Leveraging Language Model Multitasking To Predict C-H Borylation Selectivity.

C-H borylation is a high-value transformation in the synthesis of lead candidates for the pharmaceutical industry because a wide array of downstream coupling reactions is available. However, predicting its regioselectivity, especially in drug-like molecules that may contain multiple heterocycles, is not a trivial task. Using a data set of borylation reactions from Reaxys, we explored how a language model originally trained on USPTO_500_MT, a broad-scope set of patent data, can be used to predict the C-H borylation reaction product in different modes: product generation and site reactivity classification. Our fine-tuned T5Chem multitask language model can generate the correct product in 79% of cases. It can also classify the reactive aromatic C-H bonds with 95% accuracy and 88% positive predictive value, exceeding purpose-developed graph-based neural networks.

Effect of proteasome inhibitors on canine lymphoma cell response to CHOP chemotherapy in vitro.

The standard treatment for canine lymphoma is the CHOP chemotherapy regimen. Proteasome inhibitors have been employed with CHOP for the treatment of human haematological malignancies but remain to be fully explored in canine lymphoma. We identified an association between poor response to CHOP chemotherapy and high mRNA expression levels of proteasomal subunits in a cohort of 15 canine lymphoma patients, and sought to determine the effect of proteasome inhibitors on the viability of a canine B-cell lymphoma cell line (CLBL-1). The aim of this study was to investigate whether proteasome inhibitors sensitize these cells to the CHOP agents doxorubicin, vincristine and cyclophosphamide (as 4-hydroxycyclophosphamide/4-HC). CLBL-1 cells were sensitive to proteasome inhibition by bortezomib and ixazomib. The IC50 of bortezomib was 15.1 nM and of ixazomib was 59.14 nM. Proteasome inhibitors plus doxorubicin had a synergistic effect on CLBL-1 viability; proteosome inhibitors plus vincristine showed different effects depending on the combination ratio, and there was an antagonistic effect with 4-HC. These results may have clinical utility, as proteasome inhibition could potentially be used with a synergizing CHOP compound to improve responsiveness to chemotherapy for canine lymphoma patients.

Renal tubule-specific Atgl deletion links kidney lipid metabolism to glucagon-like peptide 1 and insulin secretion independent of renal inflammation or lipotoxicity.

OBJECTIVE: Lipotoxic injury from renal lipid accumulation in obesity and type 2 diabetes (T2D) is implicated in associated kidney damage. However, models examining effects of renal ectopic lipid accumulation independent of obesity or T2D are lacking. We generated renal tubule-specific adipose triglyceride lipase knockout (RT-SAKO) mice to determine if this targeted triacylglycerol (TAG) over-storage affects glycemic control and kidney health. METHODS: Male and female RT-SAKO mice and their control littermates were tested for changes in glycemic control at 10-12 and 16-18 weeks of age. Markers of kidney health and blood lipid and hormone concentrations were analyzed. Kidney and blood lysophosphatidic acid (LPA) levels were measured, and a role for LPA in mediating impaired glycemic control was evaluated using the LPA receptor 1/3 inhibitor Ki-16425. RESULTS: All groups remained insulin sensitive, but 16- to 18-week-old male RT-SAKO mice became glucose intolerant, without developing kidney inflammation or fibrosis. Rather, these mice displayed lower circulating insulin and glucagon-like peptide 1 (GLP-1) levels. Impaired first-phase glucose-stimulated insulin secretion was detected and restored by Exendin-4. Kidney and blood LPA levels were elevated in older male but not female RT-SAKO mice, associated with increased kidney diacylglycerol kinase epsilon. Inhibition of LPA-mediated signaling restored serum GLP-1 levels, first-phase insulin secretion, and glucose tolerance. CONCLUSIONS: TAG over-storage alone is insufficient to cause renal tubule lipotoxicity. This work is the first to show that endogenously derived LPA modulates GLP-1 levels in vivo, demonstrating a new mechanism of kidney-gut-pancreas crosstalk to regulate insulin secretion and glucose homeostasis.

