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Acute myelogenous leukemia (AML), the most prevalent acute and aggressive leukemia diagnosed in adults, often recurs as a difficult-to-treat, chemotherapy-resistant disease. Because chemotherapy resistance is a major obstacle to successful treatment, novel therapeutic intervention is needed. Upregulated ceramide clearance via accelerated hydrolysis and glycosylation has been shown to be an element in chemotherapy-resistant AML, a problem considering the crucial role ceramide plays in eliciting apoptosis. Herein we employed agents that block ceramide clearance to determine if such a "reset" would be of therapeutic benefit. SACLAC was utilized to limit ceramide hydrolysis, and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-threo-PDMP) was used to block the glycosylation route. The SACLAC D-threo-PDMP inhibitor combination was synergistically cytotoxic in drug-resistant, P-glycoprotein-expressing (P-gp) AML but not in wt, P-gp-poor cells. Interestingly, P-gp antagonists that can limit ceramide glycosylation via depression of glucosylceramide transit also synergized with SACLAC, suggesting a paradoxical role for P-gp in the implementation of cell death. Mechanistically, cell death was accompanied by a complete drop in ceramide glycosylation, concomitant, striking increases in all molecular species of ceramide, diminished sphingosine 1-phosphate levels, resounding declines in mitochondrial respiratory kinetics, altered Akt, pGSK-3β, and Mcl-1 expression, and caspase activation. Although ceramide was generated in wt cells upon inhibitor exposure, mitochondrial respiration was not corrupted, suggestive of mitochondrial vulnerability in the drug-resistant phenotype, a potential therapeutic avenue. The inhibitor regimen showed efficacy in an in vivo model and in primary AML cells from patients. These results support the implementation of SL enzyme targeting to limit ceramide clearance as a therapeutic strategy in chemotherapy-resistant AML, inclusive of a novel indication for the use of P-gp antagonists., This work was supported by grants from the National Institutes of Health (National Cancer Institute NIH P01 CA171983 (T.P.L., M.K., M.C.C.), DOD-W81XWH-19-1-0213 (K.H.F.-W.), Spanish Ministry of Science and Innovation PID2020-113813RB-100 (G.F.), and the Brody Brothers Foundation (K.H.F.-W.), Kinston, NC.

In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

In conventional flowcytometry one detector (primary) is dedicated for one fluorochrome. However, photons usually end up in other detectors too (fluorescence spillover). ‘Compensation’ is a process that corrects the spillover signal from all detectors except the primary detector. Post ‘compensation’, the photon counting error of spillover signals become evident as spreading of the data. The spreading induced by spillover impairs the ability to resolve stained cell population from the unstained one, potentially reducing or completely losing cell populations. For successful multi-color panel design, it is important to know the expected spillover to maximize the data resolution. The Spillover Spreading Matrix (SSM) can be used to estimate the spread, but the outcome is dependent on detector sensitivity. Simply, the same single stained sample produces different spillover spread values when detector(s) sensitivity is altered. Many researchers mistakenly use this artifact to “reduce” the spread by decreasing detector sensitivity. This can result in diminished capacity to resolve dimly expressing cell populations. Here, we introduce SQI (Spread Quantification Index), that can quantify the spillover spread independent of detector sensitivity and independent of dynamic range. This allows users to compare spillover spread between instruments having different types of detectors, which is not possible using SSM.

Cytometer characterization is critical to define operational bounds within which the data generated are reliable and reproducible. Existing instrument optimization and characterization protocols were developed for cytometers relying on photomultiplier tubes (PMTs) for photon detection. Recently, instrument manufacturers have begun incorporating avalanche photodiodes (APDs) in place of PMTs. Differences in noise and signal amplification properties of the two detector types make many of the established PMT characterization protocols inappropriate for APD-based instruments. In this article, we tested (three machines on two different sites) a variety of approaches to determine the best method for APD optimization on the Beckman Coulter CytoFLEX™ (CytoFLEX). From this, we propose easy-to-implement guidelines for CytoFLEX characterization and operation. These protocols are not designed to compare APD versus PMT based systems, nor are they designed to directly compare different CytoFlex instruments. Following these protocols will allow CytoFLEX users to characterize their instruments and help to identify optimized settings that allow for the generation of consistent and reproducible data. © 2020 International Society for Advancement of Cytometry.

