Disease-specific alterations in the enteric virome in inflammatory bowel disease.

Decreases in the diversity of enteric bacterial populations are observed in patients with Crohn's disease (CD) and ulcerative colitis (UC). Less is known about the virome in these diseases. We show that the enteric virome is abnormal in CD and UC patients. In-depth analysis of preparations enriched for free virions in the intestine revealed that CD and UC were associated with a significant expansion of Caudovirales bacteriophages. The viromes of CD and UC patients were disease and cohort specific. Importantly, it did not appear that expansion and diversification of the enteric virome was secondary to changes in bacterial populations. These data support a model in which changes in the virome may contribute to intestinal inflammation and bacterial dysbiosis. We conclude that the virome is a candidate for contributing to, or being a biomarker for, human inflammatory bowel disease and speculate that the enteric virome may play a role in other diseases.

Transcriptomic mapping uncovers Purkinje neuron plasticity driving learning.

Cellular diversification is critical for specialized functions of the brain including learning and memory1. Single-cell RNA sequencing facilitates transcriptomic profiling of distinct major types of neuron2-4, but the divergence of transcriptomic profiles within a neuronal population and their link to function remain poorly understood. Here we isolate nuclei tagged5 in specific cell types followed by single-nucleus RNA sequencing to profile Purkinje neurons and map their responses to motor activity and learning. We find that two major subpopulations of Purkinje neurons, identified by expression of the genes Aldoc and Plcb4, bear distinct transcriptomic features. Plcb4+, but not Aldoc+, Purkinje neurons exhibit robust plasticity of gene expression in mice subjected to sensorimotor and learning experience. In vivo calcium imaging and optogenetic perturbation reveal that Plcb4+ Purkinje neurons have a crucial role in associative learning. Integrating single-nucleus RNA sequencing datasets with weighted gene co-expression network analysis uncovers a learning gene module that includes components of FGFR2 signalling in Plcb4+ Purkinje neurons. Knockout of Fgfr2 in Plcb4+ Purkinje neurons in mice using CRISPR disrupts motor learning. Our findings define how diversification of Purkinje neurons is linked to their responses in motor learning and provide a foundation for understanding their differential vulnerability to neurological disorders.

MeCP2 Represses Enhancers through Chromosome Topology-Associated DNA Methylation.

The genomes of mammalian neurons contain uniquely high levels of non-CG DNA methylation that can be bound by the Rett syndrome protein, MeCP2, to regulate gene expression. How patterns of non-CG methylation are established in neurons and the mechanism by which this methylation works with MeCP2 to control gene expression is unclear. Here, we find that genes repressed by MeCP2 are often located within megabase-scale regions of high non-CG methylation that correspond with topologically associating domains of chromatin folding. MeCP2 represses enhancers found in these domains that are enriched for non-CG and CG methylation, with the strongest repression occurring for enhancers located within MeCP2-repressed genes. These alterations in enhancer activity provide a mechanism for how MeCP2 disruption in disease can lead to widespread changes in gene expression. Hence, we find that DNA topology can shape non-CG DNA methylation across the genome to dictate MeCP2-mediated enhancer regulation in the brain.

Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome.

Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.

Blocking CHOP-dependent TXNIP shuttling to mitochondria attenuates albuminuria and mitigates kidney injury in nephrotic syndrome.

