 0.2 M appear to delay germination, while higher concentrations disrupt development completely, leading to seed death. The identities of 96 proteins with expression levels altered by NaCl-incubation were established using 2-DE-MALDI-TOF-MS and 2-DE-MALDI-TOF-MS/MS. Of these 96 proteins, 79 were altered greater than twofold when incubated with a 0.2 M salt solution, while 51 were altered when incubated with a 0.1 M salt solution. According to their functional annotations in the Swiss-Prot protein-sequence databases, these proteins are mainly involved in seed storage, energy metabolism, stress response, and protein metabolism. Notably, the expression of proteins that respond to abscisic acid signals increased in response to salt stress. The results of this study provide important clues as to how NaCl stresses the physiology of germinating maize seeds.

Stepwise and Controllable Synthesis of Mesoporous Heterotrimetallic Catalysts Based on Predesigned Al4 Ln4 Metallocycles.

The motivation for making heterometallic compounds stemmed from their emergent synergistic properties and enhanced capabilities for applications. However, the atomically precisely controlled synthesis of heterometallic compounds remains a daunting challenge of the complications that arise when applying several metals and linkers. Herein, a stepwise and controlled method is reported for the accurate addition of second and third metals to homometallic aluminum macrocycles based on the synergistic coordination and hard-soft acid-base theory. These heterometallic compounds showed a good Lewis acid catalytic effect, and the addition of hetero-metals significantly improved the catalytic effect and rate, among that the conversion rate of compound AlOC-133 reached 99.9% within half an hour. This method combines both the independent controllability of stepwise assembly with the universality of one-step methods. Based on the large family of clusters, the establishment of this method paves the way for the controllable and customized molecular-level synthesis of heterometallic materials and creates materials customized for preferential application.

Large-scale analysis of phosphorylated proteins in maize leaf.

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.

Genetic Control Diversity Drives Differences Between Cadmium Distribution and Tolerance in Rice.

Rice, a staple crop for nearly half the planet's population, tends to absorb and accumulate excessive cadmium (Cd) when grown in Cd-contaminated fields. Low levels of Cd can degrade the quality of rice grains, while high levels can inhibit the growth of rice plants. There is genotypic diversity in Cd distribution and Cd tolerance in different rice varieties, but their underlying genetic mechanisms are far from elucidated, which hinders genetic improvements. In this study, a joint study of phenotypic investigation with quantitative trait loci (QTLs) analyses of genetic patterns of Cd distribution and Cd tolerance was performed using a biparent population derived from japonica and indica rice varieties. We identified multiple QTLs for each trait and revealed that additive effects from various loci drive the inheritance of Cd distribution, while epistatic effects between various loci contribute to differences in Cd tolerance. One pleiotropic locus, qCddis8, was found to affect the Cd distribution from both roots to shoots and from leaf sheaths to leaf blades. The results expand our understanding of the diversity of genetic control over Cd distribution and Cd tolerance in rice. The findings provide information on potential QTLs for genetic improvement of Cd distribution in rice varieties.

Analysis of Risk Factors for Lymph Node Metastases in Elderly Patients with Papillary Thyroid Micro-Carcinoma.

BACKGROUND: With guidance from the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 8th edition, we explored the characteristics of central lymph node metastasis (CLNM) of papillary thyroid micro-carcinoma (PTMC) in elderly patients ≥55 years of age. Our goal was to provide references for establishing a lymph node dissection scheme in such patients. METHODS: We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital, Chengdu, China, from January 2015 to September 2018. Then, we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteria. RESULTS: The study included 107 patients, including 24 men and 83 women. Median age was 59.99 ± 4.58 years. The maximum diameter range of the cancer foci was 4-10 mm, and the median was 7.59 ± 1.78 mm. Unilateral lobectomy had been performed in 32 cases, total thyroidectomy in 75 cases and lateral cervical lymph node dissection in 21 cases. There were 60 cases of CLNM (56.07%) and 13 cases of lateral cervical lymph node metastasis (12.10%). The sensitivity of preoperative ultrasound in predicting CLNM was 100%, but its accuracy was only 50.47%. Multivariate logistic regression analysis showed that multiple cancer foci (area under the curve [AUC] = 0.632), extra-thyroidal expansion of cancer focus (AUC = 0.721), and irregular nodules (AUC = 0.603) were independent risk factors for CLNM of PTMC in elderly patients (P < 0.05). Overall predictability for PTMC-CLNM was 80.30%. CONCLUSION: 1) Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients. 2) For multiple cancer foci, irregular nodules, and elderly patients with PTMC extra-thyroidal expansion, we recommend a prophylactic central lymph node dissecting. 3) Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selected.

