Rapid-onset, short-duration induction of colorectal contractions in anesthetized, adult, male rats.

Substantial clinical and preclinical evidence indicates that transient receptor potential vanilloid 1 (TRPV1) receptors are expressed on terminals of colorectal chemoreceptors and mechanoreceptors and are involved in various rectal hypersensitivity disorders with common features of colorectal overactivity. These stimulatory properties of TRPV1 receptors on colorectal function suggested that brief stimulation of TRPV1 might provide a means of pharmacologically activating the colorectum to induce defecation in patients with an "unresponsive" colorectum. The current studies explored the basic features of TRPV1 receptor-induced contractions of the colorectum in anesthetized rats with and without acute spinal cord injury (aSCI). Cumulative concentration-response curves to intrarectal (IR) capsaicin (CAP) solutions (0.003% to 3.0%) were performed in anesthetized aSCI and spinal intact rats. CAP produced an "inverted U", cumulative concentration-response curve with a threshold for inducing colorectal contractions at 0.01% and a peak response at 0.1% and slight decreases in responses up to 3%. Decreases in responses with concentrations > 0.1% are due to a rapid desensitization (i.e. < 30 min) of TRPV1 receptors to each successive dose. Desensitization appeared fully recovered within 24 hours in spinal intact rats. Colorectal contractions were completely blocked by atropine, indicating a reflexogenic activation of parasympathetic neurons, and responses were completely unaffected by a neurokinin 2 receptor antagonist, indicating that release of neurokinin A (NKA) from afferent terminals and subsequent direct contractions of the smooth muscle was not involved. IR administration of 3 other TRPV1 receptor agonists produced similar results as CAP. Significance Statement Individuals with spinal cord injury often lose control of defecation. Time consuming and inconvenient bowel programs using digital stimulation of the rectum and manual extraction of stool are used to empty the bowel. We show that intrarectal administration of the TRPV1 receptor agonist, capsaicin, can induce rapid onset, short duration colorectal contractions capable of inducing defecation in spinal cord injured and intact rats. Therefore, TRPV1 agonists show promise as a potential therapeutics to induce defecation in individuals with neurogenic bowel.

New Evidence on the Cycle in the Women, Infants, and Children Program: What Happens When Benefits Expire.

We provide evidence of a benefit redemption cycle in the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC). Using novel administrative data on item-level redemptions, we show that unlike SNAP, WIC redemptions peak at both the beginning and end of the month. Beginning-of-the-month excess redemptions are concentrated among more popular items such as infant formula, while end-of-the-month excess redemptions are concentrated among less popular items such as infant meats. We document that a substantial share of beneficiaries go at least one month without redeeming anything and discuss how administrative burdens may drive the WIC cycle.

Left atrial appendage dimension predicts elevated brain natriuretic peptide in nonvalvular atrial fibrillation.

INTRODUCTION: BNP elevation in patients with AF is observed in the absence of heart failure; however, prior mechanistic studies have not included direct left atrial pressure measurements. This study sought to understand how emptying function of the left atrial appendage (LAA) and LAA dimension contributes to brain-natriuretic peptide elevations (BNP) in atrial fibrillation (AF) accounting for left atrial pressure (LAP). METHODS: 132 patients referredfor left atrial appendage occlusion (LAAO) were prospectively enrolled in this study. BNP levels and LAP were measured just before LAAO. Statistical analysis considered BNP, rhythm at time of procedure, LAP, LAA morphology, LAA size (ostial diameter, depth, volume), LAA emptying velocity, CHADS2-VASc score, body mass index (BMI), left ventricular ejection fraction (LVEF), estimated glomerular filtration rate (eGFR), and obstructive sleep apnea (OSA) diagnosis as covariates. RESULTS: Bivariate statistical analysis demonstrated positive associations with age, LAA ostial diameter, depth, and volume, LAP, AF status at time of measurement, OSA, and CHADS2-VASc score. BNP was negatively associated with LVEF, eGFR, LAA emptying velocity and BMI. With multivariate logistic regression including LAP as covariate, significant relationships between BNP and AF/AFL(OR 1.99 [1.03, 3.85]), LAP (OR 1.13 [1.06, 1.20]), LAA diameter (OR 1.14 [1.03, 1.27]), LAA depth (OR 1.14 [1.07, 1.22]), and LAA emptying velocity (OR 0.97 [0.96,0.99]) were observed; however, no significant associations were seen with LAA morphology or CHADS2-VASc score. CONCLUSIONS: BNP elevations in AF are associated with LAA size and function, but not CHADS2-VASc score or appendage morphology after accounting for changes in LAP.