Development and Comprehensive Benchmark of a High-Quality AMBER-Consistent Small Molecule Force Field with Broad Chemical Space Coverage for Molecular Modeling and Free Energy Calculation.

Biomolecular simulations have become an essential tool in contemporary drug discovery, and molecular mechanics force fields (FFs) constitute its cornerstone. Developing a high quality and broad coverage general FF is a significant undertaking that requires substantial expert knowledge and computing resources, which is beyond the scope of general practitioners. Existing FFs originate from only a limited number of groups and organizations, and they either suffer from limited numbers of training sets, lower than desired quality because of oversimplified representations, or are costly for the molecular modeling community to access. To address these issues, in this work, we developed an AMBER-consistent small molecule FF with extensive chemical space coverage, and we provide Open Access parameters for the entire modeling community. To validate our FF, we carried out benchmarks of quantum mechanics (QM)/molecular mechanics conformer comparison and free energy perturbation calculations on several benchmark data sets. Our FF achieves a higher level of performance at reproducing QM energies and geometries than two popular open-source FFs, OpenFF2 and GAFF2. In relative binding free energy calculations for 31 protein-ligand data sets, comprising 1079 pairs of ligands, the new FF achieves an overall root-mean-square error of 1.19 kcal/mol for ΔΔG and 0.92 kcal/mol for ΔG on a subset of 463 ligands without bespoke fitting to the data sets. The results are on par with those of the leading commercial series of OPLS FFs.

Kinetic analysis of oncolytic OrfV-induced innate and adaptive immune responses in a murine model of late-stage ovarian cancer.

Immunotherapies revive host immune responses against tumors by stimulating innate and adaptive immune effector cells with antitumor functions. Thus, detailed studies of immunological cell phenotypes and functions within the tumor microenvironment (TME) following immunotherapy treatments is critical to identifying the determinants of therapeutic success, optimizing treatment regimens, and driving curative outcomes. Oncolytic viruses such as Orf virus (OrfV) are multifunctional biologics that preferentially infect and kill cancer cells while simultaneously causing inflammation that drives anticancer immune responses. Here, we describe the immunological impact of OrfV on the ascites TME in a preclinical model of advanced-stage epithelial ovarian cancer. OrfV promoted the infiltration of several immune effector cells with increased expression of activation markers and effector cytokines into the ascites TME, which correlated with reduced ascites tumor burden and improved survival. The kinetics of the immune response and change in tumor burden following OrfV therapy revealed an optimal re-administration time to sustain antitumor immunity, further extending survival. The data presented highlight the importance of investigating immune response kinetics following immunotherapy and demonstrate that detailed kinetic profiling of immune responses can reveal novel insights into mechanisms of action that can guide the development of more effective therapies.

Quantification of circulating tumour cells over time in dogs with appendicular osteosarcoma.

Enumeration of circulating tumour cells (CTC) has shown promise for prognostication and guidance of therapeutic decisions in human cancers. The objective of this study was to enumerate CTC over time in dogs with naturally occurring osteosarcoma (OSA), and to determine correlation with patient outcome. Twenty-six dogs with OSA and no evidence of metastatic disease at the time of amputation were enrolled. Dogs were assessed for lung metastases and CTC prior to and following amputation, and at each chemotherapy visit. Twenty-one dogs completed the study. Nineteen dogs were euthanized and two were alive and free of metastases. Overall survival time ranged from 88 to 1058 days (median survival time (MST) 374 days). Increased serum alkaline phosphatase activity, advanced age, and higher body weight were significantly associated with lower MST. Dogs with OSA had a mean of 356 (0 to 4443) CTC/106 leukocytes. In 12 of 15 dogs that developed radiographic evidence of metastasis, a pre-metastatic CTC spike was retrospectively detectable on average 36.5 (1-100 days) days prior to metastasis and was associated with significantly shorter MST (301 ± 64 vs. 626 ± 55 days; p = .0107). In a multivariable analysis, dogs with a CTC spike were 10× more likely to die compared with those without. These results suggest that a spike in CTC frequency precedes detection of metastasis in dogs with OSA and is associated with shorter survival. More frequent enumeration of CTC in a larger cohort of dogs with OSA may be warranted.

