Incidence, detection and outcome of differentiated thyroid cancer in Western Sweden.

BACKGROUND: It is unclear whether the increasing incidence of thyroid cancer (TC) due to increased diagnosis of small and indolent tumours might mask a real increase of clinically significant cancers. The aim of this study was to correlate surgery, pathology and outcome data of individual patients to the mode of primary detection (palpation, by imaging or incidental) to assess if TC incidence has increased. METHODS: The Swedish Cancer Registry identified all patients with TC in Västra Götaland County representing approximately 1.6 million inhabitants. Clinical information was retrieved from medical records of patient cohorts from three study intervals (2001-2002, 2006-2007 and 2011-2014) comprising 60 per cent of all TC patients. Data were also obtained from the NORDCAN registry to compare of TC incidence with other Nordic countries. RESULTS: Between 2001 and 2014, the annualized standard incidence rate/100 000 population (ASR) of TC increased from 3.14 to 10.71 in women and from 1.12 to 3.77 in men. This was higher than the mean incidence for Sweden but similar to that in Norway and Finland. Differentiated TC (DTC) increased more than threefold. The majority of tumours (64 per cent) were detected by palpation. Larger tumours (10-20, 21-40 and greater than 40 mm) increased as much as microcarcinomas (less than 10 mm). Only 5 per cent of the tumours were detected by imaging. All disease-specific deaths (8.5 per cent of DTC in the first two cohorts) and most patients with recurrent or persistent disease (6.6 per cent of DTC cases) were diagnosed due to tumour-related symptoms. CONCLUSION: DTC in Western Sweden gradually increased between 2001 and 2014. The majority of tumours were detected by palpation suggesting a real increase in the incidence of clinically significant thyroid malignancies.

Microbiota of bovine milk, teat skin, and teat canal: Similarity and variation due to sampling technique and milk fraction.

The aim of this study was to evaluate the effect of sampling technique and milk fraction on bovine milk microbiota data and to compare the microbiota in milk to microbiota on the teat end and in the teat canal. Representative milk samples are highly important for assessment of bacteriological findings and microbiota in milk. Samples were obtained from 5 healthy lactating dairy cows at udder quarter level during 1 milking. Swab samples from the teat end and teat canal, and milk samples collected using different techniques and in different milk fractions were included. Milk was collected by hand stripping and through a teat canal cannula before and after machine milking, through a trans-teat wall needle aspirate after milking, and from udder quarter composite milk. The microbiota of the samples was analyzed with sequencing of the V1-V3 region of the 16S rRNA gene. In addition, somatic cell counts and bacterial cultivability were analyzed in the milk samples. Microbiota data were analyzed using multivariate methods, and differences between samples were tested using analysis of similarity (ANOSIM). Differences between samples were further explored via individual studies of the 10 most abundant genera. The microbiota on the teat end, in the teat canal, and in udder quarter composite milk, collected using a milking machine, differed in composition from the microbiota in milk collected directly from the udder quarter. No differences in milk microbiota composition were detected between hand-stripped milk samples, milk samples taken through a teat canal cannula, or milk samples taken as a trans-teat wall needle aspirate before or after milking. We conclude that for aseptic milk samples collected directly from the lactating udder quarter, sampling technique or milk fraction has minor effect on the microbiota composition.

Evaluating risk factors for re-exploration due to postoperative neck hematoma after thyroid surgery: a nested case-control study.

PURPOSE: Postoperative bleeding after thyroid surgery remains a potentially lethal complication. Outpatient thyroidectomy is an increasing trend in the high volume centers. There is a need to identify risk factors for postoperative bleeding in order to select proper patients for outpatient thyroidectomy. This study aimed to investigate this issue using a national population-based register. MATERIAL AND METHOD: A nested case-control study on patients registered in the Swedish national register for endocrine surgery (SQRTPA) was performed. Patients with postoperative bleeding were matched 1:1 by age and gender to controls. Additional information on cases and controls was obtained from attending surgeons using a questionnaire. Risk factors for postoperative bleeding were evaluated with logistic regression and are presented as odds ratios (ORs) with 95% confidence intervals (CIs). The time of bleeding in relation to surgery was also investigated. RESULTS: There were 9494 operations, and 174 (1.8%) of them involved postoperative bleeding. In the whole cohort, patients with postoperative bleeding were older, 58 (46-69) vs. 49 (37-62) years, than patients without, p < 0.01. Male patients had a higher risk of bleeding, OR 2.18 (95% CI 1.58-2.99). In the case-control cohort, drain was an independent risk factor for bleeding, OR 1.64 (1.05-2.57). Two-thirds of patients bled within 6 h after surgery. The incidence of bleeding after 24 h was 10%. CONCLUSION: High age, male gender, and drain are independent risk factors for bleeding after thyroid surgery. Even with careful patient selection, prolonged observation might be necessary in thyroid surgery.

