Construction and evaluation of a transformant library of Lasiodiplodia theobromae generated through Restriction Enzyme-Mediated Integration

Grapevine dieback, caused by Lasiodiplodia theobromae, is an important trunk disease worldwide. Transformants of L. theobromae were generated in an attempt to identify potential pathogenicity-related genes. Lasiodiplodia theobromae strain JZB 0300251, a highly virulent isolate, was selected for the genetic transformation. Based on optimised conditions, the Restriction Enzyme-Mediated Integration (REMI) methodology was established in L. theobromae using pUCATPH (a plasmid carrying a hygromycin B marker). A total of 6,036 transformants were generated with four restriction enzymes, respectively and the transformant library was evaluated based on 200 randomly selected transformants. Mutants that exhibited various degrees of virulence and different growth rates were obtained. The study provides basic results that will lead to increased understanding of the role of the pathogenicity-related genes involved in the infection process of L. theobromae

Heat Shock Protein 40 (HSP40) in Pacific White Shrimp (Litopenaeus vannamei): Molecular Cloning, Tissue Distribution and Ontogeny, Response to Temperature, Acidity/Alkalinity and Salinity Stresses, and Potential Role in Ovarian Development

Heat shock proteins (HSPs), a family of conserved proteins that are produced by cells in response to stresses, are known as molecular chaperones with a range of housekeeping and cellular protective functions. The 40 kD heat shock protein (HSP40) is a co-chaperone for HSP70 in the regulation of ATP hydrolysis. Unlike its well-documented cofactor HSP70, little is currently known regarding the biological functions of HSP40 in crustacean species such as penaeid shrimp. In the present study, the cDNA encoding HSP40 (Lv-HSP40) was identified from the Pacific white shrimp Litopenaeus vannamei, a highly significant commercial culture species. The structural organization indicates that Lv-HSP40 belongs to the type-I HSP40s. The muscle, gill, and hepatopancreas are the main sites of Lv-HSP40 transcript expression. Within these tissues, Lv-HSP40 mRNA were predominantly exhibited in the myocytes, epithelial cells and hepatopancreatic cells, respectively. Under acute thermal stress in the culture environment, Lv-HSP40 transcript levels are significantly induced in these three tissues, while low pH stress only upregulates Lv-HSP40 mRNA in the hepatopancreas and gill. During ontogenesis, Lv-HSP40 transcript levels are high at early embryonic stages and drop sharply at late embryonic and early larval stages. The ovary is another major organ of Lv-HSP40 mRNA expression in female shrimp, and Lv-HSP40 transcripts were mainly presented in the follicle cells but only weekly detected in the oocytes. Ovarian Lv-HSP40 mRNA levels increase continuously during gonadal development. Silencing of the Lv-HSP40 gene by RNA interference may effectively delay ovarian maturation after unilateral eyestalk ablation. The roles of Lv-HSP40 in ovarian development are speculated to be independent of its cofactor HSP70, and the vitellogenesis factor vitellogenin (Vg) and vitellogenin receptor (VgR). Our study, as a whole, provides new insights into the roles of HSP40 in multiple physiological processes in L. vannamei: (1) HSP40 is a responding factor during stressful conditions; and (2) HSP40 participates in embryonic and ovarian development

Growth, biochemical composition and photosynthetic performance of Scenedesmus acuminatus under different initial sulfur supplies

The effects of sulfur availability on growth, biochemical composition and photosynthetic capacity of the freshwater green microalga Scenedesmus acuminatus were investigated. Modified BG-11 media with Na2SO4 concentrations of 0.076 mM (0.25S), 0.153 mM (0.5S), 0.306 mM (control group), 0.459 mM (1.5S) and 0.612 mM (2.0S) were used to culture S. acuminatus. Sulfur repletion promoted the growth and reproduction of this species, while low sulfur supply markedly decreased the reproduction, nitrogen uptake, soluble protein and chlorophyll content, and photosynthetic activity. Elemental analysis showed that nitrogen, sulfur and carbon content per cell was significantly higher in the sulfur-limitation groups than in the control group on day 18. The metabolic response to sulfur limitation was divided into two stages: first, carbon equivalents were diverted from soluble protein to carbohydrates; then, carbohydrates were converted into neutral lipids. The total lipid content in the 0.25S and 0.5S groups reached 55.2% dry weight (DW) and 52.5% DW, respectively; neutral lipids accounted for 92.1% and 90.3% of the total lipids. Low-sulfur (0.5S) and sulfur-replete conditions both enhanced the lipid productivity. The initial sulfur level caused significant changes in the fatty acid composition of S. acuminatus. The sulfur-limited groups accumulated C16:0 and C18:1, while the sulfur-replete groups were mainly enriched in C16:0. Levels of free leucine, isoleucine, valine, lysine, glycine, alanine, aspartic acid and proline were closely related to the initial sulfur concentration. Changes in free phenylalanine, tryptophan, threonine and serine were due more to the duration of culturing. The results of this study will be useful as a reference for regulation of the initial sulfur supply to enhance lipid productivity of S. acuminatus for use in applications such as biodiesel production

