254 research outputs found
Gene silencing in mammalian cells by PCR-based short hairpin RNA
AbstractRNA interference (RNAi) provides a powerful tool to silence genes in a sequence-specific manner in a variety of systems. However, not all sequences are effective in the RNAi-mediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)-based RNAi strategy for a quick screening of small interfering RNA (siRNA) efficiency. This method utilized a two-step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in specific depressions of exogenous (luciferase, green fluorescent protein and β-galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning
Dimethyl 2,6-dihydroxybenzene-1,4-dicarboxylate
The title compound, C10H10O6, was obtained from an esterification reaction of 2,6-dihydroxyterephthalic acid and methanol. In the molecular structure, all of the C atoms are nearly coplanar. The two hydroxy groups have C2 symmetry. Intramolecular O—H⋯O hydrogen bonds are observed. In the crystal, weak O—H⋯O interactions link the molecules
Three-dimensional controlled growth of monodisperse sub-50 nm heterogeneous nanocrystals
The ultimate frontier in nanomaterials engineering is to realize their composition control with atomic scale precision to enable fabrication of nanoparticles with desirable size, shape and surface properties. Such control becomes even more useful when growing hybrid nanocrystals designed to integrate multiple functionalities. Here we report achieving such degree of control in a family of rare-earth-doped nanomaterials. We experimentally verify the co-existence and different roles of oleate anions (OA-) and molecules (OAH) in the crystal formation. We identify that the control over the ratio of OA- to OAH can be used to directionally inhibit, promote or etch the crystallographic facets of the nanoparticles. This control enables selective grafting of shells with complex morphologies grown over nanocrystal cores, thus allowing the fabrication of a diverse library of monodisperse sub-50 nm nanoparticles. With such programmable additive and subtractive engineering a variety of three-dimensional shapes can be implemented using a bottom-up scalable approach
Protectin conjugates in tissue regeneration 1 alleviates sepsis-induced acute lung injury by inhibiting ferroptosis
Background: Acute lung injury (ALI) is a common and serious complication of sepsis with high mortality. Ferroptosis, categorized as programmed cell death, contributes to the development of lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is an endogenous lipid mediator that exerts protective effects against multiorgan injury. However, the role of PCTR1 in the ferroptosis of sepsis-related ALI remains unknown. Methods: A pulmonary epithelial cell line and a mouse model of ALI stimulated with lipopolysaccharide (LPS) were established in vitro and in vivo. Ferroptosis biomarkers, including ferrous (Fe2+), glutathione (GSH), malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE), were assessed by relevant assay kits. Glutathione peroxidase 4 (GPX4) and prostaglandin-endoperoxide synthase 2 (PTGS2) protein levels were determined by western blotting. Lipid peroxides were examined by fluorescence microscopy and flow cytometry. Cell viability was determined by a CCK-8 assay kit. The ultrastructure of mitochondria was observed with transmission electron microscopy. Morphology and inflammatory cytokine levels predicted the severity of lung injury. Afterward, related inhibitors were used to explore the potential mechanism by which PCTR1 regulates ferroptosis. Results: PCTR1 treatment protected mice from LPS-induced lung injury, which was consistent with the effect of the ferroptosis inhibitor ferrostatin-1. PCTR1 treatment decreased Fe2+, PTGS2 and lipid reactive oxygen species (ROS) contents, increased GSH and GPX4 levels and ameliorated mitochondrial ultrastructural injury. Administration of LPS or the ferroptosis agonist RSL3 resulted in reduced cell viability, which was rescued by PCTR1. Mechanistically, inhibition of the PCTR1 receptor lipoxin A4 (ALX), protein kinase A (PKA) and transcription factor cAMP-response element binding protein (CREB) partly decreased PCTR1 upregulated GPX4 expression and a CREB inhibitor blocked the effects ofPCTR1 on ferroptosis inhibition and lung protection. Conclusion: This study suggests that PCTR1 suppresses LPS-induced ferroptosis via the ALX/PKA/CREB signaling pathway, which may offer promising therapeutic prospects in sepsis-related ALI
Molecular characterization and genetic diversity of different genotypes of Oryza sativa and Oryza glaberrima
Background: Availability of related rice species is critical for rice
breeding and improvement. Two distinct species of domesticated rice
exist in the genus Oryza: Oryza sativa (Asian rice) and Oryza
glaberrima (African rice). New rice for Africa (NERICA) is derived from
interspecific crosses between these two species. Molecular profiling of
these germplasms is important for both genetics and breeding studies.