A Nested Case-Control Study on the Risk of Surgical Site Infection After Thyroid Surgery.

INTRODUCTION: It is unclear if antibiotic prophylaxis reduces the risk of surgical site infection (SSI) in thyroid surgery. This study assessed risk factors for SSI and antibiotic prophylaxis in subgroups of patients. METHOD AND DESIGN: A nested case-control study on patients registered in the Swedish National Register for Endocrine Surgery was performed. Patients with SSI were matched 1:1 by age and gender to controls. Additional information on patients with SSI and controls was queried from attending surgeons using a questionnaire. Risk factors for SSI were evaluated by logistic regression analysis and presented as odds ratio (OR) with 95% confidence interval (CI). RESULTS: There were 9494 operations; 109 (1.2%) patients had SSI. Patients with SSI were older (median 53 vs. 49 years) than patients without SSI p = 0.01 and more often had a cancer diagnosis 23 (21.1%) versus 1137 (12.1%) p = 0.01. In the analysis of patients with SSI versus controls, patients with SSI more often had post-operative drainage 68 (62.4%) versus 46 (42.2%) p = 0.01 and lymph node surgery 40 (36.7%) versus 14 (13.0%) p < 0.01, and both were independent risk factors for SSI, drain OR 1.82 (CI 1.04-3.18) and lymph node dissection, OR 3.22 (95% CI 1.32-7.82). A higher number of 26(62%) patients with independent risk factors for SSI and diagnosed with SSI did not receive antibiotic prophylaxis. Data were missing for 8 (31%) patients. CONCLUSION: Lymph node dissection and drain are independent risk factors for SSI after thyroidectomy. Antibiotic prophylaxis might be considered in patients with these risk factors.

Changing seasonal variation in births by sociodemographic factors: a population-based register study.

STUDY QUESTION: Have seasonal variations in births by factors related to maternal education, age, parity and re-partnering changed over a 72-year period? SUMMARY ANSWER: Seasonal variation in births has been reduced overall but also changed its pattern over the last seven decades. WHAT IS KNOWN ALREADY: The number of births varies markedly by season, but the causes of this variation are not fully understood. Seasonality of births is, in some populations, strongly influenced by sociodemographic factors. STUDY DESIGN SIZE DURATION: A longitudinal study design was used by analysing the seasonal variation in live births between 1940 and 2012, and relating it to mothers' sociodemographic characteristics at the time of childbirth (maternal education, age, parity and re-partnering). PARTICIPANTS/MATERIALS SETTING METHODS: Register data on 6 768 810 live births in Sweden between 1940 and 2012 were used. Information on biological parents are available for more than 95% of all births. Multinomial logistic regressions were used to calculate predicted probabilities of giving birth for each calendar month. MAIN RESULTS AND THE ROLE OF CHANCE: Between 1940 and 1999, Swedish birth rates showed the typical seasonal variation with high numbers of births during the spring, and low numbers of births during the last quarter of the year. However, during the 21st century, the seasonal variation in fertility declined so that only minor variation in birth rates between February and September now remains. Still, the pattern of low birth rates at the end of the year remains and has even become more pronounced from the 1980s onwards. The characteristic 'Christmas effect' that used to be visible in September has vanished over the last 30 years. The roles in seasonal variation of maternal education, the mother's age, parity and instances where the mother has re-partnered between subsequent births changed during the second half of the 20th century. From 1980s onwards, the decline in birth rates during the last quarter of the year became particularly pronounced among highly educated mothers. Over the 72 years studied, the seasonal variation among first-time mothers declined steadily and has almost disappeared at the end of the study period. Using data that cover ~180 000 births in each month, all meaningful results are statistically significant. LIMITATIONS REASONS FOR CAUTION: The study uses data from one Nordic country only, making it difficult to draw conclusions that may hold for other countries. WIDER IMPLICATIONS OF THE FINDINGS: The typical seasonal variation reported for Sweden between 1940 and 1999, with high numbers of births during the spring and low numbers of births during the last quarter of the year, is in line with results from most other European countries during the same time period. However, the significant decline in seasonal variation in the early 21st century is a novel development. The study underlines that in a society with low fertility and efficient birth control, active choices and behaviours associated with an individual's sociodemographic characteristics tend to matter more for the seasonal timing of childbearing than environmental factors related to the physiological ability to reproduce and cultural-behavioural factors related to the frequency of intercourse. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Swedish Research Council (Vetenskapsrådet) via the Swedish Initiative for Research on Microdata in the Social and Medical Sciences (SIMSAM): Stockholm University SIMSAM Node for Demographic Research (grant registration number 340-2013-5164). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Repetitive acute intermittent hypoxia increases growth/neurotrophic factor expression in non-respiratory motor neurons.

