Chromosome-level genome assembly of the European green woodpecker Picus viridis.

The European green woodpecker, Picus viridis, is a widely distributed species found in the Western Palearctic region. Here, we assembled a highly contiguous genome assembly for this species using a combination of short- and long-read sequencing and scaffolded with chromatin conformation capture (Hi-C). The final genome assembly was 1.28 Gb and features a scaffold N50 of 37 Mb and a scaffold L50 of 39.165 Mb. The assembly incorporates 89.4% of the genes identified in birds in OrthoDB. Gene and repetitive content annotation on the assembly detected 15,805 genes and a ∼30.1% occurrence of repetitive elements, respectively. Analysis of synteny demonstrates the fragmented nature of the P. viridis genome when compared to the chicken (Gallus gallus). The assembly and annotations produced in this study will certainly help for further research into the genomics of P. viridis and the comparative evolution of woodpeckers. Five historical and seven contemporary samples have been resequenced and may give insights on the population history of this species.

The Crown Pearl V2: an improved genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758).

Contiguous assemblies are fundamental to deciphering the composition of extant genomes. In molluscs, this is considerably challenging owing to the large size of their genomes, heterozygosity, and widespread repetitive content. Consequently, long-read sequencing technologies are fundamental for high contiguity and quality. The first genome assembly of Margaritifera margaritifera (Linnaeus, 1758) (Mollusca: Bivalvia: Unionida), a culturally relevant, widespread, and highly threatened species of freshwater mussels, was recently generated. However, the resulting genome is highly fragmented since the assembly relied on short-read approaches. Here, an improved reference genome assembly was generated using a combination of PacBio CLR long reads and Illumina paired-end short reads. This genome assembly is 2.4 Gb long, organized into 1,700 scaffolds with a contig N50 length of 3.4 Mbp. The ab initio gene prediction resulted in 48,314 protein-coding genes. Our new assembly is a substantial improvement and an essential resource for studying this species' unique biological and evolutionary features, helping promote its conservation.

IT/QA and Regulatory Aspects of Digital Pathology: Results of the 8th ESTP International Workshop.

Digital toxicologic histopathology has been broadly adopted in preclinical compound development for informal consultation and peer review. There is now increased interest in implementing the technology for good laboratory practice-regulated study evaluations. However, the implementation is not straightforward because systems and work processes require qualification and validation, with consideration also given to security. As a result of the high-throughput, high-volume nature of safety evaluations, computer performance, ergonomics, efficiency, and integration with laboratory information management systems are further key considerations. The European Society of Toxicologic Pathology organized an international expert workshop with participation by toxicologic pathologists, quality assurance/regulatory experts, and information technology experts to discuss qualification and validation of digital histopathology systems in a good laboratory practice environment, and to share the resulting conclusions broadly in the toxicologic pathology community.

Scientific and Regulatory Policy Committee Points to Consider: Primary Digital Histopathology Evaluation and Peer Review for Good Laboratory Practice (GLP) Nonclinical Toxicology Studies.

The Society of Toxicologic Pathology's Scientific and Regulatory Policy Committee formed a working group to consider the present and future use of digital pathology in toxicologic pathology in general and specifically its use in primary evaluation and peer review in Good Laboratory Practice (GLP) environments. Digital histopathology systems can save costs by reducing travel, enhancing organizational flexibility, decreasing slide handling, improving collaboration, increasing access to historical images, and improving quality and efficiency through integration with laboratory information management systems. However, the resources to implement and operate a digital pathology system can be significant. Given the magnitude and risks involved in the decision to adopt digital histopathology, this working group used pertinent previously published survey results and its members' expertise to create a Points-to-Consider article to assist organizations with building and implementing digital pathology workflows. With the aim of providing a comprehensive perspective, the current publication summarizes aspects of digital whole-slide imaging relevant to nonclinical histopathology evaluations, and then presents points to consider applicable to both primary digital histopathology evaluation and digital peer review in GLP toxicology studies. The Supplemental Appendices provide additional tabulated resources.

Scientific and Regulatory Policy Committee Brief Communication: 2019 Survey on Use of Digital Histopathology Systems in Nonclinical Toxicology Studies.

Histopathologic evaluation and peer review using digital whole-slide images (WSIs) is a relatively new medium for assessing nonclinical toxicology studies in Good Laboratory Practice (GLP) environments. To better understand the present and future use of digital pathology in nonclinical toxicology studies, the Society of Toxicologic Pathology (STP) formed a working group to survey STP members with the goal of creating recommendations for implementation. The survey was administered in December 2019, immediately before the COVID-19 pandemic, and the results suggested that the use of digital histopathology for routine GLP histopathology assessment was not widespread. Subsequently, in follow-up correspondence during the pandemic, many responding institutions either began investigating or adopting digital WSI systems to reduce employee exposure to COVID-19. Therefore, the working group presents the survey results as a pre-pandemic baseline data set. Recommendations for use of WSI systems in GLP environments will be the subject of a separate publication.

The Application, Challenges, and Advancement Toward Regulatory Acceptance of Digital Toxicologic Pathology: Results of the 7th ESTP International Expert Workshop (September 20-21, 2019).

With advancements in whole slide imaging technology and improved understanding of the features of pathologist workstations required for digital slide evaluation, many institutions are investigating broad digital pathology adoption. The benefits of digital pathology evaluation include remote access to study or diagnostic case materials and integration of analysis and reporting tools. Diagnosis based on whole slide images is established in human medical pathology, and the use of digital pathology in toxicologic pathology is increasing. However, there has not been broad adoption in toxicologic pathology, particularly in the context of regulatory studies, due to lack of precedence. To address this topic, as well as practical aspects, the European Society of Toxicologic Pathology coordinated an expert international workshop to assess current applications and challenges and outline a set of minimal requirements needed to gain future regulatory acceptance for the use of digital toxicologic pathology workflows in research and development, so that toxicologic pathologists can benefit from digital slide technology.

Linking diverse salinity responses of 14 almond rootstocks with physiological, biochemical, and genetic determinants.

Fourteen commercial almond rootstocks were tested under five types of irrigation waters to understand the genetic, physiological, and biochemical bases of salt-tolerance mechanisms. Treatments included control (T1) and four saline water treatments dominant in sodium-sulfate (T2), sodium-chloride (T3), sodium-chloride/sulfate (T4), and calcium/magnesium-chloride/sulfate (T5). T3 caused the highest reduction in survival rate and trunk diameter, followed by T4 and T2, indicating that Na and, to a lesser extent, Cl were the most toxic ions to almond rootstocks. Peach hybrid (Empyrean 1) and peach-almond hybrids (Cornerstone, Bright's Hybrid 5, and BB 106) were the most tolerant to salinity. Rootstock's performance under salinity correlated highly with its leaf Na and Cl concentrations, indicating that Na+ and Cl- exclusion is crucial for salinity tolerance in Prunus. Photosynthetic rate correlated with trunk diameter and proline leaf ratio (T3/T1) significantly correlated with the exclusion of Na+ and Cl-, which directly affected the survival rate. Expression analyses of 23 genes involved in salinity stress revealed that the expression differences among genotypes were closely associated with their performance under salinity. Our genetic, molecular, and biochemical analyses allowed us to characterize rootstocks based on component traits of the salt-tolerance mechanisms, which may facilitate the development of highly salt-tolerant rootstocks.

Evaluation of the Relative Performance of Pancreas-Specific MicroRNAs in Rat Plasma as Biomarkers of Pancreas Injury.

Drug-induced pancreatic injury (DIPI) has become linked in recent years to many commonly prescribed medications from several pharmacological classes. Diagnosis is currently most often focused on identification of acute pancreatitis and generally based on subjective clinical assessment and serum amylase and lipase enzymatic activity, which have been criticized as being insufficiently sensitive and specific. The lack of novel noninvasive biomarkers of DIPI can impede the advancement of drug candidates through nonclinical development and translation into clinical settings. Pancreas-specific microRNAs (miRNAs) are currently being evaluated as biomarkers of DIPI that may outperform and/or add value to the interpretation of amylase and lipase. To assess the relative performance of these novel miRNAs, a comprehensive evaluation was conducted to determine the sensitivity and specificity of detecting DIPI in rats. Four miRNAs were evaluated (miR-216a-5p, miR-216b-5p, miR-217-5p, and miR-375-3p) in plasma from 10 studies in which rats were treated with known pancreatic toxicants to assess sensitivity, and from 10 different studies in which toxicity was evident in tissues other than pancreas to assess specificity. The candidate miRNA biomarker performance was compared with amylase and lipase, and receiver operator characteristics (ROC) were determined. Analysis of ROCs demonstrated that all four miRNAs outperformed amylase and lipase in monitoring acute pancreatic injury defined as acinar cell degeneration/necrosis. Specifically, miR-217-5p had the highest performance among all biomarkers assessed. The increased sensitivity and specificity of these miRNAs support their use as biomarkers of DIPI, thereby adding value to the interpretation of amylase and lipase measurements in nonclinical studies. The potential for miRNAs to serve as translational biomarkers in the clinic for the monitoring of DIPI is also supported by this investigation.

Brain findings associated with risperidone in rhesus monkeys: magnetic resonance imaging and pathology perspectives.

Brain changes associated with risperidone, a dopamine-2/serotonin-2 receptor antagonist, have been documented in rats and humans, but not in nonhuman primates. This study characterized brain changes associated with risperidone in nonhuman primates. Rhesus monkeys were orally administered risperidone in a dose-escalation paradigm up to a maximum tolerated dose of 0.5 mg/kg/day for 3 weeks, or 3 months followed by a 3-month recovery period. Transient and fully reversible neurological signs consistent with risperidone pharmacology were observed. The results of a magnetic resonance imaging evaluation after 3 months of treatment and at the end of the 3-month recovery period showed no meaningful changes in the brain. There were no risperidone-related brain weight changes or gross findings. Histomorphological evaluation of brain sections stained with hematoxylin and eosin, ionized calcium binding adaptor molecule 1 (Iba1), and luxol fast blue/cresyl violet double staining showed no notable differences between control and risperidone groups. However, evaluation of the brain after glial fibrillary acidic protein (GFAP) immunohistochemical staining revealed increased staining in the cell bodies and processes of astrocytes in the putamen without apparent alterations in numbers or distribution. The increase in GFAP staining was present after 3 weeks and 3 months of treatment, but no increase in staining was observed after the 3-month recovery period, demonstrating the reversibility of this finding. The reversible increase in GFAP expression was likely an adaptive, non-adverse response of astrocytes, associated with the pharmacology of risperidone. These observations are valuable considerations in the nonclinical risk assessment of new drug candidates for psychiatric disorders.

Chronic Verubecestat Treatment Suppresses Amyloid Accumulation in Advanced Aged Tg2576-AßPPswe Mice Without Inducing Microhemorrhage.

Verubecestat is a potent BACE1 enzyme inhibitor currently being investigated in Phase III trials for the treatment of mild-to-moderate and prodromal Alzheimer's disease. Multiple anti-amyloid immunotherapies have been dose-limited by adverse amyloid related imaging abnormalities such as vasogenic edema (ARIA-E) and microhemorrhage (ARIA-H) observed in human trials and mice. Verubecestat was tested in a 12-week nonclinical study for the potential to exacerbate microhemorrhage (ARIA-H) profiles in 18-22-month-old post-plaque Tg2576-AßPPswe mice. Animals were treated with verubecestat or controls including the anti-Aß antibody analog of bapineuzumab (3D6) as a positive control for ARIA induction. ARIA-H was measured using in-life longitudinal T2*-MRI and Prussian blue histochemistry at study end. Verubecestat reduced plasma and cerebrospinal fluid Aß40 and Aß42 by >90% and 62% to 68%, respectively. The ARIA-H profile of verubecestat-treated mice was not significantly different than controls. Anti-Aß treatment significantly increased ARIA-H detected by Prussian blue staining; however, anti-Aß antibody treatment did not impact plaque status. Verubecestat treatment significantly suppressed the accumulation of total levels of brain Aß40 and Aß42 and Thioflavin S positive plaque load. Stereological analysis of cortex and hippocampus plaque load similarly revealed significantly reduced area of Aß immunoreactivity and reduced plaque number in verubecestat-treated animals compared to controls. The absence of elevated ARIA events in verubecestat-treated mice was associated with a significant reduction in the level of accumulated CNS amyloid pathology and brain Aß peptides; effects consistent with the desired therapeutic mechanism of verubecestat in AD patients. These data will be compared with longitudinal MRI profiles from ongoing clinical trials.

