Real-Time Imaging of Single Viral mRNA Translation in Live Cells Using CRISPR/dCas13.

Translation is one of the many critical cellular activities regulated by viruses following host-cell invasion, and studies of viral mRNA translation kinetics and subcellular localization require techniques for the dynamic, real-time visualization of translation. However, conventional tools for imaging mRNA translation often require coding region modifications that may affect native translation. Here, we achieve dynamic imaging of translation with a tool that labels target mRNAs with unmodified coding regions using a CRISPR/dCas13 system with specific complementary paired guide RNAs. This system enables a real-time dynamic visualization of the translation process and is a promising tool for further investigations of the mechanisms of translation.

Four Low Expression LncRNAs are Associated with Prognosis of Human Lung Adenocarcinoma.

BACKGROUND: Lung cancer is the most prevalent and deadliest cancer worldwide. The present study aims to determine the prognosis value of low expression long non-coding RNAs (lncRNAs) in LUAD. METHODS: RNA-seq data and clinical information were downloaded from The Cancer Genome Atlas (TCGA) data-base. Dysregulated genes between LUAD and paracancerous tissue were screened by GeneSpringGX. Prognostic lncRNAs which were low expressed in LUAD were filtrated by Ualcan, then further verified through the TCGA database. The association between clinicopathological features and the expression level of these lncRNAs was tested by chi-square test. Cox regression analysis was performed to test independent prognosis risk factors. Diagnostic efficiency was predicted by receiver operating characteristic (ROC) analysis. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore potential functions of these prognostic signatures. RESULTS: Nine prognostic lncRNAs (LINC00092, LINC00908, WWC2-AS2, RPL13AP17, CHIAP2, SFTA1P, SIGLEC17P, CYP2B7P1, CYP4Z2P) were screened out through Ualcan and further verified by TCGA. Among them, six lncRNAs (RPL13AP17, CHIAP2, SFTA1P, SIGLEC17P, CYP2B7P1, CYP4Z2P) were pseudogene transcripts. Multivariate Cox regression analysis showed that three lnRNAs (LINC00908, WWC2-AS2 CYP2B7P) were independent prognostic risk factors for OS and two lncRNAs (WWC2-AS2, SIGLEC17P) were independent prognostic risk factors for RFS in LUAD patients. Meanwhile, they showed powerful diagnostic value by ROC curve analysis. GO analysis revealed correlation genes of prognostic signatures were mainly enriched in plasma membrane, plasma membrane part, purine nucleotide binding, cytoskeleton and ribonucleotide binding and KEGG pathway analysis showed mainly enriched in cell adhesion molecules. CONCLUSIONS: The results illuminated that four lncRNAs (LINC00908, WWC2-AS2, CYP2B7P, SIGLEC17P) may be a powerful diagnostic and prognostic assessment tool for human LUAD.

Uncovering the Rab5-Independent Autophagic Trafficking of Influenza A Virus by Quantum-Dot-Based Single-Virus Tracking.

Autophagy is closely related to virus-induced disease and a comprehensive understanding of the autophagy-associated infection process of virus will be significant for developing more effective antiviral strategies. However, many critical issues and the underlying mechanism of autophagy in virus entry still need further investigation. Here, this study unveils the involvement of autophagy in influenza A virus entry. The quantum-dot-based single-virus tracking technique assists in real-time, prolonged, and multicolor visualization of the transport process of individual viruses and provides unambiguous dissection of the autophagic trafficking of viruses. These results reveal that roughly one-fifth of viruses are ferried into cells for infection by autophagic machineries, while the remaining are not. A comprehensive overview of the endocytic- and autophagic-trafficking process indicates two distinct trafficking pathway of viruses, either dependent on Rab5-positive endosomes or autophagosomes, with striking similarities. Expressing dominant-negative mutant of Rab5 suggests that the autophagic trafficking of viruses is independent on Rab5. The present study provides dynamic, precise, and mechanistic insights into the involvement of autophagy in virus entry, which contributes to a better understanding of the relationship between autophagy and virus entry. The quantum-dot-based single-virus tracking is proven to hold promise for autophagy-related fundamental research.

Globally visualizing the microtubule-dependent transport behaviors of influenza virus in live cells.

