The role of spliceosomal intronic structures played in evolution has only begun to be elucidated. Comparative genomic analyses of fungal snoRNA sequences, which are often contained within introns and/or exons, revealed that about one-third of snoRNA-associated introns in three major snoRNA gene clusters manifested polymorphisms, likely resulting from intron loss and gain events during fungi evolution. Genomic deletions can clearly be observed as one mechanism underlying intron and exon loss, as well as generation of complex introns where several introns lie in juxtaposition without intercalating exons. Strikingly, by tracking conserved snoRNAs in introns, we found that some introns had moved from one position to another by excision from donor sites and insertion into target sties elsewhere in the genome without needing transposon structures. This study revealed the origin of many newly gained introns. Moreover, our analyses suggested that intron-containing sequences were more prone to sustainable structural changes than DNA sequences without introns due to intron's ability to jump within the genome via unknown mechanisms. We propose that splicing-related structural features of introns serve as an additional motor to propel evolution.
Hearing loss is the most prevalent sensory perception deficit in humans, affecting 1/500 newborns, can be syndromic or nonsyndromic and is genetically heterogeneous. Nearly 80% of inherited nonsyndromic bilateral sensorineural hearing loss (NBSNHI) is autosomal recessive. Although many causal genes have been identified, most are minor contributors, except for GJB2, which accounts for nearly 50% of all recessive cases of severe to profound congenital NBSNHI in some populations. More than 60% of children with a NBSNHI do not have an identifiable genetic cause. To identify genetic contributors, we genotyped 659 GJB2 mutation negative pediatric probands with NBSNHI and assayed for copy number variants (CNVs). After identifying 8 mild-moderate NBSNHI probands with a Chr15q15.3 deletion encompassing the Stereocilin (STRC) gene amongst this cohort, sequencing of STRC was undertaken in these probands as well as 50 probands and 14 siblings with mild-moderate NBSNHI and 40 probands with moderately severe-profound NBSNHI who were GJB2 mutation negative. The existence of a STRC pseudogene that is 99.6% homologous to the STRC coding region has made the sequencing interpretation complicated. We identified 7/50 probands in the mild-moderate cohort to have biallelic alterations in STRC, not including the 8 previously identified deletions. We also identified 2/40 probands to have biallelic alterations in the moderately severe-profound NBSNHI cohort, notably no large deletions in combination with another variant were found in this cohort. The data suggest that STRC may be a common contributor to NBSNHI among GJB2 mutation negative probands, especially in those with mild to moderate hearing impairment.
Bilateral sensorineural hearing loss; SNHI; Chr15q15.3; Stereocilin; STRC; DFNB16; SNP genotyping array; copy number variation; CNV
Cross-linking of the IgE receptor (FcεRI) on mast cells plays a critical role in IgE-dependent allergy, including allergic rhinitis, asthma, anaphylaxis, and immediate-type hypersensitivity reactions. Previous studies have demonstrated that the K+ channel, KCa3.1, plays a critical role in IgE-stimulated Ca2+ entry and degranulation in both human and mouse mast cells. We now have shown that the class II phosphatidylinositol-3-kinase C2β (PI3KC2β) is necessary for FcεRI-stimulated activation of KCa3.1, Ca2+ influx, cytokine production, and degranulation of bone marrow-derived mast cells (BMMC). In addition, we found that the E3 ubiquitin ligase, tripartite motif containing protein 27 (TRIM27), negatively regulates FcεRI activation of KCa3.1 and downstream signaling by ubiquitinating and inhibiting PI3KC2β. TRIM27−/− mice are also more susceptible in vivo to acute anaphylaxis. These findings identify TRIM27 as an important negative regulator of mast cells in vivo and suggest that PI3KC2β is a potential new pharmacologic target to treat IgE-mediated disease.
Higher-order chromosomal organization for transcription regulation is poorly understood in eukaryotes. Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET), we mapped long-range chromatin interactions associated with RNA polymerase II in human cells and uncovered widespread promoter-centered intra-genic, extra-genic and inter-genic interactions. These interactions further aggregated into higher-order clusters, wherein proximal and distal genes were engaged through promoter-promoter interactions. Most genes with promoter-promoter interactions were active and transcribed cooperatively, and some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls. Comparative analyses of different cell lines showed that cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription, and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions. Furthermore, genetically-identified disease-associated non-coding elements were found to be spatially engaged with corresponding genes through long-range interactions. Overall, our study provides insights into the transcription regulation by three-dimensional chromatin interactions for both housekeeping and cell-specific genes in human cells.
DNA methylation at proximal promoters facilitates lineage restriction by silencing cell type–specific genes. However, euchromatic DNA methylation frequently occurs in regions outside promoters. The functions of such nonproximal promoter DNA methylation are unclear. Here we show that the de novo DNA methyltransferase Dnmt3a is expressed in postnatal neural stem cells (NSCs) and is required for neurogenesis. Genome-wide analysis of postnatal NSCs indicates that Dnmt3a occupies and methylates intergenic regions and gene bodies flanking proximal promoters of a large cohort of transcriptionally permissive genes, many of which encode regulators of neurogenesis. Surprisingly, Dnmt3a-dependent nonproximal promoter methylation promotes expression of these neurogenic genes by functionally antagonizing Polycomb repression. Thus, nonpromoter DNA methylation by Dnmt3a may be used for maintaining active chromatin states of genes critical for development.
Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity1. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state2,3. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance2. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.
Radiotherapy is used in locally advanced pancreatic cancers where it can improve survival in combination with gemcitabine. However, prognosis is still poor in this setting where more effective therapies remain needed. MLN4924 is an investigational small molecule currently in Phase I clinical trials. MLN4924 inhibits NAE (NEDD8 Activating Enzyme), a pivotal regulator of the E3 ubiquitin ligase SCF (SKP1, Cullins, and F-box protein), that has been implicated recently in DNA repair. In this study, we provide evidence that MLN4924 can be used as an effective radiosensitizer in pancreatic cancer. Specifically, MLN4924 (20–100 nM) effectively inhibited cullin neddylation and sensitized pancreatic cancer cells to ionizing radiation in vitro with a sensitivity enhancement ratio (SER) of ~1.5. Mechanistically, MLN4924 treatment stimulated an accumulation of several SCF substrates, including CDT1, WEE1 and NOXA, in parallel with an enhancement of radiation-induced DNA damage, aneuploidy, G2/M phase cell cycle arrest and apoptosis. RNAi-mediated knockdown of CDT1 and WEE1 partially abrogated MLN4924-induced aneuploidy, G2/M arrest, and radiosensization, indicating a causal effect. Further, MLN4924 was an effective radiosensitizer in mouse xenograft models of human pancreatic cancer. Our findings offer proof of concept for use of MLN4924 as a novel class of radiosensitizer for the treatment of pancreatic cancer.
NAE inhibitor; MLN4924; CRL/SCF E3 ubiquitin ligase; radiosensitization; DNA damage; pancreatic cancer cells
To examine the relationship between angiogenesis and lymphangigenesis in recurrent pterygia.
Tissues from 34 excised recurrent pterygia (including 12 Grade 1, 10 Grade 2, and 12 Grade 3) were involved in the study and tissues from 7 nasal epibulbar conjunctivae segments were used as controls. Sections from each pterygium were immunostained with CD31 and LYVE-1 monoclonal antibodies to evaluate lymphatic microvessel density (LMVD) and blood microvessel density (BMVD), and the relationship between LMVD and BMVD in the pterygium was examined.
There was a large number of CD31(+)LYVE-1(−) blood vessels but only a few CD31(+)LYVE-1(+) lymphatic vessels in grades 1 and 2 pterygium. However, lymphatic vessels were dramatically increased in grade 3 pterygium. LMVD correlated closely with BMVD in all pterygia, including grades 1, 2 and 3 peterygium patients (all P values <0.01). Although both the density of blood and lymphatic vessels increased in recurrent pterygia, lymphatic vessels developed much faster than blood vessels, especially in grade 3 pterygia.
There is a significant but not parallel relationship between angiogenesis and lymphangiogenesis in recurrent pterygium. The outgrowth of blood and lymphatic vessels provide evidence that immunological mechanism may play a role in the development and recurrence of pterygium.
angiogenesis; lymphangiogenesis; recurrent pterygium
SAG/RBX/ROC protein is an essential RING component of SCF E3 ubiquitin ligase. The role of SAG during embryogenesis remains unknown. We report here a critical role for SAG in controlling vascular and neural development by modulating RAS activity via promoting degradation of neurofibromatosis type 1 (NF1). Mice mutant for Sag died at embryonic day 11.5-12.5 with severe abnormalities in vascular and nervous system. Sag inactivation caused Nf1 accumulation and Ras inhibition, which blocks embryonic stem (ES) cells from undergoing endothelial differentiation and inhibits angiogenesis and proliferation in teratomas. Simultaneous Nf1 deletion fully rescues the differentiation defects in Sag−/− ES cells, and partially rescues vascular and neural defects in Sag−/− embryos, suggesting that the effects of Sag deletion may not be solely explained by Nf1 misregulation. Collectively, our study identifies NF1 as a physiological substrate of SAG-CUL1-FBXW7 E3 ligase and establishes a ubiquitin-dependent regulatory mechanism for the NF1-RAS pathway during embryogenesis.
Apoptosis; Endothelial differentiation; mouse knockout; NF1-RAS; SAG-CUL1-FBXW7 E3 ubiquitin ligase; vascular and neural development
HIV infection related to commercial sexual contact is a serious public health issue in China. The objectives of the present study are to explore the predictors of condom use among female sex workers (FSWs) in China and examine the relationship between Health Belief Model (HBM) constructs.
A cross-sectional study was conducted in two cities (Wuhan and Suizhou) in Hubei Province, China, between July 2009 and June 2010. A total of 427 FSWs were recruited through mediators from the ‘low-tier’ entertainment establishments. Data were obtained by self-administered questionnaires. Structural equation models were constructed to examine the association. We collected 363 valid questionnaires. Within the context of HBM, perceived severity of HIV mediated through perceived benefits of condom use had a weak effect on condom use (r = 0.07). Perceived benefits and perceived barriers were proximate determinants of condom use (r = 0.23 and r = −0.62, respectively). Self-efficacy had a direct effect on perceived severity, perceived benefits, and perceived barriers, which was indirectly associated with condom use behaviors (r = 0.36).
The HBM provides a useful framework for investigating predictors of condom use behaviors among FSWs. Future HIV prevention interventions should focus on increasing perceived benefits of condom use, reducing barriers to condoms use, and improving self-efficacy among FSWs.
The endoribonuclease, Dicer, is indispensible for generating the majority of mature microRNAs (miRNAs), which are posttranscriptional regulators of gene expression involved in a wide range of developmental and pathological processes in mammalian central nervous system. While functions of Dicer-dependent miRNA pathways in neurons and oligodendrocytes have been extensively investigated, little is known about the role of Dicer in astrocytes. Here we report the effect of Cre-loxP mediated conditional deletion of Dicer selectively from postnatal astroglia on brain development. Dicer-deficient mice exhibited normal motor development and neurological morphology prior to postnatal week 5. Thereafter mutant mice invariably developed a rapidly fulminant neurological decline characterized by ataxia, severe progressive cerebellar degeneration, seizures, uncontrollable movements and premature death by postnatal week 9–10. Integrated transcription profiling, histological and functional analyses of cerebella showed that deletion of Dicer in cerebellar astrocytes altered the transcriptome of astrocytes to be more similar to an immature or reactive-like state prior to the onset of neurological symptoms or morphological changes. As a result, critical and mature astrocytic functions including glutamate uptake and antioxidant pathways were substantially impaired, leading to massive apoptosis of cerebellar granule cells and degeneration of Purkinje cells. Collectively, our study demonstrates the critical involvement of Dicer in normal astrocyte maturation and maintenance. Our findings also reveal non-cell autonomous roles of astrocytic Dicer-dependent pathways in regulating proper neuronal functions and implicate that loss of or dysregulation of astrocytic Dicer-dependent pathways may be involved in neurodegeneration and other neurological disorders.
Many lineage-specific genes are poised and silenced in stem cells. Upon differentiation, genes that are related to self-renewal and alternative lineages are stably silenced. CpG methylation at proximal promoters and PRC2-mediated H3K27me3 play a role in silencing genes temporarily or permanently, with or without coexistence of active epigenetic marks, respectively. Interestingly, DNA methylation on neuronal genes that is distal to transcription start site enable transcription activation owing to its ability to repel PRC2-mediated inhibition. In addition, DNA demethylase Tet proteins play a role in regulation of changes in DNA methylation and related H3K27me3 during differentiation. Collectively, a complex epigenetic network formed by H3K4me3, histone acetylation/deacetylation, H3K27me3 and DNA methylation/demethylation act together to regulate stem cell self-renewal and differentiation.
With voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain of Aspergillus flavus, the second leading cause of IA after Aspergillus fumigatus, has emerged. The VRC-resistant strain of A. flavus was isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain of A. flavus were cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in the cyp51C gene. To further determine the role of this mutated gene for VRC resistance in A. flavus, an Agrobacterium tumefaciens-mediated gene replacement approach was applied. Consequently, the mutated cyp51C gene from this A. flavus strain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in the cyp51C gene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in the cyp51C gene was identified as responsible for VRC resistance in A. flavus. These findings indicated that the detection of this mutation in A. flavus could serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance in A. flavus causing IA, would be required to fully understand this mechanism.
DEPTOR, an inhibitor of mTORC1 and mTORC2, is degraded via ubiquitin-proteasome pathway by an unknown E3 ubiquitin ligase. Here we report that DEPTOR is a physiological substrate of SCFβTrCP E3 ligase for targeted degradation. Upon growth factor stimulation, RSK1 and S6K1 kinases are activated to phosphorylate DEPTOR, which is then recognized by the F-box protein, βTrCP via its degron sequence for subsequent ubiquitination and degradation by SCF E3. Endogenous DEPTOR levels are negatively regulated by βTrCP. DEPTOR half-life is shortened by βTrCP but extended by a dominant negative mutant of βTrCP, by RSK1/S6K1 inhibition, and by βTrCP degron site mutations. Biologically, DEPTOR accumulation upon βTrCP knockdown inactivates mTORC1 and activates AKT in cancer cells to confer resistance to rapamycin and paclitaxel. Furthermore, DEPTOR accumulates upon glucose deprivation and mTOR inhibition, to induce autophagy. Thus, βTrCP-DEPTOR-mTOR intertwine to regulate cell survival and autophagy.
Autophagy; βTrCP; DEPTOR; mTOR; SCF E3 ligase; survival
General anesthesia has been likened to a state in which anesthetized subjects are locked out of access to both rapid eye movement (REM) sleep and wakefulness. Were this true for all anesthetics, one might expect a significant REM rebound following anesthetic exposure. However, for the intravenous anesthetic propofol, studies demonstrate that no sleep debt accrues. Moreover, pre-existing sleep debts dissipate during propofol anesthesia. To determine whether these effects are specific to propofol or are typical of volatile anesthetics we tested the hypothesis that REM sleep debt would accrue in rodents anesthetized with volatile anesthetics.
Electroencephalographic and electromyographic electrodes were implanted in 10 mice. After 9–11 days of recovery and habituation to a 12h:12h light:dark cycle, baseline states of wakefulness, non-rapid eye movement sleep, and REM sleep were recorded in mice exposed to 6 hours of an oxygen control and on separate days to 6 hours of isoflurane, sevoflurane, or halothane in oxygen. All exposures were conducted at the onset of light.
Mice in all three anesthetized groups exhibited a significant doubling of REM sleep during the first six-hours of the dark phase of the circadian schedule while only mice exposed to halothane displayed a significant increase in non-rapid eye movement sleep that peaked at 152% of baseline.
REM sleep rebound following exposure to volatile anesthetics suggests that these volatile anesthetics do not fully substitute for natural sleep. This result contrasts with the published actions of propofol for which no REM sleep rebound occurred.
Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.
Although radiotherapy represents one of the most effective treatment modalities for patients with cancer, inherent and/or acquired resistance of cancer cells to radiotherapy is often an impediment to effective treatment. Diverse strategies have been developed to improve the efficacy of radiotherapy. The ubiquitin-proteasome system (UPS) operates in numerous vital biologic processes by controlling the protein turnover in cells. Ubiquitination is central to the UPS pathway, and it relies on the E3 ubiquitin ligases to catalyze the covalent attachment of ubiquitin to its protein substrates. Cullin-based RING ligases (CRLs) are the largest family of E3 ligases that are responsible for the ubiquitination and destruction of numerous cancer-relevant proteins. Its deregulation has been linked to many human cancers, making it an attractive target for therapeutic intervention. This review discusses how targeting the ubiquitin-proteasome system, particularly CRLs, is an exciting new strategy for radiosensitization in cancer and, specifically, focuses on MLN4924, a recently discovered small-molecule inhibitor of the NEDD8-activating enzyme, which is being characterized as a novel radiosensitizing agent against cancer cells by inactivating CRL E3 ubiquitin ligases.
Bioimpedance analysis (BIA) has been reported as helpful in identifying hypervolemia. Observation data showed that hypervolemic maintenance hemodialysis (MHD) patients identified using BIA methods have higher mortality risk. However, it is not known if BIA-guided fluid management can improve MHD patients’ survival. The objectives of the BOCOMO study are to evaluate the outcome of BIA guided fluid management compared with standard care.
This is a multicenter, prospective, randomized, controlled trial. More than 1300 participants from 16 clinical sites will be included in the study. The enrolment period will last 6 months, and minimum length of follow-up will be 36 months. MHD patients aged between 18 years and 80 years who have been on MHD for at least 3 months and meet eligibility criteria will be invited to participate in the study. Participants will be randomized to BIA arm or control arm in a 1:1 ratio. A portable whole body bioimpedance spectroscopy device (BCM—Fresenius Medical Care D GmbH) will be used for BIA measurement at baseline for both arms of the study. In the BIA arm, additional BCM measurements will be performed every 2 months. The primary intent-to-treat analysis will compare outcomes for a composite endpoint of death, acute myocardial infarction, stroke or incident peripheral arterial occlusive disease between groups. Secondary endpoints will include left ventricular wall thickness, blood pressure, medications, and incidence and length of hospitalization.
Previous results regarding the benefit of strict fluid control are conflicting due to small sample sizes and unstable dry weight estimating methods. To our knowledge this is the first large-scale, multicentre, prospective, randomized controlled trial to assess whether BIS-guided volume management improves outcomes of MHD patients. The endpoints of the BOCOMO study are of utmost importance to health care providers. In order to obtain that aim, the study was designed with very careful important considerations related to the endpoints, sample size, inclusion criteria, exclusion criteria and so on. For example, annual mortality of Beijing MHD patients was around 10%. To reach statistical significance, the sample size will be very large. By using composite endpoint, the sample size becomes reasonable and feasible. Limiting inclusion to patients with urine volume less than 800 ml/day the day before dialysis session will limit confounding due to residual renal function effects on the measured parameters. Patients who had received BIS measurement within 3 months prior to enrolment are excluded as data from such measurements might lead to protocol violation. Although not all patients enrolled will be incident patients, we will record the vintage of dialysis in the multivariable analysis.
Current Controlled Trials NCT01509937
Hemodialysis; Bioimpedance; Dry weight; Body composition monitor; Randomized controlled trial
For decades, it has remained unknown whether artiodactyls, such as cattle, pigs, and sheep, express immunoglobulin D (IgD), although the δ gene was identified in these species nearly 10 years ago. By developing a mouse anti-bovine IgD heavy chain monoclonal antibody (13C2), we show that secreted bovine IgD was present mainly as a monomer in serum and was heavily glycosylated by N-linked saccharides. Nonetheless, IgD was detectable in some but not all of the Holstein cattle examined. Membrane-bound IgD was detected in the spleen by western blotting. Flow cytometric analysis demonstrated that IgD-positive B cells constituted a much lower percentage of B cells in the bovine spleen (∼6.8% of total B cells), jejunal Peyer's patches (∼0.8%), and peripheral blood leukocytes (∼1.2%) than in humans and mice. Furthermore, IgD-positive B cells were almost undetectable in bovine bone marrow and ileal Peyer's patches. We also demonstrated that the bovine δ gene can be expressed via class switch recombination. Accordingly, bovine δ germline transcription, which involves an Iδ exon and is highly homologous to Iμ, was confirmed. However, we could not identify an Iδ promoter, despite bovine Eμ demonstrating both enhancer and promoter activity. This study has answered a long-standing question in cattle B cell biology and significantly contributes to our understanding of B cell development in this species.
The histamine H3 receptor (H3R) has been recognized as a promising target for the treatment of various central and peripheral nervous system diseases. In this study, a non-imidazole compound, ZEL-H16, was identified as a novel histamine H3 receptor agonist. ZEL-H16 was found to bind to human H3R with a Ki value of approximately 2.07 nM and 4.36 nM to rat H3R. Further characterization indicated that ZEL-H16 behaved as a partial agonist on the inhibition of forskolin-stimulated cAMP accumulation (the efficacy was 60% of that of histamine) and activation of ERK1/2 signaling (the efficacy was 50% of that of histamine) at H3 receptors, but acted as a full agonist just like histamin in the guinea-pig ileum contraction assay. These effects were blocked by pertussis toxin and H3 receptor specific antagonist thioperamide. ZEL-H16 showed no agonist or antagonist activities at the cloned human histamine H1, H2, and H4 receptors and other biogenic amine GPCRs in the CRE-driven reporter assay. Furthermore, our present data demonstrated that treatment of ZEL-H16 resulted in intensive H3 receptor internalization and delayed recycling to the cell surface as compared to that of control with treatment of histamine. Thus, ZEL-H16 is a novel and potent nonimidazole agonist of H3R, which might serve as a pharmacological tool for future investigations or as possible therapeutic agent of H3R.
Retinal pigment epithelium (RPE) cells can be obtained through in vitro differentiation of both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We have previously identified 87 signature genes relevant to RPE cell differentiation and function through transcriptome analysis of both human ESC- and iPSC-derived RPE as well as normal fetal RPE. Here, we profile miRNA expression through small RNA-seq in human ESCs and their RPE derivatives. Much like conclusions drawn from our previous transcriptome analysis, we find that the overall miRNA landscape in RPE is distinct from ESCs and other differentiated somatic tissues. We also profile miRNA expression during intermediate stages of RPE differentiation and identified unique subsets of miRNAs that are gradually up- or down-regulated, suggesting that dynamic regulation of these miRNAs is associated with the RPE differentiation process. Indeed, the down-regulation of a subset of miRNAs during RPE differentiation is associated with up-regulation of RPE-specific genes, such as RPE65, which is exclusively expressed in RPE. We conclude that miRNA signatures can be used to classify different degrees of in vitro differentiation of RPE from human pluripotent stem cells. We suggest that RPE-specific miRNAs likely contribute to the functional maturation of RPE in vitro, similar to the regulation of RPE-specific mRNA expression.
We investigated the antifungal effect of non-thermal plasma, as well as its combination with common antifungal drugs, against Candida biofilms. A direct current atmospheric pressure He/O2 (2%) plasma microjet (PMJ) was used to treat Candida biofilms in a 96-well plate. Inactivation efficacies of the biofilms were evaluated by XTT assay and counting colony forming units (CFUs). Morphological properties of the biofilms were evaluated by Scanning Electron Microscope (SEM). The sessile minimal inhibitory concentrations (SMICs) of fluconazole, amphotericin B, and caspofungin for the biofilms were also tested. Electron Spin Resonance (ESR) spectroscopy was used to detect the reactive oxygen species (ROS) generated directly and indirectly by PMJ. The Candida biofilms were completely inactivated after 1 min PMJ treatment, where severely deformed fungal elements were observed in SEM images. The SMICs of the tested antifungal drugs for the plasma-treated biofilms were decreased by 2–6 folds of dilution, compared to those of the untreated controls. ROS such as hydroxyl radical (•OH), superoxide anion radical (•O2-) and singlet molecular oxygen (1O2) were detected by ESR. We hence conclude that He/O2 (2%) plasma alone, as well as in combination with common antifungal drugs, is able to inactivate Candida biofilms rapidly. The generation of ROS is believed to be one of the underlying mechanisms for the fungicidal activity of plasma.
Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.
This review focuses on the diversity of immunoglobulin (Ig) genes and Ig isotypes that are expressed in domestic animals. Four livestock species—cattle, sheep, pigs, and horses—express a full range of Ig heavy chains (IgHs), including μ, δ, γ, ϵ, and α. Two poultry species (chickens and ducks) express three IgH isotypes, μ, υ, and α, but not δ. The κ and λ light chains are both utilized in the four livestock species, but only the λ chain is expressed in poultry. V(D)J recombination, somatic hypermutation (SHM), and gene conversion (GC) are three distinct mechanisms by which immunoglobulin variable region diversity is generated. Different domestic animals may use distinct means to diversify rearranged variable regions of Ig genes.
Diversity; Domestic animals; Immunoglobulin gene