In 2010, the international community, under the auspices of the Convention on Biological Diversity, agreed on 20 biodiversity-related "Aichi Targets" to be achieved within a decade. We provide a comprehensive mid-term assessment of progress toward these global targets using 55 indicator data sets. We projected indicator trends to 2020 using an adaptive statistical framework that incorporated the specific properties of individual time series. On current trajectories, results suggest that despite accelerating policy and management responses to the biodiversity crisis, the impacts of these efforts are unlikely to be reflected in improved trends in the state of biodiversity by 2020. We highlight areas of societal endeavor requiring additional efforts to achieve the Aichi Targets, and provide a baseline against which to assess future progress.
Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.
To present a case of endodontic failure obturated with a methacrylate-based root filling material, Resilon/RealSeal (RS). To determine if RS is susceptible to biodegradation by endodontically relevant microbes by a method known to show RS degradation.
Biodiversity is undergoing unprecedented global decline. Efforts to slow this rate have focused foremost on rarer species, which are at most risk of extinction. Less interest has been paid to more common species, despite their greater importance in terms of ecosystem function and service provision. How rates of decline are partitioned between common and less abundant species remains unclear. Using a 30-year data set of 144 bird species, we examined Europe-wide trends in avian abundance and biomass. Overall, avian abundance and biomass are both declining with most of this decline being attributed to more common species, while less abundant species showed an overall increase in both abundance and biomass. If overall avian declines are mainly due to reductions in a small number of common species, conservation efforts targeted at rarer species must be better matched with efforts to increase overall bird numbers, if ecological impacts of birds are to be maintained.
Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here, we show that YAP, the downstream target of the tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding posttranscriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.
Emerging data support that RNA methylation plays important roles in RNA processing and metabolism. The methyltransferases Mettl3 and Mettl14 are shown to catalyse N(6)-methyladenosine (m(6)A) RNA modification in embryonic stem cells (ESCs). This m(6)A modification controls RNA metabolism and functions to destabilize mRNAs encoding developmental regulators to help sustain ESC self-renewal.
The purposes of this study were to fabricate biodegradable polydioxanone (PDS II®) electrospun periodontal drug delivery systems (hereafter referred to as matrices) containing either metronidazole (MET) or ciprofloxacin (CIP) and to investigate the effects of antibiotic incorporation on both periodontopathogens and commensal oral bacteria.
Eliminating and/or inhibiting bacterial growth within the root canal system has been shown to play a key role in the regenerative outcome. The aim of this study was to synthesize and determine in vitro both the antimicrobial effectiveness and cytocompatibility of bimix antibiotic-containing polydioxanone-based polymer scaffolds.
The identification of nucleic acid aptamers would be advanced if they could be obtained after fewer rounds of selection and amplification. In this paper the identification of bivalent aptamers for thrombin by SELEX and single-step selection are compared using next generation sequencing and motif finding informatics. Results show that similar aptamers are identified by both methods. This is significant because it shows that next generation sequencing and motif finding informatics have the potential to simplify the selection of aptamers by avoiding multiple rounds of enzymatic transcription and amplification.
Concern that European forest biodiversity is depleted and declining has provoked widespread efforts to improve management practices. To gauge the success of these actions, appropriate monitoring of forest ecosystems is paramount. Multi-species indicators are frequently used to assess the state of biodiversity and its response to implemented management, but generally applicable and objective methodologies for species' selection are lacking. Here we use a niche-based approach, underpinned by coarse quantification of species' resource use, to objectively select species for inclusion in a pan-European forest bird indicator. We identify both the minimum number of species required to deliver full resource coverage and the most sensitive species' combination, and explore the trade-off between two key characteristics, sensitivity and redundancy, associated with indicators comprising different numbers of species. We compare our indicator to an existing forest bird indicator selected on the basis of expert opinion and show it is more representative of the wider community. We also present alternative indicators for regional and forest type specific monitoring and show that species' choice can have a significant impact on the indicator and consequent projections about the state of the biodiversity it represents. Furthermore, by comparing indicator sets drawn from currently monitored species and the full forest bird community, we identify gaps in the coverage of the current monitoring scheme. We believe that adopting this niche-based framework for species' selection supports the objective development of multi-species indicators and that it has good potential to be extended to a range of habitats and taxa.
