A full understanding of gene regulation requires an understanding of the contributions that the various regulatory regions have on gene expression. Although it is well established that sequences downstream of the main promoter can affect expression, our understanding of the scale of this effect and how it is encoded in the DNA is limited. Here, to measure the effect of native S. cerevisiae 3′ end sequences on expression, we constructed a library of 85 fluorescent reporter strains that differ only in their 3′ end region. Notably, despite being driven by the same strong promoter, our library spans a continuous twelve-fold range of expression values. These measurements correlate with endogenous mRNA levels, suggesting that the 3′ end contributes to constitutive differences in mRNA levels. We used deep sequencing to map the 3′UTR ends of our strains and show that determination of polyadenylation sites is intrinsic to the local 3′ end sequence. Polyadenylation mapping was followed by sequence analysis, we found that increased A/T content upstream of the main polyadenylation site correlates with higher expression, both in the library and genome-wide, suggesting that native genes differ by the encoded efficiency of 3′ end processing. Finally, we use single cells fluorescence measurements, in different promoter activation levels, to show that 3′ end sequences modulate protein expression dynamics differently than promoters, by predominantly affecting the size of protein production bursts as opposed to the frequency at which these bursts occur. Altogether, our results lead to a more complete understanding of gene regulation by demonstrating that 3′ end regions have a unique and sequence dependent effect on gene expression.
A basic question in gene expression is the relative contribution of different regulatory layers and genomic regions to the differences in protein levels. In this work we concentrated on the effect of 3′ end sequences. For this, we constructed a library of yeast strains that differ only by a native 3′ end region integrated downstream to a reported gene driven by a constant inducible promoter. Thus we could attribute all differences in reporter expression between the strains to the different 3′ end sequences. Interestingly, we found that despite being driven by the same strong, inducible promoter, our library spanned a wide and continuous range of expression levels of more than twelve-fold. As these measurements represent the sole effect of the 3′ end region, we quantify the contribution of these sequences to the variance in mRNA levels by comparing our measurements to endogenous mRNA levels. We follow by sequence analysis to find a simple sequence signature that correlates with expression. In addition, single cell analysis reveals distinct noise dynamics of 3′ end mediated differences in expression compared to different levels of promoter activation leading to a more complete understanding of gene expression which also incorporates the effect of these regions.
Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV–R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV–R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism while particles displaying 10 R5 peptides per CPMV (CPMV–R5L) are only slowly taken up. The fate of CPMV–R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV–R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30–50% of the CPMV–R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV–R5.
(See the editorial commentary by Johnson, on pages 353–4.)
Background. Clostridium difficile infection (CDI) can cause a wide range of disease, from mild diarrhea to fulminant systemic disease. The incidence of systemic CDI with fatal consequence has increased rapidly in recent years.
Methods. Using an ultrasensitive cytotoxicity assay, we measured C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB) in sera and body fluids of piglets and mice exposed to C. difficile to investigate the relationship between the presence of toxins in body fluids and systemic manifestations of CDI.
Results. We found that both TcdA and TcdB disseminate systemically, with toxins present in the sera and body fluids of infected animals, and toxemia is significantly correlated with the development of systemic CDI. The systemic administration of neutralizing antibodies against both toxins blocked the development of systemic disease in mice. We measured cytokine concentrations in the sera of mice and piglets with systemic and nonsystemic CDI and found that proinflammatory mediators were considerably elevated in animals with systemic CDI.
Conclusion. Our study demonstrates the existence of a strong correlation between toxemia and the occurrence of systemic disease, supporting the hypothesis that systemic CDI is most likely due to the toxicity of TcdA and TcdB and the induction of proinflammatory cytokines by the toxins.
The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.
Background and Purpose
White matter injury (WMI) is the leading cause of brain injury in preterm survivors and results in myelination failure. Although axonal degeneration occurs in necrotic lesions, the role of axonopathy in myelination failure remains controversial for diffuse non-necrotic WMI, which is currently the major form of WMI. We determined the burden of axonopathy in diffuse lesions.
We analyzed WMI in a preterm fetal sheep model of global cerebral ischemia that replicates the relative burden of necrotic and non-necrotic human WMI. WMI was analyzed at 1-or 2-weeks after ischemia and identified by ex vivo high-field (11.7 Tesla) MRI of fixed brain tissue. Axonal integrity was analyzed by immunohistochemical detection of axon injury markers and by transmission electron microscopy (EM) to quantify axon loss and degeneration in MRI-defined lesions.