A very British state capitalism: Variegation, political connections and bailouts during the COVID-19 crisis.

The COVID-19 pandemic has resulted in governments playing increasingly prominent roles as active economic agents. However, state capitalism does not necessarily serve broad developmental purposes, and rather can be directed to supporting sectional and private interests. As the literature on variegated capitalism alerts us, governments and other actors regularly devise fixes in response to a systemic crisis, but the focus, scale, and scope of the interventions vary considerably, according to the constellation of interests. Rapid progress with vaccines notwithstanding, the UK government's response to COVID-19 has been associated with much controversy, not only because of an extraordinarily high death rate, but also because of allegations of cronyism around the granting of government contracts and bailouts. We focus on the latter, investigating more closely who got bailed out. We find that badly affected sectors (e.g. hospitality, transportation) and larger employers were more likely to get bailouts. However, the latter also favored the politically influential and those who had run up debt profligately. Although, as with state capitalism, crony capitalism is most often associated with emerging markets, we conclude that the two have coalesced into a peculiarly British variety, but one that has some common features with other major liberal markets. This might suggest that the eco-systemic dominance of the latter is coming to an end, or, at the least, that this model is drifting towards one that assumes many of the features commonly associated with developing nations.

Feline myocardial transcriptome in health and in hypertrophic cardiomyopathy-A translational animal model for human disease.

Hypertrophic cardiomyopathy (HCM) is the most common heart disease in cats, characterized by primary left ventricular hypertrophy. Feline HCM closely resembles human HCM and is suggested as translational animal model for the human disease. A genetic cause is established in humans and suspected for cats, but little is known about the gene expression and pathways involved in the pathogenesis of HCM. To investigate the myocardial transcriptome changes in HCM, RNA sequencing was conducted on left ventricle (LV) and left atrium (LA) samples of healthy cats and cats with HCM (each n = 5; 20 samples). Ingenuity Pathway Analysis was used to determine functional pathways, regulators, and networks. Distinct gene expression profiles were identified in the LV and LA of the feline healthy and HCM myocardium. Analysis of differentially expressed mRNAs (>2 fold; FDR < 0.01) found chamber-specific (LV vs. LA) expression in both healthy and HCM groups, with higher transcriptional activity in the LA. Genes that contribute to the distinct structure and function of each chamber in health and HCM were identified in the regional comparison. The gene expression profiles of HCM compared to healthy hearts revealed disease related genes, including THBS4 and KLHL33 (LV), FAM177B and THRSP (LA), the latter 3 have not been reported for the myocardium so far, as the top differently expressed genes in the HCM heart. Differently expressed genes and functional pathways found in the HCM heart are associated with cardiac remodeling and fibrosis, inflammation, microvascular changes, calcium signaling and cardiac metabolism, with some regional differences. RhoGDI-RhoGTPase signaling, integrin and ILK signaling pathways, the LXR/RXR pathway in the LA, and the PPARα/RXRα, HIF1α and CXCR4 pathways in the LV might be of particular importance in the HCM disease process. This study identified region-specific myocardial gene transcription patterns as well as novel genes and pathways associated with HCM.

Prognostic value of epigenetic markers for canine mast cell cancer.