During the diversification of living organisms, novel adaptive traits usually evolve through the co-option of preexisting genes. However, most enzymes are encoded by gene families, whose members vary in their expression and catalytic properties. Each may therefore differ in its suitability for recruitment into a novel function. In this work, we test for the presence of such a gene recruitment bias using the example of C4 photosynthesis, a complex trait that evolved recurrently in flowering plants as a response to atmospheric CO2 depletion. We combined the analysis of complete nuclear genomes and high-throughput transcriptome data for three grass species that evolved the C4 trait independently. For five of the seven enzymes analyzed, the same gene lineage was recruited across the independent C4 origins, despite the existence of multiple copies. The analysis of a closely related C3 grass confirmed that C4 expression patterns were not present in the C3 ancestors but were acquired during the evolutionary transition to C4 photosynthesis. The significant bias in gene recruitment indicates that some genes are more suitable for a novel function, probably because the mutations they accumulated brought them closer to the characteristics required for the new function.
Botulinum neurotoxin type A (BoNTA) is one of seven serotypes produced by Clostridium botulinum (types A thru G) and is the serotype most widely used to treat both cosmetic and medical conditions. Potency for botulinum toxin preparations is expressed in mouse LD50 units. There is a need to develop a non-animal based replacement for this potency assay.
Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly.
Both Streptococcus mutans and Streptococcus sanguinis are normal bacterial inhabitants of dental plaque. Streptococcus mutans is the major agent causing dental caries. It has been well documented that nicotine affects the growth of S. mutans. This study investigated the effect of nicotine on mono- and dual-species growth of S. mutans and S. sanguinis. The results indicate that nicotine has no significant effect on S. sanguinis grown in either mono- or dual-species biofilms. However, nicotine significantly increased (P < 0.05) the growth of S. mutans in dual-species biofilm formation. In addition, the CFU level of S. sanguinis was higher than S. mutans without nicotine in the culture. With the addition of nicotine, the level of S. mutans biofilm was significantly enhanced as the nicotine concentration increased over the level of S. sanguinis in dual-species biofilm, and we also got the same result from the fluorescence in situ hybridization detecting the two bacteria grown in biofilm formation. The exopolysaccharide (EPS) of S. mutans has also been increased by the increasing nicotine concentration, while the EPS of S. sanguinis was decreased or inhibited by the affected nicotine. The data further confirm that nicotine is able to enhance the growth of S. mutans.
Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs.
In this study we compared the antibacterial effect of triple antibiotic paste (TAP), double antibiotic paste (DAP), and calcium hydroxide [Ca(OH)2] against Enterococcus faecalis and Porphyromonas gingivalis biofilm.
Streptococcus mutans is a common Gram-positive bacterium and plays a significant role in dental caries. Tobacco and/or nicotine have documented effects on S. mutans growth and colonization. Sortase A is used by many Gram-positive bacteria, including S. mutans, to facilitate the insertion of certain cell surface proteins, containing an LPXTGX motif such as antigen I/II. This study examined the effect of nicotine on the function of sortase A to control the physiology and growth of S. mutans using wild-type S. mutans NG8, and its isogenic sortase-defective and -complemented strains. Briefly, the strains were treated with increasing amounts of nicotine in planktonic growth, biofilm metabolism, and sucrose-induced and saliva-induced antigen I/II-dependent biofilm formation assays. The strains exhibited no significant differences with different concentrations of nicotine in planktonic growth assays. However, they had significantly increased (P?0.05) biofilm metabolic activity (2- to 3-fold increase) as the concentration of nicotine increased. Furthermore, the sortase-defective strain was more sensitive metabolically to nicotine than the wild-type or sortase-complemented strains. All strains had significantly increased sucrose-induced biofilm formation (2- to 3-fold increase) as a result of increasing concentrations of nicotine. However, the sortase-defective strain was not able to make as much sucrose- and saliva-induced biofilm as the wild-type NG8 did with increasing nicotine concentrations. These results indicated that nicotine increased metabolic activity and sucrose-induced biofilm formation. The saliva-induced biofilm formation assay and qPCR data suggested that antigen I/II was upregulated with nicotine but biofilm was not able to be formed as much as wild-type NG8 without functional sortase A.
LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here, we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO could be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling.
The pluripotency factor Lin28 blocks the expression of let-7 microRNAs in undifferentiated cells during development, and functions as an oncogene in a subset of cancers. Lin28 binds to let-7 precursor (pre-let-7) RNAs and recruits 3 terminal uridylyl transferases to selectively inhibit let-7 biogenesis. Uridylated pre-let-7 is refractory to processing by Dicer, and is rapidly degraded by an unknown RNase. Here we identify Dis3l2 as the 3-5 exonuclease responsible for the decay of uridylated pre-let-7 in mouse embryonic stem cells. Biochemical reconstitution assays show that 3 oligouridylation stimulates Dis3l2 activity in vitro, and knockdown of Dis3l2 in mouse embryonic stem cells leads to the stabilization of pre-let-7. Our study establishes 3 oligouridylation as an RNA decay signal for Dis3l2, and identifies the first physiological RNA substrate of this new exonuclease, which is mutated in the Perlman syndrome of fetal overgrowth and causes a predisposition to Wilms tumour development.