Repetitive acute intermittent hypoxia (rAIH) increases growth/trophic factor expression in respiratory motor neurons, thereby eliciting spinal respiratory motor plasticity and/or neuroprotection. Here we demonstrate that rAIH effects are not unique to respiratory motor neurons, but are also expressed in non-respiratory, spinal alpha motor neurons and upper motor neurons of the motor cortex. In specific, we used immunohistochemistry and immunofluorescence to assess growth/trophic factor protein expression in spinal sections from rats exposed to AIH three times per week for 10weeks (3×wAIH). 3×wAIH increased brain-derived neurotrophic factor (BDNF), its high-affinity receptor, tropomyosin receptor kinase B (TrkB), and phosphorylated TrkB (pTrkB) immunoreactivity in putative alpha motor neurons of spinal cervical 7 (C7) and lumbar 3 (L3) segments, as well as in upper motor neurons of the primary motor cortex (M1). 3×wAIH also increased immunoreactivity of vascular endothelial growth factor A (VEGFA), the high-affinity VEGFA receptor (VEGFR-2) and an important VEGF gene regulator, hypoxia-inducible factor-1α (HIF-1α). Thus, rAIH effects on growth/trophic factors are characteristic of non-respiratory as well as respiratory motor neurons. rAIH may be a useful tool in the treatment of disorders causing paralysis, such as spinal injury and motor neuron disease, as a pretreatment to enhance motor neuron survival during disease, or as preconditioning for cell-transplant therapies.

Mode of delivery and the probability of subsequent childbearing: a population-based register study.

OBJECTIVE: To investigate the relationship between mode of first delivery and probability of subsequent childbearing. DESIGN: Population-based study. SETTING: Nationwide study in Sweden. POPULATION: A cohort of 771 690 women who delivered their first singleton infant in Sweden between 1992 and 2010. METHODS: Using Cox's proportional-hazards regression models, risks of subsequent childbearing were compared across four modes of delivery. Hazard ratios (HRs) were calculated, using 95% confidence intervals (95% CIs). MAIN OUTCOME MEASURES: Probability of having a second and third child; interpregnancy interval. RESULTS: Compared with women who had a spontaneous vaginal first delivery, women who delivered by vacuum extraction were less likely to have a second pregnancy (HR 0.96, 95% CI 0.95-0.97), and the probabilities of a second childbirth were substantially lower among women with a previous emergency caesarean section (HR 0.85, 95% CI 0.84-0.86) or an elective caesarean section (HR 0.82, 95% CI 0.80-0.83). There were no clinically important differences in the median time between first and second pregnancy by mode of first delivery. Compared with women younger than 30 years of age, older women were more negatively affected by a vacuum extraction with respect to the probability of having a second child. A primary vacuum extraction decreased the probability of having a third child by 4%, but having two consecutive vacuum extraction deliveries did not further alter the probability. CONCLUSIONS: A first delivery by vacuum extraction does not reduce the probability of subsequent childbearing to the same extent as a first delivery by emergency or elective caesarean section.

