Avian Influenza A Virus Polymerase Can Utilize Human ANP32 Proteins To Support cRNA but Not vRNA Synthesis.

Host restriction limits the emergence of novel pandemic strains from the influenza A virus avian reservoir. For efficient replication in mammalian cells, the avian influenza RNA-dependent RNA polymerase must adapt to use human orthologues of the host factor ANP32, which lack a 33-amino-acid insertion relative to avian ANP32A. Here, we find that influenza polymerase requires ANP32 proteins to support both steps of genome replication: cRNA and vRNA synthesis. However, avian strains are only restricted in vRNA synthesis in human cells. Therefore, avian influenza polymerase can use human ANP32 orthologues to support cRNA synthesis, without acquiring mammalian adaptations. This implies a fundamental difference in the mechanism by which ANP32 proteins support cRNA versus vRNA synthesis. IMPORTANCE To infect humans and cause a pandemic, avian influenza must first adapt to use human versions of the proteins the virus hijacks for replication, instead of the avian orthologues found in bird cells. One critical host protein is ANP32. Understanding the details of how host proteins such as ANP32 support viral activity may allow the design of new antiviral strategies that disrupt these interactions. Here, we use cells that lack ANP32 to unambiguously demonstrate ANP32 is needed for both steps of influenza genome replication. Unexpectedly, however, we found that avian influenza can use human ANP32 proteins for the first step of replication, to copy a complementary strand, without adaptation but can only utilize avian ANP32 for the second step of replication that generates new genomes. This suggests ANP32 may have a distinct role in supporting the second step of replication, and it is this activity that is specifically blocked when avian influenza infects human cells.

Ste20-like kinase activity promotes meiotic resumption and spindle microtubule stability in mouse oocytes.

Ste20-like kinase (SLK) is involved in cell proliferation and migration in somatic cells. This study aims to explore SLK expression and function in mouse oocyte meiosis. Western blot, immunofluorescence, Co-immunoprecipitation, drug treatment, cRNA construct and in vitro transcription, microinjection of morpholino oilgo (MO) and cRNA were performed in oocytes. High and stable protein expression of SLK was detected in mouse oocyte meiosis, with dynamic distribution in the nucleus, chromosomes and spindle apparatus. SLK phosphorylation emerges around meiotic resumption and reaches a peak during metaphase I (MI) and metaphase II. SLK knockdown with MO or expression of kinase-dead SLK K63R dramatically delays meiotic resumption due to sequentially suppressed phosphorylation of Polo-like kinase 1 (Plk1) and cell division cycle 25C (CDC25C) and dephosphorylation of cyclin-dependent kinase 1 (CDK1). SLK depletion promotes ubiquitination-mediated degradation of paxillin, an antagonist to α-tubulin deacetylation, and thus destroys spindle assembly and chromosome alignment; these phenotypes can be substantially rescued by exogenous expression of SLK kinase active fragment. Additionally, exogenous SLK effectively promotes meiotic progression and spindle assembly in aging oocytes with reduced SLK. Collectively, this study reveals SLK is required for meiotic resumption and spindle assembly in mouse oocyte meiosis.

Cystathionine ß-synthase is required for oocyte quality by ensuring proper meiotic spindle assembly.

OBJECTIVES: Poor oocyte quality is detrimental to fertilization and embryo development, which causes infertility. Cystathionine ß-synthase (CBS) is one of the key enzymes modulating the metabolism of homocysteine (Hcy). Studies have shown that CBS plays an important role in female reproduction. However, the role of CBS in regulating oocyte quality during meiotic maturation still needs further investigation. MATERIALS AND METHODS: Immunohistochemistry, immunofluorescence, drug treatment, western blot, cRNA construct and in vitro transcription, microinjection of morpholino oligo and cRNA were performed for this study. RESULTS: We found that CBS was expressed both in human and mouse oocytes of follicles. In mouse oocytes, CBS was distributed in the nucleus at germinal vesicle (GV) stage and localized to spindle from germinal vesicle breakdown (GVBD) to metaphase II (MII). The expression of CBS was reduced in ovaries and oocytes of aged mice. CBS depletion resulted in meiotic arrest, spindle abnormality and chromosome misalignment, disrupted kinetochore-microtubule attachments and provoked spindle assembly checkpoint (SAC). CBS was disassembled when microtubules were disrupted with nocodazole, and co-localized with the stabilized microtubules after taxol treatment. Furthermore, CBS depletion decreased the acetylation of α-tubulin. CONCLUSIONS: These results reveal that CBS is required for the acetylation of α-tubulin to ensure proper spindle assembly in regulating oocyte quality during meiotic maturation.

Fluorescence-Based Measurements of Membrane-Bound Angiotensin Converting Enzyme 2 Activity Using Xenopus Laevis Oocytes.

Functional investigations of enzymes involving cellular expression systems are important for pharmacological studies. The precise control of expression is challenging in transiently transfected mammalian cell lines. Here, we explored the ability of Xenopus laevis oocytes to express a membrane-bound enzyme for functional characterization using standard 96-well plates and a fluorescence-based plate reader assay. We microinjected oocytes with cRNA encoding the angiotensin converting enzyme 2 (ACE2) and measured the enzymatic activity in single oocytes using a commercial fluorescence-based assay. The injected oocytes showed up to a 50-fold increase in fluorescence compared to uninjected oocytes. This fluorescence intensity was dose-dependent on the amount of ACE2 cRNA. These results suggest that Xenopus oocytes can be used for the functional evaluation of membrane-bound enzymes, decreasing the experimental workload.

Maternal Prkce expression in mature oocytes is critical for the first cleavage facilitating maternal-to-zygotic transition in mouse early embryos.

OBJECTIVES: Early embryo development is dependent on the regulation of maternal messages stored in the oocytes during the maternal-to-zygote transition. Previous studies reported variability of oocyte competence among different inbred mouse strains. The present study aimed to identify the maternal transcripts responsible for early embryonic development by comparing transcriptomes from oocytes of high- or low- competence mouse strains. MATERIALS AND METHODS: In vitro fertilization embryos from oocytes of different mouse strains were subject to analysis using microarrays, RNA sequencing, real-time quantitative PCR (RT-qPCR) analysis, Western blotting, and immunofluorescence. One candidate gene, Prkce, was analysed using Prkce knockout mice, followed by a cRNA rescue experiment. RESULTS: The fertilization and 2-cell rate were significantly higher for FVB/NJ (85.1% and 82.0%) and DBA/2J (79.6% and 76.7%) inbred mouse strains than those for the MRL/lpr (39.9% and 35.8%) and 129S3 (35.9% and 36.6%) strains. Thirty-nine differentially expressed genes (DEGs) were noted, of which nine were further verified by RT-qPCR. Prkce knockout mice showed a reduced 2-cell rate (Prkce+/+ 80.1% vs. Prkce-/- 32.4%) that could be rescued by Prkce cRNA injection (2-cell rate reached 76.7%). Global transcriptional analysis revealed 143 DEGs in the knockout mice, which were largely composed of genes functioning in cell cycle regulation. CONCLUSIONS: The transcription level of maternal messages such as Prkce in mature oocytes is associated with different 2-cell rates in select inbred mouse strains. Prkce transcript levels could serve as a potential biomarker to characterize high-quality mature oocytes.

Synthesis of 2'-O-alkylcarbamoylethyl-modified oligonucleotides with enhanced nuclease resistance that form isostable duplexes with complementary RNA.

To expand the variety of 2'-O-modified oligonucleotides, we synthesized 2'-O-carbamoylethyl-modified oligonucleotides bearing ethyl, n-propyl, n-butyl, n-pentyl, and n-octyl groups on their nitrogen atoms. The corresponding nucleosides were synthesized using 2'-O-benzyloxycarbonylethylthymidine, which was easily converted into the carboxylic acid through hydrogeneration; subsequent condensation with the appropriate amine gave the desired nucleoside. We evaluated the effect of the 2'-O-alkylcarbamoylethyl modifications on duplex stability by analyzing melting temperature, which revealed the formation of isostable duplexes. In addition, we also revealed that these modifications, especially octylcarbamoylethyl, endowed these oligonucleotides with resistance toward a 3'-exonuclease. These results highlight the usefulness of the 2'-O-alkylcarbamoylethyl modification for various biological applications.

Mutation of an Influenza Virus Polymerase 3' RNA Promoter Binding Site Inhibits Transcription Elongation.

Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

The identification of induction chemo-sensitivity genes of laryngeal squamous cell carcinoma and their clinical utilization.

PURPOSE: To identify potential molecular markers for induction chemotherapy of Laryngeal squamous cell carcinoma (LSCC). METHODS: Differently expressed genes between chemo-sensitive group (seven cases) and chemo-insensitive (five cases) group after induction chemotherapy by TPF were identified by microarrays. Bayes network and Random forest analyses were employed to identify core genes for induction chemotherapy. The diagnostic value of these core genes was also evaluated by ROC analysis. RESULTS: Six genes (SPP1, FOLR3, KYNU, LOC653219, ADH7 and XAGE1A) are highly expressed, while seven gene (CADM1, NDUFA4L2, CCND2, RARRES3, ERAP2, LYD6 and CNTNAP2) present significantly low expression. Among these genes, genes CADM1, FOLR3, KYNU, and CNTNAP2 are core candidates for LSCC chemo-sensitivity. And that the low expression of CADM1 may result in chemo-sensitivity, which leads to high expression of gene FOLR3 and KYNU, and low expression of gene CNTNAP2. Besides, ROC analysis shows that these four genes exhibit effective diagnostic value for induction chemo-sensitivity. CONCLUSIONS: CADM1 may be a potential molecular marker for LSCC induction chemotherapy, while CADM1, FOLR3, KYNU, and CNTNAP2 may provide essential guidance for LSCC diagnosis and follow-up treatment strategies.

Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice.

Antisense oligonucleotides that are dependent on RNase H for cleavage and subsequent degradation of complementary RNA are being developed as therapeutics. Besides the intended RNA target, such oligonucleotides may also cause degradation of unintended RNA off-targets by binding to partially complementary target sites. Here, we characterized the global effects on the mouse liver transcriptome of four oligonucleotides designed as gapmers, two targeting Apob and two targeting Pcsk9, all in different regions on their respective intended targets. This study design allowed separation of intended- and off-target effects on the transcriptome for each gapmer. Next, we used sequence analysis to identify possible partially complementary binding sites among the potential off-targets, and validated these by measurements of melting temperature and RNase H-cleavage rates. Generally, our observations were as expected in that fewer mismatches or bulges in the gapmer/transcript duplexes resulted in a higher chance of those duplexes being effective substrates for RNase H. Follow-up experiments in mice and cells show, that off-target effects can be mitigated by ensuring that gapmers have minimal sequence complementarity to any RNA besides the intended target, and that they do not have exaggerated binding affinity to the intended target.

The small RNA complement of adult Schistosoma haematobium.

BACKGROUND: Blood flukes of the genus Schistosoma cause schistosomiasis-a neglected tropical disease (NTD) that affects more than 200 million people worldwide. Studies of schistosome genomes have improved our understanding of the molecular biology of flatworms, but most of them have focused largely on protein-coding genes. Small non-coding RNAs (sncRNAs) have been explored in selected schistosome species and are suggested to play essential roles in the post-transcriptional regulation of genes, and in modulating flatworm-host interactions. However, genome-wide small RNA data are currently lacking for key schistosomes including Schistosoma haematobium-the causative agent of urogenital schistosomiasis of humans. METHODOLOGY: MicroRNAs (miRNAs) and other sncRNAs of male and female adults of S. haematobium and small RNA transcription levels were explored by deep sequencing, genome mapping and detailed bioinformatic analyses. PRINCIPAL FINDINGS: In total, 89 transcribed miRNAs were identified in S. haematobium-a similar complement to those reported for the congeners S. mansoni and S. japonicum. Of these miRNAs, 34 were novel, with no homologs in other schistosomes. Most miRNAs (n = 64) exhibited sex-biased transcription, suggestive of roles in sexual differentiation, pairing of adult worms and reproductive processes. Of the sncRNAs that were not miRNAs, some related to the spliceosome (n = 21), biogenesis of other RNAs (n = 3) or ribozyme functions (n = 16), whereas most others (n = 3798) were novel ('orphans') with unknown functions. CONCLUSIONS: This study provides the first genome-wide sncRNA resource for S. haematobium, extending earlier studies of schistosomes. The present work should facilitate the future curation and experimental validation of sncRNA functions in schistosomes to enhance our understanding of post-transcriptional gene regulation and of the roles that sncRNAs play in schistosome reproduction, development and parasite-host cross-talk.

Modulation of blood-brain barrier function by a heteroduplex oligonucleotide in vivo.

The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.

Subclassification and Detection of New Markers for the Discrimination of Primary Liver Tumors by Gene Expression Analysis Using Oligonucleotide Arrays.

BACKGROUND/AIMS: The failure to correctly differentiate between intrahepatic cholangiocarcinoma (CC) and hepatocellular carcinoma (HCC) is a significant clinical problem, particularly in terms of the different treatment goals for both cancers. In this study a specific gene expression profile to discriminate these two subgroups of liver cancer was established and potential diagnostic markers for clinical use were analyzed. METHODS: To evaluate the gene expression profiles of HCC and intrahepatic CC, Oligonucleotide arrays (AffymetrixU133A) were used. Overexpressed genes were checked for their potential use as new markers for discrimination and their expression values were validated by reverse transcription polymerase chain reaction and immunohistochemistry analyses. RESULTS: 695 genes/expressed sequence tags (ESTs) in HCC (245 up-/450 down-regulated) and 552 genes/ESTs in CC (221 up-/331 down-regulated) were significantly dysregulated (p＜0.05, fold change >2, ≥70%). Using a supervised learning method, and one-way analysis of variance a specific 270-gene expression profile that enabled rapid, reproducible differentiation between both tumors and nonmalignant liver tissues was established. A panel of 12 genes (e.g., HSP90ß, ERG1, GPC3, TKT, ACLY, and NME1 for HCC; SPT2, T4S3, CNX43, TTD1, HBD01 for CC) were detected and partly described for the first time as potential discrimination markers. CONCLUSIONS: A specific gene expression profile for discrimination of primary liver cancer was identified and potential marker genes with feasible clinical impact were described.

Chromosome biorientation and APC activity remain uncoupled in oocytes with reduced volume.

The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.

Ectopically expressed Slc34a2a sense-antisense transcripts cause a cerebellar phenotype in zebrafish embryos depending on RNA complementarity and Dicer.

Natural antisense transcripts (NATs) are complementary to protein coding genes and potentially regulate their expression. Despite widespread occurrence of NATs in the genomes of higher eukaryotes, their biological role and mechanism of action is poorly understood. Zebrafish embryos offer a unique model system to study sense-antisense transcript interplay at whole organism level. Here, we investigate putative antisense transcript-mediated mechanisms by ectopically co-expressing the complementary transcripts during early zebrafish development. In zebrafish the gene Slc34a2a (Na-phosphate transporter) is bi-directionally transcribed, the NAT predominantly during early development up to 48 hours after fertilization. Declining levels of the NAT, Slc34a2a(as), coincide with an increase of the sense transcript. At that time, sense and antisense transcripts co-localize in the endoderm at near equal amounts. Ectopic expression of the sense transcript during embryogenesis leads to specific failure to develop a cerebellum. The defect is RNA-mediated and dependent on sense-antisense complementarity. Overexpression of a Slc34a2a paralogue (Slc34a2b) or the NAT itself had no phenotypic consequences. Knockdown of Dicer rescued the brain defect suggesting that RNA interference is required to mediate the phenotype. Our results corroborate previous reports of Slc34a2a-related endo-siRNAs in two days old zebrafish embryos and emphasize the importance of coordinated expression of sense-antisense transcripts. Our findings suggest that RNAi is involved in gene regulation by certain natural antisense RNAs.

Tri-directional anaphases as a novel chromosome segregation defect in human oocytes.

STUDY QUESTION: What are the chromosome segregation errors in human oocyte meiosis-I that may underlie oocyte aneuploidy? SUMMARY ANSWER: Multiple modes of chromosome segregation error were observed, including tri-directional anaphases, which we attribute to loss of bipolar spindle structure at anaphase-I. WHAT IS KNOWN ALREADY: Oocyte aneuploidy is common and associated with infertility, but mechanistic information on the chromosome segregation errors underlying these defects is scarce. Lagging chromosomes were recently reported as a possible mechanism by which segregation errors occur. STUDY DESIGN, SIZE, DURATION: Long-term confocal imaging of chromosome dynamics in 50 human oocytes collected between January 2015 and May 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: Germinal vesicle (GV) stage oocytes were collected from women undergoing intracytoplasmic sperm injection cycles and also CD1 mice. Oocytes were microinjected with complementary RNAs to label chromosomes, and in a subset of oocytes, the meiotic spindle. Oocytes were imaged live through meiosis-I using confocal microscopy. 3D image reconstruction was used to classify chromosome segregation phenotypes at anaphase-I. Segregation phenotypes were related to spindle dynamics and cell cycle timings. MAIN RESULTS AND THE ROLE OF CHANCE: Most (87%) mouse oocytes segregated chromosomes with no obvious defects. We found that 20% of human oocytes segregated chromosomes bi-directionally with no lagging chromosomes. The rest were categorised as bi-directional anaphase with lagging chromosomes (20%), bi-directional anaphase with chromatin mass separation (34%) or tri-directional anaphase (26%). Segregation errors correlated with chromosome misalignment prior to anaphase. Spindles were tripolar when tri-directional anaphases occurred. Anaphase phenotypes did not correlate with meiosis-I duration (P = 0.73). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Oocytes were recovered at GV stage after gonadotrophin-stimulation, and the usual oocyte quality caveats apply. Whilst the possibility that imaging may affect oocyte physiology cannot be formally excluded, detailed controls and justifications are presented. WIDER IMPLICATIONS OF THE FINDINGS: This is one of the first reports of live imaging of chromosome dynamics in human oocytes, introducing tri-directional anaphases as a novel potential mechanism for oocyte aneuploidy. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by grants from Fondation Jean-Louis Lévesque (Canada), CIHR (MOP142334) and CFI (32711) to GF. JH is supported by Postdoctoral Fellowships from The Lalor Foundation and CIHR (146703). The authors have no conflict of interest.

Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ.

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.

Oligonucleotides Containing Aminated 2'-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA.

Mono- and diaminated 2'-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2'-amino-LNA monomers and the host oligonucleotide backbone.

Role of the PB2 627 Domain in Influenza A Virus Polymerase Function.

The RNA genome of influenza A viruses is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the subunits PA, PB1, and PB2. High-resolution structural data revealed that the polymerase assembles into a central polymerase core and several auxiliary highly flexible, protruding domains. The auxiliary PB2 cap-binding and the PA endonuclease domains are both involved in cap snatching, but the role of the auxiliary PB2 627 domain, implicated in host range restriction of influenza A viruses, is still poorly understood. In this study, we used structure-guided truncations of the PB2 subunit to show that a PB2 subunit lacking the 627 domain accumulates in the cell nucleus and assembles into a heterotrimeric polymerase with PB1 and PA. Furthermore, we showed that a recombinant viral polymerase lacking the PB2 627 domain is able to carry out cap snatching, cap-dependent transcription initiation, and cap-independent ApG dinucleotide extension in vitro, indicating that the PB2 627 domain of the influenza virus RNA polymerase is not involved in core catalytic functions of the polymerase. However, in a cellular context, the 627 domain is essential for both transcription and replication. In particular, we showed that the PB2 627 domain is essential for the accumulation of the cRNA replicative intermediate in infected cells. Together, these results further our understanding of the role of the PB2 627 domain in transcription and replication of the influenza virus RNA genome.IMPORTANCE Influenza A viruses are a major global health threat, not only causing disease in both humans and birds but also placing significant strains on economies worldwide. Avian influenza A virus polymerases typically do not function efficiently in mammalian hosts and require adaptive mutations to restore polymerase activity. These adaptations include mutations in the 627 domain of the PB2 subunit of the viral polymerase, but it still remains to be established how these mutations enable host adaptation on a molecular level. In this report, we characterize the role of the 627 domain in polymerase function and offer insights into the replication mechanism of influenza A viruses.

Unspecific binding of cRNA probe to plaques in two mouse models for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the pathological deposition of amyloid-ß (Aß) protein-containing plaques. Microglia and astrocytes are commonly attracted to the plaques by an unknown mechanism that may involve cell adhesion. One cell adhesion family of proteins, the cadherins, are widely expressed in the central nervous system. Therefore, our study was designed to map the expression of cadherins in AD mouse brains. A particular focus was on plaques because diverse mRNA-species were found in plaques and their surrounding area in brains of AD patients. METHODS: In this study, we used in situ hybridization to visualize cadherin expression in brains of two mouse models for AD (APP/PS1 and APP23). RESULTS: A variable number of plaques was detected in transgenic brain sections, depending on the probe used. Our first impression was that the cadherin probes visualized specific mRNA expression in plaques and that endogenous staining was unaffected. However, control experiments revealed unspecific binding with sense probes. Further experiments with variations in probe length, probe sequence, molecular tag and experimental procedure lead us to conclude that cRNA probes bind generally and in an unspecific manner to plaques. CONCLUSIONS: We demonstrate unspecific binding of cRNA probes to plaques in two mouse models for AD. The widespread and general staining of the plaques prevented us from studying endogenous expression of cadherins in transgenic brain by in situ hybridization.

The zinc spark is an inorganic signature of human egg activation.

Egg activation refers to events required for transition of a gamete into an embryo, including establishment of the polyspermy block, completion of meiosis, entry into mitosis, selective recruitment and degradation of maternal mRNA, and pronuclear development. Here we show that zinc fluxes accompany human egg activation. We monitored calcium and zinc dynamics in individual human eggs using selective fluorophores following activation with calcium-ionomycin, ionomycin, or hPLCζ cRNA microinjection. These egg activation methods, as expected, induced rises in intracellular calcium levels and also triggered the coordinated release of zinc into the extracellular space in a prominent "zinc spark." The ability of the gamete to mount a zinc spark response was meiotic-stage dependent. Moreover, chelation of intracellular zinc alone was sufficient to induce cell cycle resumption and transition of a meiotic cell into a mitotic one. Together, these results demonstrate critical functions for zinc dynamics and establish the zinc spark as an extracellular marker of early human development.