Acute myeloid leukemia (AML) is characterized by an accumulation of immature myeloid precursors in the bone marrow and, in many cases, the peripheral blood. From analysis of recurrent chromosomal translocations in bone marrow samples from AML patients, it is clear that loss-of-function mutations in transcription factors critical for hematopoiesis are involved in leukemogenesis. These balanced reciprocal translocations often result in the expression of fusion proteins that act as dominant negative inhibitors of the normal transcription factors in hematopoietic development, such as the AML1-ETO product of the t(8;21) translocation and the PML-RARα product of the t(15;17) translocation. Loss-of-function point mutations in transcription factors involved in hematopoietic differentiation, such as C/EBPα and AML1, have also been identified in AML. Although these mutations involving hematopoietic transcription factors are necessary for AML pathogenesis, they are not sufficient (1-4). It is hypothesized that the AML phenotype Oncogenic ras alleles are among the most common mutations found in patients with acute myeloid leukemia (AML). Previously, the role of oncogenic ras in cancer was assessed in model systems overexpressing oncogenic ras from heterologous promoters. However, there is increasing evidence that subtle differences in gene dosage and regulation of gene expression from endogenous promoters play critical roles in cancer pathogenesis. We characterized the role of oncogenic K-ras expressed from its endogenous promoter in the hematopoietic system using a conditional allele and IFN-inducible, Cre-mediated recombination. Mice developed a completely penetrant myeloproliferative syndrome characterized by leukocytosis with normal maturation of myeloid lineage cells; myeloid hyperplasia in bone marrow; and extramedullary hematopoiesis in the spleen and liver. Flow cytometry confirmed the myeloproliferative phenotype. Genotypic and Western blot analysis demonstrated Cre-mediated excision and expression, respectively, of the oncogenic K-ras allele. Bone marrow cells formed growth factor-independent colonies in methylcellulose cultures, but the myeloproliferative disease was not transplantable into secondary recipients. Thus, oncogenic K-ras induces a myeloproliferative disorder but not AML, indicating that additional mutations are required for AML development. This model system will be useful for assessing the contribution of cooperating mutations in AML and testing ras inhibitors in vivo.

We have investigated the impact of transmitter release site (active zone; AZ) structure on synaptic function by physically rearranging the individual AZ elements in a previously published frog neuromuscular junction (NMJ) AZ model into the organization observed in a mouse NMJ AZ. We have used this strategy, purposefully without changing the properties of AZ elements between frog and mouse models (even though there are undoubtedly differences between frog and mouse AZ elements in vivo), to directly test how structure influences function at the level of an AZ. Despite a similarly ordered ion channel array substructure within both frog and mouse AZs, frog AZs are much longer and position docked vesicles in a different location relative to AZ ion channels. Physiologically, frog AZs have a lower probability of transmitter release compared with mouse AZs, and frog NMJs facilitate strongly during short stimulus trains in contrast with mouse NMJs that depress slightly. Using our computer modeling approach, we found that a simple rearrangement of the AZ building blocks of the frog model into a mouse AZ organization could recapitulate the physiological differences between these two synapses. These results highlight the importance of simple AZ protein organization to synaptic function. NEW & NOTEWORTHY A simple rearrangement of the basic building blocks in the frog neuromuscular junction model into a mouse transmitter release site configuration predicted the major physiological differences between these two synapses, suggesting that transmitter release site structure and organization is a strong predictor of function.

Objective: To analyze serial biomarkers of the persistent inflammation, immunosuppression, and catabolism syndrome (PICS) to gain insight into the pathobiology of chronic critical illness (CCI) after surgical sepsis. Background: Although early deaths after surgical intensive care unit sepsis have decreased and most survivors rapidly recover (RAP), one third develop the adverse clinical trajectory of CCI. However, the underlying pathobiology of its dismal long-term outcomes remains unclear. Methods: PICS biomarkers over 14 days from 124 CCI and 225 RAP sepsis survivors were analyzed to determine associations and prediction models for (1) CCI (≥14 intensive care unit days with organ dysfunction) and (2) dismal 1-year outcomes (Zubrod 4/5 performance scores). Clinical prediction models were created using PIRO variables (predisposition, insult, response, and organ dysfunction). Biomarkers were then added to determine if they strengthened predictions. Results: CCI (vs RAP) and Zubrod 4/5 (vs Zubrod 0–3) cohorts had greater elevations in biomarkers of inflammation (interleukin [IL]-6, IL-8, interferon gamma-induced protein [IP-10], monocyte chemoattractant protein 1), immunosuppression (IL-10, soluble programmed death ligand-1), stress metabolism (C-reactive protein, glucagon-like peptide 1), and angiogenesis (angiopoietin-2, vascular endothelial growth factor, vascular endothelial growth factor receptor-1, stromal cell-derived factor) at most time-points. Clinical models predicted CCI on day 4 (area under the receiver operating characteristics curve [AUC] = 0.89) and 1 year Zubrod 4/5 on day 7 (AUC = 0.80). IL-10 and IP-10 on day 4 minimally improved prediction of CCI (AUC = 0.90). However, IL-10, IL-6, IL-8, monocyte chemoattractant protein 1, IP-10, angiopoietin-2, glucagon-like peptide 1, soluble programmed death ligand-1, and stromal cell-derived factor on day 7 considerably improved the prediction of Zubrod 4/5 status (AUC = 0.88). Conclusions: Persistent elevations of PICS biomarkers in the CCI and Zubrod 4/5 cohorts and their improved prediction of Zubrod 4/5 validate that PICS plays a role in CCI pathobiology.

Implementation of protocolized surveillance, diagnosis, and management of septic patients, and of surgical sepsis patients in particular, is shown to result in significantly increased numbers of patients surviving their initial hospitalization. Currently, most surgical sepsis patients will rapidly recover from sepsis; however, many patients will not rapidly recover, but instead will go on to develop chronic critical illness (CCI) and experience dismal long-term outcomes. The elderly and comorbid patient is highly susceptible to death or CCI after sepsis. Here, we review aspects of the Persistent Inflammation, Immunosuppression, and Catabolism Syndrome (PICS) endotype to explain the underlying pathobiology of a dysregulated immune system in sepsis survivors who develop CCI; then, we explore targets for immunomodulatory therapy.

Short-term synaptic facilitation occurs during high-frequency stimulation, is known to be dependent on presynaptic calcium ions, and persists for tens of milliseconds after a presynaptic action potential. We have used the frog neuromuscular junction as a model synapse for both experimental and computer simulation studies aimed at testing various mechanistic hypotheses proposed to underlie short-term synaptic facilitation. Building off our recently reported excess-calcium-binding-site model of synaptic vesicle release at the frog neuromuscular junction (Dittrich M, Pattillo JM, King JD, Cho S, Stiles JR, Meriney SD. Biophys J 104: 2751–2763, 2013), we have investigated several mechanisms of short-term facilitation at the frog neuromuscular junction. Our studies place constraints on previously proposed facilitation mechanisms and conclude that the presence of a second class of calcium sensor proteins distinct from synaptotagmin can explain known properties of facilitation observed at the frog neuromuscular junction. We were further able to identify a novel facilitation mechanism, which relied on the persistent binding of calcium-bound synaptotagmin molecules to lipids of the presynaptic membrane. In a real physiological context, both mechanisms identified in our study (and perhaps others) may act simultaneously to cause the experimentally observed facilitation. In summary, using a combination of computer simulations and physiological recordings, we have developed a stochastic computer model of synaptic transmission at the frog neuromuscular junction, which sheds light on the facilitation mechanisms in this model synapse.