Identification and Analysis of Subtypes of Liver Cancer Based on Genes Related to E3 Ubiquitin Ligases and Deubiquitinating Enzymes.

Recent studies have reported a correlation between ubiquitination or deubiquitination and cancer development. But mechanisms underlying the roles of genes associated with E3 ubiquitin ligases and deubiquitinating enzymes (DUB) in liver cancer remain to be explored. We analyzed and screened differentially expressed genes related to E3 ubiquitin ligases and DUB in liver cancer on the basis of public databases. Cluster analysis was utilized to classify liver cancer samples into different subtypes. Survival analysis, immune analysis, and pathway enrichment analysis were performed on the subtypes. We constructed a protein-protein interaction network using STRING to screen hub genes. Finally, we used the Connectivity Map (CMap) database to predict targeted small molecules. The results show that a total of 139 differentially expressed E3/DUB genes in liver cancer were screened. Then, liver cancer was classified into two subtypes, cluster 1 and cluster 2, based on E3-related and DUB-related genes. Patients in cluster 1 had higher survival rates and immune levels than those in cluster 2. Four hub genes (RPSA, RPS5, RPL30, and RPL8) significantly affecting the survival of the two subtypes of liver cancer patients were identified based on cluster 1 and cluster 2. Finally, the CMap database predicted that small-molecule drugs including probenecid, dexamethasone, and etomidate may improve the prognosis of liver cancer patients. These findings may offer a reference for risk stratification studies and drug development in liver cancer.

Hydroxyurea therapy of a murine model of sickle cell anemia inhibits the progression of pneumococcal disease by down-modulating E-selectin.

Sickle cell anemia is characterized by chronic hemolysis coupled with extensive vascular inflammation. This inflammatory state also mechanistically promotes a high risk of lethal, invasive pneumococcal infection. Current treatments to reduce vaso-occlusive complications include chronic hydroxyurea therapy to induce fetal hemoglobin. Because hydroxyurea also reduces leukocytosis, an understanding of the impact of this treatment on pneumococcal pathogenesis is needed. Using a sickle cell mouse model of pneumococcal pneumonia and sepsis, administration of hydroxyurea was found to significantly improve survival. Hydroxyurea treatment decreased neutrophil extravasation into the infected lung coincident with significantly reduced levels of E-selectin in serum and on pulmonary epithelia. The protective effect of hydroxyurea was abrogated in mice deficient in E-selectin. The decrease in E-selectin levels was also evident in human sickle cell patients receiving hydroxyurea therapy. These data indicate that in addition to induction of fetal hemoglobin, hydroxyurea attenuates leukocyte-endothelial interactions in sickle cell anemia, resulting in protection against lethal pneumococcal sepsis.

Horseradish peroxidase cDNA as a marker for electron microscopy in neurons.

A reliable and convenient marker of gene transfer in neurons is lacking for electron microscopy. To facilitate the use of molecular genetic approaches in such studies, we introduce the use of horseradish peroxidase (HRP) cDNA as a marker that identifies transfected neurons for correlated light and electron microscopy (EM). By flanking the cDNA with an endoplasmic reticulum (ER)-retention signal, expressed HRP was localized and retained inside the ER of neurons. The construct was placed under the control of the synapsin promoter resulting in neuron-specific expression of HRP. After transfection of hippocampal neurons in culture and visualization of HRP with diaminobenzidine (DAB) after fixation, both cell bodies and proximal dendrites of HRP-positive neurons were readily identifiable by light microscopy. After processing for EM, the heavily DAB-labeled ER was obvious in both cell bodies and dendrites of transfected neurons. In addition, the labeling of distal dendrites, axons, and synapses was apparent. The structural preservation of transfected neurons was optimal as is typical for glutaraldehyde-fixed tissue. Due to the ER-retention signal, HRP was contained inside the ER and the ultrastructure of all other components was not occluded and was quantitatively accessible. These characteristics make HRP co-transfection a potentially powerful tool for modern electron microscopy in both cell biology and neuroscience.

Bacterial Peptidoglycan Traverses the Placenta to Induce Fetal Neuroproliferation and Aberrant Postnatal Behavior

Maternal infection during pregnancy is associated with adverse outcomes for the fetus, including postnatal cognitive disorders. However, the underlying mechanisms are obscure. We find that bacterial cell wall peptidoglycan (CW), a universal PAMP for TLR2, traverses the murine placenta into the developing fetal brain. In contrast to adults, CW-exposed fetal brains did not show any signs of inflammation or neuronal death. Instead, the neuronal transcription factor FoxG1 was induced, and neuroproliferation leading to a 50% greater density of neurons in the cortical plate was observed. Bacterial infection of pregnant dams, followed by antibiotic treatment, which releases CW, yielded the same result. Neuroproliferation required TLR2 and was recapitulated in vitro with fetal neuronal precursor cells and TLR2/6, but not TLR2/1, ligands. The fetal neuroproliferative response correlated with abnormal cognitive behavior in CW-exposed pups following birth. Thus, the bacterial CW-TLR2 signaling axis affects fetal neurodevelopment and may underlie postnatal cognitive disorders.