Analysis of conformational variations of the cricoid cartilages in Thoroughbred horses using computed tomography.

REASONS FOR PERFORMING STUDY: Loss of arytenoid abduction is a common post operative complication of laryngoplasty without a definitive cause. It has been a clinical impression during laryngoplasty surgery that there is great conformational variability along the caudal edge of the Thoroughbred cricoid cartilage that could impact post operative retention of suture position. A change in suture position would probably lead to some loss of abduction. Defining any structural variability of the cricoid would be an initial step in determining whether this variability could impact on the retention of suture position. HYPOTHESIS: Anatomical variations in the larynx of Thoroughbred horses may be detected and measured using objective analysis and computed tomography. METHODS: Larynges were harvested from 15 mature Thoroughbred horses. Helical CT scans were performed on each specimen. Three independent observers performed a series of measurements on 2D and 3D reconstruction images using digital software. Measurements included the lateral cricoid angle, the caudal cricoid prominences, the distance to the cricoid slope, the angle of the cricoarytenoid joints (CAJ), the cricoid thickness and the suture angle. Mean, standard deviation, coefficient of variation and linear regression analysis were performed among all observers and all measurements. RESULTS: Notable conformational differences were evident on the 3D reconstructions. The highest degree of variability was found in 3 measurements: the distance to the lateral cricoid slope, the lateral cricoid angle and the cricoid thickness. A larger left CAJ angle directly and significantly correlated with a larger suture angle. CONCLUSIONS: There are notable conformational differences among cricoid specimens in the Thoroughbred larynx. POTENTIAL RELEVANCE: The morphometric differences identified may impact on optimal prosthesis placement and long-term retention. Since a larger lateral cricoid angle may facilitate abduction loss secondary to a displaced and loosened suture, alternative techniques for suture placement may be of value to prevent arytenoid abduction loss.

Differential expression of respiratory long-term facilitation among inbred rat strains.

We tested the hypotheses that: (1) long-term facilitation (LTF) following acute intermittent hypoxia (AIH) varies among three inbred rat strains: Fischer 344 (F344), Brown Norway (BN) and Lewis rats and (2) ventral cervical spinal levels of genes important for phrenic LTF (pLTF) vary in association with pLTF magnitude. Lewis and F344, but not BN rats exhibited significant increases in phrenic and hypoglossal burst amplitude 60min post-AIH that were significantly greater than control experiments without AIH, indicating strain differences in phrenic (98%, 56% and 20%, respectively) and hypoglossal LTF (66%, 77% and 5%, respectively). Ventral spinal 5-HT(2A) receptor mRNA and protein levels were higher in F344 and Lewis versus BN, suggesting that higher 5-HT(2A) receptor levels are associated with greater pLTF. More complex relationships were found for 5-HT(7), BDNF and TrkB mRNA. BN had higher 5-HT(7) and TrkB mRNA versus F344; BN and Lewis had higher BDNF mRNA levels versus F344. Genetic variations in serotonergic function may underlie strain differences in AIH-induced pLTF.

Substrate selectivity of exportin 5 and Dicer in the biogenesis of microRNAs.

Each step in the biogenesis of microRNAs (miRNAs) depends on recognition of a correct substrate and efficient transport or processing of that RNA. Exportin5 (Exp5) and Dicer are proteins that mediate two key steps in this cascade, the nuclear export and cytoplasmic processing of microRNA precursor (pre-miRNAs). Xenopus laevis oocytes, eggs, and embryos constitute convenient experimental systems in which to study the substrate specificity of these proteins because specific RNAs or proteins can be injected directly into different subcellular compartments. We have used the Xenopus system and in vitro processing to define and compare the specificities of Exp5 and Dicer. Although both proteins act on many of the same substrates, we show that they recognize different structure elements of these RNAs. Our studies also revealed several unexpected activities. For example, Exp5 can mediate export of unspliced pre-mRNAs and excised lariat introns if these RNAs contain an aptamer sequence that itself is an Exp5 export substrate. Finally, we demonstrate that maturation of Xenopus oocytes into eggs leads to a large increase in Dicer activity, suggesting that miRNA biogenesis is subject to developmental control.