Acute dissection into a large abdominal aortic aneurysm in a patient with Marfan syndrome.

OBJECTIVES: Synchronous aortic dissection (AD) with involvement of pre-existing infrarenal abdominal aortic aneurysms (AAAs) are fortunately rare. Extension of the dissection flap to involve the aneurysm is particularly dangerous with pressurization of the false lumen within a diseased aortic wall. The aim of this article is to present a unique case of an acute AD with extension to involve a large AAA in a patient with an underlying connective tissue disorder. METHODS: A 24-year-old male Marfan syndrome patient with prior type A dissection repair presented with a descending thoracic AD and a separate 7.0-cm infrarenal AAA. He was initially medically managed but developed acute extension of the dissection flap to involve the large aneurysm. RESULTS: The patient was initially treated with open infrarenal aortic replacement under left atrialfemoral artery bypass. Three years later, he developed degeneration of the residual aorta and underwent a two-stage hybrid repair. He underwent an additional stent graft for a distal anastomotic leak. At 5 years of follow-up, there was positive aneurysm remodeling and sac regression. CONCLUSION: This case illustrates the challenges in treating a patient with synchronous aortic processes. False lumen pressurization of a large aneurysm wall is fortunately rare but can be lethal. Prompt intervention is required but the presence of an underlying connective tissue disorder may limit endovascular treatment options. Careful case planning is mandatory and multiple interventions should be anticipated.

Identification and characterization of a natural polymorphism in FT-A2 associated with increased number of grains per spike in wheat.

KEY MESSAGE: We discovered a natural FT-A2 allele that increases grain number per spike in both pasta and bread wheat with limited effect on heading time. Increases in wheat grain yield are necessary to meet future global food demands. A previous study showed that loss-of-function mutations in FLOWERING LOCUS T2 (FT2) increase spikelet number per spike (SNS), an important grain yield component. However, these mutations were also associated with reduced fertility, offsetting the beneficial effect of the increases in SNS on grain number. Here, we report a natural mutation resulting in an aspartic acid to alanine change at position 10 (D10A) associated with significant increases in SNS and no negative effects on fertility. Using a high-density genetic map, we delimited the SNS candidate region to a 5.2-Mb region on chromosome 3AS including 28 genes. Among them, only FT-A2 showed a non-synonymous polymorphism (D10A) present in two different populations segregating for the SNS QTL on chromosome arm 3AS. These results, together with the known effect of the ft-A2 mutations on SNS, suggest that variation in FT-A2 is the most likely cause of the observed differences in SNS. We validated the positive effects of the A10 allele on SNS, grain number, and grain yield per spike in near-isogenic tetraploid wheat lines and in an hexaploid winter wheat population. The A10 allele is present at very low frequency in durum wheat and at much higher frequency in hexaploid wheat, particularly in winter and fall-planted spring varieties. These results suggest that the FT-A2 A10 allele may be particularly useful for improving grain yield in durum wheat and fall-planted common wheat varieties.

Incomplete program take-up during a crisis: evidence from the COVID-19 shock in one U.S. state.

In the U.S., means-tested cash, in-kind assistance, and social insurance are part of a patchwork safety net, often run with substantial involvement of state and local governments. Take-up-participation among eligible persons in this system is incomplete. A large literature points to both neo-classical and behavioral science explanations for low take-up. In this paper, we explore the response of the safety net to COVID-19 using newly-collected survey data from one U.S. state-Utah. The rich Utah data ask about income and demographics as well as use of three social safety net programs which collectively provided a large share of relief spending: the Unemployment Insurance program, a social insurance program providing workers who lose their jobs with payments; the Supplemental Nutrition Assistance Program, which provides benefit cards for purchasing unprepared food at retailers; and Economic Impact Payments, which provided relatively universal relief payments to individuals. The data do not suffice to determine eligibility for all of the programs, so we focus on participation per capita. These data also collect information on several measures of hardship and why individuals did not receive any of the 3 programs. We test for explanations that differentiate need, lack of information, transaction costs/administrative burden, stigma, and lack of eligibility. We use measures of hardship to assess targeting. We find that lack of knowledge as well as difficulty applying, and stigma in the UI program each play a role as reasons for not participating in the programs.

Neurokinin 2 receptor-mediated bladder and colorectal responses in aged spinal cord injured rats.