Canine Mast cell tumors (MCTs) constitute approximately 21% of all canine skin tumors. Despite the use of comprehensive grading systems, biological aggressiveness is sometimes difficult to predict, therefore there is a need for better prognostic markers. Progression in various cancers involves DNA hypermethylation, hypomethylation and epigenetic enzyme dysregulation. Therefore, global levels of 5-methylcytosine, 5-hydroxymethylcytosine and associated enzymes DNMT1, and IDH1 expression may predict MCT aggressiveness. A tissue microarray (TMA) with cores from 244 different tumor samples from 189 dogs was immunolabelled and used to quantify the global DNA methylation and hydroxymethylation levels as well as the levels of the enzymes involved in DNA methylation and their relationship with canine MCT outcome. From the immunolabelled TMA, H-scores were generated using QuPath (v0.1.2) and analyzed with associated patient data. High 5MC and DNMT1, and low IDH1 levels were associated with poorer outcome when looking at all canine MCT cases. High 5MC levels showed significance for shorter disease-free interval (DFI) in subcutaneous cases and high 5MC levels showed poorer DFI and overall survival (OS) in cases with Kiupel's grading system high grade. Cases with grade II in Patnaik's grading system showed better DFI with low levels of DNMT1 and better OS with low levels of 5MC and 5HMC. High levels of DNMT1 staining were also associated with shorter DFI for dermal MCTs. For cases that received adjuvant therapy in addition to surgery, all parameters except IDH1 were significantly associated with OS. Therefore, there is potential for DNA methylation status and levels of enzymes associated with DNA methylation pathways to better predict outcome in canine MCT, and to possibly influence treatment decisions.

International business and Africa: Theoretical and applied challenges, and future directions.

In response to Nachum et al.'s (J Int Bus Stud, 2023) call for further research in Africa by international business (IB) scholars, we argue that while IB scholars may have been slow to engage with Africa, the same cannot be said of related and IB-relevant business and management scholarship. There is already a substantial body of work on Africa in other domains of business and management scholarship - and relevant theorizing - that represents an important resource for IB scholarship. In contextualizing the 'interesting', we identify several contemporary theoretical strands that have so far characterized 'Africa research', interrogate ongoing challenges that mitigate these efforts, and suggest ways in which further research that speaks to theoretical, practical, and policy issues might inform IB researchers' engagement with Africa. Specifically, we set out the broader scope of the African business/management debate that might inform IB research, re-examine African diversity through the prism of 'theoretical tensions and puzzles', and consider the role of emergent indigenous theorizing such as ubuntu and Africapitalism that make Africa both 'interesting' and worthy of IB inquiry.

En réponse à l'appel lancé par Nachum et al. (2023) en faveur d'une intensification des recherches sur l'Afrique par les chercheurs en affaires internationales (International Business - IB), nous arguons que si les chercheurs en IB ont peut-être tardé à s'intéresser à l'Afrique, il n'en va pas de même pour ceux dans les disciplines du commerce et du management connexes et apparentées au domaine de l'IB. Il existe déjà un corps important de travaux sur l'Afrique dans d'autres domaines de la recherche en commerce et management - ainsi qu'une théorisation pertinente - qui constitue une grande ressource pour la recherche en IB. En contextualisant l'" intéressant ", nous identifions un certain nombre de courants théoriques contemporains qui ont jusqu'à présent caractérisé la " recherche sur l'Afrique ", nous interrogeons sur les défis actuels atténuant ces efforts, et suggérons les manières dont des recherches supplémentaires portées sur des questions politiques, pratiques et théoriques pourraient éclairer l'engagement des chercheurs en IB envers l'Afrique. Plus précisément, nous définissons la portée plus large du débat sur le commerce et le management en Afrique qui pourrait alimenter la recherche en IB, réexaminons la diversité africaine à travers le prisme des " tensions et énigmes théoriques ", et examinons le rôle de la théorisation indigène émergente, tels l'ubuntu et l'Africapitalisme qui rendent l'Afrique à la fois " intéressante " et digne d'être étudiée dans le domaine de l'IB.