The evaluation of extracorporeal shockwave therapy in naturally occurring osteoarthritis of the stifle joint in dogs.

Extracorporeal shockwave therapy (ESWT) has expanded from the original uses of human urinary calculi treatment to veterinary orthopaedic applications. This paper investigates the feasibility and efficacy of treating dogs with osteoarthritis of the stifle joint with ESWT. In this study, dogs with persistent stifle lameness despite previous surgical or medical treatment were either treated with ESWT or served as untreated controls. The more lame rear limb of each dog was determined by force platform analysis. The range of motion (ROM) of the stifle joints was assessed by goniometry. Force platform gait analysis and goniometry were performed on both groups for four visits at three-week intervals and a final examination four weeks later. Shock wave therapy was performed three times on the treated dogs, once at each of the first three examinations. A placebo treatment consisting of clipping and wetting the hair was performed on the control dogs. The vertical forces were evaluated for objective analysis of treatment response. For peak vertical force (PVF), four of seven treated dogs improved, while only one of five of control dogs improved. The PVF for the within group analysis did not show any significant change for the treated group, however, the control group has a significant decrease (p = 0.05) in PVF consistent with an increase in lameness. The range of motion (ROM) of the stifle joint improved in five of seven treated dogs and three of five controls. Dogs in the treated group had a trend toward increased ROM (p = 0.07) and a 'positive slope' when compared to dogs in the control group which did not have a significant change (p = 0.78) and had a negative slope indicating the dogs were developing a decrease in ROM. The subjective data provided by client questionnaire did not show significant difference between groups.

A placebo-controlled study of high dose buprenorphine in opiate dependents waiting for medication-assisted rehabilitation in Oslo, Norway.

AIMS: To evaluate whether buprenorphine. even without additional control and psychosocial treatment and support, alleviates the problems faced by patients waiting for medication assisted rehabilitation (MAR). DESIGN: A randomized, double-blind, 12-week study of Subutex versus placebo without additional support as an interim therapy. PARTICIPANTS: One hundred and six patients, 70 males and 36 females, waiting for MAR in Oslo. The average age was 38 years with an average history of heroin use of 20 years. Fifty-five patients were assigned to buprenorphine and 51 to a placebo. INTERVENTION: Subutex or placebo sublingual tablets were given under supervision in a daily dose of 16 mg with the exception of a double dose on Saturday and no dose on Sunday. MEASUREMENT: Retention, compliance, self-reported drug-abuse, wellbeing and mental health. FINDINGS: The average number of days of participation was significantly higher in the buprenorphine group, 42 (median: 29) compared to 14 (median: 11) for the placebo group (P < 0.001). The retention of patients after 12 weeks was 16 patients in the buprenorphine group and one patient in the placebo group. The buprenorphine group had a larger decrease in reported opioid use (p < 0.001) and in reported use of other drugs, tablets and alcohol abuse (p < 0.01). The group also showed a stronger increase in wellbeing (p < 0.01) and life satisfaction (p < 0.05). None of the participants died. CONCLUSION: The patients waiting for MAR benefited significantly from the buprenorphine as an interim therapy according to retention, self-reported use of drugs and wellbeing. However, the patients had difficulties in remaining in treatment over time without psychosocial support.

Comparative Assessment of Variation among Sorghum Germplasm Accessions Using Seed Morphology and RAPD Measurements.

The sorghum germplasm collection currently contains over 42 000 accessions, a number that is too large to manage efficiently. The specific objective of this research was to compare clusters developed from agronomic descriptors with phylogenetic groupings based on random amplified polymorphic DNA (RAPD) fingerprinting of selected sorghum [Sorghum bicolor (L.) Moench] races. Our intent was to identify one approach using agronomic descriptors that would most closely approximate the groupings produced by RAPD markers. Ninety-four accessions of sorghum were grouped into four of the five major races. Differences among accessions determined by various clustering procedures based on agronomic characteristics were compared with clusters developed by means of RAPD markers. Each race varied in the degree of similarity between the four clustering approaches taken and the information provided by RAPD fingerprinting. Test 2, standardization of data by Z-scores and cluster analysis using the complete set of data, provided the highest similarity score for the race bicolor, while Test 3, standardization of data by Z-scores and cluster analysis based on a reduced set of variables selected from principle component analysis, provided the highest similarity scores for the races guinea. Test 1, random selection, was highest for the races caudatum and durra. When averaged over all the races, Test 2 provided the highest similarity score. The results of this study indicate that no one approach to develop clusters by means of agronomic descriptors closely approximate the groupings produced by RAPD markers. These results underscore the need for further research in the evaluation of techniques used to develop core collections and their validity.

