Urban living can rescue Darwin's finches from the lethal effects of invasive vampire flies.

Human activity changes multiple factors in the environment, which can have positive or negative synergistic effects on organisms. However, few studies have explored the causal effects of multiple anthropogenic factors, such as urbanization and invasive species, on animals and the mechanisms that mediate these interactions. This study examines the influence of urbanization on the detrimental effect of invasive avian vampire flies (Philornis downsi) on endemic Darwin's finches in the Galápagos Islands. We experimentally manipulated nest fly abundance in urban and non-urban locations and then characterized nestling health, fledging success, diet, and gene expression patterns related to host defense. Fledging success of non-parasitized nestlings from urban (79%) and non-urban (75%) nests did not differ significantly. However, parasitized, non-urban nestlings lost more blood, and fewer nestlings survived (8%) compared to urban nestlings (50%). Stable isotopic values (δ15 N) from urban nestling feces were higher than those from non-urban nestlings, suggesting that urban nestlings are consuming more protein. δ15 N values correlated negatively with parasite abundance, which suggests that diet might influence host defenses (e.g., tolerance and resistance). Parasitized, urban nestlings differentially expressed genes within pathways associated with red blood cell production (tolerance) and pro-inflammatory response (innate immunological resistance), compared to parasitized, non-urban nestlings. In contrast, parasitized non-urban nestlings differentially expressed genes within pathways associated with immunoglobulin production (adaptive immunological resistance). Our results suggest that urban nestlings are investing more in pro-inflammatory responses to resist parasites but also recovering more blood cells to tolerate blood loss. Although non-urban nestlings are mounting an adaptive immune response, it is likely a last effort by the immune system rather than an effective defense against avian vampire flies since few nestlings survived.

Hepatocyte ploidy in cats with and without hepatocellular carcinoma.

BACKGROUND: Domestic cats rarely develop hepatocellular carcinoma. The reason for the low prevalence is unknown. Reductions in hepatocellular ploidy have been associated with hepatic carcinogenesis. Recent work in mice has shown that livers with more polyploid hepatocytes are protected against the development of hepatocellular carcinoma. Hepatocyte ploidy in the domestic cat has not been evaluated. We hypothesized that ploidy would be reduced in peri-tumoral and neoplastic hepatocytes compared to normal feline hepatocytes. Using integrated fluorescence microscopy, we quantified the spectra of ploidy in hepatocellular carcinoma and healthy control tissue from paraffin embedded tissue sections. RESULTS: Feline hepatocytes are predominantly mononuclear and the number of nuclei per hepatocyte did not differ significantly between groups. Normal cats have a greater number of tetraploid hepatocytes than cats with hepatocellular carcinoma. CONCLUSIONS: Total hepatocellular polyploidy in normal cat liver is consistent with values reported in humans, yet cellular ploidy (nuclei per cell) is greater in humans than in cats. Tetraploid cat hepatocytes are predominantly mononuclear.

Approach to the Diagnosis of Hepatocutaneous Syndrome in Dogs: A Retrospective Study and Literature Review.

Superficial necrolytic dermatitis (SND) is a rare and often fatal disease in dogs that has been associated with pancreatic neuroendocrine neoplasia (SND/EN) and hepatocutaneous syndrome (SND/HCS). Although various combinations of diagnostics have been used to differentiate these two causes of SND, there are currently no data on which combination would enable the most timely and noninvasive way to diagnose HCS. Medical records were reviewed retrospectively (2004-2018) for dogs with SND/HCS (n = 24) and SND/EN (n = 1). These data were compared with cases found by review of the literature of dogs with SND/HCS (n = 105) and SND/EN (n = 13). The most consistent findings with SND were dermatological lesions affecting paw pads or mucocutaneous junctions (143/143, 100%) and marked plasma hypoaminoacidemia (58/58, 100%). On ultrasound, a honeycomb liver was seen in 62/63 (98%) dogs with SND/HCS but none with SND/EN. Six out of 23 (26%) dogs in the retrospective study with SND/HCS had marked keratinocyte apoptosis, a finding that was associated with diabetes mellitus. This study suggests that in dogs with characteristic skin lesions, an amino acid profile permits a noninvasive diagnosis of SND. An abdominal ultrasound can then assist in the differentiation of SND/HCS and SND/EN.

Hydrophobic bile acid apoptosis is regulated by sphingosine-1-phosphate receptor 2 in rat hepatocytes and human hepatocellular carcinoma cells.