Albuminuria is a hallmark of glomerular disease of various etiologies. It is not only a symptom of glomerular disease but also a cause leading to glomerulosclerosis, interstitial fibrosis, and eventually, a decline in kidney function. The molecular mechanism underlying albuminuria-induced kidney injury remains poorly defined. In our genetic model of nephrotic syndrome (NS), we have identified CHOP (C/EBP homologous protein)-TXNIP (thioredoxin-interacting protein) as critical molecular linkers between albuminuria-induced ER dysfunction and mitochondria dyshomeostasis. TXNIP is a ubiquitously expressed redox protein that binds to and inhibits antioxidant enzyme, cytosolic thioredoxin 1 (Trx1), and mitochondrial Trx2. However, very little is known about the regulation and function of TXNIP in NS. By utilizing Chop-/- and Txnip-/- mice as well as 68Ga-Galuminox, our molecular imaging probe for detection of mitochondrial reactive oxygen species (ROS) in vivo, we demonstrate that CHOP up-regulation induced by albuminuria drives TXNIP shuttling from nucleus to mitochondria, where it is required for the induction of mitochondrial ROS. The increased ROS accumulation in mitochondria oxidizes Trx2, thus liberating TXNIP to associate with mitochondrial nod-like receptor protein 3 (NLRP3) to activate inflammasome, as well as releasing mitochondrial apoptosis signal-regulating kinase 1 (ASK1) to induce mitochondria-dependent apoptosis. Importantly, inhibition of TXNIP translocation and mitochondrial ROS overproduction by CHOP deletion suppresses NLRP3 inflammasome activation and p-ASK1-dependent mitochondria apoptosis in NS. Thus, targeting TXNIP represents a promising therapeutic strategy for the treatment of NS.

Efficacy and safety of tafolecimab in Chinese patients with heterozygous familial hypercholesterolemia: a randomized, double-blind, placebo-controlled phase 3 trial (CREDIT-2).

BACKGROUND: Heterozygous familial hypercholesterolemia (HeFH) is largely underdiagnosed and undertreated in China where few patients achieved recommended target levels of low density lipoprotein cholesterol (LDL-C). We conducted the first randomized, placebo-controlled clinical trial in Chinese patients with HeFH to assess the efficacy and safety of tafolecimab, a novel fully human proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibody. METHODS: Patients diagnosed with HeFH by Simon Broome criteria and on a stable lipid-lowering therapy for at least 4 weeks were randomized 2:2:1:1 to receive subcutaneous tafolecimab 150 mg every 2 weeks (Q2W), tafolecimab 450 mg every 4 weeks (Q4W), placebo Q2W or placebo Q4W in the 12-week double-blind treatment period. After that, participants received open-label tafolecimab 150 mg Q2W or 450 mg Q4W for 12 weeks. The primary endpoint was the percent change from baseline to week 12 in LDL-C levels. Secondary endpoints included proportion of participants achieving ≥50% LDL-C reductions and proportion of participants with LDL-C <1.8 mmol/L at week 12 and 24, the change from baseline to week 12 in non-high density lipoprotein cholesterol (non-HDL-C), apolipoprotein B and lipoprotein(a) levels, as well as the change from baseline to week 24 in lipid levels. RESULTS: In total, 149 participants were randomized and 148 received at least one dose of the study treatment. At week 12, tafolecimab treatment induced significant reductions in LDL-C levels (treatment difference versus placebo [on-treatment estimand]: -57.4% [97.5% CI, -69.2 to -45.5] for 150 mg Q2W; -61.9% [-73.4 to -50.4] for 450 mg Q4W; both P <0.0001). At both dose regimens, significantly more participants treated with tafolecimab achieved ≥50% LDL-C reductions or LDL-C <1.8 mmol/L at week 12 as compared with corresponding placebo groups (all P <0.0001). Meanwhile, non-HDL-C, apolipoprotein B and lipoprotein(a) levels were significantly reduced in the tafolecimab groups at week 12. The lipid-lowering effects of tafolecimab were maintained till week 24. During the double-blind treatment period, the most commonly-reported adverse events in the tafolecimab groups included upper respiratory tract infection, increased blood creatine phosphokinase, increased alanine aminotransferase, increased aspartate aminotransferase and hypertension. CONCLUSIONS: Tafolecimab administered either 150 mg Q2W or 450 mg Q4W yielded significant and persistent reductions in LDL-C levels and showed a favorable safety profile in Chinese patients with HeFH. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04179669.

Vertical transmission in Caenorhabditis nematodes of RNA molecules encoding a viral RNA-dependent RNA polymerase.

