An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.

Perceptual information supports transfer of learning in coordinated rhythmic movement.

In this paper, we trained people to produce 90° mean relative phase using task-appropriate feedback and investigated whether and how that learning transfers to other coordinations. Past work has failed to find transfer of learning to other relative phases, only to symmetry partners (identical coordinations with reversed lead-lag relationships) and to other effector combinations. However, that research has all trained people using transformed visual feedback (visual metronomes, Lissajous feedback) which removes the relative motion information typically used to produce various coordinations (relative direction, relative position; Wilson and Bingham, in Percept Psychophys 70(3):465-476, 2008). Coordination feedback (Wilson et al., in J Exp Psychol Hum Percept Perform 36(6):1508, 2010) preserves that information and we have recently shown that relative position supports transfer of learning between unimanual and bimanual performance of 90° (Snapp-Childs et al., in Exp Brain Res 233(7), 2225-2238, 2015). Here, we ask whether that information can support the production of other relative phases. We found large, asymmetric transfer of learning bimanual 90° to bimanual 60° and 120°, supported by perceptual learning of relative position information at 90°. For learning to transfer, the two tasks must overlap in some critical way; this is additional evidence that this overlap must be informational. We discuss the results in the context of an ecological, task dynamical approach to understanding the nature of perception-action tasks.

The Effect of Parathyroid Hormone Analogues When Added to Mineralized Bone Xenografts.

Implants can be a treatment option when there is sufficient quantity and quality of bone to provide support for long-term success. In the reconstruction of defects, autogenous bone remains the gold standard for its osteogenic and compatibility properties. However, the disadvantage of secondary surgery and the associated donor site morbidity prompts researchers to develop the ideal bone substitute for optimum bone reconstruction. Parathyroid hormone (PTH1-34) has provided a new option for improvement in bone regeneration. This study used a pig model to evaluate the effectiveness of parathyroid hormone when added to a xenograft, Bio-Oss, in reconstructing mandible defects. Six domestic pigs were used to create 3 posterior mandibular defects measuring 2 × 1-cm bilaterally with a total of 36 defects to simulate tooth extraction sites in humans. The defects were grafted in random order and divided into 3 groups as follows: control (no graft), Bio-Oss without PTH, and Bio-Oss with PTH. Defects were assessed with cone beam computerized tomography (CBCT), micro computerized tomography (microCT), nanoindentation, and histology. Results showed that adding PTH1-34 significantly enhanced the graft construct. CBCT showed a significant increase in the degree of bone mineralization. Nanoindentation showed increased hardness of regenerated bone and accelerated bone mineralization with PTH. MicroCT analysis revealed a trend toward higher bone regeneration and mineralization. The histological analysis showed a positive trend of the increase in cortical bone thickness and mineral apposition rate. In conclusion, the local addition of PTH1-34 to a xenograft has shown promising results to enhance bone regeneration in the reconstruction of mandibular defects.

Treatment of edentulous mandible with metal-resin fixed complete dentures: A 15- to 20-year retrospective study.

OBJECTIVES: The purpose of this retrospective study was to report the implant and prosthetic complications of mandibular metal-resin fixed complete dentures (MRFCDs) opposing a maxillary complete removable dental prosthesis (CRDP) in a 15- to 20-year post-placement follow-up period. MATERIAL AND METHODS: Dental records of 24 edentulous patients treated by a mandibular MRFCD and a maxillary CRDP were reviewed. Complications for the implants, MRFCDs, and CRDPs were recorded in four different recall periods: 0-5 years, 5-10 years, 10-15 years, and more than 15 years. The survival and failure times based on Kaplan-Meier statistics were analyzed using Lifetest procedures. Product-limit survival estimates were used for cumulative survival rates (CSRs). RESULTS: The mean service time was 18.5 years. The CSR for the implants and MRFCDs was 91.8% at 16.9 years (confidence intervals: 85.2% and 95.5%) and 80% at 19.6 years (confidence intervals: 44.1% and 94.1%), respectively. The implant failures after 15 years occurred because of a tumor resection. Acrylic resin tooth fracture (45.8% of patients) and wear (75% of patients) were the most common complications with the MRFCD. Retaining screw complications [loosening (8.1% of retaining screws) and fracture (11.3% of retaining screws)] were also common. CONCLUSIONS: The outcomes seen with MRFCD over the long term were favorable. After placement of prostheses, 1 implant loss was observed potentially due to prosthetic/hygiene/periodontal factors. However, potential maintenance complications such as acrylic resin tooth fracture and/or wear and retaining screw loosening and fracture may occur in the long term when a similar design is used for the mandible.