Coupling of two-phase flow in fractured-vuggy reservoir with filling medium

Caves in fractured-vuggy reservoir usually contain lots of filling medium, so the two-phase flow in formations is the coupling of free flow and porous flow, and that usually leads to low oil recovery. Considering geological interpretation results, the physical filled cave models with different filling mediums are designed. Through physical experiment, the displacement mechanism between un-filled areas and the filling medium was studied. Based on the experiment model, we built a mathematical model of laminar two-phase coupling flow considering wettability of the porous media. The free fluid region was modeled using the Navier-Stokes and Cahn-Hilliard equations, and the two-phase flow in porous media used Darcy's theory. Extended BJS conditions were also applied at the coupling interface. The numerical simulation matched the experiment very well, so this numerical model can be used for two-phase flow in fracture-vuggy reservoir. In the simulations, fluid flow between inlet and outlet is free flow, so the pressure difference was relatively low compared with capillary pressure. In the process of water injection, the capillary resistance on the surface of oil-wet filling medium may hinder the oil-water gravity differentiation, leading to no fluid exchange on coupling interface and remaining oil in the filling medium. But for the water-wet filling medium, capillary force on the surface will coordinate with gravity. So it will lead to water imbibition and fluid exchange on the interface, high oil recovery will finally be reached at last

Suppression of mitochondrial complex I influences cell metastatic properties.

Despite the fact that mitochondrial dysfunction has an important role in tumorigenesis and metastasis, the underlying mechanism remains to be elucidated. Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) is the first and the largest protein complex of the mitochondrial electron-transport chain (ETC),which has an essential role in maintaining mitochondrial function and integrity. In this study, we separately knocked down two subunits of mitochondrial complex I, GRIM-19 or NDUFS3, and investigated their effects on metastatic behaviors and explored the possible mechanisms. Our data showed that stable down-modulation of GRIM-19 or NDUFS3 decreased complex I activity and reactive oxygen species (ROS) production; led to enhanced cell adhesion, migration, invasion, and spheroid formation; and influenced the expressions of extracellular matrix (ECM) molecules and its related proteins. We also observed that the expressions of GRIM-19, NDUFS3, and ECM elements were correlated with invasive capabilities of breast cancer cell lines. These results suggest that inhibition of complex I affects metastatic properties of cancer cells, and mitochondrial ROS might play a crucial role in these processes by regulating ECM

Reference Gene Selection for qRT-PCR Normalization Analysis in kenaf (Hibiscus cannabinus L.) under Abiotic Stress and Hormonal Stimuli

Kenaf (Hibiscus cannabinus L.), an environmental friendly and economic fiber crop, has a certain tolerance to abiotic stresses. Identification of reliable reference genes for transcript normalization of stress responsive genes expression by quantitative real-time PCR (qRT-PCR) is important for exploring the molecular mechanisms of plants response to abiotic stresses. In this study, nine candidate reference genes were cloned, and their expression stabilities were assessed in 132 abiotic stress and hormonal stimuli samples of kenaf using geNorm, NormFinder, and BestKeeper algorithms. Results revealed that HcPP2A (Protein phosphatase 2A) and HcACT7 (Actin 7) were the optimum reference genes across all samples; HcUBC (Ubiquitin-conjugating enzyme like protein) was the worst reference gene for transcript normalization. The reliability of the selected reference genes was further confirmed by evaluating the expression profile of HcWRKY28 gene at different stress durations. This work will benefit future studies on discovery of stress-tolerance genes and stress-signaling pathways in this important fiber crop

ADAMTS4 Enhances Oligodendrocyte Differentiation and Remyelination by Cleaving NG2 Proteoglycan and Attenuating PDGFRα Signaling

Although NG2 is known to be selectively expressed in oligodendrocyte precursor cells (OPCs) for many years, its expressional regulation and functional involvement in oligodendrocyte differentiation have remained elusive. Here, we report that the surface-bound NG2 proteoglycan can physically bind to PDGF-AA and enhances PDGF receptor alpha (PDGFRα) activation of downstream signaling. During differentiation stage, NG2 protein is cleaved by A disintegrin and metalloproteinase with thrombospondin motifs type 4 (Adamts4), which is highly upregulated in differentiating OPCs but gradually downregulated in mature myelinating oligodendrocytes. Genetic ablation of Adamts4 gene impedes NG2 proteolysis, leading to elevated PDGFRα signaling but impaired oligodendrocyte differentiation and axonal myelination in both sexes of mice. Moreover, Adamts4 deficiency also lessens myelin repair in adult brain tissue following Lysophosphatidylcholine-induced demyelination. Thus, Adamts4 could be a potential therapeutic target for enhancing oligodendrocyte differentiation and axonal remyelination in demyelinating diseases. SIGNIFICANCE STATEMENT: NG2 is selectively expressed in OPCs and downregulated during differentiation stage. To date, the molecular mechanism underlying the progressive removal of NG2 surface proteoglycan in differentiating OPCs has been unknown. In this study, we demonstrate that ADAMTS4 released by differentiating OPCs cleaves surface NG2 proteoglycan, attenuates PDGFRα signaling, and accelerates oligodendrocyte differentiation. In addition, our study also suggests ADAMTS4 as a potential therapeutic target for promoting myelin recovery in demyelinating diseases

A 9‐month‐old Chinese patient with Gabriele‐de Vries syndrome due to novel germline mutation in the YY1

Abstract Background Gabriele‐de Vries syndrome (GADEVS), also known as YY1 haploinsufficiency syndrome, is a very rare autosomal dominant neurodevelopmental disorder (NDD) due to YY1 mutation characterized by mild‐to‐profound developmental delay (DD)/intellectual disability (ID), a wide spectrum of functional and morphologic abnormalities, and intrauterine growth restriction or low birth weight and feeding difficulties are common in the patients. However, NDDs, such as language development disorder and ID, could hardly be assessed in patients younger than 2 years old. Methods We describe a 9‐month‐old female with DD, failure to thrive, and facial dysmorphism. Genetic analysis was conducted by whole exome sequencing (WES) and confirmed by Sanger sequencing. Results In addition to DD and dysmorphic facial features, this patient had urinary tract infection, acute pyelonephritis, bilateral vesicoureteral reflux (grade III), gastroesophageal reflux, and malnutrition. She was found to have foramen ovale or atrial septal defect, and enlarged left lateral ventricle in the brain. After performing WES, a novel heterozygous mutation NM_003403.5:c.1124G>A, p.Arg375Gln in the YY1 gene was identified. Conclusion Our findings suggest that genetic tests are critical technique for diagnosis of GADEVS, especially in patients with early‐childhood, unexplained developmental or growth disorders, thus, the prevalence of GADEVS may be underestimated. The clinical features and identified YY1 mutation in our patient expand the spectra of phenotypes and genotypes of GADEVS, respectively

Evidence That DDR1 Promotes Oligodendrocyte Differentiation during Development and Myelin Repair after Injury

Oligodendrocytes generate myelin sheaths vital for the formation, health, and function of the central nervous system. Mounting evidence suggests that receptor tyrosine kinases (RTKs) are crucial for oligodendrocyte differentiation and myelination in the CNS. It was recently reported that discoidin domain receptor 1 (Ddr1), a collagen-activated RTK, is expressed in oligodendrocyte lineage. However, its specific expression stage and functional role in oligodendrocyte development in the CNS remain to be determined. In this study, we report that Ddr1 is selectively upregulated in newly differentiated oligodendrocytes in the early postnatal CNS and regulates oligodendrocyte differentiation and myelination. Ddr1 knock-out mice of both sexes displayed compromised axonal myelination and apparent motor dysfunction. Ddr1 deficiency alerted the ERK pathway, but not the AKT pathway in the CNS. In addition, Ddr1 function is important for myelin repair after lysolecithin-induced demyelination. Taken together, the current study described, for the first time, the role of Ddr1 in myelin development and repair in the CNS, providing a novel molecule target for the treatment of demyelinating diseases