Oncologic Safety of Immediate Oncoplastic Surgery Compared with Standard Breast-Conserving Surgery for Patients with Invasive Lobular Carcinoma.

BACKGROUND: Invasive lobular carcinoma (ILC) of the breast grows in a diffuse pattern, resulting in a high risk of positive margins at surgical resection. Oncoplastic approaches have been shown to reduce this risk, but concerns persist around the safety of immediate oncoplastic surgery for those with ILC. This study evaluated the short- and long-term oncologic outcomes of immediate oncoplastic surgery for patients with ILC. METHODS: This study retrospectively analyzed an institutional database of stages I to III ILC patients who underwent breast-conserving surgery (BCS) with or without immediate oncoplastic surgery (oncoplastic closure or oncoplastic reduction mammoplasty [ORM]). The study compared positive margin rates, rates of successful BCS, and recurrence-free survival (RFS) by type of surgery. RESULTS: For 494 patients the findings showed that the use of immediate ORM was associated with significantly lower odds of positive margins (odds ratio [OR], 0.34; 95 % confidence interval [CI], 0.17-0.66; p = 0.002). Both lumpectomy with oncoplastic closure and ORM were significantly associated with higher rates of successful BCS than standard lumpectomy (94.2 %, 87.8 %, and 73.9 %, respectively; p < 0.001). No difference in RFS was observed between those undergoing immediate oncoplastic surgery and those undergoing standard lumpectomy alone. CONCLUSIONS: The patients with stages I to III ILC who underwent immediate oncoplastic surgery had significant benefits including lower odds of positive margins and higher rates of successful BCS, with both types of immediate oncoplastic surgery showing similar RFS compared with lumpectomy alone. This supports the oncologic safety of immediate oncoplastic surgery for diffusely growing tumors such as ILC, providing it an ideal option for patients desiring BCS.

Cyclophilin D knockout significantly prevents HCC development in a streptozotocin-induced mouse model of diabetes-linked NASH.

A family of Peptidyl-prolyl isomerases (PPIases), called Cyclophilins, localize to numerous intracellular and extracellular locations where they contribute to a variety of essential functions. We previously reported that non-immunosuppressive pan-cyclophilin inhibitor drugs like reconfilstat (CRV431) or NV556 decreased multiple aspects of non-alcoholic fatty liver disease (NAFLD) in mice under two different non-alcoholic steatohepatitis (NASH) mouse models. Both CRV431 and NV556 inhibit several cyclophilin isoforms, among which cyclophilin D (CypD) has not been previously investigated in this context. It is unknown whether it is necessary to simultaneously inhibit multiple cyclophilin family members to achieve therapeutic benefits or if loss-of-function of one is sufficient. Furthermore, narrowing down the isoform most responsible for a particular aspect of NAFLD/NASH, such as hepatocellular carcinoma (HCC), would allow for more precise future therapies. Features of human diabetes-linked NAFLD/NASH can be reliably replicated in mice by administering a single high dose of streptozotocin to disrupt pancreatic beta cells, in conjunction with a high sugar, high fat, high cholesterol western diet over the course of 30 weeks. Here we show that while both wild-type (WT) and Ppif-/- CypD KO mice develop multipe severe NASH disease features under this model, the formation of HCC nodules was significantly blunted only in the CypD KO mice. Furthermore, of differentially expressed transcripts in a qPCR panel of select HCC-related genes, nearly all were downregulated in the CypD KO background. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NAFLD/NASH. This study highlights the impact of CypD loss-of-function on the development of HCC, one of the most severe disease outcomes.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in Ontario broodmares.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in broodmares in Ontario were investigated. Sixty of the 219 (27.4%) study broodmares were seropositive for N. caninum and 65/219 (29.7%) for N. hughesi with cut-offs of ≥1:40 and ≥1:160, respectively. Thirty-one of 63 participating farms (49.2%) had at least 1 broodmare seropositive for N. caninum. Thirty-three of the 63 (52.4%) participating farms had at least 1 broodmare positive for N. hughesi. Risk factors for N. caninum included presence of farm dogs (OR = 6.70; 95% CI = 2.14-20.97; p = 0.001), and high stocking density (OR = 2.83; 95% CI = 1.27-6.30; p = 0.011). Presence of livestock, excluding cattle, was associated with reduced risk of exposure (OR = 0.17; 95% CI = 0.06-0.53; p = 0.002). The only risk factor for exposure to N. hughesi was feeding hay on the ground in the paddock (OR = 4.31; 95% CI = 1.65-11.22; p = 0.003). This study demonstrated widespread exposure to Neospora spp. in broodmares in Ontario.

