Recent studies in our laboratory have shown that galectin‐3, a β‐galactoside binding lectin, mediates re‐epithelialization of corneal wounds. In an effort to characterize the molecular mechanism by which galectin‐3 stimulates the cell migration, the goals of the present study are to: (i) characterize the lectin‐induced migratory phenotypes and Rac1 activation in corneal epithelial cells in vitro, and (ii) determine whether carbohydrate‐based interactions between galectin‐3 and integrins play a role in the lectin‐induced lamellipodia formation in corneal epithelium. Specifically, exogenous galectin‐3 but not galectin‐1, and ‐8 induced formation of lamellipodia in human corneal epithelial cells in a β‐lactose‐inhibitable manner and increased Rac1 activity. α3β1, α6β1, α6β4 and & αv‐integrins and laminin‐5 were identified as major galectin‐3‐binding proteins in corneal epithelial cells by affinity chromatography of corneal epithelial cell lysates on a galectin‐3‐Sepharose column, followed by MALDI‐TOF MS analysis. Furthermore, preincubation of cells with anti‐α3 integrin function‐blocking antibody (P1B5) significantly inhibited the galectin‐3‐induced lamellipodia. These data suggest that galectin‐3 may influence re‐epithelialization of corneal wounds by initiating the formation of lamellipodia, at least in part, through its interaction with α3β1 integrins.
Toxicologic pathology is transitioning from analog to digital methods. This transition seems inevitable due to a host of ongoing social and medical technological forces. Of these, artificial intelligence (AI) and in particular machine learning (ML) are globally disruptive, rapidly growing sectors of technology whose impact on the long-established field of histopathology is quickly being realized. The development of increasing numbers of algorithms, peering ever deeper into the histopathological space, has demonstrated to the scientific community that AI pathology platforms are now poised to truly impact the future of precision and personalized medicine. However, as with all great technological advances, there are implementation and adoption challenges. This review aims to define common and relevant AI and ML terminology, describe data generation and interpretation, outline current and potential future business cases, discuss validation and regulatory hurdles, and most importantly, propose how overcoming the challenges of this burgeoning technology may shape toxicologic pathology for years to come, enabling pathologists to contribute even more effectively to answering scientific questions and solving global health issues. [Box: see text]
Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.
It is generally accepted that the glycans on the cell surface and extracellular matrix proteins play a pivotal role in the events that mediate re-epithelialization of wounds. Yet, the global alteration in the structure and composition of glycans, specifically occurring during corneal wound closure remains unknown. In this study, GLYCOv2 glycogene microarray technology was used for the first time to identify the differentially expressed glycosylation-related genes in healing mouse corneas. Of approximately 2000 glycogenes on the array, the expression of 11 glycosytransferase and glycosidase enzymes was upregulated and that of 19 was downregulated more than 1.5-fold in healing corneas compared with the normal, uninjured corneas. Among them, notably, glycosyltransferases, beta3GalT5, T-synthase, and GnTIVb, were all found to be induced in the corneas in response to injury, whereas, GnTIII and many sialyltransferases were downregulated. Interestingly, it appears that the glycan structures on glycoproteins and glycolipids, expressed in healing corneas as a result of differential regulation of these glycosyltransferases, may serve as specific counter-receptors for galectin-3, a carbohydrate-binding protein, known to play a key role in re-epithelialization of corneal wounds. Additionally, many glycogenes including a proteoglycan, glypican-3, cell adhesion proteins dectin-1 and -2, and mincle, and mucin 1 were identified for the first time to be differentially regulated during corneal wound healing. Results of glycogene microarray data were confirmed by qRT-PCR and lectin blot analyses. The differentially expressed glycogenes identified in the present study have not previously been investigated in the context of wound healing and represent novel factors for investigating the role of carbohydrate-mediated recognition in corneal wound healing.
Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-β (Aβ), there is a theoretical risk of Aβ accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aβ isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aβ methodology. The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aβ-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aβ. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aβ1-42 (65.3%; p=0.026), Aβ1-40 (35.2%; p=0.04) and Aβtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aβ (AUC) indicated elevations in the more aggregable form Aβ1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aβ1-40 (23.4%; p=0.009), Aβ1-38 (64.1%; p=0.0001) and Aβtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aβ isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aβ deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.
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