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Simple Summary: We present flow cytometry and clonality evaluation as a reliable and useful method for characterizing feline lymphomas. Neoplastic cell size is a crucial factor for prognosis. We have developed a novel system for cell sizing by flow cytometry that was 82–90% concordant with the gold standard of cytology. Moreover, from our retrospective analysis of survival for small versus large cells, we saw consistency between both methodologies. These results highlight the utility of our approach in providing prognostic insights. Feline lymphoma, a prevalent cancer in cats, exhibits varied prognoses influenced by anatomical site and cellular characteristics. In this study, we investigated the utility of flow cytometry and clonality analysis via PCR for antigen receptor rearrangement (PARR) with respect to characterizing the disease and predicting prognosis. For this purpose, we received fine needle aspirates and/or blood from 438 feline patients, which were subjected to flow cytometry analysis and PARR. We used a subset of the results from patients with confirmed B- or T-cell lymphomas for comparison to cytological or histological evaluation (n = 53). Using them as a training set, we identified the optimal set of flow cytometry parameters, namely forward scatter thresholds, for cell size categorization by correlating with cytology-defined sizes. Concordance with cytological sizing among this training set was 82%. Furthermore, 90% concordance was observed when the proposed cell sizing was tested on an independent test set (n = 24), underscoring the reliability of the proposed approach. Additionally, lymphoma subtypes defined by flow cytometry and PARR demonstrated significant survival differences, validating the prognostic utility of these methods. The proposed methodology achieves high concordance with cytological evaluations and provides an additional tool for the characterization and management of feline lymphoproliferative diseases. [ABSTRACT FROM AUTHOR]

First-line treatments of cancer do not always work, and even when they do, they cure the disease at unequal rates mostly owing to biological and clinical heterogeneity across patients. Accurate prediction of clinical outcome and survival following the treatment can support and expedite the process of comparing alternative treatments. We describe the methodology to dynamically determine remission probabilities for individual patients, as well as their prospects of progressionfree survival (PFS). The proposed methodology utilizes the ex vivo drug sensitivity of cancer cells, their immunophenotyping results, and patient information, such as age and breed, in training machine learning (ML) models, as well as the Cox hazards model to predict the probability of clinical remission (CR) or relapse across time for a given patient. We applied the methodology using the three types of data obtained from 242 canine lymphoma patients treated by (L)-CHOP chemotherapy. The results demonstrate substantial enhancement in the predictive accuracy of the ML models by utilizing features from all the three types of data. They also highlight superior performance and utility in predicting survival compared to the conventional stratification method. We believe that the proposed methodology can contribute to improving and personalizing the care of cancer patients. [ABSTRACT FROM AUTHOR]

We report a precision medicine platform that evaluates the probability of chemotherapy drug efficacy for canine lymphoma by combining ex vivo chemosensitivity and immunophenotyping assays with computational modelling. We isolated live cancer cells from fresh fine needle aspirates of affected lymph nodes and collected post‐treatment clinical responses in 261 canine lymphoma patients scheduled to receive at least 1 of 5 common chemotherapy agents (doxorubicin, vincristine, cyclophosphamide, lomustine and rabacfosadine). We used flow cytometry analysis for immunophenotyping and ex vivo chemosensitivity testing. For each drug, 70% of treated patients were randomly selected to train a random forest model to predict the probability of positive Veterinary Cooperative Oncology Group (VCOG) clinical response based on input variables including antigen expression profiles and treatment sensitivity readouts for each patient's cancer cells. The remaining 30% of patients were used to test model performance. Most models showed a test set ROC‐AUC > 0.65, and all models had overall ROC‐AUC > 0.95. Predicted response scores significantly distinguished (P <.001) positive responses from negative responses in B‐cell and T‐cell disease and newly diagnosed and relapsed patients. Patient groups with predicted response scores >50% showed a statistically significant reduction (log‐rank P <.05) in time to complete response when compared to the groups with scores <50%. The computational models developed in this study enabled the conversion of ex vivo cell‐based chemosensitivity assay results into a predicted probability of in vivo therapeutic efficacy, which may help improve treatment outcomes of individual canine lymphoma patients by providing predictive estimates of positive treatment response. [ABSTRACT FROM AUTHOR]

Contribution of the [Na.sup.+]-[K.sup.+]-[2Cl.sup.-] cotransporter NKCC1 to [Cl.sup.-] secretion in rat OMCD. Am J Physiol Renal Physiol 280: F913-F921, 2001.--In rat kidney the 'secretory' isoform of the [Na.sup.+]-[K.sup.+]-[2Cl.sup.-] cotransporter (NKCC1) localizes to the basolateral membrane of the [Alpha]-intercalated cell. The purpose of this study was to determine whether rat outer medullary collecting duct (OMCD) secretes [Cl.sup.-] and whether transepithelial [Cl.sup.-] transport occurs, in part, through [Cl.sup.-] uptake across the basolateral membrane mediated by NKCC1 in series with [Cl.sup.-] efflux across the apical membrane. OMCD tubules from rats treated with deoxycorticosterone pivalate were perfused in vitro in symmetrical [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] /[CO.sub.2]-buffered solutions. [Cl.sup.-] secretion was observed in this segment, accompanied by a lumen positive transepithelial potential. Bumetanide (100 [micro]M), when added to the bath, reduced [Cl.sup.-] secretion by 78%, although the lumen positive transepithelial potential and fluid flux were unchanged. Bumetanide-sensitive [Cl.sup.-] secretion was dependent on extracellular [Na.sup.+] and either [K.sup.+] or [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], consistent with the ion dependency of NKCC1-mediated [Cl.sup.-] transport. In conclusion, OMCD tubules from deoxycorticosterone pivalate-treated rats secrete [Cl.sup.-] into the luminal fluid through NKCC1-mediated [Cl.sup.-] uptake across the basolateral membrane in series with [Cl.sup.-] efflux across the apical membrane. The physiological role of NKCC1-mediated [Cl.sup.-] uptake remains to be determined. However, the role of NKCC1 in the process of fluid secretion could not be demonstrated. ammonium; type 1 bumetanide-sensitive sodium-potassium-2 chloride cotransporter; type 2 bumetanide-sensitive sodiumpotassium-2 chloride cotransporter; fluid flux; chloride-bicarbonate exchange; outer medullary collecting duct

Wall, Susan M., Bradley S. Davis, Kathryn A. Hassell, Pramod Mehta, and Stanley J. Park. In rat tIMCD, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake by [Na.sup.+]-[K.sup.+]-ATPase is critical to net acid secretion during chronic hypokalemia. Am. J. Physiol. 277 (Renal Physiol. 46): F866-F874, 1999.--The purpose of this study was to determine the magnitude of [Na.sup.+] pump-mediated [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake in the terminal inner medullary collecting duct (tIMCD) at [K.sup.+] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentrations observed in vivo in the inner medullary interstitium of normal and in [K.sup.+]-restricted rats. Interstitial [K.sup.+] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] concentrations in the terminal half of the inner medulla were taken to be 10 and 6 mM in [K.sup.+]-restricted rats, but 30 and 6 mM in [K.sup.+]-replete rats. In tubules from [K.sup.+]-restricted rats, when perfused at a [K.sup.+] concentration of 10 mM, addition of ouabain to the bath reduced total bicarbonate flux ([J.sub.tCO2]) by 40% and increased intracellular pH ([pH.sub.i]), indicating significant [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake by the [Na.sup.+]-[K.sup.+]-ATPase. In tubules from [K.sup.+]-restricted rats, [J.sub.tCO2] was reduced with increased extracellular [K.sup.+]. At a [K.sup.+] concentration of 30 mM, ouabain addition neither reduced [J.sub.tCO2] nor increased [pH.sub.i] in tubules from rats of either treatment group. In conclusion, in the tIMCD from hypokalemic rats, 1) acute changes in extracellular [K.sup.+] concentration modulate net acid secretion, and 2) [Na.sup.+] pump-mediated [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] uptake should be an important pathway mediating transepithelial net acid secretion in vivo. collecting duct; sodium-potassium-adenosinetriphosphatase; ouabain; ammonium; potassium

Reports a study which determined whether rat outer medullary collecting duct secretes chlorine and whether transepithelial chlorine transport occurs through uptake across the basolateral membrane. How the study was conducted; Results; Discussion of the results.

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was <10 CFU/mL, while ddPCR showed quantification from 1 to 104 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations. [ABSTRACT FROM AUTHOR]

Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.

Von Hippel-Lindau (VHL) disease is a highly penetrant autosomal dominant systemic malignancy that gives rise to cystic and highly vascularized tumors in a constellation of organs. Patients with VHL disease commonly present with hemangioblastomas in the central nervous system and the eye while other manifestations include pheochromocytoma, clear cell renal cell carcinoma, endolymphatic sac tumors of the middle ear, pancreatic cystadenomas, epididymal and broad ligament cystadenomas. Animal models inactivating the VHL gene product in various organ tissues have been constructed over the past 15 years to parse its HIF-associated mechanisms and its link to tumorigenesis. These models, despite advancing our understanding the molecular role of VHL, are by and large unable to recapitulate the more common features of human VHL disease. Up to date, no model exists that develop retinal hemangioblastomas, the most common clinical manifestation. The purpose of this review is: (1) to discuss the need for an ocular VHL model, (2) to review the animal models that recapitulate clinical VHL disease and (3) to propose potential mechanisms of tumorigenesis for the development of ocular VHL.

Background: To report the tumor, patient, and treatment characteristics of long-term metastatic uveal melanoma survivors., Methods: A non-comparative, retrospective case series of patients from a single institution surviving >24 months with metastatic uveal melanoma (UM)., Results: Nine patients met the study criteria and their charts were reviewed. The mean age at diagnosis of UM was 44.1 years (SD +/- 14.4 years). Initial treatment modalities included enucleation (67%), brachytherapy (22%), and proton beam radiation (11%). The average time from primary tumor diagnosis to detection of metastasis was 125.9 months (SD +/- 95 months). The most common location for initial metastasis was the liver. All patients underwent treatment for metastatic disease including systemic therapy, surgical resection, and isolated hepatic perfusion. The majority of patients received treatment with a tyrosine kinase inhibitor (sorafenib, sunitinib, and/or imatinib). The median survival with metastasis was 51 months (range 27-123 months). Patients had a long disease-free interval before presentation of metastatic disease., Conclusions: A small subset of patients with metastatic UM has prolonged survival. Identification of these patients may be helpful for future clinical trial design.