Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.

Technical variant techniques expand the pediatric donor pool and reduce time from listing to transplant, but they are associated with increased morbidity and mortality.

Background: Refractometry is used to assess transfer of passive immunity (TPI), but studies evaluating different refractometers and appropriate thresholds for recommended target immunoglobulin G (IgG) concentrations for beef calves are limited. Objectives: To evaluate test performance of digital (DSTP) and optical (OSTP) serum total protein (STP) refractometers and a digital Brix (DBRIX) refractometer for assessment of passive immunity in beef calves. Animals: A total of 398 beef calves from 6 herds, 1 to 7 days of age. Methods: Serum IgG concentration was estimated by DSTP, OSTP, and DBRIX, and measured by radial immunodiffusion (RID). Correlation coefficients (r) among results were calculated. Optimal STP and Brix thresholds for identification of IgG <10, <16, and <24 g/L were determined using interval likelihood ratios. Refractometer performance and agreement were assessed using areas under the curve (AUC), diagnostic test characteristics, Cohen's kappa (κ), and Bland-Altman analysis. Results: Refractometer results were highly correlated with RID (r = 0.82-0.91) and with each other (r = 0.91-0.95), and overall test performance was excellent (AUC = 0.93-0.99). The STP concentrations of ≤5.1, ≤5.1, and ≤5.7 g/dL and Brix percentages of ≤7.9%, ≤8.3%, and ≤8.7% indicated IgG concentrations <10, <16, and <24 g/L, respectively. Agreement of refractometers with RID was variable (κ = 0.46-0.80) and among refractometers was substantial (κ = 0.62-0.89). Conclusions and Clinical Importance: All refractometers showed good utility as monitoring tools for assessment of TPI in beef calves.

Acute myeloid leukemia (AML) is a disease characterized by uncontrolled proliferation of immature myeloid cells in the blood and bone marrow. The 5-year survival rate is approximately 25%, and recent therapeutic developments have yielded little survival benefit. Therefore, there is an urgent need to identify novel therapeutic targets. We previously demonstrated that acid ceramidase (ASAH1, referred to as AC) is upregulated in AML and high AC activity correlates with poor patient survival. Here, we characterized a novel AC inhibitor, SACLAC, that significantly reduced the viability of AML cells with an EC50 of approximately 3 μmol/L across 30 human AML cell lines. Treatment of AML cell lines with SACLAC effectively blocked AC activity and induced a decrease in sphingosine 1-phosphate and a 2.5-fold increase in total ceramide levels. Mechanistically, we showed that SACLAC treatment led to reduced levels of splicing factor SF3B1 and alternative MCL-1 mRNA splicing in multiple human AML cell lines. This increased proapoptotic MCL-1S levels and contributed to SACLAC-induced apoptosis in AML cells. The apoptotic effects of SACLAC were attenuated by SF3B1 or MCL-1 overexpression and by selective knockdown of MCL-1S. Furthermore, AC knockdown and exogenous C16-ceramide supplementation induced similar changes in SF3B1 level and MCL-1S/L ratio. Finally, we demonstrated that SACLAC treatment leads to a 37% to 75% reduction in leukemic burden in two human AML xenograft mouse models. Implications: These data further emphasize AC as a therapeutic target in AML and define SACLAC as a potent inhibitor to be further optimized for future clinical development.

Lipins 1, 2, and 3 are Mg-dependent phosphatidic acid phosphatases and catalyze the penultimate step of triacylglycerol synthesis. We have previously investigated the biochemistry of lipins 1 and 2 and shown that di-anionic phosphatidic acid (PA) augments their activity and lipid binding and that lipin 1 activity is negatively regulated by phosphorylation. In the present study, we show that phosphorylation does not affect the catalytic activity of lipin 3 or its ability to associate with PA The lipin proteins each contain a conserved polybasic domain (PBD) composed of nine lysine and arginine residues located between the conserved N- and C-terminal domains. In lipin 1, the PBD is the site of PA binding and sensing of the PA electrostatic charge. The specific arrangement and number of the lysines and arginines of the PBD vary among the lipins. We show that the different PBDs of lipins 1 and 3 are responsible for the presence of phosphoregulation on the former but not the latter enzyme. To do so, we generated lipin 1 that contained the PBD of lipin 3 and vice versa. The lipin 1 enzyme with the lipin 3 PBD lost its ability to be regulated by phosphorylation but remained downstream of phosphorylation by mammalian target of rapamycin. Conversely, the presence of the lipin 1 PBD in lipin 3 subjected the enzyme to negative intramolecular control by phosphorylation. These results indicate a mechanism for the observed differences in lipin phosphoregulation.