En respuesta al llamamiento de Nachum et al. (2023) para que los especialistas en negocios internacionales realicen más investigaciones en África, afirmamos que, aunque los académicos en negocios internacionales hayan tardado en comprometerse con África, no puede decirse lo mismo de la investigación en negocios internacionales y de gestión. Ya existe un conjunto sustancial de trabajos sobre África en otros ámbitos de los negocios y la gestión -y una teorización relevante- que representa un recurso importante para los estudiosos de negocios internacionales. Al contextualizar lo "interesante", identificamos una serie de corrientes teóricas contemporáneas que han caracterizado hasta ahora la "investigación sobre África", interrogamos los desafíos actuales que mitigan estos esfuerzos y sugerimos formas en las que una mayor investigación que hable de cuestiones teóricas, prácticas y políticas podría informar el compromiso de los investigadores de negocios internacionales con África. En concreto, exponemos el alcance más amplio del debate sobre los negocios y la gestión en África que podría servir de base para la investigación sobre negocios internacionales, reexaminamos la diversidad africana a través del prisma de las "tensiones y los rompecabezas teóricos" y consideramos el papel de las teorías autóctonas emergentes, como el ubuntu y el africapitalismo, que hacen que África sea "interesante" y digna de ser investigada por negocios internacionales.

Em resposta ao pedido de Nachum et al. (2023) para pesquisas adicionais na África por acadêmicos de negócios internacionais (IB), argumentamos que, embora acadêmicos de IB possam ter sido tardios ao se envolver com a África, o mesmo não pode ser dito sobre estudos relevantes e relacionados a IB sobre gestão e negócios. Já existe um conjunto substancial de trabalhos sobre África em outros domínios da pesquisa em gestão e negócios - e desenvolvimento teórico relevante - que representa um recurso importante para a pesquisa em IB. Ao contextualizar o "interessante", identificamos uma série de vertentes teóricas contemporâneas que até agora caracterizaram a "pesquisa sobre África", interrogamos desafios correntes que mitigam esses esforços e sugerimos maneiras pelas quais pesquisas adicionais que tratem de questões teóricas, práticas e políticas possam balizar o envolvimento de pesquisadores em IB com a África. Especificamente, estabelecemos o escopo mais amplo do debate africano sobre gestão e negócios que pode balizar a pesquisa em IB, reexaminamos a diversidade africana através do prisma de "tensões teóricas e enigmas" e consideramos o papel da emergente teorização nativa, como o ubuntu e o africapitalismo, que tornam a África "interessante" e digna de investigação em IB.

PCSK9 facilitates melanoma pathogenesis via a network regulating tumor immunity.

BACKGROUND: PCSK9 regulates cholesterol homeostasis and promotes tumorigenesis. However, the relevance of these two actions and the mechanisms underlying PCSK9's oncogenic roles in melanoma and other cancers remain unclear. METHODS: PCSK9's association with melanoma was analysed using the TCGA dataset. Empty vector (EV), PCSK9, gain-of-function (D374Y), and loss-of-function (Q152H) PCSK9 mutant were stably-expressed in murine melanoma B16 cells and studied for impact on B16 cell-derived oncogenesis in vitro and in vivo using syngeneic C57BL/6 and Pcsk9-/- mice. Intratumoral accumulation of cholesterol was determined. RNA-seq was performed on individual tumor types. Differentially-expressed genes (DEGs) were derived from the comparisons of B16 PCSK9, B16 D374Y, or B16 Q152H tumors to B16 EV allografts and analysed for pathway alterations. RESULTS: PCSK9 expression and its network negatively correlated with the survival probability of patients with melanoma. PCSK9 promoted B16 cell proliferation, migration, and growth in soft agar in vitro, formation of tumors in C57BL/6 mice in vivo, and accumulation of intratumoral cholesterol in a manner reflecting its regulation of the low-density lipoprotein receptor (LDLR): Q152H, EV, PCSK9, and D374Y. Tumor-associated T cells, CD8 + T cells, and NK cells were significantly increased in D374Y tumors along with upregulations of multiple immune checkpoints, IFNγ, and 143 genes associated with T cell dysfunction. Overlap of 36 genes between the D374Y DEGs and the PCSK9 DEGs predicted poor prognosis of melanoma and resistance to immune checkpoint blockade (ICB) therapy. CYTH4, DENND1C, AOAH, TBC1D10C, EPSTI1, GIMAP7, and FASL (FAS ligand) were novel predictors of ICB therapy and displayed high level of correlations with multiple immune checkpoints in melanoma and across 30 human cancers. We observed FAS ligand being among the most robust biomarkers of ICB treatment and constructed two novel and effective multigene panels predicting response to ICB therapy. The profiles of allografts produced by B16 EV, PCSK9, D374Y, and Q152H remained comparable in C57BL/6 and Pcsk9-/- mice. CONCLUSIONS: Tumor-derived PCSK9 plays a critical role in melanoma pathogenesis. PCSK9's oncogenic actions are associated with intratumoral cholesterol accumulation. PCSK9 systemically affects the immune system, contributing to melanoma immune evasion. Novel biomarkers derived from the PCSK9-network effectively predicted ICB therapy responses.