STUDY DESIGN: Animal proof of principle study. OBJECTIVES: Bladder and bowel dysfunction are common after spinal cord injury (SCI) and in the elderly. Neurokinin 2 receptor agonists are known to produce on-demand urination and defecation in adult SCI rats. This study compared the ability of a neurokinin 2 receptor (NK2R) agonist to produce bladder and colorectal contractions in both young adult and aged SCI rats. SETTING: Dignify Therapeutics and Integrated Laboratory Systems, Durham, NC USA. METHODS: Bladder and colorectal pressure and voiding efficiency were measured in response to the NK2R agonist, [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA), in anesthetized animals. The potency and efficacy of LMN-NKA was examined in young adult and aged SCI (T3 or T9 transected) rats, with young adult and aged spinal intact rats included as controls. RESULTS: LMN-NKA (3-300 µg/kg i.v.) produced dose-dependent increases in bladder and colorectal pressure in all anesthetized rats. No differences in the bladder or colorectal pressure responses or voiding efficiency were observed with age or after SCI. The level of SCI did not change the pharmacodynamic responses to the agonist. CONCLUSIONS: An NK2R agonist produced similar responses in young adult and aged SCI rats, suggesting this class of agonists could be used as a potential therapy to induce on-demand urination and defecation in aged populations, with or without SCI.

Multi-stage open surgical and endovascular treatment of progressive aortic degeneration in a patient with Marfan syndrome.

Aortic interventions in patients with underlying connective tissues disorders present a unique challenge. The inevitable degeneration of the native aorta can lead to the need for multiple staged interventions with significant risk of complications associated with reoperative aortic procedures. We present a challenging case of progressive aortic degeneration in a patient with Marfan syndrome treated with multi-staged open surgical and endovascular procedures.

Photomagnetic Prussian blue nanocubes: Synthesis, characterization, and biomedical applications.

Nanoparticles play an important role in biomedicine. We have developed a method for size-controlled synthesis of photomagnetic Prussian blue nanocubes (PBNCs) using superparamagnetic iron oxide nanoparticles (SPIONs) as precursors. The developed PBNCs have magnetic and optical properties desired in many biomedical diagnostic and therapeutic applications. Specifically, the size-tunable photomagnetic PBNCs exhibit high magnetic saturation, strong optical absorption with a peak at approximately 700 nm, and superior photostability. Our studies demonstrate that PBNCs can be used as MRI and photoacoustic imaging contrast agents in vivo. We also showed the utility of PBNCs for labeling and magnetic manipulation of cells. Dual magnetic and optical properties, together with excellent biocompatibility, render PBNCs an attractive contrast agent for both diagnostic and therapeutic applications. The use SPIONs as precursors for PBNCs provides flexibility and allows researchers to design theranostic agents according to required particle size, optical, and magnetic properties.

Analysis of recombinant inbred line populations derived from wheat landraces to identify new genes for wheat stem sawfly resistance.

Wheat landrace accessions were chosen from areas of the world with historical European wheat stem sawfly (Cephus pygmaeus L.) selection pressure to develop six recombinant inbred line (RIL) populations. Molecular maps were constructed, and resistance due to antibiosis and antixenosis was assessed at sites in Montana naturally infested by Cephus cinctus Norton, the wheat stem sawfly (WSS). Novel QTLs were identified along with QTL previously identified in elite germplasm. A newly identified QTL on chromosome 1B provided a new source for pith-filled solid stems. An allele for resistance on chromosome 4A unrelated to solid stems was identified in four of the six RIL populations. A landrace from Turkey, PI 166471, contained alleles at three QTLs causing high levels of larval mortality. None of the QTLs were related to stem solidness, but their combined effect provided resistance similar to that observed in a solid-stemmed check cultivar. These results show the utility of genetic populations derived from geographically targeted landrace accessions to identify new alleles for insect resistance. New PCR-based molecular markers were developed for introgression of novel alleles for WSS resistance into elite lines. Comparison of results with previous analysis of elite cultivars addresses changes in allele frequencies during the wheat breeding process.

Cell Type-Specific Contributions of the Angiotensin II Type 1a Receptor to Aorta Homeostasis and Aneurysmal Disease-Brief Report.