Here, we report on the discovery in Caenorhabditis nematodes of multiple vertically transmitted RNAs coding for putative RNA-dependent RNA polymerases. Their sequences share similarity to distinct RNA viruses, including bunyaviruses, narnaviruses, and sobemoviruses. The sequences are present exclusively as RNA and are not found in DNA form. The RNAs persist in progeny after bleach treatment of adult animals, indicating vertical transmission of the RNAs. We tested one of the infected strains for transmission to an uninfected strain and found that mating of infected animals with uninfected animals resulted in infected progeny. By in situ hybridization, we detected several of these RNAs in the cytoplasm of the male and female germline of the nematode host. The Caenorhabditis hosts were found defective in degrading exogenous double-stranded RNAs, which may explain retention of viral-like RNAs. Strikingly, one strain, QG551, harbored three distinct virus-like RNA elements. Specific patterns of small RNAs complementary to the different viral-like RNAs were observed, suggesting that the different RNAs are differentially recognized by the RNA interference (RNAi) machinery. While vertical transmission of viruses in the family Narnaviridae, which are known as capsidless viruses, has been described in fungi, these observations provide evidence that multicellular animal cells harbor similar viruses.

Cooperative P-Wave Velocity Measurement with Full Waveform Moment Tensor Inversion in Transversely Anisotropic Media.

Precise stochastic approaches to quantitatively calculate the source uncertainties offers the opportunity to eliminate the influence of anisotropy on moment tensor inversion. The effects of ignoring anisotropy were tested by using homogeneous Green's functions. Results indicate the influence of anisotropy and noise on fault plane rotation is very small for a pure shear source whether it is restricted to double couple solution or full moment tensor solution. Green's functions with different prior rough anisotropy information were tested, indicating that the complex source is more sensitive to velocity models than the pure shear source and the fault plane rotation caused by full moment tensor solution is larger than the pure double couple solution. Collaborative P-wave velocity inversion with active measurements and passive acoustic emission data using the fast-marching method were conducted, and new Green's functions established based on the tomography results. The resolved fault plane solution rotated only 3.5° when using the new Green's functions, but the presence of spurious isotropic and compensated linear vector dipole components was not completely eliminated. It is concluded that the cooperative inversion is capable of greatly improving the accuracy of the fault plane solutions and reducing the spurious components in the full moment tensor solution.

Therapeutic efficacy of favipiravir against Bourbon virus in mice.

Bourbon virus (BRBV) is an emerging tick-borne RNA virus in the orthomyxoviridae family that was discovered in 2014. Although fatal human cases of BRBV have been described, little is known about its pathogenesis, and no antiviral therapies or vaccines exist. We obtained serum from a fatal case in 2017 and successfully recovered the second human infectious isolate of BRBV. Next-generation sequencing of the St. Louis isolate of BRBV (BRBV-STL) showed >99% nucleotide identity to the original reference isolate. Using BRBV-STL, we developed a small animal model to study BRBV-STL tropism in vivo and evaluated the prophylactic and therapeutic efficacy of the experimental antiviral drug favipiravir against BRBV-induced disease. Infection of Ifnar1-/- mice lacking the type I interferon receptor, but not congenic wild-type animals, resulted in uniformly fatal disease 6 to 10 days after infection. RNA in situ hybridization and viral yield assays demonstrated a broad tropism of BRBV-STL with highest levels detected in liver and spleen. In vitro replication and polymerase activity of BRBV-STL were inhibited by favipiravir. Moreover, administration of favipiravir as a prophylaxis or as post-exposure therapy three days after infection prevented BRBV-STL-induced mortality in immunocompromised Ifnar1-/- mice. These results suggest that favipiravir may be a candidate treatment for humans who become infected with BRBV.

Epigenetic regulator UHRF1 inactivates REST and growth suppressor gene expression via DNA methylation to promote axon regeneration.

Injured peripheral sensory neurons switch to a regenerative state after axon injury, which requires transcriptional and epigenetic changes. However, the roles and mechanisms of gene inactivation after injury are poorly understood. Here, we show that DNA methylation, which generally leads to gene silencing, is required for robust axon regeneration after peripheral nerve lesion. Ubiquitin-like containing PHD ring finger 1 (UHRF1), a critical epigenetic regulator involved in DNA methylation, increases upon axon injury and is required for robust axon regeneration. The increased level of UHRF1 results from a decrease in miR-9. The level of another target of miR-9, the transcriptional regulator RE1 silencing transcription factor (REST), transiently increases after injury and is required for axon regeneration. Mechanistically, UHRF1 interacts with DNA methyltransferases (DNMTs) and H3K9me3 at the promoter region to repress the expression of the tumor suppressor gene phosphatase and tensin homolog (PTEN) and REST. Our study reveals an epigenetic mechanism that silences tumor suppressor genes and restricts REST expression in time after injury to promote axon regeneration.

