Point-of-care vertical flow immunoassay system for ultra-sensitive multiplex biothreat-agent detection in biological fluids.

This paper presents simple, fast, and sensitive detection of multiple biothreat agents by paper-based vertical flow colorimetric sandwich immunoassay for detection of Yersinia pestis (LcrV and F1) and Francisella tularensis (lipopolysaccharide; LPS) antigens using a vertical flow immunoassay (VFI) prototype with portable syringe pump and a new membrane holder. The capture antibody (cAb) printing onto nitrocellulose membrane and gold-labelled detection antibody (dAb) were optimized to enhance the assay sensitivity and specificity. Even though the paper pore size was relaxed from previous 0.1 µm to the current 0.45 µm for serum samples, detection limits as low as 0.050 ng/mL for LcrV and F1, and 0.100 ng/mL for FtLPS have been achieved in buffer and similarly in diluted serum (with LcrV and F1 LODs remained the same and LPS LOD reduced to 0.250 ng/mL). These were 40, 80, and 50X (20X for LPS in serum) better than those from lateral flow configuration. Furthermore, the comparison of multiplex format demonstrated low cross-reactivity and equal sensitivity to that of the singleplex assay. The optimized VFI platform thus provides a portable and rapid on-site monitoring system for multiplex biothreat detection with the potential for high sensitivity, specificity, reproducibility, and multiplexing capability, supporting its utility in remote and resource-limited settings.

A Review of Brooke Army Medical Center Chaplaincy Service During the SARS-COV2 Pandemic: Implications for Service Structure and Patient Needs.

INTRODUCTION: We aimed to evaluate the effect of the SARS-COV2 pandemic on chaplain utilization at Brooke Army Medical Center. Our hypothesis was that multiple pandemic-related factors led to a care environment with increased mental and spiritual stress for patients and their families, leading to an increased need for adjunct services such as chaplaincy. MATERIALS AND METHODS: This was a single-institution retrospective chart review study that evaluated the records of 10,698 patients admitted between July 1, 2019, and January 31, 2020, or between July 1, 2020, and January 31, 2021. Our primary study outcomes included the number of chaplain consultations, the number of visits per consultation, and the time of visits between the two study cohorts. Secondary outcomes included inpatient mortality and the number of end-of-life visits. We also isolated a subgroup of patients admitted with COVID-19 and compared their outcomes with the two larger cohorts. Statistical analysis included t-test or chi-squared test, based on the variable. This study was reviewed and approved by the Brooke Army Medical Center Institutional Review Board (IRB ID C.2021.010e). RESULTS: Fewer consults were performed during the study period affected by the SARS-COV2 pandemic (4814 vs. 5884, P-value <.01). There were fewer individual visits per consult during the study period affected by the SARS-COV2 pandemic (1.44 vs. 1.64, P-value <.01), which led to fewer overall time spent per consult (37.41 vs. 41.19 minutes, P-value <.01). The 2020 cohort (without COVID-19 cases) demonstrated a higher mortality rate than the 2019 cohort (2.8% vs. 1.9%, P-value <.01). The COVID-19 diagnosis cohort demonstrated a much higher mortality rate compared to other patients in the 2020 cohort (19.3% vs. 2.8%, P-value <.01). We demonstrated the relative need for EOL consults by presenting the ratio of EOL consults to inpatient deaths. This ratio was highest for the COVID-19 diagnosis cohort (0.76) compared to the 2020 cohort (0.50) and the 2019 cohort (0.60). CONCLUSIONS: This study demonstrates that factors related to the SARS-COV2 pandemic resulted in fewer chaplaincy consults in our inpatient setting. We did not find other reports of a change in the rate of chaplaincy consultation, but available reports suggest that many centers have had difficulty balancing the spiritual needs of patients with local exposure guidelines. Although fewer individual chaplain consults occurred during the SARS-COV2 pandemic, our chaplain service innovated by utilizing various phone, video, and web-based platforms to deliver spiritual support to our community. Our study also suggests that the patients most greatly affected by the pandemic have an increased need for spiritual support, especially at the end of life. Future studies in this subject should examine the effect of various types of chaplain services as they relate to the health and well-being of hospitalized patients.

The SSLepR mutant rat represents a novel model to study obesity-induced renal injury before puberty.