Caspase-dependent and -independent panc-1 cell death due to actinomycin D and MK 886 are additive but increase clonogenic survival.

In human panc-1 pancreatic cancer cells, actinomycin D (act D) induces a type 1 (apoptotic, extrinsic, death domain, receptor-dependent, and caspase-positive) form of programmed cell death (PCD) and MK 886, a 5-lipoxygenase inhibitor serving among other functions as a surrogate for increasing oxidative stress, a type 2 form, defined as an intrinsic, mitochondria-dependent, autophagic form of cellular suicide. Using both agents simultaneously should allow for examination of their interaction in cells able to express either form of PCD. Activation of both forms might result in synergistic, additive, null, or inhibitory effects on the reduction in proliferation, PCD, and clonogenicity of surviving cells. Co-culture of panc-1 cells with act D and MK 886, which both inhibit their proliferation, had an additive effect on increasing the development of these forms of PCD, as determined by morphology, a nucleosome assay, and flow cytometry. Initially, laddering on agarose detected with propidium iodide, present in act D, and act D plus MK 886-treated cells was partially obscured by randomly degraded DNA. With the use of the more sensitive SYBR green dye and reduced exposure of detached cells to 37 degrees C, a limited laddering of DNA from MK 886-treated cells was also detected. Caspase activity was present in act-D-cultured cells but was absent in cells cultured with MK 886. Combined culture reduced caspase activity in act D-treated cells, consistent with interference from type 2 of type 1 PCD. Removal after 48 hr of act D or MK 886 allowed regrowth of residual cells, the latter agent to a greater extent than the former. In combination, the number of clones was increased compared with act D alone. These features distinguish two forms of PCD. In therapeutic settings in which the modes of cell death have not been identified, unintentional activation of several cellular suicide pathways with "crosstalk" between them occurs. Their intentional simultaneous activation and responses, as modulated by the history of cells in or out of cycle, could reduce the intended therapeutic outcome with survival of additional clonogenic cells due to various forms of mutual interference.

Statins protect against fulminant pneumococcal infection and cytolysin toxicity in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is characterized by intravascular hemolysis and inflammation coupled to a 400-fold greater incidence of invasive pneumococcal infection resulting in fulminant, lethal pneumococcal sepsis. Mechanistically, invasive infection is facilitated by a proinflammatory state that enhances receptor-mediated endocytosis of pneumococci into epithelial and endothelial cells. As statins reduce chronic inflammation, in addition to their serum cholesterol-lowering effects, we hypothesized that statin therapy might improve the outcome of pneumococcal infection in SCD. In this study, we tested this hypothesis in an experimental SCD mouse model and found that statin therapy prolonged survival following pneumococcal challenge. The protective effect resulted in part from decreased platelet-activating factor receptor expression on endothelia and epithelia, which led to reduced bacterial invasion. An additional protective effect resulted from inhibition of host cell lysis by pneumococcal cholesterol-dependent cytotoxins (CDCs), including pneumolysin. We conclude therefore that statins may be of prophylactic benefit against invasive pneumococcal disease in patients with SCD and, more broadly, in settings of bacterial pathogenesis driven by receptor-mediated endocytosis and the CDC class of toxins produced by Gram-positive invasive bacteria.

Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function.

Synaptic vesicle (SV) size is one parameter that controls the amount of neurotransmitter released from individual SVs and, therefore, is fundamental to our understanding of synaptic function. The recently discovered variability of mean SV size among excitatory hippocampal synapses -- if actively regulated -- is a potential mechanism for the regulation of transmitter release. Here, we investigated which parameters influence mean SV size. First, we revealed that synapse-to-synapse variability of SV size is a general phenomenon in several species and brain regions. In addition, we determined the relationship between mean SV size and synaptic morphology. In three-dimensional reconstructions from serial ultrathin sections, we found that SV size did not correlate with the area of the postsynaptic density (a measure for synaptic size and synaptic cleft volume) nor with the total number of SVs within a bouton or bouton volume. We tested the long-held hypothesis that a change in osmotic pressure (potentially caused by a change in neurotransmitter concentration) affects SV size. When we reduced the osmotic pressure, SVs became significantly smaller; however, an increase in osmotic pressure had no effect on SV size. Furthermore, we found that SV size does not adapt to chronic changes in activity and that the SV cycle is capable of providing constant SV size during long-lasting, high-frequency stimulation.