OBJECTIVE: Two were the aims of this study: first, to translate whole-genome expression profiles into computational predictions of functional associations between signaling pathways that regulate aorta homeostasis and the activity of angiotensin II type 1a receptor (At1ar) in either vascular endothelial or smooth muscle cells; and second, to characterize the impact of endothelial cell- or smooth muscle cell-specific At1ar disruption on the development of thoracic aortic aneurysm in fibrillin-1 hypomorphic (Fbn1mgR/mgR ) mice, a validated animal model of early onset progressively severe Marfan syndrome. APPROACH AND RESULTS: Cdh5-Cre and Sm22-Cre transgenic mice were used to inactivate the At1ar-coding gene (Agt1ar) in either intimal or medial cells of both wild type and Marfan syndrome mice, respectively. Computational analyses of differentially expressed genes predicted dysregulated signaling pathways of cell survival and matrix remodeling in Agt1arCdh5-/- aortas and of cell adhesion and contractility in Agt1arSm22-/- aortas. Characterization of Fbn1mgR/mgR;Agt1arCdh5-/- mice revealed increased median survival associated with mitigated aneurysm growth and media degeneration, as well as reduced levels of phosphorylated (p-) Erk1/2 but not p-Smad2. By contrast, levels of both p-Erk1/2 and p-Smad2 proteins were normalized in Fbn1mgR/mgR;Agt1arSm22-/- aortas in spite of them showing no appreciable changes in thoracic aortic aneurysm pathology. CONCLUSIONS: Physiological At1ar signaling in the intimal and medial layers is associated with distinct regulatory processes of aorta homeostasis and function; improper At1ar activity in the vascular endothelium is a significant determinant of thoracic aortic aneurysm development in Marfan syndrome mice.

Dimorphic effects of transforming growth factor-ß signaling during aortic aneurysm progression in mice suggest a combinatorial therapy for Marfan syndrome.

OBJECTIVE: Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFß signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFß neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFß neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1(mgR/mgR) mice). APPROACH AND RESULTS: Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1(mgR/mgR) mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFß-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFß neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFß hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1(mgR/mgR) mice from ruptured TAA. CONCLUSIONS: By demonstrating that promiscuous AT1r and TGFß drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFß.

Overexpression of the steroidogenic enzyme cytochrome P450 side chain cleavage in the ventral tegmental area increases 3α,5α-THP and reduces long-term operant ethanol self-administration.

Neuroactive steroids are endogenous neuromodulators capable of altering neuronal activity and behavior. In rodents, systemic administration of endogenous or synthetic neuroactive steroids reduces ethanol self-administration. We hypothesized this effect arises from actions within mesolimbic brain regions that we targeted by viral gene delivery. Cytochrome P450 side chain cleavage (P450scc) converts cholesterol to pregnenolone, the rate-limiting enzymatic reaction in neurosteroidogenesis. Therefore, we constructed a recombinant adeno-associated serotype 2 viral vector (rAAV2), which drives P450scc expression and neuroactive steroid synthesis. The P450scc-expressing vector (rAAV2-P450scc) or control GFP-expressing vector (rAAV2-GFP) were injected bilaterally into the ventral tegmental area (VTA) or nucleus accumbens (NAc) of alcohol preferring (P) rats trained to self-administer ethanol. P450scc overexpression in the VTA significantly reduced ethanol self-administration by 20% over the 3 week test period. P450scc overexpression in the NAc, however, did not alter ethanol self-administration. Locomotor activity was unaltered by vector administration to either region. P450scc overexpression produced a 36% increase in (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP, allopregnanolone)-positive cells in the VTA, but did not increase 3α,5α-THP immunoreactivity in NAc. These results suggest that P450scc overexpression and the resultant increase of 3α,5α-THP-positive cells in the VTA reduces ethanol reinforcement. 3α,5α-THP is localized to neurons in the VTA, including tyrosine hydroxylase neurons, but not astrocytes. Overall, the results demonstrate that using gene delivery to modulate neuroactive steroids shows promise for examining the neuronal mechanisms of moderate ethanol drinking, which could be extended to other behavioral paradigms and neuropsychiatric pathology.

Ethanol administration produces divergent changes in GABAergic neuroactive steroid immunohistochemistry in the rat brain.