Application of Computer-Aided Design (CAD) and Three-Dimensional (3D) Visualization Technologies in the Diagnosis and Treatment of Refractory Thyroid Tumors.

BACKGROUND: To evaluate the application of computer-aided design (CAD) and three-dimensional (3D) visualization techniques in the diagnosis and treatment of refractory thyroid tumors. MATERIALS AND METHODS: The clinical data from 12 cases of refractory thyroid tumors treated with CAD and 3D visualization techniques from September 2016 to January 2018 were analyzed retrospectively. Ten cases were malignant, while two cases were benign. All tracheas in the 12 cases were invaded or oppressed by the thyroid tumors. Six of the cases experienced type III dyspnea, while the other six cases had type II dyspnea. All patients underwent contrast-enhanced computed tomography (CT) imaging. RESULTS: CAD and 3D visualization technologies were used to guide the surgeries. Ten cases of malignant thyroid tumors underwent total thyroidectomy, cervical lymph node dissection, and invaded organ resection. Among the ten cases, five underwent sleeve resections and anastomosis reconstructions, one underwent a tracheal sleeve resection and total laryngectomy, two underwent tracheal window resections, one case underwent a sternotomy, and one case underwent a laryngopharyngectomy, invaded skin resection, and pectoralis major muscle flap reconstruction. The two cases of benign thyroid tumors underwent subtotal thyroidectomies. Using CAD and 3D visualization techniques, surgeons can visually observe the relationship of the tumor with the respiratory tract and essential blood vessels, which can lead to optimized surgical plans and improved surgical outcomes. CONCLUSION: CAD and 3D visualization technologies have an important role in the personalized surgical intervention of thyroid cancer, which will likely have important clinical implications.

The Susceptibility of Primary Dermis Fibroblasts from the Chinese Tree Shrew to Human Cytomegalovirus Infection.

As a universal pathogen leading to neonatal defects and transplant failure, human cytomegalovirus (HCMV) has strict species specificity and this has prevented the development of a suitable animal model for the pathogenesis study. The mechanism of cross-species barrier remains elusive and there are so far no non-human cell culture models that support HCMV replication. The Chinese tree shrew (Tupaia belangeri chinensis) is a small laboratory animal and evolutionary closely related with primates. We investigated the susceptibility of primary tree shrew dermis fibroblasts (TSDF) to HCMV infection. Infection with a GFP-expressing HCMV virus resulted in green fluorescence in infected cells with the expression of IE1, UL44 and pp28. The titers of cell-free viruses reached 103 PFU/mL at 96 hpi, compared to titers of 104 PFU/mL observed in primary human foreskin fibroblasts. Our results suggested that TSDF was semi-permissive for HCMV infection. The TSDF model could be further used to investigate key factors influencing cross-species multiplication of HCMV.

Comparative proteomics of leaves found at different stem positions of maize seedlings.

To better understand the roles of leaves at different stem positions during plant development, we measured the physiological properties of leaves 1-4 on maize seedling stems, and performed a proteomics study to investigate the differences in protein expression in the four leaves using two-dimensional difference gel electrophoresis and tandem mass spectrometry in conjunction with database searching. A total of 167 significantly differentially expressed protein spots were found and identified. Of these, 35% are involved in photosynthesis. By further analysis of the data, we speculated that in leaf 1 the seedling has started to transition from a heterotroph to an autotroph, development of leaf 2 is the time at which the seedling fully transitions from a heterotroph to an autotroph, and leaf maturity was reached only with fully expanded leaves 3 and 4, although there were still some protein expression differences in the two leaves. These results suggest that the different leaves make different contributions to maize seedling growth via modulation of the expression of the photosynthetic proteins. Together, these results provide insight into the roles of the different maize leaves as the plant develops from a heterotroph to an autotroph.