The hepatotoxic bile acid glycochenodeoxycholate (GCDC) modulates hepatocyte cell death through activation of JNK, Akt, and Erk. The nonhepatotoxic bile acid taurocholate activates Akt and Erk through the sphingosine-1-phosphate receptor 2 (S1PR2). The role of the S1PR2 in GCDC-mediated apoptosis and kinase activation is unknown. Studies were done in rat hepatocytes, HUH7 cells, and HUH7 cells stably transfected with rat Ntcp (HUH7-Ntcp). Cells were treated with GCDC and apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for the active cleaved fragment of caspase 3. Kinase activation was determined by immunoblotting with phospho-specific antibodies. JTE-013, an inhibitor of S1PR2, significantly attenuated morphological evidence of GCDC-induced apoptosis and prevented caspase 3 cleavage in rat hepatocytes and HUH7-Ntcp cells. In hepatocytes, JTE-013 mildly suppressed, augmented, and had no effect on GCDC-induced JNK, Akt, and Erk phosphorylation, respectively. Similar results were seen in HUH7-Ntcp cells except for mild suppression of JNK and Erk phosphorylation. Knockdown of S1PR2 in HUH7-Ntcp augmented Akt, inhibited JNK, and had no effect on Erk phosphorylation. GCDC failed to induce apoptosis or kinase activation in HUH7 cells. In conclusion, SIPR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes to mediate cell death or activate kinases. These results suggest that SIPR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid.

p38 MAPK α and ß isoforms differentially regulate plasma membrane localization of MRP2.

In hepatocytes, cAMP both activates p38 mitogen-activated protein kinase (MAPK) and increases the amount of multidrug resistance-associated protein-2 (MRP2) in the plasma membrane (PM-MRP2). Paradoxically, taurolithocholate (TLC) activates p38 MAPK but decreases PM-MRP2 in hepatocytes. These opposing effects of cAMP and TLC could be mediated via different p38 MAPK isoforms (α and ß) that are activated differentially by upstream kinases (MKK3, MKK4, and MKK6). Thus we tested the hypothesis that p38α MAPK and p38ß MAPK mediate increases and decreases in PM-MRP2 by cAMP and TLC, respectively. Studies were conducted in hepatocytes isolated from C57BL/6 wild-type (WT) and MKK3-knockout (MKK3(-/-)) mice and in a hepatoma cell line (HuH7) that overexpresses sodium-taurocholate cotransporting polypeptide (NTCP) (HuH-NTCP). Cyclic AMP activated MKK3, p38 MAPK, and p38α MAPK and increased PM-MRP2 in WT hepatocytes, but failed to activate p38α MAPK or increase PM-MRP2 in MKK3(-/-) hepatocytes. In contrast to cAMP, TLC activated total p38 MAPK but decreased PM-MRP2, and did not activate MKK3 or p38α MAPK in WT hepatocytes. In MKK3(-/-) hepatocytes, TLC still decreased PM-MRP2 and activated p38 MAPK, indicating that these effects are not MKK3-dependent. Additionally, TLC activated MKK6 in MKK3(-/-) hepatocytes, and small interfering RNA knockdown of p38ß MAPK abrogated TLC-mediated decreases in PM-MRP2 in HuH-NTCP cells. Taken together, these results suggest that p38α MAPK facilitates plasma membrane insertion of MRP2 by cAMP, whereas p38ß MAPK mediates retrieval of PM-MRP2 by TLC.

Replication of a whole school ethos-changing intervention: different context, similar effects, additional insights.

BACKGROUND: Whole school, ethos-changing interventions reduce risk behaviours in middle adolescence, more than curriculum-based approaches. Effects on older ages are not known. We set out to replicate one of these interventions, Australia's Gatehouse Project, in a rural Canadian high school. METHODS: A guided, whole school change process sought to make students feel more safe, connected, and valued by: changes in teaching practices, orientation processes, professional development of staff, recognition and reward mechanisms, elevating student voice, and strategies to involve greater proactivity and participation. We conducted risk behaviour surveys in grades 10 to 12 before the intervention and 2 years afterwards, and social network analyses with the staff. Changes in health and health risk behaviours were assessed using chi-square. Interactions between the intervention and gender and between the intervention and school engagement were assessed using interaction terms in logistic regression models. Changes in the density of relationships among staff were tested with methods analogous to paired t-tests. RESULTS: Like Gatehouse, there was no statistically significant reduction in depressive symptoms or bullying, though the trend was in that direction. Among girls, there was a statistically significant decrease in low school engagement (45% relative reduction), and decreases in drinking (46% relative reduction), unprotected sex (61% relative reduction) and poor health (relative reduction of 73%). The reduction in drinking matched the national trend. Reductions in unprotected sex and poor health went against the national trend. We found no statistically significant changes for boys. The effects coincided with statistically significant increases in the densities of staff networks, indicating that part of the mechanism may be through relationships at school. CONCLUSIONS: A non-specific, risk protective intervention in the social environment of the school had a significant impact on a cluster of risk behaviours for girls. Results were remarkably like reports from similar school environment interventions elsewhere, albeit with different behaviours being affected. It may be that this type of intervention activates change processes that interact highly with context, impacting different risks differently, according to the prevalence, salience and distribution of the risk and the interconnectivity of relationships between staff and students. This requires further exploration.