We used 30 polymorphic SSR markers to assess the genetic diversity and
molecular fingerprints of 53 rice genotypes of O. sativa, O.
glaberrima, and NERICA. Results: In total, 180 alleles were detected.
Average polymorphism information content and Shannon's information
index were 0.638 and 1.390, respectively. Population structure and
neighbor-joining phylogenetic tree revealed that 53 genotypes grouped
into three distinct subpopulations conforming to the original three
groups, except three varieties (IR66417, WAB450-4, MZCD74), and that
NERICA showed a smaller genetic distance from O. sativa genotypes
(0.774) than from O. glaberrima genotypes (0.889). A molecular
fingerprint map of the 53 accessions was constructed with a novel
encoding method based on the SSR polymorphic alleles. Ten specific SSR
markers displayed different allelic profiles between the O. glaberrima
and O. sativa genotypes. Conclusions: Genetic diversity studies
revealed that 50 rice types were clustered into different
subpopulations whereas three genotypes were admixtures. Molecular
fingerprinting and 10 specific markers were obtained to identify the 53
rice genotypes. These results can facilitate the potential utilization
of sibling species in rice breeding and molecular classification of O.
sativa and O. glaberrima germplasms
Effect of Advanced HIV Infection on the Respiratory Microbiome
RATIONALE:
Previous work found the lung microbiome in healthy subjects infected with HIV was similar to that in uninfected subjects. We hypothesized the lung microbiome from subjects infected with HIV with more advanced disease would differ from that of an uninfected control population.
OBJECTIVES:
To measure the lung microbiome in an HIV-infected population with advanced disease.
METHODS:
16s RNA gene sequencing was performed on acellular bronchoalveolar lavage (BAL) fluid from 30 subjects infected with HIV with advanced disease (baseline mean CD4 count, 262 cells/mm(3)) before and up to 3 years after starting highly active antiretroviral therapy (HAART) and compared with 22 uninfected control subjects.
MEASUREMENTS AND MAIN RESULTS:
The lung microbiome in subjects infected with HIV with advanced disease demonstrated decreased alpha diversity (richness and diversity) and greater beta diversity compared with uninfected BAL. Differences improved with HAART, but still persisted up to 3 years after starting therapy. Population dispersion in the group infected with HIV was significantly greater than in the uninfected cohort and declined after treatment. There were differences in the relative abundance of some bacteria between the two groups at baseline and after 1 year of therapy. After 1 year on HAART, HIV BAL contained an increased abundance of Prevotella and Veillonella, bacteria previously associated with lung inflammation.
CONCLUSIONS:
The lung microbiome in subjects infected with HIV with advanced disease is altered compared with an uninfected population both in diversity and bacterial composition. Differences remain up to 3 years after starting HAART. We speculate an altered lung microbiome in HIV infection may contribute to chronic inflammation and lung complications seen in the HAART era
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Endothelium-specific SIRT7 targeting ameliorates pulmonary hypertension through Krüpple-like factor 4 deacetylation.
AIMS: Pulmonary hypertension (PH) is a pulmonary vascular disease characterized by a high mortality rate. Pulmonary arterial endothelium cells (PAECs) serve as a primary sensor of various environmental cues, such as shear stress and hypoxia, but PAEC dysfunction may trigger vascular remodelling during the onset of PH. This study aimed to illustrate the role of Sirtuin 7 (SIRT7) in endothelial dysfunction during PH and explore the potential therapeutic strategy for PH. METHODS AND RESULTS: SIRT7 levels were measured in human and murine experimental PH samples. Bioinformatic analysis, immunoprecipitation, and deacetylation assay were used to identify the association between SIRT7 and Krüpple-like factor 4 (KLF4), a key transcription factor essential for endothelial cell (EC) homeostasis. Sugen5416 + hypoxia (SuHx)-induced PH mouse models and cell cultures were used for the study of the therapeutic effect of SIRT7 for PH. SIRT7 level was significantly reduced in lung tissues and PAECs from PH patients and the SuHx-induced PH mouse model as compared with healthy controls. Pulmonary endothelium-specific depletion of Sirt7 increased right ventricular systolic pressure and exacerbated right ventricular hypertrophy in the SuHx-induced PH model. At the molecular level, we identified KLF4 as a downstream target of SIRT7, which deacetylated KLF4 at K228 and inhibited the ubiquitination-proteasome degradation. Thus, the SIRT7/KLF4 axis maintained PAEC homeostasis by regulating proliferation, migration, and tube formation. PAEC dysfunction was reversed by adeno-associated virus type 1 vector-mediated endothelial overexpression of Sirt7 or supplementation with nicotinamide adenine dinucleotide (NAD)+ intermediate nicotinamide riboside which activated Sirt7; both approaches successfully reversed PH phenotypes. CONCLUSION: The SIRT7/KLF4 axis ensures PAEC homeostasis, and pulmonary endothelium-specific SIRT7 targeting might constitute a PH therapeutic strategy
A combined transmission spectrum of the Earth-sized exoplanets TRAPPIST-1 b and c
Three Earth-sized exoplanets were recently discovered close to the habitable
zone of the nearby ultracool dwarf star TRAPPIST-1. The nature of these planets
has yet to be determined, since their masses remain unmeasured and no
observational constraint is available for the planetary population surrounding
ultracool dwarfs, of which the TRAPPIST-1 planets are the first transiting
example. Theoretical predictions span the entire atmospheric range from
depleted to extended hydrogen-dominated atmospheres. Here, we report a
space-based measurement of the combined transmission spectrum of the two inner
planets made possible by a favorable alignment resulting in their simultaneous
transits on 04 May 2016. The lack of features in the combined spectrum rules
out cloud-free hydrogen-dominated atmospheres for each planet at 10-
levels; TRAPPIST-1 b and c are hence unlikely to harbor an extended gas
envelope as they lie in a region of parameter space where high-altitude
cloud/haze formation is not expected to be significant for hydrogen-dominated
atmospheres. Many denser atmospheres remain consistent with the featureless
transmission spectrum---from a cloud-free water vapour atmosphere to a
Venus-like atmosphere.Comment: Early release to inform further the upcoming review of HST's Cycle 24
proposal
SPECULOOS exoplanet search and its prototype on TRAPPIST
One of the most significant goals of modern science is establishing whether
life exists around other suns. The most direct path towards its achievement is
the detection and atmospheric characterization of terrestrial exoplanets with
potentially habitable surface conditions. The nearest ultracool dwarfs (UCDs),
i.e. very-low-mass stars and brown dwarfs with effective temperatures lower
than 2700 K, represent a unique opportunity to reach this goal within the next
decade. The potential of the transit method for detecting potentially habitable
Earth-sized planets around these objects is drastically increased compared to
Earth-Sun analogs. Furthermore, only a terrestrial planet transiting a nearby
UCD would be amenable for a thorough atmospheric characterization, including
the search for possible biosignatures, with near-future facilities such as the
James Webb Space Telescope. In this chapter, we first describe the physical
properties of UCDs as well as the unique potential they offer for the detection
of potentially habitable Earth-sized planets suitable for atmospheric
characterization. Then, we present the SPECULOOS ground-based transit survey,
that will search for Earth-sized planets transiting the nearest UCDs, as well
as its prototype survey on the TRAPPIST telescopes. We conclude by discussing
the prospects offered by the recent detection by this prototype survey of a
system of seven temperate Earth-sized planets transiting a nearby UCD,
TRAPPIST-1.Comment: Submitted as a chapter in the "Handbook of Exoplanets" (editors: H.
Deeg & J.A. Belmonte; Section Editor: N. Narita). 16 pages, 4 figure
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