Orexinergic Neurotransmission in Temperature Responses to Methamphetamine and Stress: Mathematical Modeling as a Data Assimilation Approach

Experimental Data

Orexinergic neurotransmission is involved in mediating temperature responses to methamphetamine (Meth). In experiments in rats, SB-334867 (SB), an antagonist of orexin receptors (OX1R), at a dose of 10 mg/kg decreases late temperature responses (t>60 min) to an intermediate dose of Meth (5 mg/kg). A higher dose of SB (30 mg/kg) attenuates temperature responses to low dose (1 mg/kg) of Meth and to stress. In contrast, it significantly exaggerates early responses (t<60 min) to intermediate and high doses (5 and 10 mg/kg) of Meth. As pretreatment with SB also inhibits temperature response to the stress of injection, traditional statistical analysis of temperature responses is difficult.

Mathematical Modeling

We have developed a mathematical model that explains the complexity of temperature responses to Meth as the interplay between excitatory and inhibitory nodes. We have extended the developed model to include the stress of manipulations and the effects of SB. Stress is synergistic with Meth on the action on excitatory node. Orexin receptors mediate an activation of on both excitatory and inhibitory nodes by low doses of Meth, but not on the node activated by high doses (HD). Exaggeration of early responses to high doses of Meth involves disinhibition: low dose of SB decreases tonic inhibition of HD and lowers the activation threshold, while the higher dose suppresses the inhibitory component. Using a modeling approach to data assimilation appears efficient in separating individual components of complex response with statistical analysis unachievable by traditional data processing methods.     

Production of Bio-Energy from Pig Manure: A Focus on the Dynamics Change of Four Parameters under Sunlight-Dark Conditions

This study investigated the effect of sunlight-dark conditions on volatile fatty acids (VFAs), total ammonium nitrogen (TAN), total alkalinity (TA) and pH during pig manure (PM) digestion and then the subsequent influence on biogas yield of PM. PM₁ and PM₂ were performed in a transparent reactor and a non-transparent reactor, respectively. Two sets of experiments were conducted with a temperature of 35.0±2.0 °C and a total solid concentration of 8.0% to the digestion material. The dynamic change of the four parameters in response to sunlight-dark conditions resulted in variations of the physiological properties in the digester and affected the cumulative biogas production (CBP). PM₁ obtained higher CBP (15020.0 mL) with a more stable pH and a lower TAN concentration (1414.5 mg/L) compared to PM₂ (2675.0 mL and 1670.0 mg/L, respectively). The direct path coefficients and indirect path coefficients between the four parameters and CBP were also analyzed.     

Understanding the Role of Dicer in Astrocyte Development

The Dicer1 allele is used to show that microRNAs (miRNAs) play important roles in astrocyte development and functions. While it is known that astrocytes that lack miRNAs are dysregulated, the in vivo phenotypes of these astrocytes are not well understood. In this study, we use Aldh1l1-EGFP transgene, a marker of astrocytes, to characterize mouse models with conditional Dicer1 ablation (via either human or mouse GFAP-Cre). This transgene revealed novel features of the defective astrocytes from the absence of miRNA. Although astrocyte miRNAs were depleted in both lines, we found histological and molecular differences in the Aldh1l1-EGFP cells between the two Cre lines. Aldh1l1-EGFP cells from hGFAP-Cre mutant lines displayed up-regulation of Aldh1l1-EGFP with increased proliferation and a genomic profile that acquired many features of wildtype primary astrocyte cultures. In the young mGFAP-Cre mutant lines we found that Aldh1l1-EGFP cells were disorganized and hyperproliferative in the developing cerebellum. Using the Aldh1l1-EGFP transgene, our work provides new insights into the roles of miRNAs in astrocyte development and the features of astrocytes in these two mouse models.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.