Understanding the microtubule-dependent behaviors of viruses in live cells is very meaningful for revealing the mechanisms of virus infection and endocytosis. Herein, we used a quantum dots-based single-particle tracking technique to dynamically and globally visualize the microtubule-dependent transport behaviors of influenza virus in live cells. We found that the intersection configuration of microtubules can interfere with the transport behaviors of the virus in live cells, which lead to the changing and long-time pausing of the transport behavior of viruses. Our results revealed that most of the viruses moved along straight microtubules rapidly and unidirectionally from the cell periphery to the microtubule organizing center (MTOC) near the bottom of the cell, and the viruses were confined in the grid of microtubules near the top of the cell and at the MTOC near the bottom of the cell. These results provided deep insights into the influence of entire microtubule geometry on the virus infection.

Three-dimensional tracking of Rab5- and Rab7-associated infection process of influenza virus.

Three-dimensional (3D) single-particle tracking (SPT) techniques have been widely reported. However, the 3D SPT technique remains poorly used for solving actual biological problems. In this work, a quantum dots (QDs)-based single-particle tracking technique is utilized to explore the Rab5- and Rab7-associated infection behaviors of influenza virus in three dimensions with a set of easily-attained equipment by the fast and accurate centroid method for 3D SPT. The experimental results indicate that Rab5 protein takes part in the virus infection process from the cell periphery to the perinuclear region, while Rab7 protein is mainly involved in the intermittent and confined movements of the virus in the perinuclear region. Evidently, the transition process of the virus-containing vesicles from early to late endosomes might occur during the intermittent movement in the perinuclear region. These findings reveal distinct dynamic behaviors of Rab5- and Rab7-positive endosomes in the course of the intracellular transport of viruses. This work is helpful in understanding the intracellular transport of cargoes.

[Research Progress and Prospect of Herbicide Residue Characteristics in Black Soil Region of China].

Food security is the top priority of a country. As an important granary in China, the northeast black land is a "ballast" to ensure national food security. However, the long-term and high-intensity application of herbicides in black land farmland has led to the accumulation and migration of herbicides in the soil, which affects soil quality, crop yield, and quality and hinders sustainable agricultural development in the black soil. To solve the problem of herbicide residues in black land farmland, it is necessary to control the application of herbicides from the source, as well as to elucidate the current situation, spatial and temporal evolution, and driving factors of herbicide residues, in order to achieve scientific prevention and control and precise policy implementation. The main contents of this study were as follows:1systematically summarize the application status and problems of herbicides in the farmland of black soil in China, suggesting that there are currently problems such as irregular application and insufficient product innovation of herbicides in the farmland of black soil; 2 comprehensively analyze the current status of herbicide residues, identify the deficiencies in recent studies on herbicide residue characteristics, spatial distribution, and pollution diagnosis in the farmland of black soil, and clarify the gaps in the research on the residue characteristics of herbicides in the farmland of black soil; and 3 propose the research prospect and key orientation for the herbicide residue diagnosis and risk management in the farmland of the black soil region of China. The results of this study can provide science and technology support for guaranteeing soil health, food security, and ecosystem security of black land farmland in China.

[Characteristics of Heavy Metal Pollution in Farmland Soil of the Yangtze River Economic Belt Based on Bibliometric Analysis].