Bovine astrovirus and its role in lymphocytic encephalitis in cattle in Ontario, Canada, 1988-2019.

Astroviruses have been found in cattle and other species with encephalitis. Our objective was to determine the frequency of neurotropic bovine astrovirus (BoAstV) in cases of encephalitis in cattle ≥ 4-mo-old. Of 56 cases of idiopathic lymphocytic encephalitis examined retrospectively (1988-2019), fixed brain from 11 cases (19%) tested positive by semi-quantitative RT-PCR for BoAstV CH13/NeuroS1. None of the control cases tested positive, including 32 with other forms of encephalitis and 40 with no neurologic disease. Most astrovirus-positive cases were 1-2-y-old, with a range of 7 mo to 7 y, and affected both beef and dairy breeds with wide geographic distribution. BoAstV-positive cases had acute onset of neurologic signs of 12 h to 7 d before death or euthanasia. Affected cattle had lymphocytic inflammation throughout the brain including cerebrum, thalamus, midbrain, cerebellum, medulla oblongata, and spinal cord, and affecting gray and white matter. Further PCR testing identified a possible cause in 9 of the 45 (20%) remaining idiopathic cases of lymphocytic encephalitis, including eastern equine encephalitis virus, Listeria monocytogenes, bovine viral diarrhea virus, bovine alphaherpesvirus 1, and ovine gammaherpesvirus 2 (malignant catarrhal fever); we found no cases of infection by West Nile virus, rabies virus, or Chlamydia spp. No cause was identified in 36 of 56 (64%) cases of lymphocytic encephalitis. We frequently identified neurotropic BoAstV in cases of lymphocytic encephalitis that had no previously identified cause. Neurotropic BoAstV infections had gone undetected for decades, but the frequency of BoAstV infections has not increased among contemporary cases.

Ocular Manifestations of Multiple Sclerosis: A Population-Based Study.

BACKGROUND: To evaluate the population-based frequency and severity of multiple sclerosis (MS)-related ocular diseases. METHODS: Retrospective, population-based study examining patients with MS between January 1, 1998 and December 31, 2011. Patients were identified using the Rochester Epidemiology Project, which is a record-linkage system of medical records for all patient-physician encounters among Olmsted County, Minnesota residents. Diagnosis of MS was confirmed based on neuroimaging, cerebrospinal fluid studies, and serum studies for each patient according to the 2017 McDonald criteria. Patient data were obtained using the medical records and followed through April 1, 2018. RESULTS: Of the 116 patients with MS, 66% were female and the median age of onset was 36 years (interquartile range 27.5-43.5 years). About half (61/116, 53%) had MS-related neuro-ophthalmic manifestations during their disease course, and about one-fourth (33/116, 28%) had visual symptoms as their presenting symptom of MS, most commonly as optic neuritis (26/116, 22%). Optic neuritis was the leading MS-related ocular condition (37%), followed by internuclear ophthalmoplegia (16%) and nystagmus (13%). Optic neuritis was mostly unilateral (40/43, 93%), with 16% (6/43) having a visual acuity of 20/200 or worse at nadir but ultimately 95% (35/37) improving to a visual acuity of 20/40 or better. CONCLUSIONS: This study provides the population-based frequency of MS-related ocular disease, which demonstrates a high frequency of ocular manifestations in MS both at disease onset and during the disease course, emphasizing the utility of neuro-ophthalmologists, or collaboration between neurologists and ophthalmologists, in the care of patients with MS.

