Caspase Inhibition Blocks Cell Death and Enhances Mitophagy but Fails to Promote T-Cell Lymphoma

Caspase-9 is a component of the apoptosome that mediates cell death following release of cytochrome c from mitochondria. Inhibition of Caspase-9 with a dominant negative construct (Casp9DN) blocks apoptosome function, promotes viability and has been implicated in carcinogenesis. Inhibition of the apoptosome in vitro impairs mitochondrial function and promotes mitophagy. To examine whether inhibition of the apoptosome would enhance mitophagy and promote oncogenesis in vivo, transgenic mice were generated that express Casp9DN in the T cell lineage. The effects of Casp9DN on thymocyte viability, mitophagy and thymic tumor formation were examined. In primary thymocytes, Casp9DN delayed dexamethasone (Dex)-induced cell death, altered mitochondrial structure, and decreased oxidant production. Transmission electron microscopy (TEM) revealed that inhibition of the apoptosome resulted in structurally abnormal mitochondria that in some cases were engulfed by double-membrane structures resembling autophagosomes. Consistent with mitochondria being engulfed by autophagosomes (mitophagy), confocal microscopy showed colocalization of LC3-GFP and mitochondria. However, Casp9DN did not significantly accelerate T-cell lymphoma alone, or in combination with Lck-Bax38/1, or with Beclin 1+/− mice, two tumor-prone strains in which altered mitochondrial function has been implicated in promoting tumor development. In addition, heterozygous disruption of Beclin 1 had no effect on T-cell lymphoma formation in Lck-Bax38/1 mice. Further studies showed that Beclin 1 levels had no effect on Casp9DN-induced loss of mitochondrial function. These results demonstrate that neither inhibition of apoptosome function nor Beclin 1 haploinsufficiency accelerate T-cell lymphoma development in mice

Agouti-related peptide neural circuits mediate adaptive behaviors in the starved state

In the face of starvation, animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents, for example, will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression and fear. Hypothalamic agouti-related peptide (AgRP)-expressing neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques in mice, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principal bed nucleus of the stria terminalis, which suppresses territorial aggression and reduces contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues

Casp9DN expression has no effect on thymic cellularity and proliferation in Lck-Bax38/1 mice.

<p><b>A</b>) DNA content analysis was performed on freshly isolated thymocytes from mice of the indicated genotypes between 8 and 10 weeks of age. Each symbol corresponds to the percentage of cycling cells (%S/G2/M) observed from an individual mouse. The mean ± SD of at least 6 mice per group is shown. <b>B</b>) Total thymic cellularity from mice of the indicated genotypes between 8 and 10 weeks of age is shown. Each symbol corresponds to the total viable thymocytes number observed from an individual mouse. The mean ± SD of at least 6 mice per group is shown.</p

Beclin 1+/− has no effect on cell death, proliferation and ROS levels in thymocytes.

<p><b>A</b>) Isolated thymocytes from mice of the indicated genotypes were treated with Dex (1μM), using Q-VD-OPh (50 μM) as positive control for Caspase inhibition. Viability was determined over time. The means ± SD of at least 2 mice per group are shown. B) DNA content analysis of thymocytes (top panel) and thymic cellularity (lower panel) were performed on freshly isolated thymocytes from mice of the indicated genotypes between 8 and 12 weeks of age. Each symbol corresponds to the percentage of cycling cells (%S/G2/M) and the total viable thymocytes observed from an individual mouse. The mean ± SD of each group is shown. C) Representative histograms overlays of DHE and MitoSOX Red staining of the viable thymocytes isolated from Beclin 1+/+ (gray shaded) and Beclin 1+/− (black line) mice following the indicated treatment are shown. The viability of thymocytes following 24h of Dex treatment: 7.3% (Beclin 1+/+) and 8.9% (Beclin 1+/−). These results are representative of at least 3 independent experiments. D) Representative histogram overlays of the DHE and MitoSOX Red staining of the viable thymocytes isolated from Beclin 1+/+ and Beclin 1+/− mice are shown. For each graph, Beclin 1+/+ cells without caspase inhibition (gray shaded), Beclin 1+/+ cells with caspase inhibition (black line) and Beclin 1+/− cells with caspase inhibition (green line) either by Casp9DN expression or Q-VD-OPh treatment are shown. The viability of thymocytes following 24h of Dex treatment was as follows: 7.3% (Beclin 1+/+), 20% (Beclin 1+/+ x Casp9DN), and 22% (Beclin 1+/− x Casp9DN), 42% (Beclin 1+/+ Q-VD-OPh), and 43% (Beclin 1+/− Q-VD-OPh).</p

Caspase inhibition increases abnormal mitochondria and activates mitophagy.

<p><b>A</b>) TEM images showed morphologic changes in mitochondrial structures (normal mitochondria marked with black arrows and abnormal mitochondria marked with yellow arrows) in isolated thymocytes from mice of the indicated genotypes following the indicated treatment. One of examples of mitophagy showed that a mitochondrial cluster was engulfed within double-membrane autophagosome in bottom-right image. Four images have the same magnification. Scale Bar  = 0.2 µm. N = nucleus. <b>B</b>) Quantitation of the average number of normal and abnormal mitochondria per viable cell (left panel) and the percentages of cells with at least 1 normal mitochondrion or with at least 1 abnormal mitochondrion (right panel) in isolated thymocytes from mice of indicated genotypes following 4 h of the indicated treatment. <b>C</b>) Quantitation of the percentage of cells with at least 1 abnormal mitochondria in isolated thymocytes from mice of the indicated genotypes following various times of Dex treatment. Table shows that no abnormal mitochondria were observed in WT thymocytes following various treatments. In the WT thymocytes treated with 24 h of Dex, 5% of the cells were viable, so this sample is not shown (@). WT thymocytes treated with 0 h of Q-VD-OPh were not available (NA). <b>D</b>) In FL5-Neo cells, quantitation of the average number of normal and abnormal mitochondria per viable cell (left panel) and the percentages of viable cells with at least 1 normal mitochondrion or with at least 1 abnormal mitochondrion (right panel) following 24 h of the indicated treatments. <b>E</b>) FL5-Neo cells transfected with LC3-GFP (green) were stained with MitoTracker Red (red, MR) for mitochondria and TO-PRO-3 probe (blue) for nuclear counter staining following 48 h of the indicated treatment. The viability of FL5 cells: 97% (+IL-3), 9% (-IL-3), and 66% (-IL-3+QVD). Colocalization of MR and LC3-GFP indicated mitophagy activation. <b>F</b>) FL5 cells with the indicated expression were immuno-stained with p62 (green), MR (red) for mitochondria and TO-PRO-3 probe (blue) for nuclear counter staining following 48 h of IL-3 deprivation and BafA (20 nM). Pearson's correlation coefficient (PCC, R(r)) which statistically measures colocalization of p62 and MR is shown in each image. Quantitation of PCC of the treated FL5 cells is shown. Each symbol corresponds to PCC of each viable cell of the indicated cell lines. The mean ± SD of at least 22 cells per cell line in each treatment is shown. ** p<0.0001.</p

An unusual case of primary hepatic lymphoma with dramatic but unsustained response to bendamustine plus rituximab and literature review

Objectives: Primary hepatic lymphoma is an uncommon cause of hepatic space-occupying lesions. Methods: We describe the case of a 73-year-old man with primary hepatic lymphoma, who presented with a low-grade fever and lower limb weakness which had progressed in the past 2 months. Results: Abdominal ultrasound and computed tomography showed multiple small hepatic tumors. Echo-guided biopsy of the hepatic tumor demonstrated primary hepatic diffuse large B cell lymphoma. Moreover, bone marrow was uninvolved, but the bone marrow smear disclosed hemophagocytosis, which is uncommon in diffuse large B cell lymphoma. Chemotherapy with bendamustine and rituximab treatment was initiated with a dramatic response: hepatic tumors markedly shrank in size shown by follow-up computed tomography and the patient returned to his normal life. Nevertheless, the response was sustained for only 8 months. Finally, the disease resisted further chemotherapy and this patient died of a severe Klebsiella pneumoniae infection. Conclusion: Chemotherapy with bendamustine and rituximab has shown a dramatic, but not durable, response in the present case with old age and multiple comorbidities

A 110-GHz Push-Push Balanced Colpitts Oscillator Using 0.15-μm GaN HEMT Technology

This paper presents a sub-THz oscillator using 0.15-µm GaN HEMT technology. A push-push balanced Colpitts topology is proposed to achieve a very high operating frequency while also with high output power under low DC power consumption. The source-floating transistor with an in-house built three-terminal transistor model was employed to allow using the transmission line (TL) at the source node for Colpitts feedback capacitor with improved negative resistance. In addition, the layout is in Grounded-CPW (GCPW) configuration with backside vias to achieve an optimized low-loss TL structure. The measured results show that the GaN oscillator can reach a peak output power of -2.1 dBm with a tuning range from 107.9~109.5 GHz, and the measured phase noise is -110.8 dBc/Hz at a 10 MHz offset