Economic consequences of an implanted neuroprosthesis for bladder and bowel management.

OBJECTIVE: To determine whether an implanted neuroprosthesis for bladder and bowel management is less costly than conventional techniques. DESIGN: Retrospective cost-identification analysis with comparison before and after implantation of the neuroprosthesis. SETTING: Life-care planning interviews in patients' homes. PATIENTS: Twelve patients with complete suprasacral spinal cord injuries and neurogenic bladder and bowel. INTERVENTION: Implantation of a neuroprosthesis for electric stimulation of the sacral nerves and posterior sacral rhizotomy. MAIN OUTCOME MEASURES: Annual costs of bladder and bowel care with and without the neuroprosthesis, projected over 10 years. RESULTS: Bladder and bowel care costs were reduced by over 80%, from a median of 8152 dollars a year for conventional care to a median of 948 dollars a year. With the neuroprosthesis, median annual costs for bladder supplies were reduced from 3368 dollars to 58 dollars; for medications, from 1866 dollars to 108 dollars; for medical care, from 656 dollars to 96 dollars; and for bowel care supplies, from 205 dollars to 87 dollars. After 5 years, the cumulative costs of treatment with the neuroprosthesis, including the cost of the device and its implantation and maintenance, equaled those of conventional care. Thereafter, savings from the implanted neuroprosthesis are projected to increase progressively throughout the patient's life. CONCLUSION: A neuroprosthesis implant with posterior rhizotomy greatly reduces the cost of managing the neurogenic bladder and bowel.

Multiple vesiculoviral matrix proteins inhibit both nuclear export and import.

The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex.

Reduction of costs of disability using neuroprostheses.

The lifetime costs associated with spinal cord injury are substantial. Assistive technology that reduces complications, increases independence, or decreases the need for attendant services can provide economic as well as medical or functional benefit. This study describes two approaches for estimating the economic consequences of implanted neuroprostheses utilizing functional electrical stimulation. Life care plan analysis was used to estimate the costs of bladder and bowel care with and without a device restoring bladder and bowel function and to compare these with the costs of implementing the device. For a neuroprosthesis restoring hand grasp, the costs of implementation were compared to the potential savings in attendant care costs that could be achieved by the use of the device. The results indicate that the costs of implementing the bladder and bowel system would be recovered in 5 years, primarily from reduced costs of supplies, medications, and procedures. The costs of the hand grasp neuroprosthesis would be recovered over the lifetime of the user if attendant time was reduced only 2 hours per day and in a shorter time if attendant care was further reduced. Neither analysis includes valuation of the quality of life, which is further enhanced by the neuroprostheses through restoration of greater independence and dignity. Our results demonstrate that implantable neuroprosthetic systems provide good health care value in addition to improved independence for the disabled individual.

The matrix protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport when it is in the nucleus and associated with nuclear pore complexes.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor of bidirectional nuclear transport. Here we demonstrate that inhibition occurs when M protein is in the nucleus of Xenopus laevis oocytes and that M activity is readily reversed by a monoclonal antibody (alphaM). We identify a region of M protein, amino acids 51 to 59, that is required both for inhibition of transport and for efficient recognition by alphaM. When expressed in transfected HeLa cells, M protein colocalizes with nuclear pore complexes (NPCs) at the nuclear rim. Moreover, mutation of a single amino acid, methionine 51, eliminates both transport inhibition and targeting to NPCs. We propose that M protein inhibits bidirectional transport by interacting with a component of the NPC or an NPC-associated factor that participates in nucleocytoplasmic transport.

Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10(7) reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.