Mice lacking cyclophilin B, but not cyclophilin A, are protected from the development of NASH in a diet and chemical-induced model.

Cyclophilins are a diverse family of peptidyl-prolyl isomerases (PPIases) of importance in a variety of essential cellular functions. We previously reported that the pan-cyclophilin inhibitor drug reconfilstat (CRV431) decreased disease in mice under the western-diet and carbon tetrachloride (CCl4) non-alcoholic steatohepatitis (NASH) model. CRV431 inhibits several cyclophilin isoforms, among which cyclophilin A (CypA) and B (CypB) are the most abundant. It is not known whether simultaneous inhibition of multiple cyclophilin family members is necessary for the observed therapeutic effects or if loss-of-function of one is sufficient. Identifying the responsible isoform(s) would enable future fine-tuning of drug treatments. Features of human liver fibrosis and complete NASH can be reliably replicated in mice by administration of intraperitoneal CCl4 alone or CCl4 in conjunction with high sugar, high cholesterol western diet, respectively. Here we show that while wild-type (WT) and Ppia-/- CypA KO mice develop severe NASH disease features under these models, Ppib-/- CypB KO mice do not, as measured by analysis of picrosirius red and hematoxylin & eosin-stained liver sections and TNFα immuno-stained liver sections. Cyclophilin inhibition is a promising and novel avenue of treatment for diet-induced NASH. In this study, mice without CypB, but not mice without CypA, were significantly protected from the development of the characteristic features of NASH. These data suggest that CypB is necessary for NASH disease progression. Further investigation is necessary to determine whether the specific role of CypB in the endoplasmic reticulum secretory pathway is of significance to its effect on NASH development.

Reexamining the Incidence of Nonarteritic Anterior Ischemic Optic Neuropathy: A Rochester Epidemiology Project Study.

BACKGROUND: This study aims to determine the population-based incidence and characterize the features of nonarteritic anterior ischemic optic neuropathy (NAION) using the Rochester Epidemiology Project (REP). METHODS: All patients diagnosed with an optic neuropathy from January 1, 1990, to December 31, 2016, were retrospectively reviewed to identify incident cases of NAION using the REP database, which is a record-linkage system of medical records for all patient-physician encounters among Olmsted County, Minnesota residents. The overall incidence of NAION was estimated using the age-specific and sex-specific population figures for Olmsted County census data for 1990 through 2016. Visual outcomes and risk factors were evaluated. The systemic risk factors were compared with age-matched controls. RESULTS: One hundred four patients were diagnosed with NAION during the 26-year study period. The overall age and sex adjusted incidence was 3.89 per 100,000 individuals (95% confidence interval [CI]: 3.14-4.65). The incidence was 7.73 (CI: 6.24-9.22) in patients aged 40 years or older and 10.19 (CI: 8.15-12.23) in patients aged 50 years or older. Median age at diagnosis was 65 years (range, 40-90 years), and 59 (56.7%) were male. The median logMAR visual acuity at presentation was 0.35 (Snellen equivalent of 20/40) with 14 patients (13.5%) having vision of counting fingers or worse. Among the 91 patients with final visual acuity outcome data available, the median visual acuity was 0.40 (Snellen equivalent of 20/50) with 12 patients (13.2%) having vision of counting fingers or worse. Twenty-four patients (26.4%) were noted to have final acuity at least 3 Snellen lines worse than at presentation, whereas 17 patients (18.7%) were noted to improve by at least 3 lines. The median mean deviation on automated visual field testing was -10.2 dB at presentation and -11.1 dB at follow-up. Twenty-two patients (21.2%) suffered NAION in the fellow eye; the median interval between involvement of the first and second eyes was 1.39 years. Systemic diseases present in the NAION cohort included hypertension (79.8%), diabetes mellitus (39.4%), obstructive sleep apnea (23.1%), and hyperlipidemia (74.0%), which were all statistically higher than age-matched controls. CONCLUSIONS: NAION is a relatively common optic neuropathy in elderly patients with vascular risk factors. Our data indicate that the incidence of NAION has remained relatively stable in the population of Olmsted County over the past 4 decades.

Comparison of the diagnostic yield of 3 and 5 mm laparoscopic liver biopsy forceps in cats.

OBJECTIVE: To determine whether 3 and 5 mm laparoscopic cup biopsy forceps provide samples of equivalent diagnostic quality in cats. STUDY DESIGN: Experimental study. ANIMALS: Twelve colony cats undergoing a concurrent nutrition study. METHODS: Two biopsy forceps (3 and 5 mm) and three biopsy techniques (twist, pull, and twist + pull) were used to collect 68 laparoscopic liver samples. Biopsies were performed consecutively with the 3 and 5 mm biopsy sites adjacent to each other. Data analyzed included the number of portal triads and hepatic lobules, tissue crush and fragmentation, overall sample area (mm2 ), sample weight, and agreement regarding morphologic diagnosis. RESULTS: The 5 mm forceps provided more hepatic lobules, portal triads, and a larger tissue weight and histologic area (mm2 ) (p < .01). The twist and pull techniques provide more hepatic lobules and portal triads compared to the twist + pull technique while the twist + pull technique resulted in greater tissue crush compared to the twist technique (p = .0097). There was good agreement for morphological diagnosis between the 3 and 5 mm samples using the twist + pull technique but not for the twist or pull techniques. CONCLUSION: Liver samples can be safely collected with 3 or 5 mm laparoscopic biopsy forceps and provide sufficient tissue for histopathology analysis in cats, with minimal artifact. The diagnostic accuracy of 3 mm samples remains unknown. CLINICAL SIGNIFICANCE: Although 3 mm laparoscopic cup biopsy forceps provided samples of sufficient diagnostic quality for histopathologic interpretation in cats, further studies are required to assess their diagnostic accuracy.

Revisiting the Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceuticals.

N-Nitrosamines are a class of indirect acting mutagens, as their metabolic degradation leads to the formation of the DNA-alkylating diazonium ion. Following up on the in-silico identification of thousands of nitrosamines that can potentially be derived from small molecule drugs and their known impurities described in a previous publication, we have now re-analyzed this dataset to apply EMA's Carcinogenic Potency Categorization Approach (CPCA) introduced with the 16th revision of their Q&A document for Marketing Authorization Holders. We find that the majority of potential nitrosamines from secondary amine precursors belongs to potency categories 4 and 5, corresponding to an acceptable daily intake of 1500 ng, whereas nitrosamines from tertiary amine precursors distribute more evenly among all categories, resulting in a substantial number of structures that are assigned the more challenging acceptable intakes of 18 ng/day and 100 ng/day for potency categories 1 and 2, respectively. However, the nitrosative dealkylation pathway for tertiary amine is generally far slower than the direct nitrosation on secondary amines, with a direct nitrosation mechanism suspected only for structures featuring electron-rich (hetero)aromatic substituents. This allows for greater focus towards those structures that require further review, and we demonstrate that their number is not substantial. In addition, we reflect on the nitrosamine risk posed by secondary amine API impurities and demonstrate that based on the ICH Q3A/B identification threshold unknown impurities may exist that could be transformed to relevant amounts of NA. We also demonstrate that the analytical sensitivity required for the quantification of high potency nitrosamines can be problematic especially for high dose APIs. In summary, the regulatory framework rolled out with the latest Q&A document represents a substantial improvement compared with the previous situation, but further refinement through interaction between manufacturers, regulators, not-for-profit and academic institutions will be required to ensure patient access to vital medicines without compromising safety.

Recovery course of persistent posterior subretinal fluid after successful repair of rhegmatogenous retinal detachment.