Axonal degeneration, defined by staining for neurofilament protein and β-amyloid precursor protein, was restricted to discrete necrotic foci with robust microglial activation. Unexpectedly, axonal degeneration was not visualized in the major form of WMI, which comprised large non-necrotic lesions with diffuse reactive astrogliosis. In these major lesions, quantitative EM studies confirmed no significant differences in the density of intact and degenerating axons or in the distribution of axon diameters relative to controls.
The mechanism of myelination failure differs significantly in perinatal WMI dependent upon the burden of necrosis. Axonopathy is associated with focal necrotic injury but not with primary diffuse non-necrotic lesions, which supports that intact axons in the primary lesions are potential targets for myelination.
axonal injury; white matter injury; hypoxia-ischemia; prematurity; magnetic resonance imaging; electron microscopy
Converging evidence shows that monoamine oxidase A (MAO A), the key enzyme catalyzing serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) degradation, is a primary factor in the pathophysiology of antisocial and aggressive behavior. Accordingly, male MAO A-deficient humans and mice exhibit an extreme predisposition to aggressive outbursts in response to stress. As NMDARs regulate the emotional reactivity to social and environmental stimuli, we hypothesized their involvement in the modulation of aggression mediated by MAO A. In comparison with WT male mice, MAO A KO counterparts exhibited increases in 5-HT and NE levels across all brain regions, but no difference in glutamate concentrations and NMDAR binding. Notably, the prefrontal cortex (PFC) of MAO A KO mice exhibited higher expression of NR2A and NR2B, as well as lower levels of glycosylated NR1 subunits. In line with these changes, the current amplitude and decay time of NMDARs in PFC was significantly reduced. Furthermore, the currents of these receptors were hypersensitive to the action of the antagonists of the NMDAR complex (dizocilpine), as well as NR2A (PEAQX) and NR2B (Ro 25–6981) subunits. Notably, systemic administration of these agents selectively countered the enhanced aggression in MAO A KO mice, at doses that did not inherently affect motor activity. Our findings suggest that the role of MAO A in pathological aggression may be mediated by changes in NMDAR subunit composition in the PFC, and point to a critical function of this receptor in the molecular bases of antisocial personality.
MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies.
MicroRNA genes (miRNAs) are small non-coding RNAs that regulate the expression levels of mRNAs post-transcriptionally. miRNAs are critical in many important biological processes, like development, and are important markers for many diseases. Identifying the targets of miRNAs is not an easy task. Recent developments of high-throughput data collection methods for identification of all miRNA targets in a cell are promising, but they still depend on computational algorithms to identify the exact miRNA:mRNA interactions. In this paper we present a novel algorithm, ComiR, which addresses a more general question, that is, whether a given mRNA is targeted by a set of miRNAs. ComiR uses miRNA expression to improve the targeting models of four target prediction algorithms. Then it combines their predicted targets using a support vector machine. By applying ComiR to single nucleotide polymorphism (SNP) data, we identified a SNP that is likely to be causally associated to osteoporosis in women.
Monoamine oxidase (MAO)-A is a key enzyme for the degradation of brain serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE). In humans and mice, total MAO-A deficiency results in high 5-HT and NE levels, as well as elevated reactive aggression. Here we report the generation of MAO-ANeo mice, a novel line of hypomorphic MAO-A mutants featuring the insertion of a floxed neomycin-resistance cassette in intron-12 of the Maoa gene. This construct resulted in a chimeric, non-functional variant of the Maoa-Neo transcript, with a truncated C-terminus, likely due to aberrant splicing; these deficits notwithstanding, small amounts of functional Maoa transcript were found in the brain of MAO-ANeo mice. In the prefrontal cortex and amygdala, MAO-ANeo mice showed low, yet detectable, MAO-A catalytic activity, as well as 5-HT levels equivalent to WT littermates; conversely, the hippocampus and midbrain of MAO-ANeo mice featured a neurochemical profile akin to MAO-A-knockout (KO) mice, with undetectable MAO-A activity and high 5-HT concentrations. MAO-ANeo mice showed significant increases in dendritic length in the pyramidal neurons of orbitofrontal cortex, but not basolateral amygdala, in comparison with WT littermates; by contrast, the orbitofrontal cortex of MAO-A KO mice showed significant reductions in basilar dendritic length, as well as a profound increase in apical dendritic length. MAO-ANeo mice showed a unique set of behavioral abnormalities, encompassing reduced open-field locomotion, perseverative responses, such as marble burying and water mist-induced grooming, and a lack of anxiety-like behaviors in the elevated plus-maze and light–dark box paradigms. Notably, whereas MAO-ANeo and KO mice showed significant reductions in social interaction, only the latter genotype showed increases in resident–intruder aggression. Taken together, our findings indicate that MAO A hypomorphism results in behavioral and morphological alterations distinct from those featured by MAO-A KO mice.