The Yangtze River Economic Belt is one of the major strategic development regions in China. It is of great significance to clarify the characteristics and sources of heavy metal pollution in farmland soil of the Yangtze River Economic Belt for the prevention and control of heavy metal pollution and to ensure safe agricultural production. After collecting extensive literature data, we analyzed the pollution characteristics, environmental risk, and potential sources of heavy metals (Cd, Cr, Hg, As, Pb, Cu, Zn, and Ni) in farmland soil of the Yangtze River Economic Belt through the integrated use of spatial and geo-accumulation index analyses. The results showed that:① the proportion of soil samples exceeding the risk screening values for soil contamination of agricultural land of Cd, Cu, Pb, Hg, Zn, and As were 39.8%, 18.5%, 8.3%, 6.9%, 6.9%, and 6.4%, respectively. Compared with the risk standard, soil Cd had the highest rate of exceeding the standard. ② The contents of Cr, Cu, Zn, and Ni in the upper reaches were higher than those in the middle and lower reaches, and the contents of Cd, As, and Pb in the middle reaches were higher than those in the upper and lower reaches. ③ The results of the geo-accumulation index analysis showed that the contamination degree of the eight heavy metals decreased in the order of Cd(0.42)>Hg(-0.28)>Pb(-0.32)>Zn(-0.39)>Cu(-0.42)>Cr(-0.7)>As(-0.81)>Ni(-0.73), where the accumulation risk of soil Cd and Hg was relatively higher. ④ Higher environmental background and mining activities were the main factors affecting the accumulation of heavy metals in soils in the upper and middle reaches. By contrast, rapid urbanization, industrial production, and intensive agricultural activities were the main factors affecting the heavy metal accumulation in soils in the middle and lower reaches. In view of the current status and control needs of heavy metal pollution in the Yangtze River Economic Belt, it is recommended to strengthen source prevention and control and to also carry out hierarchical control and regional classification management of heavy metal pollution in farmland soils according to the degree of heavy metal pollution, geological background, and quality of agricultural products. The ultimate objective of this research was to archive the farmland soil environmental quality safety and agricultural green sustainable production in the Yangtze River Economic Belt.

[Hydrocalumite Passivation Effect and Mechanism on Heavy Metals in Different Cd-Contaminated Farmland Soils].

Hydrocalumite (Ca-Al-LDHs) is a new type of layered composite metal hydroxide that has a large specific surface area, high anion exchange performance, and high stability. This study focuses on the application of hydrocalumite to remediate different Cd-contaminated farmland soils. These were collected from the Lanping County in the Yunnan Province (highly polluted), Kunshan City in the Jiangsu Province (medium polluted), and Nanjing City in the Jiangsu Province (lowly polluted). Changes in the available Cd, Pb, Zn, and the morphological transformations of these heavy metals in the three soils were investigated through a passivation experiment; moreover, the immobilization mechanism of hydrocalumite was explored by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The results showed that hydrocalumite could increase the soil pH and reduce the content of available Cd, Pb, and Zn:the maximum reduction in the available Cd reached 97.7%, 96.3%, and 91.8% in each of the three polluted soils, respectively. The easily exchangeable heavy metals were converted into carbonates, as well as into Fe-Mn oxide organic and residual forms following the addition of hydrocalumite:the passivation effect was more evident in the highly Cd-polluted soil than in the low and medium Cd-polluted soils. Since hydrocalumite possess several adsorption sites, the presence of carbonate impurities and reactive groups (e.g., hydroxyl and carboxyl groups) easily coordinated with Cd, Pb, and Zn can lead to a considerable reduction of heavy metal availability in the soils. Therefore, we conclude that hydrocalumite can be effectively applied to the remediation of farmland soils characterized by different Cd pollution levels.

[Analysis of Gene Mutation Types of Thalassemia in Fuzhou Area of China].

OBJECTIVE: To investigate the gene mutation types and spectrum of α, ß-thalassemia in Fuzhou area of China. METHODS: Thalassemia gene screening was performed in the women receiving physical, prenatal, and pre-pregnancy examination, and the patients with suspected thalassemia in our hospital from July 2013 to March 2018.Genotypes of thalassem were detected by Gap-PCR and RDB-PCR. RESULTS: 1042 were positive among 2074 suspected cases with a positive rate of 50.24%; 618 cases were confirmed to be α-thalassemia and with a positive rate of 29.8%; 409 cases were confirmed to be ß-thalassemia with a positive rate of 19.72%. 15 cases were confirmed to be α-ß complex thalassemia with a positive rate of 0.72%. the --SEA/αα(76.54%) was the most common genotype among α-thalassemia, -α3.7/αα(10.03%) and -α4.2/αα(2.91%) in hot pursuit. In addition, IVS-II-55 (T->G) and IVS-II-119 (-G, +CTCGGCCC) were newly found alpha mutations; the IVS-2-654 (C→T) (40.83%) was the most common genotype among ß-thalassemia, CD41-42 (-TCTT) (35.94%) and CD17 (A→T) (9.78%) in hot pursuit. CONCLUSION: The genotype of thalassemia in Fuzhou area is highly heterogenic, --SEA/αα is the most common genotype among α-thalassemia, IVS-2-654 (C→T) is the most common genotype among ß-thalassemia, Meanwhile, two α-mutation sites are found in this study which were not reported in the Database of Human Hemoglobin Variants and Thalassemias.

