A Bedside Test to Detect the Presence of Embryonic or Fetal Tissue in Vaginal Blood.

OBJECTIVE: To evaluate a rapid bedside test that detects alpha-fetoprotein (AFP) and insulin-like growth factor-binding protein 1 (IGFBP-1) to identify the presence of embryonic or fetal tissue in vaginal blood. METHOD: This was a prospective cohort study. Reproductive-aged individuals were recruited into three groups: a negative control group consisting of nonpregnant individuals undergoing dilation and curettage (D&C) or experiencing vaginal bleeding; a positive control group of individuals with confirmed intrauterine pregnancy undergoing D&C; and the study group of pregnant individuals with first-trimester bleeding. Lateral flow immunoassay strips capable of detecting both AFP and IGFBP-1 were used to test vaginal blood for the presence of embryonic or fetal tissue. RESULTS: Ninety individuals were recruited: 31 in the positive control group, 23 in the negative control group, and 36 in the study group, including 12 individuals with ectopic pregnancies, 16 with active miscarriages, four with threatened miscarriages, and four with complete miscarriages. Vaginal blood from 14 of the 16 individuals with active miscarriages was correctly positive for embryonic or fetal tissue. Vaginal blood from all individuals with ectopic pregnancies, threatened miscarriages, and complete miscarriages was negative for embryonic or fetal tissue. Overall, 45 of 47 individuals with confirmed embryonic or fetal tissue in vaginal blood correctly tested positive using the test strips, a test sensitivity of 95.7% (95% CI 85.5-99.5%). Of the 43 individuals with confirmed absence of embryonic or fetal tissue in their vaginal blood, 42 were correctly negative, a test specificity of 97.7% (95% CI 87.7-99.9%). CONCLUSION: A rapid test strip detecting both AFP and IGFBP-1 can accurately identify the presence of embryonic or fetal tissue in vaginal blood. When positive, this could aid in diagnosing miscarriage and ruling out ectopic pregnancy at the bedside.

Miscarriage determination in first trimester based on alpha-fetoprotein extracted from sanitary pads.

OBJECTIVE: To determine if high alpha-fetoprotein (AFP) level in vaginal blood collected on a sanitary pad can assist with detecting an active miscarriage. DESIGN: A prospective cohort study. SETTING: Academic medical center. PATIENT(S): Five groups were evaluated: women with active miscarriage, pregnancy of unknown location, completed miscarriage or extrauterine pregnancy (EUP), ongoing pregnancy, and undergoing elective dilation and curettage (D&C). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): For each patient, AFP level in the vaginal blood collected on a sanitary pad was quantified. RESULT(S): The vaginal blood AFP median levels (and their ranges) were 3.7 IU/mL (0.5-739.2) and 4,542 IU/mL (15.6-100,000) in the active miscarriage (n = 16) and the elective D&C (n = 24) groups, respectively. Alpha-fetoprotein was detected in all elective D&C and active miscarriage cases except in 1 case. In the ongoing pregnancy group (n = 35), only 2 of 35 specimens showed detectable AFP levels. In the pregnancy of unknown location (n = 12) and the completed miscarriage or EUP (n = 10) groups, no AFP was detected. Receiver operating characteristic analysis demonstrated 93.7% sensitivity and 97.8% specificity for the detection of an active miscarriage (cutoff 0.61 IU/mL; area under the curve 0.96). CONCLUSION(S): Alpha-fetoprotein can be extracted from vaginal blood collected on sanitary pads. A high level of vaginal AFP can assist with the same-day detection of an active miscarriage. This novel test is useful in differentiating active miscarriages from ongoing pregnancies, completed miscarriages, and EUPs and, therefore, it reduces uncertainty, anxiety level, and number of repeat office visits.

Chemical intervention of influenza virus mRNA nuclear export.

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.

Oocyte activation, oolemma piercing, and real-time viability confirmation in human oocytes using electrophysiological techniques.

PURPOSE OF REVIEW: To discuss the recent applications of electrophysiological principles to the optimization and automation of the IVF laboratory. RECENT FINDINGS: There is growing evidence showing improvement of live birth rates following oocyte electro-activation. Novel applications using electrophysiological techniques are now employed to determine oocyte penetration and viability in real-time. SUMMARY: In this short review, we summarize the recent advances in the integration of electrophysiological techniques into the assisted reproductive technology laboratories. We describe the potential clinical applications and their advantages such as creation of reliable automated cell injection systems and novel manual intracytoplasmic sperm injection (ICSI) training platforms. We also discuss theoretical adverse effects and ways to mitigate them.

A step towards the automation of intracytoplasmic sperm injection: real time confirmation of mouse and human oocyte penetration and viability by electrical resistance measurement.

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.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

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.

Same-day confirmation of intrauterine pregnancy failure in women with first- and early second-trimester bleeding.