BACKGROUND: The 5α-reduced pregnane neuroactive steroid (3α,5α)-3-hydroxypregnan-20-one (3α,5α-THP or allopregnanolone) is a potent positive modulator of GABAA receptors capable of modulating neuronal activity. In rats, systemic ethanol (EtOH) administration increases cerebral cortical and hippocampal levels of 3α,5α-THP, but the effects of EtOH on 3α,5α-THP levels in other brain regions are unknown. There is a large body of evidence suggesting that 3α,5α-THP enhances EtOH sensitivity, contributes to some behavioral effects of EtOH, and modulates EtOH reinforcement and motivation to drink. In this study, we used immunohistochemistry (IHC) to determine EtOH-induced changes in cellular 3α,5α-THP expression in brain regions associated with EtOH actions and responses. METHODS: Male Wistar rats were administered EtOH (2 g/kg) or saline intraperitoneally and after 60 minutes transcardially perfused. IHC was performed on free-floating sections (3 to 4 sections/animal/brain region) using an affinity purified anti-3α,5α-THP primary antibody, and immunoreactivity was visualized with 3,3'-diaminobenzidine. RESULTS: EtOH significantly increased 3α,5α-THP immunoreactivity by 24 ± 6% in the medial prefrontal cortex, 32 ± 12% in the hippocampal Cornu Ammonis area 1 (CA1) pyramidal cell layer, 52 ± 5% in the polymorph cell layer of the dentate gyrus (DG), 44 ± 15% in the bed nucleus of the stria terminalis, and 36 ± 6% in the paraventricular nucleus of the hypothalamus. In contrast, EtOH administration significantly reduced 3α,5α-THP immunoreactivity by 25 ± 5% in the nucleus accumbens "shore" and 21 ± 3% in the central nucleus of the amygdala. No changes were observed in the ventral tegmental area, dorsomedial striatum, granule cell layer of the DG, or the lateral and basolateral amygdala. CONCLUSIONS: The results suggest acute EtOH (2 g/kg) produces divergent, brain region specific, effects on cellular 3α,5α-THP levels. Regional differences in the effects of EtOH suggest there may be regional brain synthesis of 3α,5α-THP independent of the adrenal glands and novel mechanisms that reduce cellular 3α,5α-THP. Regional differences in EtOH-induced changes in 3α,5α-THP levels likely contribute to EtOH effects on neuronal function in brain.

Chronic intermittent ethanol exposure and withdrawal alters (3α,5α)-3-hydroxy-pregnan-20-one immunostaining in cortical and limbic brain regions of C57BL/6J mice.

BACKGROUND: The GABAergic neuroactive steroid (3α,5α)-3-hydroxy-pregnan-20-one (3α,5α-THP; allopregnanolone) has been studied during withdrawal from ethanol (EtOH) in humans, rats, and mice. Serum 3α,5α-THP levels decreased, and brain levels were not altered following acute EtOH administration (2 g/kg) in male C57BL/6J mice; however, the effects of chronic intermittent ethanol (CIE) exposure on 3α,5α-THP levels have not been examined. Given that CIE exposure changes subsequent voluntary EtOH drinking in a time-dependent fashion following repeated cycles of EtOH exposure, we conducted a time-course analysis of CIE effects on 3α,5α-THP levels in specific brain regions known to influence drinking behavior. METHODS: Adult male C57BL/6J mice were exposed to 4 cycles of CIE to induce EtOH dependence. All mice were sacrificed and perfused at 1 of 2 time points, 8 or 72 hours following the final exposure cycle. Free-floating brain sections (40 µm; 3 to 5 sections/region/animal) were immunostained and analyzed to determine relative levels of cellular 3α,5α-THP. RESULTS: Withdrawal from CIE exposure produced time-dependent and region-specific effects on immunohistochemical detection of 3α,5α-THP levels across cortical and limbic brain regions. A transient reduction in 3α,5α-THP immunoreactivity was observed in the central nucleus of the amygdala 8 hours after withdrawal from CIE (-31.4 ± 9.3%). Decreases in 3α,5α-THP immunoreactivity were observed 72 hours following withdrawal in the medial prefrontal cortex (-25.0 ± 9.3%), nucleus accumbens core (-29.9 ± 6.6%), and dorsolateral striatum (-18.5 ± 6.0%), while an increase was observed in the CA3 pyramidal cell layer of the hippocampus (+42.8 ± 19.5%). Sustained reductions in 3α,5α-THP immunoreactivity were observed at both time points in the lateral amygdala (8 hours -28.3 ± 12.8%; 72 hours -27.5 ± 12.4%) and in the ventral tegmental area (8 hours -26.5 ± 9.9%; 72 hours -31.6 ± 13.8%). CONCLUSIONS: These data suggest that specific neuroadaptations in 3α,5α-THP levels may be present in regions of brain that mediate anxiety, stress, and reinforcement relevant to EtOH dependence. The changes that occur at different time points likely modulate neurocircuitry involved in EtOH withdrawal as well as the elevated drinking observed after CIE exposure.

Clinical, diagnostic, and therapeutic aspects of the Marfan syndrome.