Prepubertal obesity (PPO) has emerged as a major health problem over the past few decades and is a risk factor for the development of proteinuria. The current study investigated whether the development of renal injury in the obese SSLepR mutant strain occurs before puberty. When determining the temporal changes in serum sex hormones in female and male SS and SSLepR mutant rats between 4 and 10 wk of age, we only observed significant increases in estradiol and testosterone levels in female and male SS rats at 10 wk of age than at 4 wk of age. The results suggest that studying both strains between 4 and 8 wk of age is appropriate to study the effects of PPO on renal injury in this model. Proteinuria was significantly higher in SSLepR mutant rats as opposed to the values observed in SS rats at 8 wk of age, and we did not observe any sex differences in proteinuria in either strain. The kidneys from the SSLepR mutant rats displayed significant glomerular and tubular injury and renal fibrosis versus the values measured in SS rats without any sex differences. Overall, we observed increased immune cell infiltration in the kidneys from SSLepR mutant rats compared with SS rats. Interestingly, female SSLepR mutant rats displayed significant increases in not only M1 macrophages (proinflammatory) but also M2 macrophages (anti-inflammatory) versus male SSLepR mutant rats. These results suggest the SSLepR mutant rat may be a useful model to study early progression of obesity-related renal injury before the onset of puberty.

Magnetic particle imaging: tracer development and the biomedical applications of a radiation-free, sensitive, and quantitative imaging modality.

Magnetic particle imaging (MPI) is an emerging tracer-based modality that enables real-time three-dimensional imaging of the non-linear magnetisation produced by superparamagnetic iron oxide nanoparticles (SPIONs), in the presence of an external oscillating magnetic field. As a technique, it produces highly sensitive radiation-free tomographic images with absolute quantitation. Coupled with a high contrast, as well as zero signal attenuation at-depth, there are essentially no limitations to where that can be imaged within the body. These characteristics enable various biomedical applications of clinical interest. In the opening sections of this review, the principles of image generation are introduced, along with a detailed comparison of the fundamental properties of this technique with other common imaging modalities. The main feature is a presentation on the up-to-date literature for the development of SPIONs tailored for improved imaging performance, and developments in the current and promising biomedical applications of this emerging technique, with a specific focus on theranostics, cell tracking and perfusion imaging. Finally, we will discuss recent progress in the clinical translation of MPI. As signal detection in MPI is almost entirely dependent on the properties of the SPION employed, this work emphasises the importance of tailoring the synthetic process to produce SPIONs demonstrating specific properties and how this impacts imaging in particular applications and MPI's overall performance.

Luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, reverses cerebrovascular dysfunction and cognitive impairments in 18-mo-old diabetic animals.

Diabetes mellitus (DM) is a leading risk factor for age-related dementia, but the mechanisms involved are not well understood. We previously discovered that hyperglycemia induced impaired myogenic response (MR) and cerebral blood flow (CBF) autoregulation in 18-mo-old DM rats associated with blood-brain barrier (BBB) leakage, impaired neurovascular coupling, and cognitive impairment. In the present study, we examined whether reducing plasma glucose with a sodium-glucose cotransporter-2 inhibitor (SGLT2i) luseogliflozin can ameliorate cerebral vascular and cognitive function in diabetic rats. Plasma glucose and HbA1c levels of 18-mo-old DM rats were reduced, and blood pressure was not altered after treatment with luseogliflozin. SGLT2i treatment restored the impaired MR of middle cerebral arteries (MCAs) and parenchymal arterioles and surface and deep cortical CBF autoregulation in DM rats. Luseogliflozin treatment also rescued neurovascular uncoupling, reduced BBB leakage and cognitive deficits in DM rats. However, SGLT2i did not have direct constrictive effects on vascular smooth muscle cells and MCAs isolated from normal rats, although it decreased reactive oxygen species production in cerebral vessels of DM rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.NEW & NOTEWORTHY This study demonstrates that luseogliflozin, a sodium-glucose cotransporter-2 inhibitor, improved CBF autoregulation in association with reduced vascular oxidative stress and AGEs production in the cerebrovasculature of 18-mo-old DM rats. SGLT2i also prevented BBB leakage, impaired functional hyperemia, neurodegeneration, and cognitive impairment seen in DM rats. Luseogliflozin did not have direct constrictive effects on VSMCs and MCAs isolated from normal rats. These results provide evidence that normalization of hyperglycemia with an SGLT2i can reverse cerebrovascular dysfunction and cognitive impairments in rats with long-standing hyperglycemia, possibly by ameliorating oxidative stress-caused vascular damage.

A Sensitive, Portable Microfluidic Device for SARS-CoV-2 Detection from Self-Collected Saliva.

Since the outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in December 2019, the spread of SARS-CoV2 infection has been escalating rapidly around the world. In order to provide more timely access to medical intervention, including diagnostic tests and medical treatment, the FDA authorized multiple test protocols for diagnostic tests from nasopharyngeal swab, saliva, urine, bronchoalveolar lavage and fecal samples. The traditional diagnostic tests for this novel coronavirus 2019 require standard processes of viral RNA isolation, reverse transcription of RNA to cDNA, then real-time quantitative PCR with the RNA templates extracted from the patient samples. Recently, many reports have demonstrated a direct detection of SARS-Co-V2 genomic material from saliva samples without any RNA isolation step. To make the rapid detection of SARS-Co-V2 infection more accessible, a point-of-care type device was developed for SARS-CoV-2 detection. Herein, we report a portable microfluidic-based integrated detection-analysis system for SARS-CoV-2 nucleic acids detection directly from saliva samples. The saliva cartridge is self-contained and capable of microfluidic evaluation of saliva, from heating, mixing with the primers to multiplex real-time quantitative polymerase chain reaction, detecting SARS-CoV-2 with different primer sets and internal control. The approach has a detection sensitivity of 1000 copies/mL of SARS-CoV-2 RNA or virus, with consistency and automation, from saliva sample-in to result-out.

Critical Comparison between Large and Mini Vertical Flow Immunoassay Platforms for Yersinia Pestis Detection.

Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.

The Use of Telemedicine for Stabilization of Neonates Transferred from Rural Community Hospitals.

Background: For newborns requiring transfer to a higher level of care, stabilization before the arrival of the transport team is essential. Telemedicine consultations with a neonatologist may improve local providers' ability to stabilize a newborn during this critical interval. The purpose of this study was to describe the use of telemedicine for stabilizing newborns who were transferred from one of six rural hospitals to a regional neonatal intensive care unit in northern California and to examine the association between telemedicine use and time needed to stabilize the newborn. Materials and Methods: We collected data on all newborns who were transferred after either a telemedicine or telephone consultation with a neonatologist between April 2014 and June 2018. We used multiple regression to examine the association between the use of telemedicine and stabilization time, adjusting for gestational age, 5-min Apgar score, birth weight, site, and primary reason for consultation. Results: In total, 162 infants (77.5%) received a telephone consultation and 47 (22.5%) received a telemedicine consultation. Neonates who received telemedicine had a significantly greater severity of illness, as measured by mean 5-min Apgar score (6.9 vs. 7.8, p = 0.008) and Transport Risk Index of Physiologic Stability version II (TRIPS-II) score (14.4 vs. 6.0, p < 0.001). There was no significant difference in stabilization time for telemedicine consultations compared with telephone consultations in the adjusted analysis (adjusted mean difference: -1.80, 95% confidence interval: -16.0 to 12.4, p = 0.802). Conclusions: Although we found no difference in stabilization times between modes of consultation, telemedicine may be helpful for stabilizing infants with a higher severity of illness, particularly those in respiratory distress. Future studies should examine the impact of telemedicine on specific interventions.

Increased Levels of Renal Lysophosphatidic Acid in Rodent Models with Renal Disease.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that has been implicated in the pathophysiology of kidney disease. However, few studies have attempted to measure changes in the levels of various LPA species in the kidney after the development of renal disease. The present study measured the renal LPA levels during the development of kidney disease in rat models of hypertension, diabetes, and obstructive nephropathy using liquid chromatography/mass spectrometry/mass spectrometry. LPA levels (sum of 16:0, 18:0, 18:1, 18:2, and 20:4 LPA) were higher in the renal cortex of hypertensive Dahl salt-sensitive (Dahl S) rats fed a high-salt diet than those in normotensive rats fed a low-salt diet (296.6 ± 22.9 vs. 196.3 ± 8.5 nmol/g protein). LPA levels were elevated in the outer medulla of the kidney of streptozotocin-induced type 1 diabetic Dahl S rats compared with control rats (624.6 ± 129.5 vs. 318.8 ± 17.1 nmol/g protein). LPA levels were also higher in the renal cortex of 18-month-old, type 2 diabetic nephropathy (T2DN) rats with more severe renal injury than in 6-month-old T2DN rats (184.9 ± 20.9 vs. 116.9 ± 6.0 nmol/g protein). LPA levels also paralleled the progression of renal fibrosis in the renal cortex of Sprague-Dawley rats after unilateral ureteral obstruction (UUO). Administration of an LPA receptor antagonist, Ki16425, reduced the degree of renal fibrosis in UUO rats. These results suggest that the production of renal LPA increases during the development of renal injury and contributes to renal fibrosis. SIGNIFICANCE STATEMENT: The present study reveals that the lysophosphatidic acid (LPA) levels increase in the kidney in rat models of hypertension, diabetes, and obstructive nephropathy, and administration of an LPA receptor antagonist attenuates renal fibrosis. Therapeutic approaches that target the formation or actions of renal LPA might be renoprotective and have therapeutic potential.

Human Adipose Derived Cells in Two- and Three-Dimensional Cultures: Functional Validation of an In Vitro Fat Construct.

Obesity, defined as a body mass index of 30 kg/m2 or above, has increased considerably in incidence and frequency within the United States and globally. Associated comorbidities including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, and nonalcoholic fatty liver disease have led to a focus on the mechanisms promoting the prevention and treatment of obesity. Commonly utilized in vitro models employ human or mouse preadipocyte cell lines in a 2-dimensional (2D) format. Due to the structural, biochemical, and biological limitations of these models, increased attention has been placed on "organ on a chip" technologies for a 3-dimensional (3D) culture. Herein, we describe a method employing cryopreserved primary human stromal vascular fraction (SVF) cells and a human blood product-derived biological scaffold to create a 3D adipose depot in vitro. The "fat-on-chip" 3D cultures have been validated relative to 2D cultures based on proliferation, flow cytometry, adipogenic differentiation, confocal microscopy/immunofluorescence, and functional assays (adipokine secretion, glucose uptake, and lipolysis). Thus, the in vitro culture system demonstrates the critical characteristics required for a humanized 3D white adipose tissue (WAT) model.

Quartz-Helium Method to Estimate Fluid Flow in Thick Aquitards, Gunnedah Basin, Australia.

The hydraulic integrity of aquitards is generally assumed and relies on a few core-scale permeability measurements, drill-stem tests, or textbook values. This approach is because hydraulic data across the full aquitard thickness is generally lacking. Proper assessment of aquitard integrity should be studied at the formation (spanning its entire thickness at a single point) or regional (formation properties at multiple locations throughout the basin) scale. One formation-scale approach uses environmental tracers and advection-dispersion modeling to constrain fluid flow rates. This study demonstrates the use of helium concentrations in quartz as a method of constraining the rate of fluid flow in a 520-m thick aquitard in the Gunnedah Basin, NSW, Australia. Quartz was separated from existing core samples in the Watermark and Porcupine Formations at depths from 750 to 1200 m. The helium was released from these samples by heating and select samples were impregnated with helium to determine the rate of helium diffusion through the quartz. One-dimensional advection-dispersion modeling of the helium profile accounting for diffusive helium exchange between quartz and pore water revealed, that (1) vertical fluid velocity has been on the order of 0.02 mm/year or less for tens to thousands of years, (2) helium is in equilibrium between quartz and pore water, and (3) the helium profile is transient indicating that helium concentrations in the underlying Maules Creek Formation has varied over geological time. Further modeling identified aquitard conditions (thickness and temperature) for which equilibrium exists, a precondition for deriving formation-scale permeability.

Bone Marrow Adipocyte Developmental Origin and Biology.

PURPOSE OF REVIEW: This review explores how the relationships between bone marrow adipose tissue (BMAT) adipogenesis with advancing age, obesity, and/or bone diseases (osteopenia or osteoporosis) contribute to mechanisms underlying musculoskeletal pathophysiology. RECENT FINDINGS: Recent studies have re-defined adipose tissue as a dynamic, vital organ with functions extending beyond its historic identity restricted solely to that of an energy reservoir or sink. "State of the art" methodologies provide novel insights into the developmental origin, physiology, and function of different adipose tissue depots. These include genetic tracking of adipose progenitors, viral vectors application, and sophisticated non-invasive imaging modalities. While constricted within the rigid bone cavity, BMAT vigorously contributes to local and systemic metabolic processes including hematopoiesis, osteogenesis, and energy metabolism and undergoes dynamic changes as a function of age, diet, bone topography, or sex. These insights will impact future research and therapies relating to osteoporosis.

An Analysis of Oncotype DX Recurrence Scores and Clinicopathologic Characteristics in Invasive Lobular Breast Cancer.

The Oncotype DX breast cancer assay (Genomic Health, Redwood City, CA) is increasingly being used to guide treatment decisions for patients with early stage, hormone-positive, Her-2-negative breast cancer. The utility of the Oncotype DX in decision making for treatment of invasive lobular carcinoma (ILC) has not been investigated as the results reported by Genomic Health are largely in a population with invasive ductal carcinoma (IDC). The authors hypothesized that the Oncotype DX recurrence score (RS) distribution for ILC is different than that for IDC. We performed a retrospective analysis of early stage breast cancer patients treated at Penn State Cancer Institute from 2001 to 2011 and identified 102 patients with ILC. We also pulled RS data from our institution's prospective registry of consecutive patients with early stage IDC treated during the same time period. Median follow-up was 55 months. We found that the RS distribution for ILC differed significantly from that of IDC (p = 0.024). We also found a statistically significant difference in the RS distribution between the pure ILC and pleomorphic ILC subtypes (p = 0.027). The Oncotype DX RS distribution in ILC is unique, differing significantly from that in ductal carcinoma. Consequently, the clinical usefulness and cost-effectiveness of the Oncotype DX in guiding treatment for ILC should be further investigated.

Identification of processing elements and interactors implicate SMN, coilin and the pseudogene-encoded coilp1 in telomerase and box C/D scaRNP biogenesis.

Many cellular functions, such as translation, require ribonucleoproteins (RNPs). The biogenesis of RNPs is a multi-step process that, depending on the RNP, can take place in many cellular compartments. Here we examine 2 different RNPs: telomerase and small Cajal body-specific RNPs (scaRNPs). Both of these RNPs are enriched in the Cajal body (CB), which is a subnuclear domain that also has high concentrations of another RNP, small nuclear RNPs (snRNPs). SnRNPs are essential components of the spliceosome, and scaRNPs modify the snRNA component of the snRNP. The CB contains many proteins, including WRAP53, SMN and coilin, the CB marker protein. We show here that coilin, SMN and coilp1, a newly identified protein encoded by a pseudogene in human, associate with telomerase RNA and a subset of scaRNAs. We also have identified a processing element within box C/D scaRNA. Our findings thus further strengthen the connection between the CB proteins coilin and SMN in the biogenesis of telomeras e and box C/D scaRNPs, and reveal a new player, coilp1, that likely participates in this process.

Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets.

Dominant plant taxa predict plant productivity responses to CO2 enrichment across precipitation and soil gradients.

The Earth's atmosphere will continue to be enriched with carbon dioxide (CO2) over the coming century. Carbon dioxide enrichment often reduces leaf transpiration, which in water-limited ecosystems may increase soil water content, change species abundances and increase the productivity of plant communities. The effect of increased soil water on community productivity and community change may be greater in ecosystems with lower precipitation, or on coarser-textured soils, but responses are likely absent in deserts. We tested correlations among yearly increases in soil water content, community change and community plant productivity responses to CO2 enrichment in experiments in a mesic grassland with fine- to coarse-textured soils, a semi-arid grassland and a xeric shrubland. We found no correlation between CO2-caused changes in soil water content and changes in biomass of dominant plant taxa or total community aboveground biomass in either grassland type or on any soil in the mesic grassland (P > 0.60). Instead, increases in dominant taxa biomass explained up to 85 % of the increases in total community biomass under CO2 enrichment. The effect of community change on community productivity was stronger in the semi-arid grassland than in the mesic grassland, where community biomass change on one soil was not correlated with the change in either the soil water content or the dominant taxa. No sustained increases in soil water content or community productivity and no change in dominant plant taxa occurred in the xeric shrubland. Thus, community change was a crucial driver of community productivity responses to CO2 enrichment in the grasslands, but effects of soil water change on productivity were not evident in yearly responses to CO2 enrichment. Future research is necessary to isolate and clarify the mechanisms controlling the temporal and spatial variations in the linkages among soil water, community change and plant productivity responses to CO2 enrichment.