Protein kinase Cδ protects against bile acid apoptosis by suppressing proapoptotic JNK and BIM pathways in human and rat hepatocytes.

Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.

Rab11, but not Rab4, facilitates cyclic AMP- and tauroursodeoxycholate-induced MRP2 translocation to the plasma membrane.

Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking. Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively. Tauroursodeoxycholate (TUDC) and cAMP increase bile formation, in part, by increasing plasma membrane localization of multidrug resistance-associated protein 2 (MRP2). The goal of the present study was to determine the role of these Rab proteins in the trafficking of MRP2 by testing the hypothesis that Rab11 and/or Rab4 facilitate cAMP- and TUDC-induced MRP2 translocation to the plasma membrane. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which constitutively express MRP2. HuH-NTCP cells were transfected with Rab11-WT and GDP-locked dominant inactive Rab11-GDP or with Rab4-GDP to study the role of Rab11 and Rab4. A biotinylation method and a GTP overlay assay were used to determine plasma membrane MRP2 and activation of Rab proteins (Rab11 and Rab4), respectively. Cyclic AMP and TUDC increased plasma membrane MRP2 and stimulated Rab11 activity. Plasma membrane translocation of MRP2 by cAMP and TUDC was increased and inhibited in cells transfected with Rab11-WT and Rab11-GDP, respectively. Cyclic AMP (previous study) and TUDC increased Rab4 activity. However, cAMP- and TUDC-induced increases in MRP2 were not inhibited by Rab4-GDP. Taken together, these results suggest that Rab11 is involved in cAMP- and TUDC-induced MRP2 translocation to the plasma membrane.

Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells.

UNLABELLED: Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. CONCLUSION: Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.

Characterization of clinical presentation, histological features, ultrasonographic findings, and survival in 29 dogs with granulomatous hepatitis.

BACKGROUND: Granulomatous hepatitis (GH) is a form of chronic hepatitis (CH) in dogs for which limited information is published. HYPOTHESIS: Describe the clinical presentation, clinical pathology, ultrasound, and hepatic histopathology findings and to report survival times in dogs with GH. ANIMALS: Twenty-nine client-owned dogs with GH. METHODS: Retrospective observational study. Pathology records were searched. Inclusion criteria included a histopathologic diagnosis of GH, absence of an identified etiology or evidence of extrahepatic granulomatous disease, and a medical record available for review. Clinical presentation, clinical pathologic findings, treatment protocols, and survival times were recorded. Available hepatic biopsy material was graded and scored, and ultrasound evaluations reviewed. RESULTS: The median age was 7 years (range, 0.66-12 years). Nineteen breeds were represented. Decreased appetite (19/29), lethargy (16/29), and fever (13/29) were seen most commonly. All dogs had increased serum transaminase activities, whereas 21/29 and 12/24 had hyperbilirubinemia and neutrophilia, respectively. Ultrasonographic findings included hepatomegaly (12/22), nodular parenchymal lesions (9/22), and hyperechoic parenchymal bands (8/22). Histopathologic necroinflammatory scores were moderate to severe in 16/19 dogs, and fibrosis scores were mild in 14/19 dogs. Treatments varied and included antibiotics, immunosuppressive drugs, and hepatoprotectants. Overall median survival was 635 days (range, 1-2482 days). CONCLUSION AND CLINICAL IMPORTANCE: Granulomatous hepatitis in dogs is associated with high histopathologic grade, fever, neutrophilia, and a high incidence of hepatomegaly and focal parenchymal lesions on ultrasound examination. Despite disease severity on presentation, dogs with GH can have a good outcome with prolonged survival.

Chromosome-Level Genome Assembly and Annotation of a Periodical Cicada Species: Magicicada septendecula.

We present a high-quality assembly and annotation of the periodical cicada species, Magicicada septendecula (Hemiptera: Auchenorrhyncha: Cicadidae). Periodical cicadas have a significant ecological impact, serving as a food source for many mammals, reptiles, and birds. Magicicada are well known for their massive emergences of 1 to 3 species that appear in different locations in the eastern United States nearly every year. These year classes ("broods") emerge dependably every 13 or 17 yr in a given location. Recently, it has become clear that 4-yr early or late emergences of a sizeable portion of a population are an important part of the history of brood formation; however, the biological mechanisms by which they track the passage of time remain a mystery. Using PacBio HiFi reads in conjunction with Hi-C proximity ligation data, we have assembled and annotated the first whole genome for a periodical cicada, an important resource for future phylogenetic and comparative genomic analysis. This also represents the first quality genome assembly and annotation for the Hemipteran superfamily Cicadoidea. With a scaffold N50 of 518.9 Mb and a complete BUSCO score of 96.7%, we are confident that this assembly will serve as a vital resource toward uncovering the genomic basis of periodical cicadas' long, synchronized life cycles and will provide a robust framework for further investigations into these insects.

Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Juglans cinerea).

With the advent of affordable and more accurate third-generation sequencing technologies, and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea, commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100 × coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea's recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

Unveiling the Genetic Blueprint of a Desert Scorpion: A Chromosome-level Genome of Hadrurus arizonensis Provides the First Reference for Parvorder Iurida.

Over 400 million years old, scorpions represent an ancient group of arachnids and one of the first animals to adapt to life on land. Presently, the lack of available genomes within scorpions hinders research on their evolution. This study leverages ultralong nanopore sequencing and Pore-C to generate the first chromosome-level assembly and annotation for the desert hairy scorpion, Hadrurus arizonensis. The assembled genome is 2.23 Gb in size with an N50 of 280 Mb. Pore-C scaffolding reoriented 99.6% of bases into nine chromosomes and BUSCO identified 998 (98.6%) complete arthropod single copy orthologs. Repetitive elements represent 54.69% of the assembled bases, including 872,874 (29.39%) LINE elements. A total of 18,996 protein-coding genes and 75,256 transcripts were predicted, and extracted protein sequences yielded a BUSCO score of 97.2%. This is the first genome assembled and annotated within the family Hadruridae, representing a crucial resource for closing gaps in genomic knowledge of scorpions, resolving arachnid phylogeny, and advancing studies in comparative and functional genomics.

Abdominal ultrasonographic findings of cats with feline infectious peritonitis: an update.

OBJECTIVES: The aim of this study was to describe the abdominal ultrasonographic findings in cats with confirmed or presumed feline infectious peritonitis (FIP). METHODS: This was a retrospective study performed in an academic veterinary hospital. The diagnosis of FIP was reached on review of history, signalment, clinical presentation, complete blood count, biochemistry panel, peritoneal fluid analysis, cytology and/or histopathology results from abnormal organs, and/or molecular testing (immunohistochemical or FIP coronavirus [FCoV] RT-PCR). Cats with confirmed FIP by molecular testing or with a highly suspicious diagnosis of FIP were included. Abdominal ultrasound examination findings were reviewed. RESULTS: In total, 25 cats were included. Common clinical signs/pathology findings included hyperglobulinemia (96%), anorexia/hyporexia (80%) and lethargy (56%). Abdominal ultrasound findings included effusion in 88% and lymphadenopathy in 80%. Hepatic changes were noted in 80%, the most common being hepatomegaly (58%) and a hypoechoic liver (48%). Intestinal changes were noted in 68% of cats, characterized by asymmetric wall thickening and/or loss of wall layering, with 52% being ileocecocolic junction and/or colonic in location. Splenic changes were present in 36% of cats, including splenomegaly, mottled parenchyma and hypoechoic nodules. Renal changes were present in 32%, encompassing a hypoechoic subcapsular rim and/or cortical nodules. Mesenteric and peritoneal abnormalities were seen in 28% and 16% of cats, respectively. Most cats (92%) had two or more locations of abdominal abnormalities on ultrasound. CONCLUSIONS AND RELEVANCE: The present study documents a wider range and distribution of ultrasonographic lesions in cats with FIP than previously reported. The presence of effusion and lymph node, hepatic and/or gastrointestinal tract changes were the most common findings, and most of the cats had a combination of two or more abdominal abnormalities.

Use of short videos and case studies to enhance student confidence in biochemistry knowledge and application in a large lecture biochemistry course in first year veterinary curriculum.

Large lecture courses are an efficient way to convey material to many students but have potential limitations, most notably the tendency for them to promote passive learning opportunities rather than active pedagogies. The curriculum at Cummings School of Veterinary Medicine at Tufts University, like many veterinary schools, contains many large lecture courses in the pre-clinical curriculum. This objective of this study was to use two active pedagogical interventions in a first-year lecture course named Veterinary Biochemistry and Metabolism that drew connections between basic science and several veterinary diseases. The first intervention targeted increasing students' intrinsic motivation and their confidence with understanding biochemistry concepts using videos created via collaborations between students, staff, and clinical and basic science faculty. The second intervention targeted active and collaborative learning via the implementation of clinical case studies completed in groups to relate lecture content to clinical scenarios with the aim of further enhancing student confidence in their knowledge of the material. To assess the effectiveness of these two interventions, pre-and post-course surveys using Likert style questions were administered to evaluate student confidence in the targeted concepts. The post-survey included open-ended responses on students' perspectives on their most important takeaways from the activities and their suggestions for improvements. The data showed a positive impact of these interventions on student motivation and confidence in their knowledge. This study provides support that targeted interventions to increase active learning strategies increase student engagement and may improve learning efficacy in large lecture courses.

Welcome to the big leaves: Best practices for improving genome annotation in non-model plant genomes.

Premise: Robust standards to evaluate quality and completeness are lacking in eukaryotic structural genome annotation, as genome annotation software is developed using model organisms and typically lacks benchmarking to comprehensively evaluate the quality and accuracy of the final predictions. The annotation of plant genomes is particularly challenging due to their large sizes, abundant transposable elements, and variable ploidies. This study investigates the impact of genome quality, complexity, sequence read input, and method on protein-coding gene predictions. Methods: The impact of repeat masking, long-read and short-read inputs, and de novo and genome-guided protein evidence was examined in the context of the popular BRAKER and MAKER workflows for five plant genomes. The annotations were benchmarked for structural traits and sequence similarity. Results: Benchmarks that reflect gene structures, reciprocal similarity search alignments, and mono-exonic/multi-exonic gene counts provide a more complete view of annotation accuracy. Transcripts derived from RNA-read alignments alone are not sufficient for genome annotation. Gene prediction workflows that combine evidence-based and ab initio approaches are recommended, and a combination of short and long reads can improve genome annotation. Adding protein evidence from de novo assemblies, genome-guided transcriptome assemblies, or full-length proteins from OrthoDB generates more putative false positives as implemented in the current workflows. Post-processing with functional and structural filters is highly recommended. Discussion: While the annotation of non-model plant genomes remains complex, this study provides recommendations for inputs and methodological approaches. We discuss a set of best practices to generate an optimal plant genome annotation and present a more robust set of metrics to evaluate the resulting predictions.

Protein kinase Cδ differentially regulates cAMP-dependent translocation of NTCP and MRP2 to the plasma membrane.

Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.

Serum 25-hydroxyvitamin D and C-reactive protein and plasma von Willebrand concentrations in 23 dogs with chronic hepatopathies.

BACKGROUND: Serum concentrations of 25-hydroxyvitamin D (25(OH)VD) and C-reactive protein (CRP) and von Willebrand's factor (vWF) concentration correlate with histopathologic disease grade and stage in chronic inflammatory and fibrotic hepatopathies (CH) in humans. OBJECTIVES: To evaluate serum 25(OH)VD and serum CRP concentrations and plasma vWF concentration and determine if they correlate with histopathologic and biochemical variables in dog with CH. ANIMALS: Twenty-three client-owned dogs with a histopathologic diagnosis of CH were prospectively enrolled. METHODS: Blood samples were collected before liver biopsy. Correlations between biomarkers and clinical pathological and histopathologic variables were evaluated using Pearson's or Spearman's test. RESULTS: Serum 25(OH)VD concentration (median, 213 nmol/L; range, 42-527 nmol/L) was negatively correlated with serum aspartate aminotransferase activity (AST; rho = -0.59, P < .01), polymorphonuclear neutrophil count (PMN; r = -0.46, P < .05), and positively correlated with serum albumin concentration (r = 0.69, P < .001). Serum CRP concentration (median, 7.4 µg/L; range, 1-44.9 µg/L) was positively correlated with overall histopathologic necroinflammatory activity (r = 0.78, P < .001) and fibrosis score (rho = 0.49, P < .05). Plasma vWF concentration (median, 73.3%; range, 15-141%) was positively correlated with fibrosis score (r = 0.53, P < .05) and prothrombin time (rho = 0.67, P < .01), and negatively correlated with serum albumin concentration (r = -0.73, P < .001). CONCLUSION AND CLINICAL IMPORTANCE: In dogs with CH, serum 25(OH)VD concentration was negatively correlated with disease activity, whereas serum CRP concentration and plasma vWF concentration were positively correlated with histopathologic grade and stage. Our results provide preliminary evidence that these biomarkers may be useful to assess grade and stage of CH in dogs in the absence of liver biopsy.

Pre-operative Hemostatic Status in Dogs Undergoing Splenectomy for Splenic Masses.

Portal system thrombosis is a rare but potentially fatal complication of splenectomy in dogs. The mechanism behind development of post-operative portal system thrombosis is unclear but may include alterations of portal blood flow following surgery, acquired hypercoagulability and endothelial dysfunction. The aim of the study was to evaluate hemostatic biomarkers in hemodynamically stable (heart rate <130 beats/min, blood lactate < 2.5 mMol/L) and non-anemic (hematocrit >35%) dogs prior to splenectomy for splenic masses. Our hypothesis was that this population of stable dogs would have pre-existing laboratory evidence of hypercoagulability unrelated to shock, bleeding, anemia, or other pre-operative comorbidities. Pre-operatively, abdominal ultrasonography was performed and blood was collected for platelet enumeration, prothrombin time (PT), activated partial thromboplastin time (aPTT), kaolin-activated thromboelastography (TEG), fibrinogen, von Willebrand factor activity (vWF:Ag), antithrombin and thrombin-antithrombin complex (TAT). Histopathological diagnosis and 30-day survival were recorded. None of the 15 enrolled dogs had pre-operative sonographic evidence of portal system thrombosis. Three of fifteen dogs were thrombocytopenic, three had thrombocytosis, three were hyperfibrinogenemic, one had low vWF:Ag, three had mild prolongations of PT and none had abnormal aPTT. Based on the TEG G value, 13/15 dogs were hypercoagulable (mean ± SD 13.5 ± 5.4 kd/s). Antithrombin deficiency was identified in 9/15 dogs (mean ± SD 68.7 ± 22.7%) with 5/9 having concurrently elevated TAT suggesting active thrombin generation. No dogs developed portal system thrombosis and all achieved 30-day survival. Pre-operative hypercoagulability was recognized commonly but its association with post-operative thrombosis remains undetermined.

Gastrointestinal signs and a need for nutritional management may persist long term in dogs and cats undergoing resection of the ileocolic junction: 35 cases (2008-2020).

OBJECTIVE: To evaluate the effects of ileocecocolic junction (ICJ) resection on gastrointestinal signs, biochemical parameters, and nutritional variables in dogs and cats. ANIMALS: 20 dogs and 15 cats that underwent ICJ resection between January 2008 and June 2020. PROCEDURES: Medical records of dogs and cats that underwent ICJ resection were reviewed, and clinical signs, laboratory abnormalities, and nutritional information were obtained. Additional follow-up information was obtained by contacting primary care veterinarians or owners. A subset of dogs (n = 6) and cats (2) were evaluated in the hospital via clinical examination, clinicopathologic testing, nutritional testing, and abdominal ultrasound. RESULTS: Twenty dogs and 15 cats underwent resection of the ICJ for treatment of a variety of conditions. Ten of 20 dogs (50%) and 11/15 cats (73%) were reported by their owners to have a good long-term outcome based on the lack of long-term gastrointestinal signs or the ability to control gastrointestinal signs with diet and supplements alone. Despite owner-reported good outcomes, long-term diarrhea, weight loss, and muscle loss were common. Of the 6 dogs evaluated in the hospital, 3/6 (50%) had muscle loss, 2/6 (33%) had low taurine concentrations, and 1 dog each had low cobalamin, folate, 25-hydroxyvitamin D, and ionized calcium. Neither of the 2 cats evaluated in the hospital had nutritional abnormalities identified. CLINICAL RELEVANCE: Owners should be informed of the possibility of long-term gastrointestinal clinical signs and the potential need for long-term nutritional management after ICJ resection.