OBJECTIVE: To determine if alpha-fetoprotein (AFP) concentration in vaginal blood, in the setting of dissolved fetal tissue, is significantly higher than its concentration in the maternal serum. DESIGN: A prospective cohort study. SETTING: Medical center. PATIENT(S): Four groups of women were evaluated: 1) with missed/incomplete miscarriage with vaginal bleeding; 2) with threatened miscarriage; 3) with vaginal bleeding during cerclage placement; and 4) undergoing dilation and curettage (D&C). INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): In each patient, AFP concentration in the vaginal blood or in the liquid component of the evacuated products of conception (POC; D&C group) was compared with the AFP concentration in the maternal serum. RESULT(S): The median (range) concentration ratios of AFP in vaginal blood (or POC) to AFP in maternal serum were 24.5 (5.1-8,620) and 957 (4.6-24,216) for the missed/incomplete (n = 30) and the D&C (n = 22) groups, respectively, whereas they were only 1.2 (0.4-13.4) and 1.01 (0.7-1.5) for the threatened miscarriage (n = 15) and cerclage (n = 9) groups, respectively. Receiver operating characteristic (ROC) analysis demonstrated 100% sensitivity and 86.7% specificity for the detection of the passage of fetal tissue (ratio 4.3, area under the ROC curve 0.96). CONCLUSION(S): Higher concentrations of AFP in vaginal blood than in maternal serum may indicate the presence of dissolved fetal tissue (i.e., confirming a failed pregnancy).

Visualizing nuclear RNAi activity in single living human cells.

Nuclear RNA interference (RNAi) is mediated by the canonical RNAi machinery and can lead to transcriptional silencing, transcriptional activation, or modulation of alternative splicing patterns. These effects transpire through changes in histone and DNA modifications via RNAi-mediated recruitment of chromatin-modifying enzymes. To prove that nuclear RNAi occurs and modulates transcription in human cells, we used live-cell imaging to detect and track nuclear RNAi transcriptional repression in single living human cells. While employing reporter genes constructed with inducible promoters and cognate-inducible short hairpin RNA (shRNA) targeted against the reporter coding region, we have characterized the dynamics of the nuclear RNAi process in living human cells. We show that the silencing effect is mediated through the nascent mRNA, followed by activity of histone methylating enzymes, but not through DNA methylation.

Recent advances in the field of ovarian tissue cryopreservation and opportunities for research.

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.

Fetal proximal humeral epiphysis as an indicator of term gestation in different ethnic groups.

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.

Influenza virus mRNA trafficking through host nuclear speckles.

Influenza A virus is a human pathogen with a genome composed of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein, while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that the influenza virus uses nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies and exports the spliced M2 mRNA from the nucleus. Given that nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.

A Sanitary Pad for Self-Assessment of Rupture of Membranes.

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.

Carcinoembryonic antigen as a biomarker for meconium-stained amniotic fluid.

OBJECTIVE: To assess whether elevated carcinoembryonic antigen (CEA) concentration in amniotic fluid can indicate meconium-stained amniotic fluid (MSAF). METHODS: In a prospective cohort study, women with a term singleton pregnancy who were in labor but had intact membranes were recruited at a center in Israel over a 5-month period in 2013. Only women who subsequently underwent artificial rupture of membranes following a clear medical indication were included. Samples of amniotic fluid, urine, and serum were collected. Amniotic fluid was examined by sight and classified as clear, MSAF, or undetermined. CEA concentration in the samples was measured. RESULTS: Among 81 participants, 45 had clear amniotic fluid, 28 had MSAF, and eight had undetermined amniotic fluid. Mean CEA concentration was more than 10 times higher in MSAF (2658 µg/L, standard error 250) than in clear amniotic fluid (238 µg/L, standard error 29; P<0.001). Receiver operating characteristic curve analysis demonstrated a sensitivity of 96% and a specificity of 100% for distinguishing MSAF from clear amniotic fluid at a CEA cutoff of 799.2 µg/L. CEA concentrations in urine and serum were all within the normal range (≤5 µg/L), irrespective of amniotic fluid status. CONCLUSION: High CEA concentrations in amniotic fluid can assist in the diagnosis of MSAF. These findings could provide the basis for a bedside test to detect MSAF following rupture of membranes.

Molecular characterization of the human microbiome from a reproductive perspective.

The process of reproduction inherently poses unique microbial challenges because it requires the transfer of gametes from one individual to the other, meanwhile preserving the integrity of the gametes and individuals from harmful microbes during the process. Advances in molecular biology techniques have expanded our understanding of the natural organisms living on and in our bodies, including those inhabiting the reproductive tract. Over the past two decades accumulating evidence has shown that the human microbiome is tightly related to health and disease states involving the different body systems, including the reproductive system. Here we introduce the science involved in the study of the human microbiome. We examine common methods currently used to characterize the human microbiome as an inseparable part of the reproductive system. Finally, we consider a few limitations, clinical implications, and the critical need for additional research in the field of human fertility.

Acute presentation of gestational diabetes insipidus with pre-eclampsia complicated by cerebral vasoconstriction: a case report and review of the published work.

Gestational diabetes insipidus (GDI) is a rare, self-limited complication of pregnancy. As it is related to excess placental vasopressinase enzyme activity, which is metabolized in the liver, GDI is more common in pregnancies complicated by conditions associated with liver dysfunction. We present a case of a 41-year-old woman at 38 weeks' gestation who presented with pre-eclampsia with severe features, including impaired liver function and renal insufficiency. Following cesarean section she was diagnosed with GDI, which was further complicated by cerebral vasoconstriction as demonstrated by magnetic resonance angiography. This case raises the possibility that cerebral vasoconstriction may be related to the cause of GDI. A high index of suspicion of GDI should be maintained in patients who present with typical signs and symptoms, especially in the setting of pregnancy complications associated with liver dysfunction.

Alpha-fetoprotein as a tool to distinguish amniotic fluid from urine, vaginal discharge, and semen.

OBJECTIVE: To estimate whether alpha-fetoprotein (AFP) can be used to distinguish amniotic fluid absorbed in sanitary pads from other similarly absorbed substances (semen, urine, and normal vaginal discharge). METHODS: A prospective cohort study. Urine and amniotic fluid specimens were collected from 52 pregnant women admitted for labor. Semen specimens were collected from 17 men undergoing infertility evaluation. Alpha-fetoprotein concentrations were measured directly from urine, amniotic fluid, and semen and from pads instilled with samples from these specimens. Alpha-fetoprotein concentrations were also measured from pads absorbed with normal vaginal discharge collected from 27 pregnant women. RESULTS: Alpha-fetoprotein levels in amniotic fluid (245.38 ± 21.03 ng/mL, n = 52) were significantly higher than those measured in maternal urine (0.84 ± 0.17 ng/mL, n = 52, P < .001), or semen (1.52 ± 0.35 ng/mL, n = 17, P < .001). The same trend was seen when AFP was extracted from pads: amniotic fluid levels (19.44 ± 1.98 ng/mL, n=52) were significantly higher than those of urine (undetectable, n=52), semen (undetectable, n = 17), or normal vaginal discharge (0.53 ± 0.16 ng/mL, n = 27, P < .001). Receiver operator characteristic curve analysis demonstrated 96.2% sensitivity and 100% specificity for distinguishing the presence of amniotic fluid from normal vaginal discharge on sanitary pads (cutoff 3.88 ng/mL, area under the curve 0.99). CONCLUSION: When the diagnosis of rupture of membranes is in doubt, AFP levels can assist in differentiating amniotic fluid from other bodily fluids. A method that utilizes sanitary pads and an assay for AFP quantification may be an accurate and convenient way to confirm the diagnosis of rupture of membranes.

Nuclear trafficking in health and disease.

In eukaryotic cells, the cytoplasm and the nucleus are separated by a double-membraned nuclear envelope (NE). Thus, transport of molecules between the nucleus and the cytoplasm occurs via gateways termed the nuclear pore complexes (NPCs), which are the largest intracellular channels in nature. While small molecules can passively translocate through the NPC, large molecules are actively imported into the nucleus by interacting with receptors that bind nuclear pore complex proteins (Nups). Regulatory factors then function in assembly and disassembly of transport complexes. Signaling pathways, cell cycle, pathogens, and other physiopathological conditions regulate various constituents of the nuclear transport machinery. Here, we will discuss several findings related to modulation of nuclear transport during physiological and pathological conditions, including tumorigenesis, viral infection, and congenital syndrome. We will also explore chemical biological approaches that are being used as probes to reveal new mechanisms that regulate nucleocytoplasmic trafficking and that are serving as starting points for drug development.

Pressure-selective modulation of NMDA receptor subtypes may reflect 3D structural differences.

Professional deep-water divers exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is associated with CNS hyperexcitability. We have previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. We now report that HP (10.1 MPa) differentially affects eight specific NMDAR subtypes. GluN1(1a or 1b) was co-expressed with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. HP increased ionic currents (measured by two electrode voltage clamps) of one subtype, reduced the current in four others, and did not affect the current in the remaining three. 3D theoretical modeling was aimed at revealing specific receptor domains involved with HP selectivity. In light of the information on the CNS spatial distribution of the different NMDAR subtypes, we conclude that the NMDAR's diverse responses to HP may lead to selective HP effects on different brain regions. These discoveries call for further and more specific investigation of deleterious HP effects and suggest the need for a re-evaluation of deep-diving safety guidelines.

Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells.

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.