Zinc-finger nuclease knockout of dual-specificity protein phosphatase-5 enhances the myogenic response and autoregulation of cerebral blood flow in FHH.1BN rats.

We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.

A miniature quantitative PCR device for directly monitoring a sample processing on a microfluidic rapid DNA system.

We report a microfluidic device and measurement method to perform real-time PCR (or qPCR) in a miniaturized configuration for on-chip implementation using reaction volumes of less than 20 µL. The qPCR bioreactor is designed as a module to be embedded in an automated sample-in/profile-out system for rapid DNA biometrics or human identification. The PCR mixture is excited with a 505 nm diode-pumped solid-state laser (DPSSL) and the fluorescence build-up is measured using optical fibers directly embedded to the sidewalls of the microfluidic qPCR bioreactor. We discuss manufacturing and operating parameters necessary to adjust the internal surface conditions and temperature profiles of the bioreactor and to optimize the yield and quality of the PCR reaction for the amplification of 62 bp hTERT intron fragments using the commercial Quantifiler® kit (Life Technologies, Carlsbad, CA) commonly accepted for genotyping analysis. We designed a microfluidic device suitable for continuously processing a specimen by efficiently mixing the reagents from the kit to a set volume of DNA template on chip. Our approach relies on a calibration curve for the specific device using control DNA. We successfully applied this method to determine the concentration of genomic DNA extracted from a buccal swab on separate microfluidic devices which are operated upstream the qPCR device and perform buccal swab lysis and buccal DNA extraction. A precise correlation between the amount determined on chip and that obtained using a commercial cycler is demonstrated.

Urinary CYP eicosanoid excretion correlates with glomerular filtration in African-Americans with chronic kidney disease.

Previous studies have indicated that cytochrome P450 (CYP) metabolites of arachidonic acid (AA), i.e., 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), play an important role in the regulation of renal tubular and vascular function. The present study for the first time profiled HETEs and epoxygenase derived dihydroxyeicosatetraenoic acid diHETEs levels in spot urines and plasma in 262 African American patients from the University of Mississippi Chronic Kidney Disease Clinic and 31 African American controls. Significant correlations in eGFR and urinary 20-HETE/creatinine and 19-HETE/creatinine levels were observed. The eGFR increased by 17.47 [p=0.001] and 60.68 [(p=0.005]ml/min/for each ng/mg increase in 20-HETE and 19-HETE levels, respectively. Similar significant positive associations were found between the other urinary eicosanoids and eGFR and also with 19-HETE/urine creatinine concentration and proteinuria. We found that approximately 80% of plasma HETEs and 30% diHETEs were glucuronidated and the fractional excretion of 20-HETE was less than 1%. These results suggest that there is a significant hepatic source of urinary 20-HETE glucuronide and EETs with extensive renal biotransformation to metabolites which may play a role in the pathogenesis of CKD.

Does a decade of elevated [CO2] affect a desert perennial plant community?

Understanding the effects of elevated [CO2 ] on plant community structure is crucial to predicting ecosystem responses to global change. Early predictions suggested that productivity in deserts would increase via enhanced water-use efficiency under elevated [CO2], but the response of intact arid plant communities to elevated [CO2 ] is largely unknown. We measured changes in perennial plant community characteristics (cover, species richness and diversity) after 10 yr of elevated [CO2] exposure in an intact Mojave Desert community at the Nevada Desert Free-Air CO2 Enrichment (FACE) Facility. Contrary to expectations, total cover, species richness, and diversity were not affected by elevated [CO2]. Over the course of the experiment, elevated [CO2] had no effect on changes in cover of the evergreen C3 shrub, Larrea tridentata; alleviated decreases in cover of the C4 bunchgrass, Pleuraphis rigida; and slightly reduced the cover of C3 drought-deciduous shrubs. Thus, we generally found no effect of elevated [CO2] on plant communities in this arid ecosystem. Extended drought, slow plant growth rates, and highly episodic germination and recruitment of new individuals explain the lack of strong perennial plant community shifts after a decade of elevated [CO2].