PURPOSE: To investigate best corrected visual acuity (BCVA), subretinal fluid (SRF) absorption time or ellipsoid zone (EZ) restoration time and various variables in patients with persistent SRF after successful primary repair of rhegmatogenous retinal detachment (RRD). METHODS: This retrospective multicenter study allowed independent analysis of the healing pattern by two observers based on composite of serial cross-sectional macular optical coherence tomography (OCT) scans. Univariate and multivariate analyses were implemented. RESULTS: One hundred and three cases had persistent SRF after pars plana vitrectomy, scleral buckling, or pneumatic retinopexy. By univariate analysis, SRF resolution time correlated positively with the number of retinal breaks (p < 0.001) and with increased myopia (p = 0.011). Using multivariate analysis, final BCVA (log MAR) correlated positively with age, duration of RRD, initial BCVA (OR = 3.28; [95%CI = 1.44-7.47]; p = 0.015), and SRF resolution time (OR = 0.46 [95%CI 0.21-1.05]; p = 0.049). EZ restoration time was longer with increasing number of retinal tears (OR = 0.67; [95%CI 0.29-1.52]; p = 0.030), worse final BCVA, and presence of macula-off RRD (OR = 0.26; [95%CI 0.08-0.88]; p = 0.056). SRF resolution time correlated marginally with prone position. CONCLUSIONS: Residual posterior SRF is more common in eyes with multiple breaks or in myopic eyes. Final BCVA is better in younger subjects and in eyes with shorter duration of RRD. Persistent SRF is a self-limited disorder with a mean resolution of 11.2 months with good visual prognosis improving from a mean baseline logMAR of 1.08 to 0.25 at one year.

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-Induced Hepatocellular Carcinoma Independently of Its Antiviral Activity.

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypIs) represent such new drugs. We demonstrate that the nonimmunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we show that the HCV infection of humanized mice results in the progressive development of HCC. This is true for the four genotypes tested (1 to 4). Remarkably, we demonstrate that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks after infection when HCC is well established. Importantly, we show that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC, while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

TRAV26-2 T-Cell Receptor Expression Is Associated With Mucosal Lymphocyte Response to Wheat Proteins in Patients With Functional Dyspepsia.

INTRODUCTION: An association between functional dyspepsia (FD) and wheat-containing foods has been reported in observational studies; however, an adaptive response has not been demonstrated. We examined whether antigens present in wheat could provoke a response from FD duodenal lymphocytes. METHODS: Lamina propria mononuclear cells (LPMCs) were isolated from duodenal biopsies from 50 patients with FD and 23 controls. LPMCs were exposed to gluten (0.2 mg/mL) or gliadin (0.2 mg/mL) for 24 hours. Flow cytometry was performed to phenotype lymphocytes. Quantitative PCR was used to measure the expression of gliadin-associated T-cell receptor alpha variant ( TRAV ) 26-2. RESULTS: In response to gliadin (but not gluten) stimulation, the effector Th2-like population was increased in FD LPMCs compared with that in controls and unstimulated FD LPMCs. Duodenal gene expression of TRAV26- 2 was decreased in patients with FD compared with that in controls. We identified a positive association between gene expression of this T-cell receptor variant and LPMC effector Th17-like cell populations in patients with FD, but not controls after exposure to gluten, but not gliadin. DISCUSSION: Our findings suggest that gliadin exposure provokes a duodenal effector Th2-like response in patients with FD, supporting the notion that food antigens drive responses in some patients. Furthermore, these findings suggest that altered lymphocyte responses to wheat proteins play a role in FD pathogenesis.

Assessing the utility of common arguments used in expert review of in silico predictions as part of ICH M7 assessments.

Expert review of two predictions, made by complementary (quantitative) structure-activity relationship models, to an overall conclusion is a key component of using in silico tools to assess the mutagenic potential of impurities as part of the ICH M7 guideline. In lieu of a specified protocol, numerous publications have presented best practise guides, often indicating the occurrence of common prediction scenarios and the evidence required to resolve them. A semi-automated expert review tool has been implemented in Lhasa Limited's Nexus platform following collation of these common arguments and assignment to the associated prediction scenarios made by Derek Nexus and Sarah Nexus. Using datasets primarily donated by pharmaceutical companies, an automated analysis of the frequency these prediction scenarios occur, and the likelihood of the associated arguments assigning the correct resolution, could then be conducted. This article highlights that a relatively small number of common arguments may be used to accurately resolve many prediction scenarios to a single conclusion. The use of a standardised method of argumentation and assessment of evidence for a given impurity is proposed to improve the efficiency and consistency of expert review as part of an ICH M7 submission.

Comparing 3 mm and 5 mm laparoscopic liver biopsy samples in dogs.

OBJECTIVE: To determine whether 3 mm cup biopsy forceps (CBF) provide equivalent diagnostic samples to 5 mm CBF for histopathologic diagnosis, bacterial culture, and copper quantification. STUDY DESIGN: Clinical prospective study. ANIMALS: Ten client-owned dogs, presenting for laparoscopic liver biopsy (LLB). METHODS: Dogs underwent LLB, and paired samples were collected using 3 and 5 mm CBF. Portal triad and hepatic lobule counts, crush and fragmentation artifacts, copper concentration, bacterial culture results, and agreement on histopathologic diagnosis were compared. RESULTS: Both CBF sizes allowed for easy sample collection and resulted in minimal hemorrhage. An average of 12.13 (confidence limit (CL): 9.4-14.9) and 17.84 (CL: 15.1-20.6) portal triads were obtained using a 3 and 5 mm CBF, respectively (p = .0003). A portal triad count of 11 or more was achieved in 73.3% of the 3 mm and 93.3% of the 5 mm samples. Gwets AC1 coefficient showed a high level of agreement (0.8) for overall histopathologic diagnosis (p < .0001). The 3 mm CBF crush scores were higher (median of the differences: -1; range: -1 to 1) (p = .035). There was no difference in fragmentation scores (p = .935). CONCLUSION: The 3 mm CBF yielded smaller samples in terms of size and portal triad count compared with the 5 mm CBF. However, the portal triad count was sufficient in a majority of samples and histologic agreement with the 5 mm CBF was excellent. CLINICAL SIGNIFICANCE: In dogs, a 3 mm CBF yields adequate samples for histopathologic interpretation, copper quantification, and bacterial culture.

The Cyclophilin Inhibitor Rencofilstat Decreases HCV-induced Hepatocellular Carcinoma Independently of Its Antiviral Activity.

There is an urgent need for the identification of new drugs that inhibit HCV-induced hepatocellular carcinoma (HCC). Our work demonstrates that cyclophilin inhibitors (CypI) represent such new drugs. We demonstrated that the non-immunosuppressive cyclosporine A (CsA) analog (CsAa) rencofilstat possesses dual therapeutic activities for the treatment of HCV infection and HCV-induced HCC. Specifically, we showed that HCV infection of humanized mice results in the progressive development of HCC. This was true for four genotypes tested (1 to 4). Remarkably, we demonstrated that rencofilstat inhibits the development of HCV-induced HCC in mice even when added 16 weeks post-infection when HCC is well established. Importantly, we showed that rencofilstat drastically reduces HCC progression independently of its anti-HCV activity. Indeed, the CypI rencofilstat inhibits HCC while other anti-HCV agents such as NS5A (NS5Ai) and NS5B (NS5Bi) fail to reduce HCC. In conclusion, this study shows for the first time that the CypI rencofilstat represents a potent therapeutic agent for the treatment of HCV-induced HCC.

cDNA Cloning of Feline PIWIL1 and Evaluation of Expression in the Testis of the Domestic Cat.