Adults with type 2 diabetes have a two-to-three-fold increased risk of cardiovascular diseases. B cell activating factor (BAFF), a tumor necrosis superfamily member, is implicated both in diabetes and cardiovascular diseases. BAFF deficiency in mice retains glucose homeostasis despite high-fat diet-induced obesity. We examined if neutralization of BAFF with an anti-BAFF antibody improves glucose utilization in high-fat diet-induced obese mice. Male C57BL/6J mice fed with a high-fat diet (60% calories from fat) for 12 weeks were segregated into two groups based on their glucose intolerance (GI) determined by a glucose tolerance test (GTT). Each group received a total of 4 injections of anti-BAFF or an isotype control antibody, and GTT was performed every two weeks. BAFF neutralization was confirmed by depletion of B2 B cells in the blood two weeks after the first anti-BAFF antibody injection. Although GI increased after continuous feeding of a high-fat diet, no differences in GI was observed between the groups till 18 weeks on diet. At 20 weeks, GI was significantly higher in the anti-BAFF treated group compared to the control group. Extensive cellular phenotyping using flow cytometry revealed that BAFF neutralization depleted mature B2 B cells in the spleen and blood but did not affect the CD4+ T cells and CD8+ T cells. No differences were found in fat accumulation in the liver, whole body fat content, visceral adipose tissue (VAT) weight, or in the number of VAT-infiltrated M1 and M2 macrophages, eosinophils, natural killer T cells, CD4+ T cells, and CD8+ T cells. Unexpectedly, in the VAT of anti-BAFF antibody-treated mice, B2 cells were retained, whereas, the number of CD4+ T cells and dead adipocytes was higher. Real-time RT-PCR analysis of stromal vascular fraction from VAT revealed increased expression of both the M1 and M2 macrophage markers IL-1β, IL-6, IL-10, Arg1, and Mrc1 in the anti-BAFF treated mice. Altogether, these results suggest BAFF neutralization promotes inflammation in the VAT of obese mice and exacerbates systemic glucose intolerance. Furthermore, our results highlight the need for more studies on the use of anti-BAFF biologics for the treatment of systemic lupus erythematosus patients who are diabetic.

Summary: Adipocytes, once considered simple lipid-storing cells, are rapidly emerging as complex cells with many biologically diverse functions. A powerful high-throughput method for analyzing single cells is flow cytometry. Several groups have attempted to analyze and sort freshly isolated adipocytes; however, using an adipocyte-specific reporter mouse, we demonstrate that these studies fail to detect the majority of white adipocytes. We define critical settings required for adipocyte flow cytometry and provide a rigid strategy for analyzing and sorting white and brown adipocyte populations. The applicability of our protocol is shown by sorting mouse adipocytes based on size or UCP1 expression and demonstrating that a subset of human adipocytes lacks the β2-adrenergic receptor, particularly in the insulin-resistant state. In conclusion, the present study confers key technological insights for analyzing and sorting mature adipocytes, opening up numerous downstream research applications. : Freshly isolated adipocytes are a notoriously difficult cell type to study. Hagberg et al. provide a detailed flow cytometry protocol for the analysis and sorting of mouse and human adipocytes by defining the critical factors and conditions required for studying this specialized cell type and pinpointing common pitfalls. Keywords: adipocyte, adipose tissue, flow cytometry, FACS, mouse, human, uncoupled protein 1, beta 2 adrenergic receptor

Melanoma is one of the most aggressive forms of cancer, usually resistant to standard chemotherapeutics. Despite a huge number of clinical trials, any success to find a chemotherapeutic agent that can effectively destroy melanoma is yet to be achieved. Para-phenylenediamine (p-PD) in the hair dyes is reported to purely serve as an external dyeing agent. Very little is known about whether p-PD has any effect on the melanin producing cells. We have demonstrated p-PD mediated apoptotic death of both human and mouse melanoma cellsin vitro. Mouse melanoma tumour growth was also arrested by the apoptotic activity of intraperitoneal administration of p-PD with almost no side effects. This apoptosis is shown to occur primarily via loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and caspase 8 activation. p-PD mediated apoptosis was also confirmed by the increase in sub-G0/G1 cell number. Thus, our experimental observation suggests that p-PD can be a potential less expensive candidate to be developed as a chemotherapeutic agent for melanoma.

Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas.

Hepatitis B e-antigen negative (e(-)) chronic HBV infection (CHI) encompasses a heterogeneous clinical spectrum ranging from inactive carrier (IC) state to e(-) chronic hepatitis B (CHB), cirrhosis and hepatic decompensation. In the backdrop of dysfunctional virus-specific T cells, natural killer (NK) cells are emerging as innate effectors in CHI. We characterized CD3(-) CD56(+) NK cells in clinically well-defined, treatment-naive e(-) patients in IC, e(-)CHB or decompensated liver cirrhosis (LC) phase to appraise their role in disease progression. The NK cell frequencies increased progressively with disease severity (IC 8.2%, e(-)CHB 13.2% and LC 14.4%). Higher proportion of NK cells from LC/e(-)CHB expressed CD69, NKp46, NKp44, TRAIL and perforin, the last two being prominent features of CD56(bright) and CD56(dim) NK subsets, respectively. The frequencies of CD3(-) CD56(+) NK cells together with TRAIL(+) CD56(bright) and Perforin(+) CD56(dim) NK cells correlated positively with serum alanine transaminase levels in e(-)CHB/LC. K562 cell-stimulated NK cells from e(-)CHB/LC exhibited significantly greater degranulation but diminished interferon-γ production than IC. Further, Perforin(+) NK cell frequency inversely correlated with autologous CD4(+) T-cell count in e(-) patients and ligands of NK receptors were over-expressed in CD4(+) T cells from e(-)CHB/LC relative to IC. Co-culture of sorted CD56(dim) NK cells and CD4(+) T cells from e(-)CHB showed enhanced CD4(+) T-cell apoptosis, which was reduced by perforin inhibitor, concanamycin A, suggesting a possible perforin-dependent NK cell-mediated CD4(+) T-cell depletion. Moreover, greater incidence of perforin-expressing NK cells and decline in CD4(+) T cells were noticed intrahepatically in e(-)CHB than IC. Collectively, NK cells contribute to the progression of e(-)CHI by enhanced TRAIL- and perforin-dependent cytolytic activity and by restraining anti-viral immunity through reduced interferon-γ secretion and perforin-mediated CD4(+) T-cell lysis.

Piperine, the main alkaloid of black pepper, Piper nigrum Linn., is an important Indian spice used in traditional food and medicine in India. In the present study, we investigated the antioxidant activities of piperine against copper-ascorbate induced toxic injury to mitochondria obtained from a goat heart, in vitro. Incubation of isolated cardiac mitochondria with copper-ascorbate resulted in elevated levels of lipid peroxidation and protein carbonylation of the mitochondrial membrane, a reduced level of mitochondrial GSH and altered status of antioxidant enzymes as well as decreased activities of pyruvate dehydrogenase and the Kreb's cycle enzymes, altered mitochondrial morphology, mitochondrial swelling, di-tyrosine level and mitochondrial DNA damage. All these changes were found to be ameliorated when the cardiac mitochondria were co-incubated with copper-ascorbate and piperine, in vitro. Piperine, in our in vitro experiments, was found to scavenge hydrogen peroxide, superoxide anion free radicals, hydroxyl radicals and DPPH radicals, in a chemically defined system, indicating that this compound may provide protection to cardiac mitochondria against copper-ascorbate induced toxic injury through its antioxidant activities. The results of this study suggest that piperine may be considered as a future therapeutic antioxidant and may be used singly or as a co-therapeutic in the treatment of diseases associated with mitochondrial oxidative stress.

Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.

Summary Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5− and formed distant metastases in which Lgr5+ CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5− cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors., Graphical Abstract, Highlights • The majority of disseminating cells of colorectal cancer are Lgr5− • Lgr5− cancer cells are the main seeds of colorectal cancer metastatic lesions • Long-term metastatic growth from Lgr5− cells requires appearance of Lgr5+ cells • Lgr5− metastases have the intrinsic capacity to re-establish the cellular hierarchy, Van Rheenen and colleagues study Lgr5+ cancer stem cells during colorectal cancer metastasis. They demonstrate that the majority of metastases are seeded by Lgr5− cells, which upon arrival seed metastases in which Lgr5+ cells appear. This plasticity can occur independently of stemness-inducing factors and is indispensable for long-term metastatic growth.