Pain relief for outpatient hysteroscopy.

BACKGROUND: Hysteroscopy is increasingly performed in an outpatient setting. Pain is the primary reason for abandonment of procedure or incomplete assessment. There is no consensus upon routine use of analgesia during hysteroscopy. OBJECTIVES: To assess the effectiveness and safety of pharmacological interventions for pain relief in women undergoing outpatient hysteroscopy, compared with placebo, no treatment or other pharmacological therapies. SEARCH METHODS: In September 2016 we searched the Cochrane Gynaecology and Fertility (CGF) Trials Register, CENTRAL, MEDLINE, Embase, PsycINFO, CINAHL and two trials registers (ClinicalTrials.gov and WHO ICTRP), together with reference checking and contact with study authors and experts. SELECTION CRITERIA: We included randomised controlled trials (RCTs) comparing use of pharmacological interventions with other pharmacological interventions and pharmacological interventions versus placebo or no treatment. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. Our primary outcome was mean pain score. MAIN RESULTS: We included 32 RCTS (3304 participants), of which only 19 reported data suitable for analysis. Most studies were at unclear or high risk of bias in most of the domains assessed. The evidence was low or very low quality, mainly due to risk of bias and imprecision. Baseline pain scores were relatively low in all groups. Analgesic versus placebo or no treatment Local anaesthetics Local anaesthetics reduced mean pain scores during the procedure [(SMD) -0.29, 95% CI -0.39 to -0.19, 10 RCTs, 1496 women, I2 = 80%, low-quality evidence)] and within 30 minutes (SMD 0.50, 95% CI -0.67 to -0.33, 5 RCTs, 545 women, I2 = 43%, low-quality evidence). This translates to a difference of up to 7 mm on a 0-10 cm visual analogue scale (VAS) during the procedure and up to 13 mm within 30 minutes, which is unlikely to be clinically meaningful. There was no clear evidence of a difference between the groups in mean pain scores after > 30 minutes (SMD -0.11, 95% CI -0.30 to 0.07, 4 RCTs, 450 women, I2 = 0%, low-quality evidence), or in rates of vasovagal reactions (OR 0.70, 95% CI 0.43 to 1.13, 8 RCTs, 1309 women, I2 = 66%, very low-quality evidence). There was insufficient evidence to determine whether there was a difference in rates of non-pelvic pain (OR 1.76, 95% CI 0.53 to 5.80, 1 RCT, 99 women, very low-quality evidence). Nonsteroidal anti-inflammatory drugs (NSAIDs) There was insufficient evidence to determine whether there was a difference between the groups in mean pain scores during the procedure (SMD -0.18, 95% CI -0.35 to 0.00, 3 RCTs, 521 women, I2 = 81%, low-quality evidence). Pain scores were lower in the NSAIDs group within 30 minutes (SMD -0.25, 95% CI -0.46 to -0.04, 2 RCTs, 340 women, I2=29%, low-quality evidence) and at over 30 minutes (SMD -0.27, 95% CI -0.49 to -0.05, 2 RCTs, 321 women, I2 = 78%, low-quality evidence). This equates to maximum differences of under 7.5 mm on a 0-10 cm scale, which are unlikely to be clinically significant. One RCT (181 women) reported adverse events: there was insufficient evidence to determine whether there was a difference between the groups in vasovagal reactions (OR 0.76, 95% CI 0.20 to 2.94, very low-quality evidence). For other reported adverse events (non pelvic pain and allergic reactions) evidence was lacking. Opioids One RCT utilised sublingual buprenorphine and one utilised oral tramadol. Data on pain scores during the procedure were unsuitable for pooling due to inconsistency. Tramadol was associated with a benefit of up to 22 mm on a 0-10 cm scale (SMD -0.76, 95% CI -1.10 to -0.42, 1 RCT, 140 women). However, the effect estimate for this outcome for sublingual opioids did not support a benefit from the intervention (SMD 0.08, 95% CI -0.22 to 0.39, 164 women). Compared with placebo, the pain score within 30 minutes of the procedure was reduced in the tramadol group, with a difference of up to 17mm on a 0-10cm scale (SMD -0.57, 95% CI -0.91 to -0.23 , 1 RCT, 140 women, low-quality evidence. There was no clear evidence of a difference between the tramadol and placebo groups at over 30 minutes (SMD -0.17, 95% CI -0.51 to 0.16, 1 RCT, 140 women, low-quality evidence). Nausea and vomiting occurred in 39% of the buprenorphine group, and in none of the placebo group (OR 107.55, 95% CI 6.44 to 1796.46) Analgesic versus any other analgesic Some comparisons did not report pain scores at all time frames of interest, and none reported data on adverse events. One RCT (84 women) compared local intracervical anaesthesia versus combined intracervical and paracervical anaesthesia. Pain scores were higher in the group with local intracervical anaesthesia during the procedure (SMD 4.27, 95% CI 3.49 to 5.06, very low-quality evidence), within 30 minutes (SMD 1.55, 95% CI 1.06 to 2.05, very low-quality evidence) and at more than 30 minutes (SMD 3.47, 95% CI 2.78 to 4.15, very low-quality evidence). This translates to a possible benefit in the combined group of up to 12 mm on a 0-10 cm scale during the procedure. Benefits at longer follow-up were smaller. One RCT compared antispasmodic + NSAID versus local paracervical anaesthesia. Pain scores were lower in the NSAID group than in the local anaesthesia group (during procedure: SMD -1.40, 95% CI -1.90 to -0.91; >30 minutes after procedure: SMD -0.87, 95% CI -1.33 to -0.41; 80 women, very low-quality evidence). This suggests a possible benefit of during the procedure of up to 23 mm on a 0-10 VAS scale and up to 11 mm >30 minutes after the procedure. Other comparisons included local intracervical anaesthesia versus combined intracervical, paracervical and topical anaesthesia, and opioid versus NSAIDs. Findings were inconclusive. AUTHORS' CONCLUSIONS: There was no consistent good-quality evidence of a clinically meaningful difference in safety or effectiveness between different types of pain relief compared with each other or with placebo or no treatment in women undergoing outpatient hysteroscopy.

Reduced learning rates but successful learning of a coordinated rhythmic movement by older adults.

Previous work has investigated the information-based mechanism for learning and transfer of learning in coordinated rhythmic movements. In those papers, we trained young adults to produce either 90° or 60° and showed in both cases that learning entailed learning to use relative position as information for the relative phase. This variable then supported transfer of learning to untrained coordinations +/30° on either side. In this article, we replicate the 90° study with younger adults and extend it by training older adults (aged between 55 and 65 years). Other work has revealed a steep decline in learning rate around this age, and no follow-up study has been able to successfully train older adults to perform a novel coordination. We used a more intensive training paradigm and showed that while older adult learning rates remain about half that of younger adults, given time they are able to acquire the new coordination. They also learn to use relative position, and consequently show the same pattern of transfer. We discuss implications for attempts to model the process of learning in this task.

Pericardial Effusion: A Novel Presentation of Aplastic Anemia.

Pericardial effusion is defined as the accumulation of fluid between the visceral and parietal pericardium. The underlying etiology varies as any pathology that causes pericarditis or involves the pericardium can cause effusion. In practice, the majority of pericarditis cases are idiopathic, although these are assumed to be secondary to occult viral infection or inflammatory phenomena. Malignancy, particularly the metastatic spread of noncardiac primary tumors, has been implicated as a differential in the diagnosis of pericardial effusion. Though commonly seen in solid malignancies, effusion has been reported in hematologic malignancies such as myelodysplastic syndrome (MDS), acute leukemia, and lymphoma. Nonetheless, pericardial effusions associated with hematologic conditions are extremely rare with only one case report published describing pericardial effusion secondary to immune thrombocytopenia (ITP). We herein report the first documented case, to our knowledge, of pericardial effusion as an initial clinical manifestation of aplastic anemia in a middle-aged male presenting with pancytopenia.

Recurrent Subclavian Steal Syndrome: A Novel Case of Vasculopathy.

Subclavian steal syndrome (SSS) refers to the phenomenon of retrograde flow in an ipsilateral branch of the subclavian artery due to hemodynamically significant stenosis or occlusion of the ipsilateral proximal subclavian artery. While SSS is usually asymptomatic, it can manifest as vertebrobasilar insufficiency (VBI), ischemia of the affected extremity, or cardiac angina when an internal mammary artery (IMA) is used as a bypass graft. The underlying etiology is most often atherosclerosis but can include Takayasu arteritis, thoracic outlet syndrome, cervical rib, and stenosis secondary to surgical repair of aortic coarctation or tetralogy of Fallot. There are several case reports describing unique presentations of SSS as well as limited reports of double SSS, where the brachiocephalic steno-occlusive disease causes flow reversal in both the ipsilateral vertebral and carotid arteries. We report herein the first documented case, to our knowledge, of a patient with SSS previously treated with left subclavian artery stenting and left common carotid-subclavian bypass who developed recurrent SSS in conjunction with orthostatic cerebral hypoperfusion syndrome (OCHOS) secondary to severe vasculopathy. She presented with recurrent, paroxysmal vertigo and near-syncope associated with left upper extremity paresthesias that would only abate with sitting in the context of left subclavian artery stent restenosis and occlusion of her left common carotid-subclavian bypass graft. Interestingly, her initial presentation entailed retrograde flow from the left vertebral artery to the left subclavian artery, classic for SSS, but recurrence of her SSS involved retrograde flow from the left common carotid artery to the left subclavian artery, a phenomenon which has also not been described in the literature to our knowledge. As her symptoms of VBI appeared to be triggered by standing and not left arm movement, they were considered to be primarily secondary to OCHOS. Consequently, her primary treatment was to increase salt and fluid intake and thus increase intravascular volume for improved cerebral perfusion as she was not deemed to be a suitable candidate for regrafting of the left subclavian artery.

Radiation Treatment for Cancer of the Anal Margin.

OBJECTIVE: To identify best treatment practices by examining outcomes of anal margin cancer patients treated with radiotherapy. METHODS: Relevant literature was compared with 38 patients at our institution treated 1979 to 2019 with curative radiotherapy. Median age was 51. Four patients had T1, 22 had T2, and 12 had T3 disease based on the American Joint Committee on Cancer (AJCC) staging at time of diagnosis. Nodal staging distribution was: N0=33; N1=2; N2=2; N3=1. Median radiation dose was 56 Gy/30 fractions. Five received nodal radiation for node positivity, 29 received elective nodal radiation, and 29 had perineal boost. Twenty-seven received concurrent chemotherapy. RESULTS: Three patients experienced isolated local recurrence, 2 had isolated inguinal node recurrences, and 2 developed distant metastases, 1 of whom also had local and regional recurrence. Ten-year disease-free survival (DFS), cause-specific survival, and overall survival were 87%, 92%, and 68%, respectively. One patient did not complete radiation, and 4 had unexpected treatment breaks. Two received salvage abdominoperineal resections. At last follow-up, 17 were alive with no evidence of disease, 2 were alive with anal margin cancer present, 3 had died with anal margin cancer present at 11, 18, and 21 months from radiation therapy, and 16 had died from intercurrent disease. Median follow-up was 6.6 years (range 0.9 to 29.0 y). Age ≥51 was associated with worse locoregional control ( P =0.018) and DFS ( P =0.0233), males had worse DFS ( P =0.0311), and HIV-positive patients had worse overall survival ( P =0.006). CONCLUSIONS: Radiation provides high locoregional control of anal margin cancer with good long-term outcomes.

Impact of genotype on clinical course in sickle cell disease and the utility of neutrophil-lymphocyte ratio: a ten-year single-institution experience.

BACKGROUND: Sickle cell disease (SCD) is a diverse group of blood disorders with significant global disease burden. Contemporary interest in the underlying inflammatory paradigm of SCD has emphasized the role of the neutrophil-lymphocyte ratio (NLR) as a prognostic inflammatory marker. METHODS: We retrospectively reviewed 268 hospitalized patients with SCDs of different genotypes (HbSS, HbSß0 thalassemia, HbSß+ thalassemia, and HbSC), totaling 3329 hospital admissions over a 10-year period. Patients were stratified into SS/Sß0 and Sß+/SC groups for statistical analysis of parameters collected at steady state and at hospital admission. RESULTS: At steady state, per unit increase of hemoglobin values was associated with reduced odds of ≥ 2 hospital admissions per year in SS/Sß0 and Sß+/SC groups; per unit increase in platelet count and white blood cell count was associated with increased odds only in the SS/Sß0 group. The NLR had no association in either group. During admission, a cutoff of NLR = 3.5 discerned infection with a sensitivity of 60% and specificity of 57%. Performance improved when excluding patients on outpatient hydroxyurea therapy (cutoff of NLR = 3.5; sensitivity of 68% and specificity of 64%). CONCLUSION: This study supports the utility of NLR as an accessible adjunctive clinical tool in SCD prognostication.

Importance of carbohydrate antigen (CA 19-9) and carcinoembrionic antigen (CEA) in the prognosis of patients with duodenal adenocarcinoma: a retrospective single-institution cohort study.

BACKGROUND: Duodenal adenocarcinoma (DA) is a rare malignancy without validated tumor markers. In practice, carcinoembryonic antigen (CEA) and carbohydrate antigen (CA 19-9) are often used in the management of DA, though their prognostic value is unknown. MATERIALS AND METHODS: A single-institution retrospective review included patients diagnosed with biopsy-confirmed adenocarcinoma of the duodenum between 2006 and 2021. Peri-ampullary tumors were excluded. Levels of CA 19-9 and CEA were collected as continuous variables and were analyzed as binary variables: normal vs. high, using the maximum normal value as a cut-off (normal Ca 19-9 <35 U/ml; CEA <3 ng/ml). Survival analysis was conducted using Kaplan Meier curves, log-rank test and Cox proportional hazards model. RESULTS: There were 68 patients included in the final analysis. Median age was 67 years old and median follow-up time was 22.2 months. CA 19-9 and CEA were elevated in 36.8% and 48.5% of patients, respectively. A concomitant elevation of both tumor markers was associated with worsened OS (HR 2.140, 95% CI: 1.114-4.112; p = 0.019). After controlling for age and sex on multivariate analysis, elevation in both CA 19-9 ≥35 and CEA ≥3.0 remained significantly associated with increased mortality (HR 2.278, 95% CI: 1.162-4.466; p = 0.016). CONCLUSIONS: In summary, CA 19-9 and, to a lesser extent, CEA, show promise as prognostic markers in DA. Larger studies are needed to validate their use and to evaluate their performance as markers of recurrence.

A 26-Year-Old Woman With Retinal Telangiectasias, Onychodystrophy, and Persistent Dyspnea.

CASE PRESENTATION: A 26-year-old woman with no significant past medical history sought treatment for worsening dyspnea and hypoxia. The exertional dyspnea began 2 years prior and was associated with substernal chest discomfort. She did not report myalgia, edema, or worsening of dyspnea on supine or upright position. The patient reported no personal history of tobacco or illicit drug use. Family history was unremarkable. She was started on supplemental oxygen at 3 L/min. Initial workup included CT scan angiography of the chest, which showed no pulmonary embolism and normal lung parenchyma. Transthoracic echocardiography showed unremarkable results. She was not given a clear diagnosis for hypoxia and was treated empirically with antibiotics and bronchodilators without improvement. Over the course of 2 years, her condition progressed to requiring 6 L/min nasal canula at rest and associated dyspnea with minimal exertion and a 30-pound unintentional weight loss. During this time, pulmonary function tests noted normal spirometry results and lung volumes, but a decreased diffusing capacity for carbon monoxide of 33%. She also was discovered incidentally to be leukopenic and thrombocytopenic, with subsequent bone marrow biopsy revealing hypocellularity of 30% to 40%. The patient concurrently demonstrated bilateral visual impairment secondary to retinal telangiectasias with increased severity of deficit in the right eye. She subsequently was referred to our institution for further evaluation.

The Ecological Task Dynamics of Learning and Transfer in Coordinated Rhythmic Movement.

Research spanning 100 years has revealed that learning a novel perception-action task is remarkably task-specific. With only a few exceptions, transfer is typically very small, even with seemingly small changes to the task. This fact has remained surprising given previous attempts to formalise the notion of what a task is, which have been dominated by common-sense divisions of tasks into parts. This article lays out an ecologically grounded alternative, ecological task dynamics, which provides us with tools to formally define tasks as experience from the first-person perspective of the learner. We explain this approach using data from a learning and transfer experiment using bimanual coordinated rhythmic movement as the task, and acquiring a novel coordination as the goal of learning. 10 participants were extensively trained to perform 60° mean relative phase; this learning transferred to 30° and 90°, against predictions derived from our previous work. We use recent developments in the formal model of the task to guide interpretation of the learning and transfer results.

Improving the care of children with urinary tract infection: use of a clinical decision proforma.