Magnetic Resonance and Ultrastructural Characterization of PEGylation-associated Vacuolation in Nonclinical Models.

Conjugation with polyethylene glycol (PEG) is a strategy for improving the pharmaceutical properties of therapeutic proteins. In nonclinical studies of PEGylated compounds, microscopic tissue vacuolation is often observed, characterized ultrastructurally in this report by lysosomal distension. Although PEGylation-associated vacuolation appears to be of limited toxicologic concern when alternative therapies are limited, the risk-benefit considerations may be impacted by uncertainty about reversibility, lack of methods for monitoring PEG accumulation in vivo without biopsy, and the variability in tissues affected depending on species studied. We demonstrate the use of magnetic resonance spectroscopy (MRS) to measure PEG concentrations at multiple time points in vivo in the kidney with comparison to PEG concentrations ex vivo in body fluids and tissue extracts using nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we demonstrate the use of these techniques to study distribution and elimination of PEG in a dog model of PEGylation-associated vacuolation. This report suggests that MRS could be further investigated as a feasible imaging-based method for monitoring PEG accumulation in a clinical setting in conjunction with NMR quantitation of PEG in plasma and urine.

Spontaneous epithelioid hemangiosarcoma in a rhesus monkey (Macaca mulatta).

Epithelioid hemangiosarcoma is a rare malignant endothelial neoplasia with a unique, predominantly epithelioid morphology. A 4-y-old rhesus monkey from our laboratory had multiple neoplastic nodules in a digit, limb skin, hindlimb muscle, and visceral organs including lung, heart, and brain. The nodules were composed of pleomorphic, polygonal, epithelioid, neoplastic cells that were arranged in sheets, nests, and cords and supported by variably dense fibrovascular connective tissue. The morphologic features of this tumor were predominantly epithelioid. However, some regions contained cystic spaces, clefts, and channel-like structures, all of which were lined with morphologically distinct neoplastic endothelial cells. These neoplastic cells, with or without epithelioid morphology, were positive immunohistochemically for CD31, factor VIII-related antigen, and vimentin. The presence of multiple metastatic nodules, high mitotic rate, and extensive Ki67-positive staining were consistent with malignancy. This report is the first description of epithelioid hemangiosarcoma in a rhesus monkey.

Characterization of the exocrine pancreas in the male Zucker diabetic fatty rat model of type 2 diabetes mellitus following 3 months of treatment with sitagliptin.

Sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor-based incretin therapy intended for the treatment of type 2 diabetes mellitus (T2DM), has not been linked to adverse effects on the pancreas in prospective clinical trials or in nonclinical toxicology studies. To further assess potential pancreatic effects, sitagliptin was studied in the male Zucker diabetic fatty (ZDF) rat model of T2DM. Following 3 months of oral dosing with vehicle, or sitagliptin at doses 3- to 19-fold above the clinically therapeutic plasma concentration, which increased active plasma glucagon-like peptide-1 levels up to approximately 3-fold, or following 3 months of oral dosing with metformin, a non-incretin-based reference T2DM treatment, the pancreas of male ZDF rats was evaluated using qualitative and quantitative histopathology techniques. In the quantitative evaluation, proliferative index was calculated in exocrine pancreatic ducts and ductules using computer-based image analysis on sections stained by immunohistochemistry for cytokeratin (a cytoplasmic epithelial cell marker) and Ki-67 (a nuclear marker of recent cell division). Relative to controls, sitagliptin treatment did not alter disease progression based on detailed clinical signs and clinical pathology assessments. Sitagliptin treatment did not result in pancreatitis or any adverse effect on the pancreas based on a qualitative histopathology evaluation. Proliferative index did not increase with sitagliptin treatment based on quantitative assessment of more than 5000 sections of pancreas, where control group means ranged from 0.698-0.845% and sitagliptin-treated group means ranged from 0.679-0.701% (P = .874). Metformin treatment was similarly evaluated and found not to have adverse effects on pancreas.

Mechanism based neurotoxicity of mGlu5 positive allosteric modulators--development challenges for a promising novel antipsychotic target.

Previous work has suggested that activation of mGlu5 receptor augments NMDA receptor function and thereby may constitute a rational approach addressing glutamate hypofunction in schizophrenia and a target for novel antipsychotic drug development. Here, we report the in vitro activity, in vivo efficacy and safety profile of 5PAM523 (4-Fluorophenyl){(2R,5S)-5-[5-(5-fluoropyridin-2-yl)-1,2,4-oxadiazol-3-yl]-2-methylpiperidin-1-yl}methanone), a structurally novel positive allosteric modulator selective of mGlu5. In cells expressing human mGlu5 receptor, 5PAM523 potentiated threshold responses to glutamate in fluorometric calcium assays, but does not have any intrinsic agonist activity. 5PAM523 acts as an allosteric modulator as suggested by the binding studies showing that 5PAM523 did not displace the binding of the orthosteric ligand quisqualic acid, but did partially compete with the negative allosteric modulator, MPyEP. In vivo, 5PAM523 reversed amphetamine-induced locomotor activity in rats. Therefore, both the in vitro and in vivo data demonstrate that 5PAM523 acts as a selective mGlu5 PAM and exhibits anti-psychotic like activity. To study the potential for adverse effects and particularly neurotoxicity, brain histopathological exams were performed in rats treated for 4 days with 5PAM523 or vehicle. The brain exam revealed moderate to severe neuronal necrosis in the rats treated with the doses of 30 and 50 mg/kg, particularly in the auditory cortex and hippocampus. To investigate whether this neurotoxicity is mechanism specific to 5PAM523, similar safety studies were carried out with three other structurally distinct selective mGlu5 PAMs. Results revealed a comparable pattern of neuronal cell death. Finally, 5PAM523 was tested in mGlu5 knock-out (KO) and wild type (WT) mice. mGlu5 WT mice treated with 5PAM523 for 4 days at 100 mg/kg presented significant neuronal death in the auditory cortex and hippocampus. Conversely, mGlu5 KO mice did not show any neuronal loss by histopathology, suggesting that enhancement of mGlu5 function is responsible for the toxicity of 5PAM523. This study reveals for the first time that augmentation of mGlu5 function with selective allosteric modulators results in neurotoxicity.

Eplerenone decreases inflammatory foci in spontaneously hypertensive rat hearts with minimal effects on blood pressure.

The blood pressure (BP)-lowering effects of mineralocorticoid receptor (MR) antagonists in salt-sensitive rat models of hypertension are well understood. However, studies in salt-independent models have yielded mixed results, and therefore, we measured the hemodynamic effects of MR blockade in spontaneously hypertensive rats. We treated spontaneously hypertensive rats for 8 weeks with 30-300 mg.kg.d eplerenone or 20 mg.kg.d losartan and monitored BP using radiotelemetry and performed histopathological analyses of the hearts. Eplerenone, in contrast to losartan, caused only a small reduction in systolic BP at the highest dose tested. Both reduced left ventricular wall thickness, although eplerenone was less effective than losartan. Only losartan decreased heart weight. We observed foci of cardiomyopathy characterized by combinations of infiltrating monocytes, necrotic myocytes, and interstitial fibrosis in hearts of control animals. The number of foci seemed to be decreased in hearts of losartan- and eplerenone-treated animals. In a second study, using quantitative histomorphometry, the number of foci was significantly reduced by 20 mg.kg.d losartan (by 68%) or by 300 mg.kg.d eplerenone (by 50%). Our data support the hypothesis that a direct BP-independent effect on the progression of cardiomyopathy in the heart may be one basis for the cardiac protection afforded by MR antagonism.

Active intranuclear movement of herpesvirus capsids.

Although small molecules diffuse rapidly through the interphase nucleus, recent reports indicate that nuclear diffusion is limited for particles that are larger than 100 nm in diameter. Given the apparent size limits to nuclear diffusion, there is some debate as to whether the movement of large particles should be attributed to diffusion or to active transport. Here, we show that 125 nm-diameter herpes simplex virus 1 (HSV-1) capsids are actively transported within infected nuclei. Movement is directed, temperature- and energy-dependent, sensitive to the putative myosin inhibitor 2,3-butanedione monoxime (BDM) and to actin depolymerization with latrunculin-A, but insensitive to actin depolymerization with cytochalasin-D.