The PIWI clade of Argonaute proteins is essential for spermatogenesis in all species examined to date. This protein family binds specific classes of small non-coding RNAs known as PIWI-interacting RNAs (piRNAs) which together form piRNA-induced silencing complexes (piRISCs) that are recruited to specific RNA targets through sequence complementarity. These complexes facilitate gene silencing through endonuclease activity and guided recruitment of epigenetic silencing factors. PIWI proteins and piRNAs have been found to play multiple roles in the testis including the maintenance of genomic integrity through transposon silencing and facilitating the turnover of coding RNAs during spermatogenesis. In the present study, we report the first characterization of PIWIL1 in the male domestic cat, a mammalian system predicted to express four PIWI family members. Multiple transcript variants of PIWIL1 were cloned from feline testes cDNA. One isoform shows high homology to PIWIL1 from other mammals, however, the other has characteristics of a "slicer null" isoform, lacking the domain required for endonuclease activity. Expression of PIWIL1 in the male cat appears limited to the testis and correlates with sexual maturity. RNA-immunoprecipitation revealed that feline PIWIL1 binds small RNAs with an average size of 29 nt. Together, these data suggest that the domestic cat has two PIWIL1 isoforms expressed in the mature testis, at least one of which interacts with piRNAs.

Intraocular neuroectodermal embryonal tumor in two rabbits.

Spontaneous intraocular tumors are rarely reported in rabbits, despite their widespread use as laboratory animals. We describe two cases of intraocular neuroectodermal embryonal tumors, formerly primitive neuroectodermal tumors, in young rabbits. Histologically, both tumors exhibited prominent rosette or pseudorosettes, consistent with the histomorphology seen in human tumors. The neuroectodermal subtype is supported by immunoreactivity for the neuronal markers, SRY-box transcription factor 2, microtubule-associated protein 2, neuronal nuclear protein, and neuron-specific enolase. In one of the rabbits, there was metastasis to the contralateral conjunctiva. Intraocular neoplasms can occur in young rabbits and eyes with refractory disease should be enucleated for clinical management.

A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants.

BACKGROUND: PacBio HiFi sequencing provides highly accurate long-read sequencing datasets which are of great advantage for whole genome sequencing projects. One limitation of the method is the requirement for high quality, high molecular weight input DNA. This can be particularly challenging for plants that frequently contain common and species-specific secondary metabolites, which often interfere with downstream processes. Cape Primroses (genus Streptocarpus), are some of these recalcitrant plants and are selected here as material to develop a high quality, high molecular weight DNA extraction protocol for long read genome sequencing. RESULTS: We developed a DNA extraction method for PacBio HiFi sequencing for Streptocarpus grandis and Streptocarpus kentaniensis. A CTAB lysis buffer was employed to avoid guanidine, and the traditional chloroform and phenol purification steps were replaced with pre-lysis sample washes. Best cells/nucleus lysis was achieved with 4 h at 58 °C. The obtained high quality and high molecular weight DNAs were tested in PacBio SMRTBell™ library preparations, which resulted in circular consensus sequencing (CCS) reads from 17 to 27 Gb per cell, and a read length N50 from 14 to 17 kbp. To evaluate the quality of the reads for whole genome sequencing, they were assembled with HiFiasm into draft genomes, with N50 = 49 Mb and 23 Mb, and L50 = 10 and 11. The longest contigs were 95 Mb and 57 Mb respectively, showing good contiguity as these are longer than the theoretical chromosome length (genome size/chromosome number) of 78 Mb and 55 Mb, for S. grandis and S. kentaniensis respectively. CONCLUSIONS: DNA extraction is a critical step towards obtaining a complete genome assembly. Our DNA extraction method here provided the required high quality, high molecular weight DNA for successful standard-input PacBio HiFi library preparation. The contigs from those reads showed a high contiguity, providing a good starting draft assembly towards obtaining a complete genome. The results obtained here were highly promising, and demonstrated that the DNA extraction method developed here is compatible with PacBio HiFi sequencing and suitable for de novo whole genome sequencing projects of plants.