Urinary tract infection (UTI) is a common and important clinical problem in children. Follow up imaging is indicated in some cases to reduce the risk of long-term harm from UTI and sometimes to help guide acute management. Overuse of imaging may be harmful due to radiation exposure, as well as increasing demand on services and budgets. On the other hand under-use of imaging may leave children vulnerable to renal damage and long-term morbidity. Accepted standards propose an imaging strategy specific to age and type of UTI. The complexity of the guideline makes compliance with the standards challenging. The aim of this project was to assess current practice for imaging of children with UTI managed at The Royal Oldham Hospital and to improve compliance with accepted standards through the use of a proforma to aid clinical decision making, supported by an education programme. A retrospective audit was performed over a 6 month period both prior to and after the intervention. The baseline audit found 57.7% of children treated for UTI (n=26) had imaging compliant with the accepted standards, which improved to 75.9% (n=29) on post-measurement. The percentage of inappropriate investigations reduced from 52.4% to 10.5%. The percentage of missed investigations reduced from 35.0% to 32.0%. The proforma was used and filed in 40% of cases where practice was in line with accepted standards. It was not used in any of the cases where practice deviated. In conclusion, a clear clinical decision aid, supported by an education programme, can significantly improve compliance with accepted standards for imaging of children with UTI. This may also be transferable to other scenarios where guidelines exist but have reduced efficacy due to complexity and/or lack of understanding.

Automating bioanalytical sample analysis through enhanced system integration.

BACKGROUND: In the bioanalytical laboratory, challenges associated with manual, repetitive, labor-intensive processes can be addressed by powerful automated liquid handlers; however, their implementation has been difficult due to lack of efficient integration into laboratory workflows. Faster throughput is afforded to ligand binding assay (LBA) technologies via enhanced automation, but the upstream sample processing still remains a bottleneck. To be truly efficient, these technologies must be incorporated into a laboratory information management system (LIMS) to streamline data analysis and reporting. RESULTS: Three off-the-shelf technologies that aid in improving bioanalytical laboratory efficiencies were utilized with minimal customization to streamline the sample analysis process. Information extracted via a sequence file from the LIMS run was utilized to perform the sample processing on the automated liquid handler. A file conversion tool converted the sequence files that allowed for sample processing and preparing the assay ready plate. The plate was then transferred to the LBA microfluidics platform to run the experiments. The integration was tested using a LBA PK assay that demonstrated good sample dilution and assay performance. CONCLUSION: We successfully integrated LIMS with an automated liquid handler and a microfluidics platform to automate the sample analysis process in the bioanalytical laboratory. The utilization of off-the-shelf technologies with minimal customization requires minimal resources from laboratory scientists. It may be possible to implement this approach for other analytical platforms.

Characterization of intestinal dendritic cells in murine norovirus infection.

We have shown that respiratory viral infections drive allergic disease through dendritic cells, whether gastrointestinal viruses induce allergies is not known. Norovirus infections are a major cause of gastroenteritis in humans. We used murine norovirus (MNV) to explore the effect of MNV infection on gastrointestinal conventional DCs (cDCs) and plasmacytoid DCs (pDCs). MNV infection induced disparate effects on cDCs and pDCs in lymphoid tissues of the small intestine and draining mesenteric lymph nodes. FcεRI was transiently expressed on lamina propria cDCs, but not on pDCs. In addition, feeding ovalbumin during the viral infection led to a modest, brief induction of anti-ovalbumin IgE. Together, these data suggest that like with a respiratory viral infection, an intestinal viral infection may be sufficient to induce changes in DCs and the generation of food-specific IgE. Whether this represents a novel mechanism of food allergy remains to be determined.

20-hydroxyeicosatetraenoic acid is a vasoconstrictor in the newborn piglet pulmonary microcirculation.

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome p-450 metabolite of arachidonic acid, is a vasoconstrictor in the systemic circulation and a vasodilator in the adult pulmonary circulation. Little is known about the vasoactive properties of 20-HETE in the newborn pulmonary circulation. The objectives of this study were to determine the vascular effects of 20-HETE and to explore the signaling mechanism(s) that mediate these effects in newborn pulmonary resistance-level arteries (PRA). Our findings demonstrate that, in contrast to the adult pulmonary circulation where 20-HETE mediates vasodilation, it causes constriction in newborn PRA at resting tone. Furthermore, inhibition of cyclooxygenase (COX) with indomethacin augments 20-HETE-induced constriction. The enhanced constrictor response to 20-HETE under conditions of COX inhibition is abolished in endothelium-disrupted PRA, suggesting that 20-HETE either stimulates endothelium-derived COX to release a counteracting vasodilator or is rapidly metabolized by COX to a less potent vasoconstrictor. 20-HETE-induced constriction is significantly inhibited by blocking calcium-dependent K(+) (K(Ca)) channels and the thromboxane-PGH(2) receptor. Altogether, our data indicate that the vascular actions of 20-HETE are partially mediated via the activation of K(Ca) channels and are significantly modulated by interactions with the COX-prostaglandin pathway.