The Transcriptional Regulator SnoN Promotes the Proliferation of Cerebellar Granule Neuron Precursors in the Postnatal Mouse Brain.

Control of neuronal precursor cell proliferation is essential for normal brain development, and deregulation of this fundamental developmental event contributes to brain diseases. Typically, neuronal precursor cell proliferation extends over long periods of time during brain development. However, how neuronal precursor proliferation is regulated in a temporally specific manner remains to be elucidated. Here, we report that conditional KO of the transcriptional regulator SnoN in cerebellar granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cell cycle exit at later stages of cerebellar development in the postnatal male and female mouse brain. In laser capture microdissection followed by RNA-Seq, designed to profile gene expression specifically in the external granule layer of the cerebellum, we find that SnoN promotes the expression of cell proliferation genes and concomitantly represses differentiation genes in granule neuron precursors in vivo Remarkably, bioinformatics analyses reveal that SnoN-regulated genes contain binding sites for the transcription factors N-myc and Pax6, which promote the proliferation and differentiation of granule neuron precursors, respectively. Accordingly, we uncover novel physical interactions of SnoN with N-myc and Pax6 in cells. In behavior analyses, conditional KO of SnoN impairs cerebellar-dependent learning in a delayed eye-blink conditioning paradigm, suggesting that SnoN-regulation of granule neuron precursor proliferation bears functional consequences at the organismal level. Our findings define a novel function and mechanism for the major transcriptional regulator SnoN in the control of granule neuron precursor proliferation in the mammalian brain.SIGNIFICANCE STATEMENT This study reports the discovery that the transcriptional regulator SnoN plays a crucial role in the proliferation of cerebellar granule neuron precursors in the postnatal mouse brain. Conditional KO of SnoN in granule neuron precursors robustly inhibits the proliferation of these cells and promotes their cycle exit specifically at later stages of cerebellar development, with biological consequences of impaired cerebellar-dependent learning. Genomics and bioinformatics analyses reveal that SnoN promotes the expression of cell proliferation genes and concomitantly represses cell differentiation genes in vivo Although SnoN has been implicated in distinct aspects of the development of postmitotic neurons, this study identifies a novel function for SnoN in neuronal precursors in the mammalian brain.

Robust principal component analysis for accurate outlier sample detection in RNA-Seq data.

BACKGROUND: High throughput RNA sequencing is a powerful approach to study gene expression. Due to the complex multiple-steps protocols in data acquisition, extreme deviation of a sample from samples of the same treatment group may occur due to technical variation or true biological differences. The high-dimensionality of the data with few biological replicates make it challenging to accurately detect those samples, and this issue is not well studied in the literature currently. Robust statistics is a family of theories and techniques aim to detect the outliers by first fitting the majority of the data and then flagging data points that deviate from it. Robust statistics have been widely used in multivariate data analysis for outlier detection in chemometrics and engineering. Here we apply robust statistics on RNA-seq data analysis. RESULTS: We report the use of two robust principal component analysis (rPCA) methods, PcaHubert and PcaGrid, to detect outlier samples in multiple simulated and real biological RNA-seq data sets with positive control outlier samples. PcaGrid achieved 100% sensitivity and 100% specificity in all the tests using positive control outliers with varying degrees of divergence. We applied rPCA methods and classical principal component analysis (cPCA) on an RNA-Seq data set profiling gene expression of the external granule layer in the cerebellum of control and conditional SnoN knockout mice. Both rPCA methods detected the same two outlier samples but cPCA failed to detect any. We performed differentially expressed gene detection before and after outlier removal as well as with and without batch effect modeling. We validated gene expression changes using quantitative reverse transcription PCR and used the result as reference to compare the performance of eight different data analysis strategies. Removing outliers without batch effect modeling performed the best in term of detecting biologically relevant differentially expressed genes. CONCLUSIONS: rPCA implemented in the PcaGrid function is an accurate and objective method to detect outlier samples. It is well suited for high-dimensional data with small sample sizes like RNA-seq data. Outlier removal can significantly improve the performance of differential gene detection and downstream functional analysis.

Bioremediation of malachite green by cyanobacterium Synechococcus elongatus PCC 7942 engineered with a triphenylmethane reductase gene.

Malachite green is a carcinogenic dye that has been detected in fish tissues and freshwater. Here we evaluated the malachite green decoloring ability of a photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942 (Synechococcus), that lives in freshwater. Results show that 99.5% of the dye was removed by Synechococcus through bioabsorption and bioaccumulation; however, the dye was not degraded or chemically modified. Next, we established an engineered Synechococcus strain to degrade the dye after uptake. The triphenylmethane reductase gene katmr was heterologously expressed, resulting in high production of a soluble recombinant protein. The engineered strain showed advanced decoloring abilities at a low cell density and in stressful environments. It degraded malachite green into the smaller molecules 4-methylaminobenzoic acid and 4-hydroxyl-aniline. After treatment with the engineered cyanobacterium, the growth of wheat seeds was fully recovered in the presence of malachite green. These results demonstrate the potential application of the engineered Synechococcus as a photosynthetic cell factory for the removal of malachite green from wastewater.

Effects of Atorvastatin on Transient Sodium Currents in Rat Normal, Simulated Ischemia, and Reperfusion Ventricular Myocytes.

OBJECTIVES: (1) To detect the whether the effects of simulated ischemia on INa of rat left ventricular myocytes in a time-dependent manner and the effects of atorvastatin on ischemia INa; (2) To investigate the effects of atorvastatin on INa of rat-simulated ischemia/reperfusion (I/R) ventricular cells. MATERIALS AND METHODS: Ventricular cells were enzymatically isolated by Langendorff perfusion system. Whole-cell patch clamp was applied to detect INa level. Some elements of extracellular fluid were hanged to simulate the status of normal, I and R condition. Then the effects of atorvastatin on INa were observed. RESULTS: (1) During simulated reperfusion, INa decreased and atorvastatin further suppressed the reduction degree. (2) At test potential -40 mV, no difference was detected among peak INa amplitude of ischemia for 20 min, reperfusion phase 3/5/7/9 min in continuous ischemia (I) group (p = 0.275). In I/R group, peak INa amplitude continuously decreased at 3 min (p = 0.005) and 9 min (p = 0.041). In atorvastatin intervention + I/R (Statin + I/R) group, peak INa amplitude at reperfusion 3 min decreased compared with ischemia phase (p = 0.000), while no significant difference was detected between 3 and 9 min (p = 0.858). The differences were significant at the same time point between groups. At reperfusion 3/5/7/9 min, peak INa of the I/R group was lower than the ischemia group (all p = 0.000), same as the Statin + I/R group (p = 0.000, p = 0.003, p = 0.006, and p = 0.001). Peak INa of the Statin + I/R group was higher than the I/R group at the same time point (p = 0.011, p = 0.033, p = 0.003, p = 0.003). There was no change in the I group during reperfusion phase (p > 0.05). In I/R group, V1/2 (mV) shifted from -58.87 ± 3.36 to -54.33 ± 2.40, k (mV) shifted from 1.25 ± 0.59 to 1.91 ± 0.84 (p < 0.05). In the Statin + I/R group, V1/2 (mV) increased from -57.80 ± 2.97 to -52.76 ± 3.14 (p < 0.01), no change was observed in k (p > 0.05). CONCLUSIONS: (1) In the status of reperfusion, INa decreased more than that in the status of ischemia. (2) Atorvastatin protected the cells from reduction of INa during long-time simulated (>15 min) I/R. (3) Overall, atorvastatin affected INa of the normal, simulated ischemic/reperfusion cell in rat left ventricle by blocking sodium channel -directly.

