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Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus. Author summary: Influenza virus remains a major public health threat killing ~250,000–500,000 people yearly. In pandemic years, influenza infection can lead to even higher mortality rates, as in 1918 when at least 20 million deaths occurred worldwide. Available treatments include vaccines and a few antiviral drugs, but both are limited by mutability of the virus and the development of resistance. Here we leveraged on knowledge of our previous work that uncovered a unique influenza virus pathway inside the nucleus of the host cell, which have the potential to be targeted by a novel therapeutic strategy. We designed and performed a new screen to identify chemical compounds that inhibited viral mRNA production and transport from the nucleus to the cytoplasm of the host cell. Among these compounds, we highlight a novel nuclear export inhibitor of a subset of viral and cellular mRNAs that does not substantially alter bulk cellular RNA export or levels at non-toxic dosages. Consequently, this small molecule inhibits virus replication and therefore can be used as a lead for drug development and serve as an experimental tool for further understanding key steps of the influenza virus mRNA nuclear export pathway. [ABSTRACT FROM AUTHOR]

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein–protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export. [ABSTRACT FROM AUTHOR]

Objectives: Accurate pregnancy dating is critical for appropriate clinical management. Our aim was to determine the time of appearance of proximal humeral epiphysis (PHE), consistency of its appearance among ethnic groups and whether 3D imaging helps with its visualization.Methods: A cross-sectional study was done on 360 patients with 563 scans in different ethnic groups between August 2013 and July 2015. Inclusion criteria were singleton pregnancies (34-40+  weeks of gestation), well dated by <20 weeks sonogram.Results: PHE was not seen at 34 (n = 44) or 35 weeks (n = 36) and was present at gestational ages 36 (n = 3), 37 (n = 126), 38 (n = 96), 39 (n = 100) and 40 weeks (n = 28) in 2%, 12%, 51%, 75% and 100%, respectively. PHE was seen in 20 of 50 (60%) African-Americans, 22 of 61 (64%) south Asians, 41 of 72 (57%) Caucasians, 45 of 86 (48%) Hispanics and 41 of 80 (49%) Asians.Conclusion: Appearance of PHE did increase with gestational age, prior to 40 weeks, it was not uniformly present and was seen as early as 36 weeks independent of ethnic group. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to summarize the latest advances and successes in the field of ovarian tissue cryopreservation while identifying gaps in current knowledge that suggest opportunities for future research. Methods: A systematic review was performed according to PRISMA guidelines for all relevant full-text articles in PubMed published in English that reviewed or studied historical or current advancements in ovarian tissue cryopreservation and auto-transplantation techniques. Results: Ovarian tissue auto-transplantation in post-pubertal women is capable of restoring fertility with over 80 live births currently reported with a corresponding pregnancy rate of 23 to 37%. The recently reported successes of live births from transplants, both in orthotopic and heterotopic locations, as well as the emerging methods of in vitro maturation (IVM), in vitro culture of primordial follicles, and possibility of in vitro activation (IVA) suggest new fertility options for many women and girls. Vitrification, as an ovarian tissue cryopreservation technique, has also demonstrated successful live births and may be a more cost-effective method to freezing with less tissue injury. Further, transplantation via the artificial ovary with an extracellular tissue matrix (ECTM) scaffolding as well as the effects of sphingosine-1-phosphate (SIP) and fibrin modified with heparin-binding peptide (HBP), heparin, and a vascular endothelial growth factor (VEGF) have demonstrated important advancements in fertility preservation. As a fertility preservation method, ovarian tissue cryopreservation and auto-transplantation are currently considered experimental, but future research may pave the way for these modalities to become a standard of care for women facing the prospect of sterility from ovarian damage. [ABSTRACT FROM AUTHOR]