Marfan syndrome (MFS) is a relatively common and often lethal disease of connective tissue. Medical, surgical and basic research advances over the last two decades have had a major positive impact on the clinical management of MFS patients. Life expectancy has increased significantly, more discriminating diagnostic criteria have been developed, a number of new clinical entities have been recognized, and exciting opportunities for drug-based therapy have emerged. Despite such a remarkable progress, MFS diagnosis remains difficult and aortic disease progression is very heterogeneous and clinical outcome is unpredictable. Ongoing research efforts are therefore exploiting animal models of MFS to identify novel diagnostic and prognostic biomarkers, genetic, epigenetic and environmental modifiers and druggable biological targets.

Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels.

The maize (Zea mays) kernel plays a critical role in feeding humans and livestock around the world and in a wide array of industrial applications. An understanding of the regulation of kernel starch, protein, and oil is needed in order to manipulate composition to meet future needs. We conducted joint-linkage quantitative trait locus mapping and genome-wide association studies (GWAS) for kernel starch, protein, and oil in the maize nested association mapping population, composed of 25 recombinant inbred line families derived from diverse inbred lines. Joint-linkage mapping revealed that the genetic architecture of kernel composition traits is controlled by 21-26 quantitative trait loci. Numerous GWAS associations were detected, including several oil and starch associations in acyl-CoA:diacylglycerol acyltransferase1-2, a gene that regulates oil composition and quantity. Results from nested association mapping were verified in a 282 inbred association panel using both GWAS and candidate gene association approaches. We identified many beneficial alleles that will be useful for improving kernel starch, protein, and oil content.

[Lys5,MeLeu9,Nle10]-NKA(4-10) induces neurokinin 2 receptor mediated urination and defecation and neurokinin 1 receptor mediated flushing in rats: measured using the rapid detection voiding assay.

OBJECTIVES: Neurokinin 2 receptor (NK2R) agonists may be useful for treating bladder and bowel dysfunction via direct contraction of detrusor and gastrointestinal smooth muscle. The NK2R agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) (LMN-NKA) induces urination and defecation, but also produces the potential side effect of dermal flushing in rats. Although LMN-NKA is a NK2R agonist, it also has affinity for neurokinin 1 receptors (NK1R). Therefore, the goal of this study was to determine the neurokinin receptor (NKR) subtypes responsible for LMN-NKA-induced urination, defecation, and flushing by blocking either NK2Rs or NK1Rs before LMN-NKA administration. METHODS: To accomplish this goal, we developed a simple high-throughput 'rapid detection voiding assay' to detect rapid-onset drug-induced urination and defecation in rats. In LMN-NKA dose-response experiments, LMN-NKA (10-100 µg/kg, subcutaneous) was injected and urination, defecation, and flushing were monitored for 30 min. For NKR antagonist experiments, vehicle, the NK2R antagonist GR159897, or the NK1R antagonist CP-99,994 were injected before an acclimation period. Following acclimation, saline or 100 µg/kg LMN-NKA were injected, and behavior was observed for 30 min. RESULTS: LMN-NKA produced dose-related increases in urination, defecation, and flushing. Blocking NK2Rs reduced urination and blocked defecation, without affecting flushing. Blocking NK1Rs did not change LMN-NKA-induced urination or defecation but reduced LMN-NKA-induced flushing. CONCLUSIONS: Using the rapid detection voiding assay we show that LMN-NKA-induced urination and defecation are mediated by NK2Rs, while flushing is mediated by NK1Rs. Therefore, drugs that are more selective for NK2 vs. NK1Rs should produce rapid-onset urination and defecation without producing the potential side effect of flushing.

Design, execution, and interpretation of plant RNA-seq analyses.

Genomics has transformed our understanding of the genetic architecture of traits and the genetic variation present in plants. Here, we present a review of how RNA-seq can be performed to tackle research challenges addressed by plant sciences. We discuss the importance of experimental design in RNA-seq, including considerations for sampling and replication, to avoid pitfalls and wasted resources. Approaches for processing RNA-seq data include quality control and counting features, and we describe common approaches and variations. Though differential gene expression analysis is the most common analysis of RNA-seq data, we review multiple methods for assessing gene expression, including detecting allele-specific gene expression and building co-expression networks. With the production of more RNA-seq data, strategies for integrating these data into genetic mapping pipelines is of increased interest. Finally, special considerations for RNA-seq analysis and interpretation in plants are needed, due to the high genome complexity common across plants. By incorporating informed decisions throughout an RNA-seq experiment, we can increase the knowledge gained.