Intestinal virome changes precede autoimmunity in type I diabetes-susceptible children.

Viruses have long been considered potential triggers of autoimmune diseases. Here we defined the intestinal virome from birth to the development of autoimmunity in children at risk for type 1 diabetes (T1D). A total of 220 virus-enriched preparations from serially collected fecal samples from 11 children (cases) who developed serum autoantibodies associated with T1D (of whom five developed clinical T1D) were compared with samples from controls. Intestinal viromes of case subjects were less diverse than those of controls. Among eukaryotic viruses, we identified significant enrichment of Circoviridae-related sequences in samples from controls in comparison with cases. Enterovirus, kobuvirus, parechovirus, parvovirus, and rotavirus sequences were frequently detected but were not associated with autoimmunity. For bacteriophages, we found higher Shannon diversity and richness in controls compared with cases and observed that changes in the intestinal virome over time differed between cases and controls. Using Random Forests analysis, we identified disease-associated viral bacteriophage contigs after subtraction of age-associated contigs. These disease-associated contigs were statistically linked to specific components of the bacterial microbiome. Thus, changes in the intestinal virome preceded autoimmunity in this cohort. Specific components of the virome were both directly and inversely associated with the development of human autoimmune disease.

LysMD3 is a type II membrane protein without an in vivo role in the response to a range of pathogens.

Germline-encoded receptors recognizing common pathogen-associated molecular patterns are a central element of the innate immune system and play an important role in shaping the host response to infection. Many of the innate immune molecules central to these signaling pathways are evolutionarily conserved. LysMD3 is a novel molecule containing a putative peptidoglycan-binding domain that has orthologs in humans, mice, zebrafish, flies, and worms. We found that the lysin motif (LysM) of LysMD3 is likely related to a previously described peptidoglycan-binding LysM found in bacteria. Mouse LysMD3 is a type II integral membrane protein that co-localizes with GM130+ structures, consistent with localization to the Golgi apparatus. We describe here two lines of mLysMD3-deficient mice for in vivo characterization of mLysMD3 function. We found that mLysMD3-deficient mice were born at Mendelian ratios and had no obvious pathological abnormalities. They also exhibited no obvious immune response deficiencies in a number of models of infection and inflammation. mLysMD3-deficient mice exhibited no signs of intestinal dysbiosis by 16S analysis or alterations in intestinal gene expression by RNA sequencing. We conclude that mLysMD3 contains a LysM with cytoplasmic orientation, but we were unable to define a physiological role for the molecule in vivo.

Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors.

Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development.IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development.

Hyperexpansion of RNA Bacteriophage Diversity.

Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent.

Biodegradation of malachite green by an endophytic bacterium Klebsiella aerogenes S27 involving a novel oxidoreductase.

Endophytic microorganisms can metabolize organic contaminants and assist in plant growth, thus facilitating the phytoremediation of polluted environments. An endophytic bacterium capable of decoloring malachite green (MG) was isolated from the leaves of the wetland plant Suaeda salsa and was identified as Klebsiella aerogenes S27. Complete decolorization of MG (100 mg/l) was achieved in 8 h at 30 °C and pH 7.0. Ultraviolet-visible spectroscopy and Fourier-transform infrared spectroscopy analyses indicated the degradation of MG by the isolate. The enzymic assays of the strain showed the triphenylmethane reductase (TMR) activity. A gene encoding putative TMR-like protein (named as KaTMR) was cloned and heterologously expressed in Escherichia coli. KaTMR showed only 42.6-43.3% identities in amino acids compared with well-studied TMRs, and it phylogenetically formed a new branch in the family of TMRs. The degraded metabolites by recombinant KaTMR were detected by liquid chromatography-mass spectrometry, showing differences from the products of reported TMRs. The biotransformation pathway of MG was proposed. Phytotoxicity studies revealed the less-toxic nature of the degraded metabolites compared to the dye. This study presented the first report of an endophyte on the degradation and detoxification of triphenylmethane dye via a novel oxidoreductase, thus facilitating the study of the plant-endophyte symbiosis in the bioremediation processes.

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4+ T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7.IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties.