Postmitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal chromatin, transcriptome, and identity.

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide long-term histone replacement in postmitotic cells, including neurons. Beyond replenishment, histone variants also play active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover. Here, we uncover crucial roles for the histone H3 variant H3.3 in neuronal development. We find that newborn cortical excitatory neurons, which have only just completed replication-coupled deposition of canonical H3.1 and H3.2, substantially accumulate H3.3 immediately postmitosis. Codeletion of H3.3-encoding genes H3f3a and H3f3b from newly postmitotic neurons abrogates H3.3 accumulation, markedly alters the histone posttranslational modification landscape, and causes widespread disruptions to the establishment of the neuronal transcriptome. These changes coincide with developmental phenotypes in neuronal identities and axon projections. Thus, preexisting, replication-dependent histones are insufficient for establishing neuronal chromatin and transcriptome; de novo H3.3 is required. Stage-dependent deletion of H3f3a and H3f3b from 1) cycling neural progenitor cells, 2) neurons immediately postmitosis, or 3) several days later, reveals the first postmitotic days to be a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, codeletion of H3f3a and H3f3b does not lead to immediate H3.3 loss, but causes progressive H3.3 depletion over several months without widespread transcriptional disruptions or cellular phenotypes. Our study thus uncovers key developmental roles for de novo H3.3 in establishing neuronal chromatin, transcriptome, identity, and connectivity immediately postmitosis that are distinct from its role in maintaining total histone H3 levels over the neuronal lifespan.

Chromatin remodeler Arid1a regulates subplate neuron identity and wiring of cortical connectivity.

Loss-of-function mutations in chromatin remodeler gene ARID1A are a cause of Coffin-Siris syndrome, a developmental disorder characterized by dysgenesis of corpus callosum. Here, we characterize Arid1a function during cortical development and find unexpectedly selective roles for Arid1a in subplate neurons (SPNs). SPNs, strategically positioned at the interface of cortical gray and white matter, orchestrate multiple developmental processes indispensable for neural circuit wiring. We find that pancortical deletion of Arid1a leads to extensive mistargeting of intracortical axons and agenesis of corpus callosum. Sparse Arid1a deletion, however, does not autonomously misroute callosal axons, implicating noncell-autonomous Arid1a functions in axon guidance. Supporting this possibility, the ascending axons of thalamocortical neurons, which are not autonomously affected by cortical Arid1a deletion, are also disrupted in their pathfinding into cortex and innervation of whisker barrels. Coincident with these miswiring phenotypes, which are reminiscent of subplate ablation, we unbiasedly find a selective loss of SPN gene expression following Arid1a deletion. In addition, multiple characteristics of SPNs crucial to their wiring functions, including subplate organization, subplate axon-thalamocortical axon cofasciculation ("handshake"), and extracellular matrix, are severely disrupted. To empirically test Arid1a sufficiency in subplate, we generate a cortical plate deletion of Arid1a that spares SPNs. In this model, subplate Arid1a expression is sufficient for subplate organization, subplate axon-thalamocortical axon cofasciculation, and subplate extracellular matrix. Consistent with these wiring functions, subplate Arid1a sufficiently enables normal callosum formation, thalamocortical axon targeting, and whisker barrel development. Thus, Arid1a is a multifunctional regulator of subplate-dependent guidance mechanisms essential to cortical circuit wiring.

Next-generation fungal identification using target enrichment and Nanopore sequencing.

BACKGROUND: Rapid and accurate pathogen identification is required for disease management. Compared to sequencing entire genomes, targeted sequencing may be used to direct sequencing resources to genes of interest for microbe identification and mitigate the low resolution that single-locus molecular identification provides. This work describes a broad-spectrum fungal identification tool developed to focus high-throughput Nanopore sequencing on genes commonly employed for disease diagnostics and phylogenetic inference. RESULTS: Orthologs of targeted genes were extracted from 386 reference genomes of fungal species spanning six phyla to identify homologous regions that were used to design the baits used for enrichment. To reduce the cost of producing probes without diminishing the phylogenetic power, DNA sequences were first clustered, and then consensus sequences within each cluster were identified to produce 26,000 probes that targeted 114 genes. To test the efficacy of our probes, we applied the technique to three species representing Ascomycota and Basidiomycota fungi. The efficiency of enrichment, quantified as mean target coverage over the mean genome-wide coverage, ranged from 200 to 300. Furthermore, enrichment of long reads increased the depth of coverage across the targeted genes and into non-coding flanking sequence. The assemblies generated from enriched samples provided well-resolved phylogenetic trees for taxonomic assignment and molecular identification. CONCLUSIONS: Our work provides data to support the utility of targeted Nanopore sequencing for fungal identification and provides a platform that may be extended for use with other phytopathogens.

Implant-Based Breast Reconstruction in the Elderly: Complications and Patient-Reported Outcomes in Women Older Than 70 Years.

BACKGROUND: Advanced age is considered by many to be a relative contraindication to breast reconstruction. However, despite increased medical comorbidities and a perception that elderly patients are less concerned with body image, more women older than 70 years are choosing to undergo breast reconstruction. There is a paucity of data to guide reconstructive decision-making and counseling in this population. OBJECTIVES: The aim of this study was to evaluate patient satisfaction, complication rates, and long-term outcomes in women older than 70 years undergoing implant-based breast reconstruction. METHODS: A total of 400 patients were identified at the authors' institution and divided into 2 groups: ≥70 and <70 years old. Medical comorbidities, surgical outcomes, and patient-reported outcomes as defined by the BREAST-Q were compared using the χ2 tests for categorical variables and t tests for continuous variables. RESULTS: The cohort of patients older than 70 years was made up of 25 women, with a mean age of 73 years, and the cohort of patients younger than 70 years was made up of 375 women, with a mean age of 50 years. There was no significant difference in body mass index (P = 0.373), smoking status (P = 0.360), or history of prior ipsilateral radiation (P = 0.508) between the 2 cohorts; however, the elderly cohort was significantly more likely to have diabetes (P = 0.026). Although elderly patients were less likely to undergo bilateral mastectomy (P < 0.001), there was no significant difference in the type of mastectomy, pathological diagnosis, or method of reconstruction. There was no significant difference in complication rates when looking at minor infection (P = 0.553) or major infection (P = 0.553). The 2 groups were equally likely to undergo secondary procedures (P = 0.192). Overall satisfaction rates were high in all BREAST-Q categories in the elderly group and not significantly different when compared with the group of patients younger than 70 years. Matched-pair analysis showed a significant difference with the group of patients older than 70 years having higher levels physical well-being (P < 0.001). CONCLUSIONS: Immediate breast reconstruction can be performed safely and with similar high satisfaction rates in the elderly population as their younger counterparts. Age alone should not be used as a reason for excluding women from these life-changing operations.

Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Skin Reducing Mastectomy and Immediate Tissue Expander Reconstruction: A Critical Analysis.

BACKGROUND: Complications of implant-based reconstruction have been shown to be related to increasing body mass index (BMI) and breast size. The impact of skin reducing mastectomy (SRM) with a dermal flap is examined. METHODS: A retrospective review of a single surgeon's experience with immediate submuscular tissue expander (TE) reconstruction from 2011 to 2019 was performed. The outcomes of SRM were compared with those of skin sparing mastectomy (SSM). RESULTS: A total of 162 patients (292 breasts) were identified. Mastectomy types were as follows: SRM, 73 (136 breasts) and SSM, 89 (156 breasts). Acellular dermal matrix (ADM) was used to supplement TE coverage in 65.4% of SRM cases. Mean BMI was 29.2 among SRM patients and 25.9 in SSM patients (P < 0.001). Obesity (BMI ≥ 30) was more prevalent in the SRM group (SRM, 38.4% vs SSM, 22.5%; P = 0.03). Mean mastectomy weight was higher in the SRM group (SRM, 833.6 g vs SSM, 425.6 g; P < 0.001). Mean BMI and mastectomy weight were lower in SRM patients who were reconstructed with ADM (ADM, 28.1 vs no ADM, 30.8; P = 0.01; ADM, 746.1 g vs no ADM, 1006.3 g; P < 0.001). Minor complications were more prevalent in the SRM group (SRM, 22.8% vs SSM, 4.5%; P < 0.001). Mastectomy skin flap necrosis (MSFN) was more common in the SRM group (SRM, 22.8% vs SSM, 7.7%; P < 0.001), but MSFN necessitating operative debridement was similarly low in both groups (SRM: 1.9% vs SSM: 4.5%). Major complication rates (SRM 11.0% vs SSM 10.9%) and reconstructive failure rates (SRM 5.9% vs SSM 5.1%) were similar between groups. Mastectomy weight 800 g or higher and BMI of 30 or higher were found to be risk factors for complications on analysis of the SRM cohort (P < 0.05). CONCLUSIONS: Mastectomy weight and BMI were positive predictors of complications after immediate TE reconstruction. Mastectomy skin flap necrosis is more common after SRM than SSM. The use of SRM with a dermal flap has a similar major complication rate as SSM despite its use in obese, large-breasted women. The dermal flap provides soft tissue coverage, which prevents implant exposure and seroma. The use of ADM does not adversely affect the complication rate of SRM.

A Simplified Approach to Myelomeningocele Defect Repair.

BACKGROUND: Repair of the soft tissue defect in myelomeningoceles remains challenging. The literature currently lacks a systematic approach, reporting high rates of complications. We present outcomes from the largest series to date and describe a simplified approach that minimizes morbidity and streamlines decision making. METHODS: Patients 1 year or younger who underwent myelomeningocele repair between 2008 and 2018 were reviewed. Flap types were categorized by tissue composition. Complications were dichotomized into early and late (<30 days and >30 days postoperative, respectively). Logistic regression was used to measure the impact of flap tissue composition and skin closure technique on odds of postoperative complications. RESULTS: Ninety-seven patients met inclusion criteria. Reoperation was required in only 3 (3.0%) patients-1 for wound dehiscence and 2 for surgical site infections. Zero cases of tethered cord or cerebrospinal fluid leak occurred. The most common minor complications were early wound complications (n = 18, 18.6%) and early infection (n = 5, 5.2%). Fascia-only flaps and muscle + other tissue flaps were not associated with higher odds of complications compared with muscle-only flaps (odds ratio [OR], 2.13; 95% confidence interval [CI], 0.53-8.50, P = 0.29; OR = 2.87, 95% CI 0.66-12.51, P = 0.16, respectively). Rhomboid flaps for skin closure were associated with higher odds of complications (OR, 4.47; 95% CI, 1.00-19.97; P = 0.05). CONCLUSIONS: Our approach to myelomeningocele repair demonstrated no cases of secondary tethered cord or cerebrospinal fluid leak, and reoperative rates were extremely low. Because complications were unrelated to flap type, we recommend a simplified approach using any tissue type for dural coverage and 2-layer primary closure of the skin.

Concentrations in plasma and selected tissues of marbofloxacin after oral and intravenous administration in Bilgorajska geese (Anser anser domesticus).

Aims: To determine the pharmacokinetics and tissue depletion of 2 mg/kg marbofloxacin (MBX) in Bilgorajska geese (Anser anser domesticus) after I/V and oral administration, to calculate the daily dose from experimental data and to compare it with that calculated by allometric scaling.Methods: Eight clinically normal female Bilgorajska geese were used in a three-phase study with a 3-week wash-out period between phases. In the first phase birds received I/V administration of 2 mg/kg MBX; the same dose was given orally in the second and third phases. Blood samples were collected between 0 minutes and 48 hours in the first and second phases, and samples of liver, kidney, lung, muscle and heart were collected following slaughter of birds between 6 and 48 hours in the third phase. Concentrations of MBX in plasma and tissues were analysed using HPLC. Two additional birds served as controls. The optimal dose was calculated based on a minimal inhibitory concentration (MIC) of 0.125 µg/mL using the observed clearance, or using clearance calculated by allometric scaling.Results: Concentrations of MBX in plasma were detectable up to 24 hours following both I/V and oral administration. Mean oral bioavailability was 26.5 (SD 7.7)%. Concentrations of MBX in all tissues were highest at 6 hours and decreased constantly up to 34 hours. The mean optimal daily dose for oral administration of MBX, calculated using the observed clearance was 10.36 (SD 2.18) mg/kg, and using predicted clearance was 5.54 (SD 0.14) mg/kg. The preliminary withdrawal time for a maximum residue limit of 0.15 mg/kg calculated for muscle was 38.4 hours, heart 33.6 hours, kidney 48.3 hours, lung 47.7 hours and liver 49.3 hours.Conclusion and Clinical Relevance: There was insufficient evidence to recommend MBX orally administered to geese at a daily dose of 2 mg/kg for treatment of bacteria with an MIC of 0.125 µg/mL. Further pharmacokinetic/pharmacodynamic studies in geese are recommended to determine the MBX dose regimen and its clinical efficacy in geese.

Neuronal UCP1 expression suggests a mechanism for local thermogenesis during hibernation.

Hibernating mammals possess a unique ability to reduce their body temperature to ambient levels, which can be as low as -2.9 °C, by active down-regulation of metabolism. Despite such a depressed physiologic phenotype, hibernators still maintain activity in their nervous systems, as evidenced by their continued sensitivity to auditory, tactile, and thermal stimulation. The molecular mechanisms that underlie this adaptation remain unknown. We report, using differential transcriptomics alongside immunohistologic and biochemical analyses, that neurons from thirteen-lined ground squirrels (Ictidomys tridecemlineatus) express mitochondrial uncoupling protein 1 (UCP1). The expression changes seasonally, with higher expression during hibernation compared with the summer active state. Functional and pharmacologic analyses show that squirrel UCP1 acts as the typical thermogenic protein in vitro. Accordingly, we found that mitochondria isolated from torpid squirrel brain show a high level of palmitate-induced uncoupling. Furthermore, torpid squirrels during the hibernation season keep their brain temperature significantly elevated above ambient temperature and that of the rest of the body, including brown adipose tissue. Together, our findings suggest that UCP1 contributes to local thermogenesis in the squirrel brain, and thus supports nervous tissue function at low body temperature during hibernation.

Pharmacokinetic profiles of the two major active metabolites of metamizole (dipyrone) in cats following three different routes of administration.

This study was performed to determine pharmacokinetic profiles of the two active metabolites of the analgesic drug metamizole (dipyrone, MET), 4-methylaminoantipyrine (MAA), and 4-aminoantipyrine (AA), after intravenous (i.v., intramuscular (i.m.), and oral (p.o.) administration in cats. Six healthy mixed-breed cats were administered MET (25 mg/kg) by i.v., i.m., or p.o. routes in a crossover design. Adverse clinical signs, namely salivation and vomiting, were detected in all groups (i.v. 67%, i.m. 34%, and p.o. 15%). The mean maximal plasma concentration of MAA for i.v., i.m., and p.o. administrations was 148.63 ± 106.64, 18.74 ± 4.97, and 20.59 ± 15.29 µg/ml, respectively, with about 7 hr of half-life in all routes. Among the administration routes, the area under the plasma concentration curve (AUC) value was the lowest after i.m. administration and the AUCEV/i.v . ratio was higher in p.o. than the i.m. administration without statistical significance. The plasma concentration of AA was detectable up to 24 hr, and the mean plasma concentrations were smaller than MAA. The present results suggest that MET is converted into the active metabolites in cats as in humans. Further pharmacodynamics and safety studies should be performed before any clinical use.

Pharmacokinetic profiles of the active metamizole metabolites after four different routes of administration in healthy dogs.

Metamizole (MT), an analgesic and antipyretic drug, is rapidly hydrolyzed to the active primary metabolite 4-methylaminoantipyrine (MAA) and relatively active secondary metabolite 4-aminoantipyrine (AA). The aim of this study was to assess the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.), intramuscular (i.m.), oral (p.o.), and rectal (RC) routes in dogs. Six dogs were randomly allocated to an open, single-dose, four-treatment, four-phase, unpaired, crossover study design. Blood was collected at predetermined times within 24 hr, and plasma was analyzed by a validated HPLC-UV method. Plasma concentrations of MAA and AA after i.v., i.m., p.o., and RC administrations of MT were detectable from 5 (i.v. and i.m.) or 30 (p.o. and RC) min to 24 hr in all dogs. The highest concentrations of MAA were found in the i.v., then i.m., p.o., and RC groups. Plasma concentrations of AA were similar for i.v., i.m., and RC, and the concentrations were approximately double those in the PO groups. The AUCEV/IV ratio for MAA was 0.75 ± 0.11, 0.59 ± 0.08, and 0.32 ± 0.05, for i.m., p.o., and RC, respectively. The AUCEV/IV ratio for AA was 1.21 ± 0.33, 2.17 ± 0.62, and 1.08 ± 0.19, for i.m., p.o., and RC, respectively. Although further studies are needed, rectal administration seems to be the least suitable route of administration for MT in the dog.

Pharmacokinetics of tapentadol in laying hens and its residues in eggs after multiple oral dose administration.

1. The aim of the study was to evaluate the pharmacokinetics (PKs) of tapentadol (TAP), a novel opioid analgesic, in laying hens after intravenous (IV) and oral (PO) administration and to quantify the concentrations of TAP residues in eggs. 2. Twenty healthy laying hens were divided into three groups: A (n = 6), B (n = 6) and C (n = 8). The study was conducted in two phases. Groups A and B received TAP by IV and PO routes at the dose of 1 and 5 mg/kg, respectively. 3. No visible adverse effects were observed after administration of the drug. TAP plasma concentrations were detectable up to 4 h following administration. Following IV administration, TAP plasma concentrations were only higher than the minimal effective concentration (148 ng/ml) reported for humans for 1 h. After single PO administration, plasma concentrations of TAP would not conform to software algorithms and the PK parameters were not calculated. TAP concentration following multiple PO doses at 5 mg/kg for 5 d was found to be higher and more persistent (12 h vs. 7 h) in yolk compared with albumen. 4. This is the first PK study on the novel atypical opioid TAP in laying hens. Further studies are required to investigate the analgesic efficacy and actual effective plasma concentration of TAP in this species.

Pharmacokinetic profiles of 5 mg/kg ibudilast, a phosphodiesterase inhibitor, orally administered to dogs in fasted and non-fasted states. A preliminary study.

Ibudilast (AV-411) is a non-selective inhibitor of cyclic nucleotide phosphodiesterase (PDE). It is currently marketed for human use in Asian countries for the treatment of asthma, cerebrovascular disorders and ocular allergies. Ibudilast has also been found to have an analgesic action for neuropathic pain at doses 5-10 times higher than those used in asthma therapy. Six healthy Labrador dogs were randomly assigned to two treatment groups using an open, single-dose, two-treatment, two-phase, cross-over design (2x2 Latin-square). Dogs in group 1 (n=3) were fasted for at least 10 hours overnight before the beginning of the experiment and 4 h following dosing while dogs in group 2 (n=3) received food ad libitum. During the first phase, each dog in group 1 and 2 received a single dose of 5 mg/kg ibudilast administered orally. After 1-week washout period the groups were rotated and the experiment was repeated. The analytical method, validated for dog plasma, was shown to be linear in the range 0.10-20 µg/mL. The limit of detection (LOD) and quantification (LOQ) were 0.03 and 0.1 µg/mL, respectively. No behavioural or health alterations were observed in the animals during or after the study. Ibudilast was detectable in plasma for up to 24 h showing a wide variability between animals. Although no statistically significant differences were observed in the present study between the fed and fasted states, examination of the raw data suggests that an effect may be present. The wide degree of variation observed in area under the curve (AUC) suggests that the investigation of population pharmacokinetic modelling is warranted.

The Association Between Diabetes and Olfactory Function in Adults.

Diabetes is a significant chronic disease that in limited studies has been linked with olfactory dysfunction. We investigated the cross-sectional association between diabetes and olfactory dysfunction in 3151 adults aged ≥40 years who participated in US National Health and Nutrition Examination Survey 2013-2014 with information on olfactory dysfunction and diabetes. Diabetes was defined from fasting serum glucose ≥126 mg/dL, oral glucose tolerance test ≥200 mg/dL, HbA1c levels ≥6.5%, physician-diagnosed diabetes, or current use of oral hypoglycemic agents and/or insulin. Self-reported olfactory dysfunction was defined as a positive answer to any of the following questions: 1) "Have you had problem with smell in the past 12 months?"; 2) "Have you had a change in the ability to smell since age 25?", or 3) "Do you have phantom smells?". Participants were considered to have severe hyposmia or anosmia if they had <5 correct answers in the 8-item pocket smell test. Analyses were adjusted for the main confounders, including olfactory dysfunction risk factors. Compared to non-diabetics, diabetics under insulin treatment showed a higher prevalence of phantom odors [OR(95% CI): 2.42 (1.16; 5.06)] and a non-significant higher prevalence of severe hyposmia/anosmia [OR(95% CI): 1.57 (0.89; 2.78)]. Amongst diabetics, there was a significant trend to severe hyposmia/anosmia for those on more aggressive treatments [OR (95% CI) including oral and insulin treatment compared to those who reported no use of drug treatment, respectively: 1.33 (0.60; 2.96) and 2.86 (1.28; 6.40); P trend 0.01]. No association was observed between diabetes duration and prevalence of olfactory dysfunction.

Quality of life following fistulotomy - short term follow-up.

AIM: Anal fistula causes pain and discharge of pus and blood. Treatment by fistulotomy has the highest success, but can risk continence; treatment needs to balance cure with continence. This study assessed the impact of fistulotomy on quality of life (QOL) and continence. METHOD: Patients selected for fistulotomy prospectively completed the St Mark's Continence Score (full incontinence = 24) and Short Form-36 questionnaires preoperatively at two institutions with an interest in anal fistula. Patients were reassessed 3 months' postoperatively. RESULTS: There were 52 patients with a median age of 44 (range 19-82) years; 10 were women. Preoperative continence scores were median 0 (range 0-23) and there was no significant difference compared with postoperative scores (median 1, range 0-24). Following fistulotomy QOL was significantly improved in four of eight domains - Bodily Pain (P < 0.001), Vitality (P < 0.01), Social Functioning (P < 0.05) and Mental Health (P < 0.001) - and returned to that of the general population. QOL for patients with intersphincteric fistula improved postfistulotomy, and for those with trans-sphincteric fistula it remained the same. Data were further examined in two groups, with and without deterioration in continence score. Where continence improved postoperatively, QOL improved in three domains; where continence deteriorated QOL improved in two domains (P < 0.05). Patients with postoperative continence scores of < 5 had worse QOL than those scoring 4 or less. CONCLUSION: QOL significantly improved at 3 months' follow-up after fistulotomy where continence was maintained or a small reduction occurred.

Neuronal mechanism for acute mechanosensitivity in tactile-foraging waterfowl.

Relying almost exclusively on their acute sense of touch, tactile-foraging birds can feed in murky water, but the cellular mechanism is unknown. Mechanical stimuli activate specialized cutaneous end organs in the bill, innervated by trigeminal afferents. We report that trigeminal ganglia (TG) of domestic and wild tactile-foraging ducks exhibit numerical expansion of large-diameter mechanoreceptive neurons expressing the mechano-gated ion channel Piezo2. These features are not found in visually foraging birds. Moreover, in the duck, the expansion of mechanoreceptors occurs at the expense of thermosensors. Direct mechanical stimulation of duck TG neurons evokes high-amplitude depolarizing current with a low threshold of activation, high signal amplification gain, and slow kinetics of inactivation. Together, these factors contribute to efficient conversion of light mechanical stimuli into neuronal excitation. Our results reveal an evolutionary strategy to hone tactile perception in vertebrates at the level of primary afferents.

Ecological guild and enzyme activities of rhizosphere soil microbial communities associated with Bt-maize cultivation under field conditions in North West Province of South Africa.

Insecticidal proteins expressed by genetically modified Bt maize may alter the enzymatic and microbial communities associated with rhizosphere soil. This study investigated the structure and enzymatic activity of rhizosphere soil microbial communities associated with field grown Bt and non-Bt maize. Rhizosphere soil samples were collected from Bt and non-Bt fields under dryland and irrigated conditions. Samples were subjected to chemical tests, enzyme analyses, and next generation sequencing. Results showed that nitrate and phosphorus concentrations were significantly higher in non-Bt maize dryland soils, while organic carbon was significantly higher in non-Bt maize irrigated field soil. Acid phosphatase and ß-glucosidase activities were significantly reduced in soils under Bt maize cultivation. The species diversity differed between fields and Bt and non-Bt maize soils. Results revealed that Actinobacteria, Proteobacteria, and Acidobacteria were the dominant phyla present in these soils. Redundancy analyses indicated that some chemical properties and enzyme activities could explain differences in bacterial community structures. Variances existed in microbial community structures between Bt and non-Bt maize fields. There were also differences between the chemical and biochemical properties of rhizosphere soils under Bt and non-Bt maize cultivation. These differences could be related to agricultural practices and cultivar type.

Pharmacokinetics of grapiprant, a selective EP4 prostaglandin PGE2 receptor antagonist, after 2 mg/kg oral and i.v. administrations in cats.

Drugs that provide effective analgesia in cats are limited. The aim of the study was to assess the pharmacokinetics of grapiprant after 2 mg/kg administration via p.o. and i.v. routes in cats. Six healthy adult cats were used according to an open, single-dose, two-treatment, two-period, randomized cross-over design. Cats were assigned to two treatment groups and administered with 2 mg/kg of grapiprant (pure powder) through p.o. and i.v. administration. Blood samples were collected at preassigned times and analysed by a validated HPLC method. After both administrations, grapiprant concentrations were detectable in plasma for up to 24 hr in five of six animals. The critical parameters including clearance (173.2 ml hr-1  kg-1 , range 120-326 ml hr-1  kg-1 ) and volume of distribution (918 ml/kg, range 611-1608 ml/kg) were calculated from the i.v. group. The mean oral F% was low (39.6% range 31.5%-45.2%). If the assumption that the minimal effective concentration in dogs (164 ng/ml) applies in cats too, grapiprant orally administered at 2 mg/kg might be effective for 10 hr. Further studies are necessary to establish the minimal effective concentration in this animal species.

Pharmacokinetic/pharmacodynamic evaluation of grapiprant in a carrageenan-induced inflammatory pain model in the rabbit.

Grapiprant is the novel selective EP4 receptor inhibitor recently issued on the veterinary market for dogs affected by osteoarthritis. The aim of this study was twofold: to evaluate the pharmacokinetics and the pharmacodynamics of grapiprant in the induced inflammatory pain model in the rabbit after a single IV injection of 2 mg/kg; to compare the thermal antinociception effect after 2 mg/kg IV grapiprant, with that generated by 0.5 mg/kg meloxicam SC injected. Rabbits (n = 12) were randomly assigned to two crossover studies (single-dose, two-period crossover). The first study group A (n = 3) received a single IV dose of grapiprant at 2 mg/kg dissolved in ethanol. Group B (n = 3) received a single IV injection of ethanol (equivalent volume to grapiprant volume) at the same site. The second study group C (n = 3) received a single SC dose of meloxicam at 0.5 mg/kg. Group D (n = 3) received a single SC injection of 15% ethanol (equivalent volume to grapiprant volume) at the same site. After a 2-week washout period, the groups were rotated and the experiments repeated. Blood samples (0.7 mL) were collected from the right ear artery at assigned times and grapiprant plasma concentrations determined by a validated HPLC-FL method. Three hours prior to administration of the drugs, inflammation was induced by SC injection of lambda carrageenan (200 µL, 3% in physiological saline) under the plantar surface of the right hind paw. At a similar time to the blood collection, an infrared thermal stimuli (40 °C) was applied to the plantar surface of the rabbits' hindlimbs to evaluate the thermal withdrawal latency (TWL). The thermal antinociceptive effect was expressed as maximum possible response (% MPR). Grapiprant plasma concentrations were detectable up to the 10-h time point (concentration range 17-7495 ng/mL). The grapiprant-treated group showed a significant increase in TWL from 1 h and up to 10 h after drug administration compared to the control. In contrast, the meloxicam group showed a significant increase in TWL from 4 up to 10 h after drug administration, compared to control. The maximal MPR% was not statistically different between the grapiprant and meloxicam group from 4 to 8 h, while significant differences were shown at 1, 1.5, 2, 10 and 24 h. Given these findings, grapiprant appears to be an attractive option for antinociception in rabbits, due to its rapid onset and extended duration of effect.

Pharmacokinetics and estimated bioavailability of grapiprant, a novel selective prostaglandin E2 receptor antagonist, after oral administration in fasted and fed dogs.

AIMS: To assess the effect of food intake on the pharmacokinetics of grapiprant administered orally at 2 mg/kg, and to estimate its oral bioavailability in dogs. METHODS: Eight healthy female Labrador Retriever dogs, aged 4-10 years were used. In the initial trial two dogs were administered a 0.5 mg/kg I/V bolus of grapiprant dissolved in ethanol. In the second trial, six dogs were assigned to two treatment groups, using a randomised cross-over design, and received 2 mg/kg of grapiprant orally, as pure powder, after fasting for 12 hours or after being fed. Blood samples were collected at preassigned times up to 36 hours after administration, and concentrations of grapiprant in plasma determined using validated high performance liquid chromatography. RESULTS: After I/V administration in the two dogs the terminal half life was 5.30 and 6.06 hours, clearance was 444 and 476 mL/hours/kg, and volume of distribution was 3,642 and 3,883 mL/kg. Compared with fasted dogs, oral administration in fed dogs resulted in reduced median peak concentrations in plasma (1,598 vs. 614 ng/mL) and delayed median time of peak concentration (1.0 vs. 3.0 hours). The estimated bioavailability in fasted and fed dogs was 111.9 and 59.1%, respectively. Concentrations of grapiprant in plasma following oral administration, in either fed or fasted dogs, remained higher than 164 ng/mL for up to 6 hours. This concentration has been estimated to be the minimal effective concentration required to control pain in dogs. CONCLUSION AND RELEVANCE: Oral administration of 2 mg/kg grapiprant in fed and fasted dogs resulted in different pharmacokinetics of the drug, but did not influence the length of time when concentrations in plasma exceeded theoretical effective concentrations. Further studies are necessary to verify these findings using pharmacokinetic-pharmacodynamic studies and in clinical subjects.