Neutrophils in Dendritic Cell-Based Cancer Vaccination: The Potential Roles of Neutrophil Extracellular Trap Formation.

Neutrophils have conflicting roles in the context of cancers, where they have been associated with contributing to both anti-tumor and pro-tumor responses. Their functional heterogenicity is plastic and can be manipulated by environmental stimuli, which has fueled an area of research investigating therapeutic strategies targeting neutrophils. Dendritic cell (DC)-based cancer vaccination is an immunotherapy that has exhibited clinical promise but has shown limited clinical efficacy. Enhancing our understanding of the communications occurring during DC cancer vaccination can uncover opportunities for enhancing the DC vaccine platform. There have been observed communications between neutrophils and DCs during natural immune responses. However, their crosstalk has been poorly studied in the context of DC vaccination. Here, we review the dual functionality of neutrophils in the context of cancers, describe the crosstalk between neutrophils and DCs during immune responses, and discuss their implications in DC cancer vaccination. This discussion will focus on how neutrophil extracellular traps can influence immune responses in the tumor microenvironment and what roles they may play in promoting or hindering DC vaccine-induced anti-tumor efficacy.

Multiplex flow cytometry-based assay for quantifying tumor- and virus-associated antibodies induced by immunotherapies.

Novel immunotherapies continue to be developed and tested for application against a plethora of diseases. The clinical translation of immunotherapies requires an understanding of their mechanisms. The contributions of antibodies in driving long-term responses following immunotherapies continue to be revealed given their diverse effector functions. Developing an in-depth understanding of the role of antibodies in treatment efficacy is required to optimize immunotherapies and improve the chance of successfully translating them into the clinic. However, analyses of antibody responses can be challenging in the context of antigen-agnostic immunotherapies, particularly in the context of cancers that lack pre-defined target antigens. As such, robust methods are needed to evaluate the capacity of a given immunotherapy to induce beneficial antibody responses, and to identify any therapy-limiting antibodies. We previously developed a comprehensive method for detecting antibody responses induced by antigen-agnostic immunotherapies for application in pre-clinical models of vaccinology and cancer therapy. Here, we extend this method to a high-throughput, flow cytometry-based assay able to identify and quantify isotype-specific virus- and tumor-associated antibody responses induced by immunotherapies using small sample volumes with rapid speed and high sensitivity. This method provides a valuable and flexible protocol for investigating antibody responses induced by immunotherapies, which researchers can use to expand their analyses and optimize their own treatment regimens.

Feline Oncogenomics: What Do We Know about the Genetics of Cancer in Domestic Cats?

Cancer is a significant cause of morbidity and mortality in domestic cats. In humans, an understanding of the oncogenome of different cancer types has proven critical and is deeply interwoven into all aspects of patient care, including diagnostics, prognostics and treatments through the application of targeted therapies. Investigations into understanding the genetics of feline cancers started with cytogenetics and was then expanded to studies at a gene-specific level, looking for mutations and expression level changes of genes that are commonly mutated in human cancers. Methylation studies have also been performed and together with a recently generated high-quality reference genome for cats, next-generation sequencing studies are starting to deliver results. This review summarises what is currently known of the genetics of both common and rare cancer types in cats, including lymphomas, mammary tumours, squamous cell carcinomas, soft tissue tumours, mast cell tumours, haemangiosarcomas, pulmonary carcinomas, pancreatic carcinomas and osteosarcomas. Shining a spotlight on our current understanding of the feline oncogenome will hopefully serve as a springboard for more much-needed research into the genetics of cancer in domestic cats.