Argonaute proteins are essential components of microRNA (miRNA)- and small interfering (siRNA)-mediated post-transcriptional gene-silencing pathways. In mammals, Argonaute2 (Ago2) is the catalytic center of the RNA-induced silencing complex (RISC) that recognizes and endonucleolytically cleaves messenger RNAs of complementary sequence. Although Ago2 is essential for RISC activity, the mechanisms regulating Argonaute protein expression are largely unknown. Here we report that Ago2 expression is dependent on miRNA abundance and that unloaded Ago2 protein is unstable. We observed a low level of Ago2 protein in Dicer- or DGCR8-deficent mouse embryonic stem cells (ESCs) that could be rescued by reintroduction of the respective cDNAs or by transfection of miRNAs or siRNAs. We found expression of Ago2 protein from a transgene to be similarly regulated, further supporting a post-transcriptional control mechanism. Inhibition of Hsc70/Hsp90 led to decreased Ago2 expression consistent with the reported role of this chaperone complex in RISC assembly. We furthermore found that the degradation of Ago2 was specifically blocked by inhibition of the lysosome, but not the proteasome. Our results illuminate a novel feedback mechanism that post-transcriptionally couples Ago2 protein levels with small RNA abundance with implications for RNA-interference (RNAi) and miRNA function.
Overexpression of LIN28A is associated with human germ cell tumors and promotes primordial germ cell (PGC) development from embryonic stem cells in vitro and in chimeric mice. Knockdown of Lin28a inhibits PGC development in vitro, but how constitutional Lin28a deficiency affects the mammalian reproductive system in vivo remains unknown. Here, we generated Lin28a knockout (KO) mice and found that Lin28a deficiency compromises the size of the germ cell pool in both males and females by affecting PGC proliferation during embryogenesis. Interestingly however, in Lin28a KO males, the germ cell pool partially recovers during postnatal expansion, while fertility remains impaired in both males and females mated to wild-type mice. Embryonic overexpression of let-7, a microRNA negatively regulated by Lin28a, reduces the germ cell pool, corroborating the role of the Lin28a/let-7 axis in regulating the germ lineage.
Bacterial adherence to the acquired dental pellicle, important in dental caries (caries), is mediated by receptor-adhesins such as salivary agglutinin binding to Streptococcus mutans antigen I/II (I/II). Ten selected I/II epitopes were chosen to determine their reactivity to human salivary IgA. Previous studies suggested that a specific HLA biomarker group (HLA-DRB1*04) may have differential influence of immune responses to I/II. However, it was not known whether secretory IgA (SIgA) responses to the selected epitopes from HLA-DRB1*04 positive subjects were different compared to controls, or across other caries-related factors such as total IgA (TIgA). Thirty-two total subjects were matched according to HLA type, gender, ethnicity and age. HLA genotyping, oral bacterial, immunoglobulin and antibody analyses were performed. A large observed difference emerged with regard to the natural immune reservoir of TIgA in HLA-DRB1*04 positive subjects, specifically, a 27.6% reduction compared to controls. In contrast to all other epitopes studied, HLA-DRB1*04 positive subjects also exhibited reduced reactivity to I/II epitope 834-853. HLA-DRB1*04 positive subjects exhibited lower specific SIgA activity/TIgA to 834-853 and also a lower specific reactivity to 834-853/whole cell S. mutans UA159. Furthermore, HLA-DRB1*04 positive subjects exhibited lower responses to I/II in its entirety. The large observed difference in TIgA and the 834-853 reactivity pattern across multiple measures suggest potentially important connections pertaining to the link between HLA-DRB1*04 and caries.
The ecological impacts of changing forest management practices in Europe are poorly understood despite European forests being highly managed. Furthermore, the effects of potential drivers of forest biodiversity decline are rarely considered in concert, thus limiting effective conservation or sustainable forest management. We present a trait-based framework that we use to assess the detrimental impact of multiple land-use and management changes in forests on bird populations across Europe. Major changes to forest habitats occurring in recent decades, and their impact on resource availability for birds were identified. Risk associated with these changes for 52 species of forest birds, defined as the proportion of each species key resources detrimentally affected through changes in abundance and/or availability, was quantified and compared to their pan-European population growth rates between 1980 and 2009. Relationships between risk and population growth were found to be significantly negative, indicating that resource loss in European forests is an important driver of decline for both resident and migrant birds. Our results demonstrate that coarse quantification of resource use and ecological change can be valuable in understanding causes of biodiversity decline, and thus in informing conservation strategy and policy. Such an approach has good potential to be extended for predictive use in assessing the impact of possible future changes to forest management and to develop more precise indicators of forest health.
Transforming growth factor-? (TGF-?) promotes cancer invasion and metastasis and is therefore a potential drug target for cancer treatment. Fresolimumab, which neutralizes all mammalian active isoforms of TGF-?, was radiolabeled with (89)Zr for PET to analyze TGF-? expression, antibody tumor uptake, and organ distribution.
Because of the tradition of using honey as an antimicrobial medicament, we investigated the effect of natural honey (NH) on Streptococcus mutans growth, viability, and biofilm formation compared to that of an artificial honey (AH). AH contained the sugars at the concentrations reported for NH. NH and AH concentrations were obtained by serial dilution with tryptic soy broth (TSB). Several concentrations of NH and AH were tested for inhibition of bacterial growth, viability, and biofilm formation after inoculation with S. mutans UA159 in 96-well microtiter plates to obtain absorbance and CFU values. Overall, NH supported significantly less (P < 0.05) bacterial growth than AH at 25 and 12.5% concentrations. At 50 and 25% concentrations, both honey groups provided significantly less bacterial growth and biofilm formation than the TSB control. For bacterial viability, the results for all honey concentrations except 50% NH were not significantly different from those for the TSB control. NH was able to decrease the maximum velocity of S. mutans growth compared to AH. In summary, NH demonstrated more inhibition of bacterial growth, viability, and biofilm formation than AH. This study highlights the potential antibacterial properties of NH and could suggest that the antimicrobial mechanism of NH is not solely due to its high sugar content.
The ontogeny of neural substrates underlying episodic memory is not well described. Place cells are a surrogate for episodic memory and are important for spatial navigation in rodents. Although place cells are well described in mature brains, the nature of the maturation processes remains uncertain. We now report on the ontogeny of the place cell system in rats between P22 and P43, a time during which there is rapid improvement in spatial behavior. We found that place cells with adult like firing fields were observed at the earliest ages. However, at this age, adult like place cells were few in number and their place fields were not stable across multiple exposures to the same environment. Independently of confounding factors such as the number of exposures to the environment, the proportion of adult-like place cells, their firing rate, and their stability increased with age and the average spatial signal of all pyramidal cells improved. These findings could account for the poor spatial behavior observed at young ages (P20-P30) and suggests that a small number of adult-like place cells are insufficient to support navigation.
Biofilms are masses of microorganisms that bind to and multiply on a solid surface, typically with a fluid bathing the microbes. The microorganisms that are not attached but are free floating in an aqueous environment are termed planktonic cells. Traditionally, microbiology research has addressed results from planktonic bacterial cells. However, many recent studies have indicated that biofilms are the preferred form of growth of most microbes and particularly those of a pathogenic nature. Biofilms on animal hosts have significantly increased resistance to various antimicrobials compared to planktonic cells. These microbial communities form microcolonies that interact with each other using very sophisticated communication methods (i.e., quorum-sensing). The development of unique microbiological tools to detect and assess the various biofilms around us is a tremendously important focus of research in many laboratories. In the present review, we discuss the major biofilm mechanisms and the interactions among oral bacteria.
The Microprocessor, comprising the ribonuclease Drosha and its essential cofactor, the double-stranded RNA-binding protein, DGCR8, is essential for the first step of the miRNA biogenesis pathway. It specifically cleaves double-stranded RNA within stem-loop structures of primary miRNA transcripts (pri-miRNAs) to generate precursor (pre-miRNA) intermediates. Pre-miRNAs are subsequently processed by Dicer to their mature ?22 nt form. Thus, Microprocessor is essential for miRNA maturation, and pri-miRNA cleavage by this complex defines one end of the mature miRNA. Moreover, it is emerging that dysregulation of the Microprocessor is associated with various human diseases. It is therefore important to understand the mechanisms by which the expression of the subunits of the Microprocessor is regulated. Recent findings have uncovered a post-transcriptional mechanism that maintains the integrity of the Microprocessor. These studies revealed that the Microprocessor is involved in the processing of the messenger RNA (mRNA) that encodes DGCR8. This regulatory feedback loop, along with the reported role played by DGCR8 in the stabilization of Drosha protein, is part of a newly identified regulatory mechanism controlling Microprocessor activity.
In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding ?-galactosidase A (?-gal). As in mice, sustained expression in monkeys attenuated antibody responses to ?-gal. However, this effect was not robust, and sustained ?-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.
The let-7 tumor suppressor microRNAs are known for their regulation of oncogenes, while the RNA-binding proteins Lin28a/b promote malignancy by inhibiting let-7 biogenesis. We have uncovered unexpected roles for the Lin28/let-7 pathway in regulating metabolism. When overexpressed in mice, both Lin28a and LIN28B promote an insulin-sensitized state that resists high-fat-diet induced diabetes. Conversely, muscle-specific loss of Lin28a or overexpression of let-7 results in insulin resistance and impaired glucose tolerance. These phenomena occur, in part, through the let-7-mediated repression of multiple components of the insulin-PI3K-mTOR pathway, including IGF1R, INSR, and IRS2. In addition, the mTOR inhibitor, rapamycin, abrogates Lin28a-mediated insulin sensitivity and enhanced glucose uptake. Moreover, let-7 targets are enriched for genes containing SNPs associated with type 2 diabetes and control of fasting glucose in human genome-wide association studies. These data establish the Lin28/let-7 pathway as a central regulator of mammalian glucose metabolism.
MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene expression. Among these, members of the let-7 miRNA family control many cell-fate determination genes to influence pluripotency, differentiation, and transformation. Lin28 is a specific, posttranscriptional inhibitor of let-7 biogenesis. We report crystal structures of mouse Lin28 in complex with sequences from let-7d, let-7-f1, and let-7 g precursors. The two folded domains of Lin28 recognize two distinct regions of the RNA and are sufficient for inhibition of let-7 in vivo. We also show by NMR spectroscopy that the linker connecting the two folded domains is flexible, accommodating Lin28 binding to diverse let-7 family members. Protein-RNA complex formation imposes specific conformations on both components that could affect downstream recognition by other processing factors. Our data provide a molecular explanation for Lin28 specificity and a model for how it regulates let-7.
Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy.
The purpose of this study was to determine if three strains of bacteria could impact the mechanical or surface properties of a dental resin material. Resin material specimens were incubated at 37°C in sterile saline, tryptic soy broth supplemented with sucrose (TSBS), or TSBS inoculated with Streptococcus mutans, Streptococcus gordonii, or Streptococcus sanguis. The specimens were subjected to Fourier transform infrared spectroscopy before and after incubation. The flexural strength test was performed once a week for 6 weeks. Microhardness and scanning electron microscopy (SEM) was performed on specimens at 1 and 6 weeks. Differences in the area under the carbonyl peak were statistically significant for the specimens incubated in the media inoculated with either S. mutans or S. gordonii. To determine why S. sanguis did not produce changes as the other bacteria did, triethylene glycol dimethacrylate, methacrylic acid, and triethylene glycol were added to bacterial cultures at increasing concentrations. Both methacrylic acid and triethylene glycol reduced the number of colony-forming units of S. sanguis. Specimens incubated in TSBS, saline or in culture with S. sanguis demonstrated a decrease in peak stress in week 1 of the flexure strength test. SEM demonstrated that surface topology changed for those specimens incubated in culture with S. mutans or S. gordonii. The changes in surface topology demonstrated here could contribute to the secondary caries and changes in esthetic properties seen clinically with the use of resin materials in dental restorations.
The objective of this study was to use the newly synthesized poly(quaternary ammonium salt) (PQAS)-containing polyacid to formulate the light-curable glass-ionomer cements and study the effect of the PQAS on the compressive strength and antibacterial activity of the formed cements.
Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.
Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity.
Although RNA-mediated interference (RNAi) is a widely conserved process among eukaryotes, including many fungi, it is absent from the budding yeast Saccharomyces cerevisiae. Three human proteins, Ago2, Dicer and TRBP, are sufficient for reconstituting the RISC complex in vitro. To examine whether the introduction of human RNAi genes can reconstitute RNAi in S. cerevisiae, genes encoding these three human proteins were introduced into S. cerevisiae. We observed both siRNA and siRNA- and RISC-dependent silencing of the target gene GFP. Thus, human Ago2, Dicer and TRBP can functionally reconstitute human RNAi in S. cerevisiae, in vivo, enabling the study and use of the human RNAi pathway in a facile genetic model organism.
Pumilio proteins PUM1 and PUM2 are shown to regulate microRNA-dependent gene silencing by induction of a conformational switch in the 3 untranslated region of p27 mRNA. This conformational change is required for efficient microRNA-mediated repression of this cell-cycle regulator in rapidly proliferating cells.
This study reports the synthesis and evaluation of a novel non-leachable poly(quaternary ammonium salt) (PQAS)-containing antibacterial glass-ionomer cement. Fuji II LC cement was used for comparison. Compressive strength (CS) and Streptococcus mutans viability were used to evaluate strength and antibacterial activity, respectively. All specimens were conditioned in distilled water at 37 degrees C before testing. After the addition of 1-30% PQAS, both cements showed a reduction in CS, of 25-95% for Fuji II LC and 13-78% for the experimental cement, and a reduction in S. mutans viability, of 40-79% for Fuji II LC and 40-91% for the experimental cement. The experimental cement showed less CS reduction and higher antibacterial activity compared with Fuji II LC. The result also indicates that the cements are permanently antibacterial, with no leaching of PQAS. It appears that the experimental cement is a clinically attractive dental restorative that can be potentially used for longlasting restorations as a result of its high mechanical strength and permanent antibacterial function.
Understanding the molecular foundations of embryonic stem cell (ESC) self-renewal and pluripotency will facilitate therapeutic exploitation of these remarkable cells. Here we discuss the emerging roles of microRNAs in the establishment and maintenance of ESC identity and summarize our current understanding of the mechanisms controlling microRNA expression and function in ESCs.
In 2002, world leaders committed, through the Convention on Biological Diversity, to achieve a significant reduction in the rate of biodiversity loss by 2010. We compiled 31 indicators to report on progress toward this target. Most indicators of the state of biodiversity (covering species population trends, extinction risk, habitat extent and condition, and community composition) showed declines, with no significant recent reductions in rate, whereas indicators of pressures on biodiversity (including resource consumption, invasive alien species, nitrogen pollution, overexploitation, and climate change impacts) showed increases. Despite some local successes and increasing responses (including extent and biodiversity coverage of protected areas, sustainable forest management, policy responses to invasive alien species, and biodiversity-related aid), the rate of biodiversity loss does not appear to be slowing.
Alcohol withdrawal is associated with affective-behavioral disturbances in both human alcoholics and in animal models. In general, these phenomena are potentiated by increased alcohol exposure duration and by prior withdrawal episodes. Previous studies have also reported locomotor hypoactivity during ethanol withdrawal in rats and mice, but only in novel test environments and not in the home cage. In the present study, we examined the effects of withdrawal from chronic intermittent ethanol (CIE) vapor exposure on the level and circadian periodicity of wheel-running activity in C57BL/6J mice. CIE treatment resulted in reductions in wheel-running activity compared with plain-air controls that persisted for about 1 week after withdrawal. Analysis of circadian waveforms indicated that reduced activity occurred throughout the night phase, but that daily-activity patterns were otherwise unaltered. CIE failed to alter free-running circadian period or phase in animals maintained under constant darkness. These results show that ethanol withdrawal can result in locomotor hypoactivity even in the habitual, home-cage environment, and suggest that withdrawal-related reductions in wheel-running activity may reflect the specific motivational significance of this behavior.
We carried out six experiments to find out whether simple manual reaction time (RT) to flux-equated visual stimuli of different size is modulated by size constancy or by the retinal angle subtended by the stimuli. We found that RT decreased with the increase in perceived stimulus size rather than retinal angle and that this relationship depended on the use of familiar 3-D-like stimuli and on the availability of other size-constancy cues. Thus, a stereotyped speeded motor response, such as that employed in a simple RT paradigm, is modulated by size constancy, as is the case with perceptual judgments. The present results provide original evidence on the relationship between simple RT and perception.
Lin28 and Lin28B, two developmentally regulated RNA-binding proteins and likely proto-oncogenes, selectively inhibit the maturation of let-7 family microRNAs (miRNAs) in embryonic stem cells and certain cancer cell lines. Moreover, let-7 precursors (pre-let-7) were previously found to be terminally uridylated in a Lin28-dependent fashion. Here we identify Zcchc11 (zinc finger, CCHC domain containing 11) as the 3 terminal uridylyl transferase (TUTase) responsible for Lin28-mediated pre-let-7 uridylation and subsequent blockade of let-7 processing in mouse embryonic stem cells. We demonstrate that Zcchc11 activity is UTP-dependent, selective for let-7 and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 miRNAs and repression of let-7 target reporter genes. Our results establish a role for Zcchc11-catalyzed pre-let-7 uridylation in the control of miRNA biogenesis.
In the triangle-bisection illusion, a dot is inscribed exactly halfway up the height of an equilateral triangle, but it looks apparently far more than halfway up. The illusion is found for second-order triangles defined by stereo depth and by equiluminous texture. It is strongest for equilateral triangles, and even stronger for concave triangles with curved sides. We propose that the observers are probably responding to the centre of area or centre of gravity of the triangle rather than to its half-height.
The Microprocessor, comprising the RNase III Drosha and the double-stranded RNA binding protein DGCR8, is essential for microRNA (miRNA) biogenesis. In the miRNA processing pathway certain hairpin structures within primary miRNA (pri-miRNA) transcripts are specifically cleaved by the Microprocessor to release approximately 60-70-nucleotide precursor miRNA (pre-miRNA) intermediates. Although both Drosha and DGCR8 are required for Microprocessor activity, the mechanisms regulating the expression of these proteins are unknown. Here we report that the Microprocessor negatively regulates DGCR8 expression. Using in vitro reconstitution and in vivo studies, we demonstrate that a hairpin, localized in the 5 untranslated region (5UTR) of DGCR8 mRNA, is cleaved by the Microprocessor. Accordingly, knockdown of Drosha leads to an increase in DGCR8 mRNA and protein levels in cells. Furthermore, we found that the DGCR8 5UTR confers Microprocessor-dependent repression of a luciferase reporter gene in vivo. Our results uncover a novel feedback loop that regulates DGCR8 levels.
Rapid climatic change poses a threat to global biodiversity. There is extensive evidence that recent climatic change has affected animal and plant populations, but no indicators exist that summarise impacts over many species and large areas. We use data on long-term population trends of European birds to develop such an indicator. We find a significant relationship between interspecific variation in population trend and the change in potential range extent between the late 20(th) and late 21(st) centuries, forecasted by climatic envelope models. Our indicator measures divergence in population trend between bird species predicted by climatic envelope models to be favourably affected by climatic change and those adversely affected. The indicator shows a rapid increase in the past twenty years, coinciding with a period of rapid warming.
MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as development and cancer. Although their mode of action has attracted great attention, the principles governing their expression and activity are only beginning to emerge. Recent studies have introduced a paradigm shift in our understanding of the microRNA biogenesis pathway, which was previously believed to be universal to all microRNAs. Maturation steps specific to individual microRNAs have been uncovered, and these offer a plethora of regulatory options after transcription with multiple proteins affecting microRNA processing efficiency. Here we review the recent advances in knowledge of the microRNA biosynthesis pathways and discuss their impact on post-transcriptional microRNA regulation during tumour development.
Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.
The pluripotency factor Lin28 recruits a 3 terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.
Status epilepticus (SE) is a common neurological emergency, which has been associated with subsequent cognitive impairments. Neuronal death in hippocampal CA1 is thought to be an important mechanism of these impairments. However, it is also possible that functional interactions between surviving neurons are important. In this study we recorded in vivo single-unit activity in the CA1 hippocampal region of rats while they performed a spatial memory task. From these data we constructed functional networks describing pyramidal cell interactions. To build the networks, we used maximum entropy algorithms previously applied only to in vitro data. We show that several months following SE pyramidal neurons display excessive neuronal synchrony and less neuronal reactivation during rest compared with those in healthy controls. Both effects predict rat performance in a spatial memory task. These results provide a physiological mechanism for SE-induced cognitive impairment and highlight the importance of the systems-level perspective in investigating spatial cognition.
Streptococcus mutans is a key contributor to dental caries. Smokers have a higher number of caries-affected teeth than do nonsmokers, but the association among tobacco, nicotine, caries, and S. mutans growth has not been investigated in detail. Seven S. mutans strains--UA159, UA130, 10449, A32-2, NG8, LM7, and OMZ175--were used in the present study. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), planktonic cell growth, biofilm formation, metabolism, and structure (determined using scanning electron microscopy) of the seven strains treated with different concentrations of nicotine (0-32 mg ml(-1)) were investigated. The MIC, MBC, and MBIC were 16 mg ml(-1) (0.1 M), 32 mg ml(-1) (0.2 M), and 16 mg ml(-1) (0.1 M), respectively, for most of the S. mutans strains. Growth of planktonic S. mutans cells was significantly repressed by 2.0-8.0 mg ml(-1) of nicotine. Biofilm formation and metabolic activity of S. mutans was increased in a nicotine-dependent manner up to 16.0 mg ml(-1) of nicotine. Scanning electron microscopy revealed that S. mutans treated with a high concentration of nicotine a had thicker biofilm and more spherical bacterial cells. In summary, nicotine enhances S. mutans biofilm formation and biofilm metabolic activity. These results suggest that smoking can increase the development of caries by fostering increased formation of S. mutans biofilm on tooth surfaces.
One of the most ancient and highly conserved microRNAs (miRNAs), the let-7 family, has gained notoriety owing to its regulation of stem cell differentiation and essential role in normal development, as well as its tumor suppressor function. Mechanisms controlling let-7 expression have recently been uncovered, specifically the role of the RNA-binding protein Lin28 - a key developmental regulator - in blocking let-7 biogenesis. This review focuses on our current understanding of the Lin28-mediated control of let-7 maturation and highlights the central role of Lin28 in stem cell biology, development, control of glucose metabolism, and dysregulation in human disease. Manipulating the Lin28 pathway for the precise control of let-7 expression may provide novel therapeutic opportunities for cancer and other diseases.
Pluripotent embryonic stem cells have a shortened cell cycle that enables their rapid proliferation. The embryonic stem cell-specific miR-290 and miR-302 microRNA families promote proliferation whereas let-7 microRNAs inhibit self-renewal, and promote cell differentiation. Lin28 suppresses let-7 expression in embryonic stem cells. Here to gain further insight into mechanisms controlling embryonic stem cell self-renewal, we explore the molecular and cellular role of the let-7 target Trim71 (mLin41). We show that Trim71 associates with Argonaute2 and microRNAs, and represses expression of Cdkn1a, a cyclin-dependent kinase inhibitor that negatively regulates the G1-S transition. We identify protein domains required for Trim71 association with Argonaute2, localization to P-bodies, and for repression of reporter messenger RNAs. Trim71 knockdown prolongs the G1 phase of the cell cycle and slows embryonic stem cell proliferation, a phenotype that was rescued by depletion of Cdkn1a. Thus, we demonstrate that Trim71 is a factor that facilitates the G1-S transition to promote rapid embryonic stem cell self-renewal.
A novel furanone derivative and a polyacid constructed from it were synthesized, characterized and formulated into experimental high strength cements. The compressive strength (CS) and Streptococcus mutans viability were used to evaluate the mechanical strength and antibacterial activity of the cements. The effect of human saliva and aging were investigated. The antibacterial activity against Lactobacillus sp. and cytotoxicity to human pulp cells were also evaluated. The results show that all the formulated furanone-containing cements showed antibacterial activity, with an initial reduction in CS. The effect of the furanone derivative loading was significant. Increasing loading enhanced the antibacterial activity but reduced the initial CS of the formed cements. The derivative showed antibacterial activity against both S. mutans and Lactobacillus sp. Human saliva did not affect the antibacterial activity of the cement. The cytotoxicity study with human dental pulp cells shows that the furanone-modified cement was biocompatible. A 30 day aging study indicated that the cements may have long-lasting antibacterial activity. Within the limitations of this study it appears that the experimental cement could be a clinically attractive dental restorative due to its high mechanical strength and antibacterial function.
A novel furanone-containing antibacterial resin composite has been prepared and evaluated. compressive strength (CS) and Streptococcus mutans viability were used to evaluate the mechanical strength and antibacterial activity of the composites. The modified resin composites showed a significant antibacterial activity without substantially decreasing the mechanical strengths. With 5-30 % addition of the furanone derivative, the composite kept its original CS unchanged but showed a significant antibacterial activity with a 16-68 % reduction in the S. mutans viability. Further, the antibacterial function of the new composite was not affected by human saliva. The aging study indicates that the composite may have a long-lasting antibacterial function. Within the limitations of this study, it appears that the experimental antibacterial resin composite may potentially be developed into a clinically attractive dental restorative due to its high mechanical strength and antibacterial function.
C(4) photosynthesis is a complex trait that confers higher productivity under warm and arid conditions. It has evolved more than 60 times via the co-option of genes present in C(3) ancestors followed by alteration of the patterns and levels of expression and adaptive changes in the coding sequences, but the evolutionary path to C(4) photosynthesis is still poorly understood. The grass lineage Alloteropsis offers unparalleled opportunities for studying C(4) evolution, because it includes a C(3) taxon and five C(4) species that vary significantly in C(4) anatomy and biochemistry. Using phylogenetic analyses of nuclear genes and leaf transcriptomes, we show that fundamental elements of the C(4) pathway in the grass lineage Alloteropsis were acquired via a minimum of four independent lateral gene transfers from C(4) taxa that diverged from this group more than 20 million years ago. The transfer of genes that were already fully adapted for C(4) function has occurred periodically over at least the last 10 million years and has been a recurrent source for the optimization of the C(4) pathway. This report shows that plant-plant lateral nuclear gene transfers can be a potent source of genetic novelty and adaptation in flowering plants.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.