Objective: To evaluate if oocyte penetration and viability can be confirmed by an electrical resistance increase. Automated (robotic) intracytoplasmic sperm injection (ICSI) requires confirmation of oolemma penetration before sperm injection. Visual assessment using image processing algorithms have been developed but remain unreliable. We hypothesized that an increase in electrical resistance upon oolemma piercing during ICSI can serve as an objective tool to confirm oocyte penetration and viability.Design: Experimental study.Setting: Research laboratory in an academic center.Patients/animals: Oocytes from female mice and women undergoing oocyte retrieval procedure.Intervention: Oolemma piercing attempts with the ICSI pipette were performed by advancing the pipette towards mature (metaphase II) oocytes collected from 6 to 12-week-old mice and immature (germinal vesicle stage and metaphase I) oocytes donated by women who underwent oocyte retrieval. Electrical resistance was measured using a conventional electrophysiological setup that includes an electrical resistance meter and two electrical wires located in the lumina of the holding and ICSI pipettes.Main Outcome Measure(s): The measure of interest was the change in electrical resistance (ΔR) before and after advancing the ICSI pipette in an attempt to penetrate an oocyte. The experiments of resistance measurements were done in 3 steps: Step 1 (proof of concept), penetrated vs. non-penetrated mouse oocytes. Step 2, mouse oocytes with visually intact oolemma vs. fragmented mouse oocytes. Step 3, human oocytes with visually intact oolemma vs. fragmented human oocytes. For each group, median and range (in parenthesis) of ΔR were determined in MΩ. Mann-Whitney test was performed to compare the two groups in each step.Results: In Step 1, the penetrated mouse oocytes showed a statistically significant resistance increase compared to the non-penetrated ones (n = 20, median ΔR = 7.79 [2.57 - 106.00] vs. n = 15, median ΔR = 0.10 [-0.06 - 0.69], respectively. In Step 2, the mouse oocytes with visually intact oolemma showed a statistically significant resistance increase compared to the fragmented ones (n = 45, median ΔR = 6.5 [0.1 - 191.7] vs. n = 13, median ΔR = 0.1 [-0.3 - 2.2], respectively. In Step 3, the human oocytes with visually intact oolemma showed a statistically significant resistance increase compared to the fragmented ones (n = 96, median ΔR = 1.92 [-0.05 - 6.70] vs. n = 17, median ΔR = 0.11 [0.00 - 0.30], respectively.Conclusions: An electrical resistance increase can serve as a reliable tool to confirm oocyte penetration and viability, independent of optical visualization. Following further validation and safety assessment, this technology can potentially be integrated into manual and robotic ICSI systems. [ABSTRACT FROM AUTHOR]

Background: Multistep immunoassay kits for the diagnosis of rupture of membranes are relatively complex and are not designed to be used by pregnant women themselves. These kits require procedural steps of specimen extraction and preparation. We evaluated the ability of a sanitary pad containing a qualitative immunoassay for alpha-fetoprotein (AFP) to serve as a one-step self-test to detect amniotic fluid leakage.Technique: Four sets of pads were evaluated. The pads in the study set were worn by 288 pregnant women with confirmed rupture of membranes. Three controls were evaluated: 1) pads worn by 93 pregnant women with intact membranes, 2) additional pads instilled with urine specimens obtained from the 381 women described previously (study set plus control set 1), and 3) pads instilled with semen collected from 40 men.Experience: All 288 pads that absorbed amniotic fluid had positive results. Approximately half of the pads absorbed with normal vaginal discharge had a sufficient amount to yield valid results, which were all negative. All 381 pads with instilled urine and all 40 pads with instilled semen had negative results.Conclusion: An immunoassay for AFP, embedded in a pad, appears to be a feasible and reproducible self-test for the detection of rupture of membranes. [ABSTRACT FROM AUTHOR]

The flow of genetic information in eukaryotic cells occurs through the nucleocytoplasmic translocation of mRNAs. Knowledge of in vivo messenger RNA export kinetics remains poor in comparison with that of protein transport. We have established a mammalian system that allowed the real-time visualization and quantification of large single mRNA–protein complexes (mRNPs) during export. The in vivo dynamics of bulk mRNP transport and export, from transcription to the nuclear pore complex (NPC), occurred within a 5–40 minute time frame, with no NPC pile-up. mRNP export was rapid (about 0.5 s) and kinetically faster than nucleoplasmic diffusion. Export inhibition demonstrated that mRNA–NPC interactions were independent of ongoing export. Nucleoplasmic transport dynamics of intron-containing and intronless mRNAs were similar, yet an intron did increase export efficiency. Here we provide visualization and analysis at the single mRNP level of the various steps in nuclear gene expression and the inter-chromatin tracks through which mRNPs diffuse, and demonstrate the kinetics of mRNP–NPC interactions and translocation. [ABSTRACT FROM AUTHOR]

High pressure, which induces central nervous system (CNS) dysfunction (high-pressure neurological syndrome) depresses synaptic transmission at all synapses examined to date. Several lines of evidence indicate an inhibitory effect of pressure on Ca2+ entry into the presynaptic terminal. In the present work we studied for the first time the effect of pressure on the cerebellar climbing fiber (CF) synaptic responses. Pressure modulation of cerebellar synaptic plasticity was tested in both the CF and parallel fiber (PF) pathways using paired-pulse protocols. CF synapses, which normally operate at a high baseline release probability, demonstrate paired-pulse depression (PPD). High pressure reduced CF synaptic responses at 5.1 and 10.1 MPa but did not affect its PPD. High extracellular Ca2+ concentration ([Ca2+]o) could not antagonize the effect of pressure on the CF response, whereas low [Ca2+]o, in contrast to pressure, decreased both the response amplitude and the observed PPD. PF synapses, which usually operate at low release probability, exhibit paired-pulse facilitation (PPF). Pressure increased PF PPF at all interstimulus intervals (ISIs) tested (20-200 ms). Several Ca2+ channel blockers as well as low [Ca2+]o could mimic the effect of pressure on the PF response but significantly increased the PPF only at the 20-ms ISI. These results, together with previous data, show that the CF synapse is relatively resistant to pressure. The lack of pressure effect on CF PPD is surprising and may suggest that the PPD is not directly linked to synaptic depletion, as generally suggested. The increase in PPF of the PF at pressure, which is mimicked by Ca2+ channel blockers or low [Ca2+]o, further supports pressure involvement in synaptic release mechanism(s). These results also indicate that pressure effects may be selective for various types of synapses in the CNS. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the N-methyl-D-aspartate receptor (NMDAR) component alone without the contribution of the central nervous system (CNS) network. Mammalian cRNA of the most common hippocampal NMDAR compositions NR1-1a/NR2A and NR1-1b/NR2A were injected into Xenopus laevis oocytes. According to the researchers, increased NMDAR response, in addition to other mechanisms, plays in important role in high pressure neurological syndrome (HPNS).

Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed β-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic β-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the ?-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell. [ABSTRACT FROM AUTHOR]

Objectives: Accurate pregnancy dating is critical for appropriate clinical management. Our aim was to determine the time of appearance of proximal humeral epiphysis (PHE), consistency of its appearance among ethnic groups and whether 3D imaging helps with its visualization.Methods: A cross-sectional study was done on 360 patients with 563 scans in different ethnic groups between August 2013 and July 2015. Inclusion criteria were singleton pregnancies (34-40+  weeks of gestation), well dated by <20 weeks sonogram.Results: PHE was not seen at 34 (n = 44) or 35 weeks (n = 36) and was present at gestational ages 36 (n = 3), 37 (n = 126), 38 (n = 96), 39 (n = 100) and 40 weeks (n = 28) in 2%, 12%, 51%, 75% and 100%, respectively. PHE was seen in 20 of 50 (60%) African-Americans, 22 of 61 (64%) south Asians, 41 of 72 (57%) Caucasians, 45 of 86 (48%) Hispanics and 41 of 80 (49%) Asians.Conclusion: Appearance of PHE did increase with gestational age, prior to 40 weeks, it was not uniformly present and was seen as early as 36 weeks independent of ethnic group. [ABSTRACT FROM AUTHOR]