monoamine oxidase-A; transgenic mice; social interaction; aggression; anxiety; repetitive behaviors; animal models; behavioral science; serotonin; neurochemistry; monoamine oxidase-A; aggression; anxiety; transgenic mice
Monoamine oxidase A (MAO-A) is the key enzyme for the degradation of brain serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE) and dopamine (DA). We recently generated and characterized a novel line of MAO-A hypormorphic mice (MAO-ANeo), featuring elevated monoamine levels, social deficits and perseverative behaviors as well as morphological changes in the basolateral amygdala and orbitofrontal cortex. Here we showed that MAO-ANeo mice displayed deficits in motor control, manifested as subtle disturbances in gait, motor coordination, and balance. Furthermore, magnetic resonance imaging of the cerebellum revealed morphological changes and a moderate reduction in the cerebellar size of MAO- ANeo mice compared to wild type (WT) mice. Histological and immunohistochemical analyses using calbindin-D-28k (CB) expression of Purkinje cells revealed abnormal cerebellar foliation with vermal hypoplasia and decreased in Purkinje cell count and their dendritic density in MAO- ANeo mice compared to WT. Our current findings suggest that congenitally low MAO-A activity leads to abnormal development of the cerebellum.
Monoamine oxidase A; Hypomorphism; Serotonin; Cerebellum; Purkinje cells
Poly(ADP-ribose) polymerase-1 (PARP-1) is a mammalian enzyme that attaches long branching chains of ADP-ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP-1 plays an important role in DNA strand-break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single-stranded to double-strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single-strandedness were used for measurements of both PARP-1/DNA binding kinetics and PARP-1’s enzyme activities. We found that binding correlated with activity, but was unrelated to single-strandedness of the ODN. Instead, PARP-1 binding and activity were highest on ODNs that modeled a DNA double-strand break (DSB). These results provide support for PARP-1 recognizing and binding DSBs in a manner that is independent of single-stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP-1 binding and PARP-1 auto-poly(ADP-ribosyl)ation activities within the same in vitro system.
poly(ADP-ribose) polymerase-1; PARP-1; DNA repair; DNA double-strand breaks; surface plasmon resonance; poly(ADP-ribosyl)ation; binding kinetics; non-homologous end joining
There are elevated rates of comorbid psychiatric disorders among individuals with substance dependence; however, little research examines these rates within inpatient settings, particularly in relation to gender and type of substance. The current study aimed to fill this gap.
465 patients (71.4% male) were recruited from an inpatient substance use treatment facility from 2006 to 2009. These patients were interviewed and diagnosed using the Structure Clinical Interview for DSM-IV and the Diagnostic Interview for Personality Disorders.
60.6% of patients with substance dependence had a current comorbid psychiatric disorder, and more than 30% had at least two psychiatric disorders. The most common current Axis I diagnosis was major depressive disorder (25.8%), followed by PTSD (14%). Comparable rates were found for Antisocial and Borderline Personality Disorders. Females were significantly more likely to meet diagnostic criteria for a psychiatric disorder than were males (73.7% versus 55.4%). When examining comorbidities across different substance dependences, the highest rates of comorbid psychiatric disorders were found among individuals with alcohol dependence (76.8%) and cannabis dependence (76%), although rates were above 60% for cocaine and opioid dependence. Rates of psychiatric diagnoses were significantly lower (27%) among patients who did not meet diagnostic criteria for substance dependence.
There are particularly elevated rates of psychiatric disorders among individuals with substance dependence in inpatient treatment. These rates differ as a function of substance dependence type and gender, making these factors important to consider when researching and treating this type of population.
Comorbidity; substance dependence; psychiatric disorders; inpatient treatment; gender differences
Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNA found in animals. PiRNAs are primarily expressed in the germline where their best understood function is to repress transposable elements. Unlike previous studies that investigated the evolution of piRNA-generating loci at the level of nucleotide substitutions, here we studied the evolution of piRNA-generating loci at the level of copy number variation (i.e. duplications and deletions) using genome-wide copy number variation data from three human populations. Our analysis shows that at the level of copy number variation there is strong selective constraint and a very high mutation rate in human piRNA-generating loci. Our results differ from a model of positive selection on copy number variation in piRNA-generating loci previously proposed in rodents. We discuss possible reasons for this difference based on the transposable element insertion histories in the rodent and primate lineages.
In rodents, noradrenergic (NE) locus coeruleus (LC) neurons are well known to express tyrosine hydroxylase (TH) immunoreactivity. However, due to its very low enzyme activity, NE cortical fibers do not typically express TH immunoreactivity, thus dopamine-beta-hydroxylase (DBH) immunoreactivity is commonly utilized as a marker for NE cortical fibers. In this study, we performed double and/or triple immunofluorescent staining using antibodies against TH, DBH, and/or norepinephrine transporter (NET) to investigate the altered noradrenergic TH expression of cortical fibers in citalopram (CTM) exposed rats and monoamine oxidase (MAO) A knock out (KO) mice. We have noted the following novel findings: 1) neonatal exposure to the selective serotonin reuptake inhibitor (SSRI) CTM enhanced noradrenergic TH immunoreactive fibers throughout the entire neocortex, and a few of them appeared to be hypertrophic; 2) slightly enhanced noradrenergic cortical TH immunoreactive fibers were also noted in MAO A KO mice, and many of them revealed varicosities compared to the rather smooth noradrenergic cortical TH immunoreactive fibers in wild type (WT) mice; 3) LC dendrites of MAO A KO mice exhibited beaded morphology compared to the smooth LC dendrites in WT mice. Our findings suggest that both genetic and environmental factors during early development may play a critical role in the regulation and proper function of noradrenergic TH expression in the neocortex.
norepinephrine; tyrosine hydroxylase; monoamine oxidase; neonates; antidepressants; knock out mice
The culture of human embryonic stem cells (hESCs) is limited, both technically and with respect to clinical potential, by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. The concern over xenogeneic contaminants from the mouse feeder cells may restrict transplantation to humans and the variability in MEFs from batch-to-batch and laboratory-to-laboratory may contribute to some of the variability in experimental results. Finally, use of any feeder layer increases the work load and subsequently limits the large-scale culture of human ES cells. Thus, the development of feeder-free cultures will allow more reproducible culture conditions, facilitate scale-up and potentiate the clinical use of cells differentiated from hESC cultures. In this review, we describe various methods tested to culture cells in the absence of MEF feeder layers and other advances in eliminating xenogeneic products from the culture system.
Human embryonic stem cells; MEF; Feeder-free cell culture; Matrigel
Monoamine oxidase (MAO) A is the major metabolizing enzyme of serotonin (5-hydroxytryptamine, 5-HT) which regulates early brain development. In this study, wild-type (WT) and MAO Aneo embryonic stem (ES) cell lines were established from the inner cell mass of murine blastocysts and their characteristics during ES and differentiating stages were studied. Our results show that the differentiation to neural cells in MAO Aneo ES cells was reduced compared to WT, suggesting MAO A played a regulatory role in stem cells neural differentiation.
Embryonic stem cells; Neural differentiation; Neurogenesis; Monoamine oxidase (MAO) A
A novel line of mutant mice [monoamine oxidase A knockout (MAOAA863T KO)] harboring a spontaneous point nonsense mutation in exon 8 of the MAO A gene was serendipitously identified in a 129/SvEvTac colony. This mutation is analogous to the cause of a rare human disorder, Brunner syndrome, characterized by complete MAO A deficiency and impulsive aggressiveness. Concurrent with previous studies of MAO A KO mice generated by insertional mutagenesis (‘Tg8’), MAOAA863T KO lack MAO A enzyme activity and display enhanced aggression toward intruder mice. MAOAA863T KO, however, exhibited lower locomotor activity in a novel, inescapable open field and similar immobility during tail suspension compared with wild type, observations which differ from reports of Tg8. These findings consolidate evidence linking MAO A to aggression and highlight subtle yet distinctive phenotypical characteristics.
Brunner syndrome; monoamine oxidase; nonsense mutation
Rich evidence indicates that monoamine oxidase (MAO) A, the major enzyme catalysing the degradation of monoamine neurotransmitters, plays a key role in emotional regulation. Although MAOA deficiency is associated with reactive aggression in humans and mice, the involvement of this enzyme in defensive behaviour remains controversial and poorly understood. To address this issue, we tested MAOA knockout (KO) mice in a spectrum of paradigms and settings associated with variable degrees of threat. The presentation of novel inanimate objects induced a significant reduction in exploratory approaches and increase in defensive behaviours, such as tail-rattling, biting and digging. These neophobic responses were context-dependent and particularly marked in the home cage. In the elevated plus- and T-mazes, MAOA KO mice and wild-type (WT) littermates displayed equivalent locomotor activity and time in closed and open arms; however, MAOA KO mice featured significant reductions in risk assessment, as well as unconditioned avoidance and escape. No differences between genotypes were observed in the defensive withdrawal and emergence test. Conversely, MAOA KO mice exhibited a dramatic reduction of defensive and fear-related behaviours in the presence of predator-related cues, such as predator urine or an anaesthetized rat, in comparison with those observed in their WT littermates. The behavioural abnormalities in MAOA KO mice were not paralleled by overt alterations in sensory and microvibrissal functions. Collectively, these results suggest that MAOA deficiency leads to a general inability to appropriately assess contextual risk and attune defensive and emotional responses to environmental cues.
Anxiety; defensive behaviour; exploration; monoamine oxidase A; predator urine
Although MRI is the optimal imaging modality to define cerebral white-matter injury (WMI) in preterm survivors, the histopathological features of MRI-defined chronic lesions are poorly defined. We hypothesized that chronic WMI is related to a combination of delayed oligodendrocyte (OL) lineage cell death and arrested maturation of pre-oligodendrocytes (preOLs). We determined whether ex vivo MRI can distinguish distinct microglial and astroglial responses related to WMI progression and arrested preOL differentiation.
We employed a preterm fetal sheep model of global cerebral ischemia where acute WMI results in selective preOL degeneration. We developed novel algorithms to register histopathologically defined lesions with contrast- and diffusion-weighted high-field ex vivo MRI data.
Despite mild delayed preOL degeneration, preOL density recovered to control levels by 7 days after ischemia and was ~2 fold greater at 14 days. However, pre-myelinating OLs were significantly diminished at 7 and 14 days. WMI evolved to mostly gliotic lesions where arrested preOL differentiation was directly proportional to the magnitude of astrogliosis. A reduction in cerebral WM volume was accompanied by four classes of MRI-defined lesions. Each lesion type displayed unique astroglial and microglial responses that corresponded to distinct forms of necrotic or non-necrotic injury. High-field MRI defined two novel hypo-intense signal abnormalities on T2-weighted images that coincided with microscopic necrosis or identified astrogliosis with high sensitivity and specificity.
These studies support the potential of high-field MRI for early identification of microscopic necrosis and gliosis with preOL maturation arrest, a common form of WMI in preterm survivors.
A major challenge in the analysis of population genomics data consists of isolating signatures of natural selection from background noise caused by random drift and gene flow. Analyses of massive amounts of data from many related populations require high-performance algorithms to determine the likelihood of different demographic scenarios that could have shaped the observed neutral single nucleotide polymorphism (SNP) allele frequency spectrum. In many areas of applied mathematics, Fourier Transforms and Spectral Methods are firmly established tools to analyze spectra of signals and model their dynamics as solutions of certain Partial Differential Equations (PDEs). When spectral methods are applicable, they have excellent error properties and are the fastest possible in high dimension; see . In this paper we present an explicit numerical solution, using spectral methods, to the forward Kolmogorov equations for a Wright-Fisher process with migration of K populations, influx of mutations, and multiple population splitting events.
Monoamine oxidase (MAO) A and MAO B are a crucial pair of isoenzymes, which oxidatively deaminate monoamine neurotransmitters and dietary amines with a production of hydrogen peroxide. These two isoenzymes have different but overlapping substrate and inhibitor specificities. MAO A and MAO B share 70% amino acid sequence identity and show different temporal and spatial expressions in both humans and mice. Abnormal MAO A or MAO B activity has been implicated in numerous neurological and psychiatric disorders. A better understanding of the transcriptional regulation of MAO A and MAO B genes may help explain the differential tissue-specific expression of these two isoenzymes and provide insights into the molecular basis of the disorders associated with MAO dysfunction. This review discusses the recent progress in the transcriptional regulation and multiple functions of MAO A and MAO B genes.
Monoamine oxidase; Promoter; Transcriptional regulation; Hormone; Sp1
Type IV pili (TFP) are membrane-anchored filaments with a number of important biological functions. In the model organism Myxococcus xanthus, TFP act as molecular engines that power social (S) motility through cycles of extension and retraction. TFP filaments consist of several thousand copies of a protein called PilA or pilin. PilA contains an N-terminal α-helix essential for TFP assembly and a C-terminal globular domain important for its activity. The role of the PilA sequence and its structure–function relationship in TFP-dependent S motility remain active areas of research. In this study, we identified an M. xanthus PilA mutant carrying an alanine to valine substitution at position 32 in the α-helix, which produced structurally intact but retraction-defective TFP. Characterization of this mutant and additional single-residue variants at this position in PilA demonstrated the critical role of alanine 32 in PilA stability, TFP assembly and retraction.
Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids—piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.
piRNA; CRISPR; co-evolution; transposable elements; phage; plasmids
Clostridium difficile is the causative agent of primary and recurrent antibiotic-associated diarrhea and colitis in hospitalized patients. The disease is caused mainly by two exotoxins, TcdA and TcdB, produced by the bacteria. Recurrent C. difficile infection (CDI) constitutes one of the most significant clinical issues of this disease, occurs in more than 20% of patients after the first episode, and may be increasing in frequency. However, there is no well-established animal model of CDI relapse currently available for studying disease pathogenesis, prevention, and therapy. Here we report the establishment of a conventional mouse model of recurrence/relapse CDI. We found that the primary episode of CDI induced little or no protective antibody response against C. difficile toxins and mice continued shedding C. difficile spores. Antibiotic treatment of surviving mice induced a second episode of diarrhea, while a simultaneous reexposure of animals to C. difficile bacteria or spores elicited a full spectrum of CDI similar to that of the primary infection. Moreover, mice treated with immunosuppressive agents were prone to more severe and fulminant recurrent disease. Finally, utilizing this model, we demonstrated that vancomycin only delayed disease recurrence, whereas neutralizing polysera against both TcdA and TcdB completely protected mice against CDI relapse. In conclusion, we have established a mouse relapse CDI model that allows for future investigations of the role of the host immune response in the disease's pathogenesis and permits critical testing of new therapeutics targeting recurrent disease.
To describe ocular findings in 3 cases of solar retinopathy using high definition, spectral domain optical coherence tomography (SD-OCT) and review the literature for optical coherence tomography (OCT) characteristics associated with worse vision.
Case series and retrospective review of clinical features and Spectralis SD-OCT (Heidelberg Engineering, Vista, California, United States of America). A literature review of OCT findings in cases of solar retinopathy reported on MEDLINE was also performed and analyzed.
Six eyes of 3 patients with solar retinopathy revealed significant foveal pathology. Visual acuity ranged from Snellen 20/30 to 20/50. High definition SD-OCT demonstrated defects at the level of the inner and outer segment junction of the photoreceptors as well as in the inner high reflective layer. There was a significant correlation between chronic disruption of the inner photoreceptor junction with worse vision based on the current case series and literature review.
Screening patients with exposure to central foveal damage from solar retinopathy with high definition SD-OCT improves diagnosis and assessment of photoreceptor damage and vision loss.
Solar retinopathy; fovea; spectral domain optical coherence tomography; Spectralis SD-OCT.
Serotonin (5-hydroxytryptamine; 5-HT) is thought to regulate neurodevelopmental processes through maternal-fetal interactions that have long-term mental health implications. Dogma states that beyond fetal 5-HT neurons, there are significant maternal contributions to fetal 5-HT during pregnancy1,2, but this has not been tested empirically. To examine putative central and peripheral sources of embryonic brain 5-HT, we used the Pet-1−/− mice in which most dorsal raphe (DR) neurons lack 5-HT3. Measures of 5-HT revealed previously unknown differences in accumulation between the fore- and hindbrain during early and late fetal stages, through an exogenous source of 5-HT. We show that this source is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo, and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source and uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor, in both mice and humans. This study reveals a new, direct role for placental metabolic pathways in modulating fetal brain development and implicates novel maternal-placental-fetal interactions that could underlie the pronounced impact of 5-HT on long-lasting mental health outcomes.