A "Driver Switchover" Mechanism of Influenza Virus Transport from Microfilaments to Microtubules.

When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a "driver switchover" mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.

Enhanced directional cell migration induced by vaccinia virus on a microfluidic-based multi-shear cell migration assay platform.

Virus-induced cell migration plays important roles in the direct and rapid spread of virus particles. As cell migration is also regulated by shear stress, it is necessary to explore the cell migration behavior influenced by multiple factors including a virus and shear stress. In this report, a three-layer microfluidic chip with symmetric channels was designed and fabricated to investigate vaccinia virus-induced cell migration in different shear stress environments. Regular "exclusion zones" without cell damage were formed by microvalves. The results showed that infected cells were more elongated and tended to migrate along the flow direction compared to the random cell migration under static conditions. Meanwhile, shear stress enhanced the natural directional persistence and accelerated the velocity of infected cell migration. Moreover, reduced peripheral lamellae and the axial lamella being confined to the flow direction were responsible for the increased directionality of cell migration under shear stress. Interestingly, in the presence of shear stress, the Golgi complex reoriented and relocated behind the nucleus and aligned to the flow direction in infected migratory cells accompanied by the rearrangement of the cytoskeleton. Our report reveals the cell migration behavior in the multi-environment of virus infection and shear stress based on the microfluidic cell migration assay platform. It helps us to deeply understand the interactions between the virus, host cells, and surrounding microenvironment.

Our Clinical Experience of Self-Expanding Metal Stent for Malignant Central Airway Obstruction.

BACKGROUND: We studied the safety, effectiveness, and limitations of airway stenting using self-expanding metal stent (SEMS) in patients with malignant central airway obstruction (CAO). METHODS: A retrospective review of records of patients undergoing SEMS placement for malignant CAO during year 2013 - 2014 was done. RESULTS: Sixteen patients (11 males and five females) underwent SEMS placement for malignant CAO. Median (range) age was 66 (54 - 78) years. No perioperative or immediate postoperative complications were seen except acute myocardial infarction (AMI) in one patient. Three patients were transferred to intensive care unit (ICU) for closer monitoring after the procedure and were discharged the next day. All four patients with lung atelectasis on presentation experienced complete re-expansion of the lung post-stenting. The dyspnea was substantially relieved in 14 (87.5%) patients. Two of the three patients who had been intubated were weaned off from the ventilator following stent insertion. Stent patency was maintained until death in all patients except one. Median survival from the date of diagnosis and the date of stent placement in lung cancer, esophageal cancer, and thyroid cancer were 140 (21 - 564) and 85 (15 - 361), 288 (80 - 419) and 61 (60 - 171), and 129 (71 - 187) and 67 (16 - 118) days, respectively. This survival was similar to reported expected survival associated with the underlying malignancy. During follow-up, granulation tissue (n = 1), mucostasis (n = 1), and tumor ingrowth (n = 2) were the most frequently encountered complications. CONCLUSION: SEMSs are safe and effective in reversing respiratory failure caused by malignant CAO, averting premature death, allowing application of cancer targeted therapy, and restoring impending shortened survival to expected life expectancy associated with the underlying malignancy.

High-efficiency dual labeling of influenza virus for single-virus imaging.

Many viruses invade host cells by entering the cells and releasing their genome for replication, which are remarkable incidents for viral infection. Therefore, the viral internal and external components should be simultaneously labeled and dynamically tracked at single-virus level for further understanding viral infection mechanisms. However, most of the previously reported methods have very low labeling efficiency and require considerable time and effort, which is laborious and inconvenient for researchers. In this work, we report a general strategy to high-efficiently label viral envelope and genome for single-virus imaging with quantum dots (QDs) and Syto 82, respectively. It was found that nearly all viral envelopes could be labeled with QDs with superior stability, which makes it possible to realize global and long-term tracking of single virus in individual cells. Effectively labeling their genome with Syto 82, about 90% of QDs-labeled viruses could be used to monitor the viral genome signal, which may provide valuable information for deeply studying viral genome transport. This is very important and meaningful to investigate the viral infection mechanism. Our labeling strategy has advantage in commonality, convenience and efficiency, which is expected to be widely used in biological research.