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Background: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders., Methodology/principal Findings: In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles., Conclusion: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LT was the recipient of a conference travel award from ONT., (Copyright: © 2024 McCabe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary ( Rosmarinus officinalis ) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3 H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Background: Ultrasound-determined gallbladder wall thickness is widely used to aid in the diagnosis of gallbladder disease, but no reference values supported by published measurement data are available in dogs., Hypothesis/objective: Establish normal thickness of the gallbladder wall in dogs., Animals: Fifty-three dogs presented to a referral hospital and required abdominal ultrasound examination for reasons unrelated to primary hepatobiliary disease., Methods: Cross-sectional observational study recruiting dogs requiring abdominal ultrasound examination. A standard sequence of gallbladder wall images was recorded for later review. Inclusion criteria were normal ultrasonographic hepatobiliary, pancreatic, and small intestinal findings. Exclusion was determined by 2 European College of Veterinary Internal Medicine (ECVIM)-certified veterinary internists blinded to gallbladder wall thickness data. Dogs were excluded if they had inadequate medical records, a previous history of hepatobiliary, gastrointestinal, or pancreatic disease likely to impact the biliary system (eg, chronic vomiting, nausea, jaundice, diarrhea), unexplained increases in liver enzyme activities, hypoalbuminemia, or ascites. Gallbladder wall thickness was determined by 2 European College of Veterinary Diagnostic Imaging (ECVDI)-certified veterinary radiologists working together to generate a consensus for each dog. The final output was the maximum normal wall thickness for this population of dogs., Results: The upper limit for gallbladder wall thickness in 53 fasted (8 hours) dogs <40 kg was 1.30 mm (90% confidence interval, 1.19-1.41)., Conclusions and Clinical Importance: Normal gallbladder wall thickness in dogs is lower than previously reported. Additional studies are required to determine potential effects of body weight and the optimal cut-off to distinguish between healthy and diseased gallbladders., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC. on behalf of the American College of Veterinary Internal Medicine.)

Formalin-Fixed Paraffin Embedded (FFPE) biopsies would provide a critical mass of cases to allow investigation of canine liver disease, however their use is often limited by challenges typically associated with transcriptomic analysis. This study evaluates the capability of NanoString® to measure the expression of a broad panel of genes in FFPE liver samples. RNA was isolated from matched histopathologically normal liver samples using FFPE (n = 6) and snap frozen in liquid nitrogen (n = 6) and measured using a custom NanoString® panel. Out of the 40 targets on the panel, 27 and 23 targets were above threshold for non-diseased snap frozen and FFPE tissue respectively. The binding density and total counts were significantly reduced in the FFPE samples relative to the snap frozen samples (p = 0.005, p = 0.01, respectively), confirming a reduction in sensitivity. The concordance between the snap frozen and FFPE samples was high, with correlations (R) ranging between 0.88 and 0.99 between the paired samples. An additional 14 immune-related targets, undetectable the non-diseased FFPE liver, were above threshold when the technique was applied to a series of diseased samples, further supporting their inclusion on this panel. This use of NanoString® based analysis opens up huge opportunity for retrospective evaluation of gene signatures in larger caseloads through harnessing the capacity of archived FFPE samples This information used alongside clinical and histological data will not only afford a way to explore disease etiopathogenesis, it may also offer insight into sub-types of liver disease in dogs, which cannot be discerned using more traditional diagnostic methods., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The pancreas is a glandular organ with endocrine and exocrine functions necessary for the maintenance of blood glucose homeostasis and secretion of digestive enzymes. Pancreatitis is characterized by inflammation of the pancreas leading to temporary or permanent pancreatic dysfunction. Inflammation and fibrosis caused by chronic pancreatitis exacerbate malignant transformation and significantly increase the risk of developing pancreatic cancer, the world's most aggressive cancer with a 5-year survival rate less than 10%. Berberine (BBR) is a naturally occurring plant-derived polyphenol present in a variety of herbal remedies used in traditional medicine to treat ulcers, infections, jaundice, and inflammation. The current review summarizes the existing in vitro and in vivo evidence on the effects of BBR against pancreatitis and pancreatic cancer with a focus on the signalling mechanisms underlying the effects of BBR.

Non-small cell lung cancer (NSCLC) represents an aggressive form of lung cancer which often develops resistance to chemo- and radiotherapy emphasizing a need to identify novel treatment agents to combat it. Many plants contain compounds with anti-inflammatory, antimicrobial, antidiabetic, and anticancer properties and some plant-derived chemicals are used in the treatment of cancer. A limited number of in vitro and in vivo animal studies provide evidence of anticancer effects of rosemary ( Rosmarinus officinalis ) extract (RE); however, no studies have explored its role in H1299 NSCLC cells, and its underlying mechanism(s) of action are not understood. The current study examined the effects of RE on H1299 cell proliferation, survival, and migration using specific assays. Additionally, immunoblotting was used to investigate the effects of RE treatment on signalling molecules implicated in cell growth and survival. Treatment with RE dose-dependently inhibited H1299 proliferation with an IC 50 value of 19 µg/mL. Similarly, RE dose-dependently reduced cell survival, and this reduction correlated with increased levels of cleaved poly (ADP-ribose) polymerase (PARP), a marker of apoptosis. RE was also able to inhibit cell migration as assessed with a wound healing assay. These cellular effects of RE were associated with an increase in phosphorylated levels of extracellular signal-regulated kinase (ERK), AMP-activated protein kinase (AMPK), and its downstream targets ACC, the mTORC1 protein raptor, and decreased p70S6K phosphorylation. More studies are required to fully examine the effects of RE against NSCLC.