Background Brix refractometry can be used to assess colostral immunoglobulin G (IgG) concentration, but studies identifying Brix percentages to detect high‐ and low‐IgG colostrum are lacking for beef cows and interlaboratory agreement is unknown. Objectives Evaluate Brix refractometer performance and interlaboratory agreement for assessing beef cow colostrum IgG concentration, including determination of thresholds to identify colostrum containing IgG concentrations <100 g/L and ≥150 g/L. Animals Beef cows (n = 416) from 11 cow‐calf operations in Alberta, Canada. Methods Colostral IgG concentrations were measured using radial immunodiffusion (RID) and estimated by Brix refractometry for this retrospective study. Spearman correlation coefficients were assessed between RID and Brix refractometry. Likelihood ratios and misclassification cost‐term analysis were used to determine optimal Brix percentages for detecting colostrum containing IgG concentrations <100 g/L and ≥150 g/L. Concordance correlation coefficient (CCC) and Bland‐Altman analyses were performed for Brix percentages obtained at 3 different laboratories. Results Brix percentages obtained at 3 laboratories were positively correlated with IgG results ( r = 0.72, 0.68, and 0.76, respectively). Colostrum Brix percentages of <24% and ≥30% were optimal for indicating IgG concentrations of <100 g/L and ≥150 g/L, respectively. Interlaboratory agreement was substantial, with CCC ranging from 0.89 to 0.96 and Bland‐Altman analysis showing small mean differences (−1.2% to 0.09% Brix) and narrow limits of agreements (−4.8% to 2.4% Brix) among laboratories. Conclusions and Clinical Importance Brix refractometry shows good potential for reliably estimating IgG concentrations in beef cow colostrum across multiple laboratories and can be recommended to aid colostrum management decisions on farms.

This cross-sectional study quantifies subclinical trauma associated with calving difficulty, calf vigour, and passive immunity (PI) in newborn beef calves. The degree of calving difficulty was categorised as: unassisted, easy assist (one or two people manually pulling to deliver the calf) and difficult assist (more than two people pulling, a fetal extractor (ie, calf jack), or caesarean section). Vigour assessment occurred at 10 minutes and blood sampling at 24 hours after birth in 77 beef calves. The measured blood parameters associated with trauma were creatine kinase (CK), aspartate aminotransferase (AST), and haptoglobin. Serum IgG concentration was measured, and an IgG concentration at least 24 g/l was considered as adequate PI. Calving difficulty was associated with elevated levels of CK (P=0.002) and AST (P=0.01), weak suckle reflex (P=0.001), abnormal mucous membrane colour (P<0.0001), and decreased odds of adequate PI (P=0.004). Elevated levels of CK and AST were associated with abnormal mucous membrane colour, incomplete tongue withdrawal and weak suckle reflex at birth (P<0.001). An incomplete tongue withdrawal (P=0.005) and weak suckle reflex (P=0.02) were associated with decreased IgG concentrations. Abnormal mucous membrane colour, incomplete tongue withdrawal, and a weak suckle reflex were associated with decreased odds of having adequate PI (P<0.05). Haptoglobin was not associated with any of the parameters measured. Subclinical trauma was associated with calving difficulty, decreased vigour and decreased odds of having adequate PI. Understanding the impacts of a traumatic birth may aid the development of management strategies for compromised newborn beef calves.

Summary Sphingolipid metabolism is increasingly recognised as a therapeutic target in cancer due to its regulation of cell proliferation and apoptosis. The sphingolipid rheostat is proposed to control cell fate through maintaining balance between pro‐apoptotic and pro‐survival sphingolipids. This balance is regulated by metabolising enzymes involved in sphingolipid production. One such enzyme, sphingosine kinase‐2 (SPHK2), produces pro‐survival sphingosine 1‐phosphate (S1P) by phosphorylation of pro‐apoptotic sphingosine. Elevated SPHK2 has been found in multiple cancer types and contributes to cell survival, chemotherapeutic resistance and apoptosis resistance. We have previously shown elevation of S1P in large granular lymphocyte (LGL) leukaemia serum and cells isolated from patients. Here, we examined SPHK2 expression in LGL leukaemia and found SPHK2 mRNA and protein upregulation in a majority of LGL leukaemia patient samples. Knockdown of SPHK2 with siRNA in LGL leukaemia cell lines decreased proliferation. Additionally, the use of ABC294640 or K145, both SPHK2‐specific inhibitors, decreased viability of LGL leukaemia cell lines. ABC294640 selectively induced apoptosis in LGL cell lines and freshly isolated LGL leukaemia patient cells compared to normal controls. Mechanistically, SPHK2 inhibition downregulated pro‐survival myeloid cell leukaemia‐1 (Mcl‐1) protein through proteasomal degradation. Targeting of SPHK2 therefore provides a novel therapeutic approach for the treatment of LGL leukaemia.