Prognostic significance of the urokinase plasminogen activator system in tissue and serum of dogs with appendicular osteosarcoma.

Urokinase plasminogen activator (uPA) and its receptor uPAR promote cancer invasion and metastasis and are emerging therapeutic targets in both human and canine malignancies. While their clinical significance is well-characterized in multiple human tumor types, studies investigating their roles in osteosarcoma are lacking. The objectives of this study were to characterize serum and tissue uPA/uPAR expression in dogs with osteosarcoma and assess the prognostic significance. Serum samples and a tissue microarray of canine appendicular osteosarcoma were analyzed for uPA and uPAR expression by ELISA (n = 49) and immunohistochemistry (n = 38), respectively. Serum uPA activity was also measured by a chromogenic assay (n = 25). Survival analysis was performed by Kaplan-Meier survival analysis, log rank test, and Cox regression analysis. Serum uPA level was significantly higher in dogs with osteosarcoma than clinically healthy control dogs (median 1905 vs 1440 pg/ml, p = 0.008). The majority of canine osteosarcoma tissues expressed uPA (75.9%) or uPAR (77.6%), with 70.7% dual-positivity, indicating autocrine/paracrine activation of the pathway. Survival analysis revealed shorter progression free survival (PFS) in dogs with high serum uPA level in a discovery cohort (n = 29; median PFS 94 vs 266 days, p = 0.003) but not in a validation cohort (n = 23; median PFS 167 vs 490 days, p = 0.16). The difference was significant when both cohorts were combined (n = 49; median PFS 128 vs 266 days, p = 0.003). Serum uPAR and tissue uPA/uPAR levels were not prognostic. In Cox multivariate analysis, high serum uPA level and activity were both associated with poor prognosis, independent of serum ALP, tumor location, and peripheral lymphocyte/monocyte counts. These results indicate high utilization of the uPA pathway and association with disease progression in canine osteosarcoma. Further study involving prospective evaluation to confirm the prognostic significance is warranted. The high prevalence of tissue uPA and uPAR expression suggests the uPA system as a potential therapeutic target in canine osteosarcoma.

Binary isobaric phase diagrams of stearyl alcohol-poloxamer 407 formulations in the molten and solid state.

Multiparticulate formulations allow for the design of specialized pharmaceutical dosage forms that cater to the needs of a wide range of patient demographics, such as pediatric and geriatric populations, by affording control over the release rate and facilitating the formulation of fixed-dose combination drugs. Melt spray-congealing (MSC) is a method for preparing multiparticulate dosage forms from a suspension or solid solution of active pharamaceutical ingredients (API) and a molten carrier matrix. Stearyl alcohol and poloxamer 407 mixtures are widely used as carrier matrices in MSC microsphere formulations. In this report, the phase equilibria of stearyl alcohol-poloxamer 407 mixtures were investigated by generating binary phase diagrams of composition, i.e. weight/weight percent of poloxamer 407 in stearyl alcohol, and temperature in the molten form and the solid state. The phase equilibria of the molten state were characterized by 1H NMR measurements. The miscibility curves of stearyl alcohol-poloxamer 407 molten mixtures revealed that stearyl alcohol and poloxamer 407 are not miscible in all proportions and that miscibility substantially increases with temperature. The phase equilibria of the solid state were characterized by DSC and PXRD experiments. The phase diagrams of the solid state indicate that stearyl alcohol and poloxamer 407 crystallize and melt separately and, thus, do not form a eutectic or a single phase. The phases equilibria of the bulk mixtures were compared to the phases observed in placebo MSC microspheres and it was determined that the microspheres consist of a mixture of thermodynamically stable and metastable stearyl alcohol crystals immediately after manufacture.

Investigating the Role of Solvent in the Formation of Vacancies on Ibuprofen Crystal Facets.

Surface defects play a crucial role in the process of crystal growth, as incorporation of growth units generally takes place on undercoordinated sites on the growing crystal facet. In this work, we use molecular simulations to obtain information on the role of the solvent in the roughening of three morphologically relevant crystal faces of form I of racemic ibuprofen. To this aim, we devise a computational strategy to evaluate the energetic cost associated with the formation of a surface vacancy for a set of ten solvents, covering a range of polarities and hydrogen bonding propensities. We find that the mechanism as well as the work of defect formation are markedly solvent and facet dependent. Based on Mean Force Integration and Well Tempered Metadynamics, the methodology developed in this work has been designed with the aim of capturing solvent effects at the atomistic scale while maintaining the computational efficiency necessary for implementation in high-throughput in-silico screenings of crystallization solvents.

Crystal structures of anhydrous and hydrated ceftibuten.

Ceftibuten, C15H14N4O6S2, with the systematic name (6R,7R)-7-{[(Z)-2-(2-amino-1,3-thia-zol-4-yl)-4-carb-oxy-but-2-eno-yl]amino}-8-oxo-5-thia-1-aza-bicyclo-[4.2.0]oct-2-ene-2-carb-oxy-lic acid, is a third generation, orally administered cephalosporin anti-biotic with broad anti-microbial activity and stability against extended spectrum ß-lactamases. Ceftibuten can exist in various hydration states and to better understand the location of the water mol-ecules of crystallization and their effect on the structure, the crystal structures of anhydrous (I) and hydrated (II) ceftibuten were determined and both occur as zwitterions with proton transfer from the carboxyl-ate group adjacent to the ß-lactam ring to the N atom of the thia-zole ring. The ß-lactam ring in (I) is almost planar but the equivalent grouping in (II) is slightly buckled. In the extended structure of (I), O-H⋯O and N-H⋯O hydrogen bonds link the mol-ecules into a three-dimensional network. In (II), O-H⋯Oc, N-H⋯Oc, O-H⋯Ow, N-H⋯Ow and Ow-H⋯Ow (c = ceftibuten, w = water) hydrogen bonds link the components into a three-dimensional network. A large void space is present within the anhydrous crystal structure that can accommodate between two and three mol-ecules of water.

Kinetic Modeling of API Oxidation: (2) Imipramine Stress Testing.

Gauging the chemical stability of active pharmaceutical ingredients (APIs) is critical at various stages of pharmaceutical development to identify potential risks from drug degradation and ensure the quality and safety of the drug product. Stress testing has been the major experimental method to study API stability, but this analytical approach is time-consuming, resource-intensive, and limited by API availability, especially during the early stages of drug development. Novel computational chemistry methods may assist in screening for API chemical stability prior to synthesis and augment contemporary API stress testing studies, with the potential to significantly accelerate drug development and reduce costs. In this work, we leverage quantum chemical calculations and automated reaction mechanism generation to provide new insights into API degradation studies. In the continuation of part one in this series of studies [Grinberg Dana et al., Mol. Pharm. 2021 18 (8), 3037-3049], we have generated the first ab initio predictive chemical kinetic model of free-radical oxidative degradation for API stress testing. We focused on imipramine oxidation in an azobis(isobutyronitrile) (AIBN)/H2O/CH3OH solution and compared the model's predictions with concurrent experimental observations. We analytically determined iminodibenzyl and desimipramine as imipramine's two major degradation products under industry-standard AIBN stress testing conditions, and our ab initio kinetic model successfully identified both of them in its prediction for the top three degradation products. This work shows the potential and utility of predictive chemical kinetic modeling and quantum chemical computations to elucidate API chemical stability issues. Further, we envision an automated digital workflow that integrates first-principle models with data-driven methods that, when actively and iteratively combined with high-throughput experiments, can substantially accelerate and transform future API chemical stability studies.

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle.

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients.