HIF3A gene disruption causes abnormal alveoli structure and early neonatal death.

Transcriptional response to changes in oxygen concentration is mainly controlled by hypoxia-inducible transcription factors (HIFs). Besides regulation of hypoxia-responsible gene expression, HIF-3α has recently been shown to be involved in lung development and in the metabolic process of fat tissue. However, the precise mechanism for such properties of HIF-3α is still largely unknown. To this end, we generated HIF3A gene-disrupted mice by means of genome editing technology to explore the pleiotropic role of HIF-3α in development and physiology. We obtained adult mice carrying homozygous HIF3A gene mutations with comparable body weight and height to wild-type mice. However, the number of litters and ratio of homozygous mutation carriers born from the mating between homozygous mutant mice was lower than expected due to sporadic deaths on postnatal day 1. HIF3A gene-disrupted mice exhibited abnormal configuration of the lung such as a reduced number of alveoli and thickened alveolar walls. Transcriptome analysis showed, as well as genes associated with lung development, an upregulation of stearoyl-Coenzyme A desaturase 1, a pivotal enzyme for fatty acid metabolism. Analysis of fatty acid composition in the lung employing gas chromatography indicated an elevation in palmitoleic acid and a reduction in oleic acid, suggesting an imbalance in distribution of fatty acid, a constituent of lung surfactant. Accordingly, administration of glucocorticoid injections during pregnancy resulted in a restoration of normal alveolar counts and a decrease in neonatal mortality. In conclusion, these observations provide novel insights into a pivotal role of HIF-3α in the preservation of critically important structure and function of alveoli beyond the regulation of hypoxia-mediated gene expression.

Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody.

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

Ca2+ -Calmodulin-Calcineurin Signaling Modulates α-Synuclein Transmission.

BACKGROUND: The intercellular transmission of pathogenic proteins plays a crucial role in the progression of neurodegenerative diseases. Previous research has shown that the neuronal uptake of such proteins is activity-dependent; however, the detailed mechanisms underlying activity-dependent α-synuclein transmission in Parkinson's disease remain unclear. OBJECTIVE: To examine whether α-synuclein transmission is affected by Ca2+ -calmodulin-calcineurin signaling in cultured cells and mouse models of Parkinson's disease. METHODS: Mouse primary hippocampal neurons were used to examine the effects of the modulation of Ca2+ -calmodulin-calcineurin signaling on the neuronal uptake of α-synuclein preformed fibrils. The effects of modulating Ca2+ -calmodulin-calcineurin signaling on the development of α-synuclein pathology were examined using a mouse model injected with α-synuclein preformed fibrils. RESULTS: Modulation of Ca2+ -calmodulin-calcineurin signaling by inhibiting voltage-gated Ca2+ channels, calmodulin, and calcineurin blocked the neuronal uptake of α-synuclein preformed fibrils via macropinocytosis. Different subtypes of voltage-gated Ca2+ channel differentially contributed to the neuronal uptake of α-synuclein preformed fibrils. In wild-type mice inoculated with α-synuclein preformed fibrils, we found that inhibiting calcineurin ameliorated the development of α-synuclein pathology. CONCLUSION: Our data suggest that Ca2+ -calmodulin-calcineurin signaling modulates α-synuclein transmission and has potential as a therapeutic target for Parkinson's disease. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges.

The advancement in epigenetics research over the past several decades has led to the potential application of epigenome-editing technologies for the treatment of various diseases. In particular, epigenome editing is potentially useful in the treatment of genetic and other related diseases, including rare imprinted diseases, as it can regulate the expression of the epigenome of the target region, and thereby the causative gene, with minimal or no modification of the genomic DNA. Various efforts are underway to successfully apply epigenome editing in vivo, such as improving target specificity, enzymatic activity, and drug delivery for the development of reliable therapeutics. In this review, we introduce the latest findings, summarize the current limitations and future challenges in the practical application of epigenome editing for disease therapy, and introduce important factors to consider, such as chromatin plasticity, for a more effective epigenome editing-based therapy.

Prediction of Single Trial Somatosensory Evoked Potentials From Mechanical Stimulation Intensity.

Somatosensory evoked potentials (SSEPs) are important for both scientific research and the evaluation of treatment efficacy in neurorehabilitation. SSEPs measure the response in the sensorimotor cortex to a peripheral stimulation. Individual responses are often noisy, so SSEPs have typically required hundreds of stimulation-response trials to produce a single measurement. This work presents a method to estimate single trial SSEPs from tendon hammer accelerations. While predictions from the input side can not completely replace actual measurements of SSEPs, the results produced may help to provide clinicians insight where full scale SSEP measurement is not practical.

Towards Image Guided Magnetic Resonance Elastography via Active Driver Positioning Robot.

Magnetic Resonance Elastography (MRE) is a developing imaging technique that enables non-invasive estimation of tissue mechanical properties through the combination of induced mechanical displacements in the tissue and Magnetic Resonance Imaging (MRI). The mechanical drivers necessary to produce shear waves in the tissue have been a focus of engineering effort in the development and refinement of MRE. The potential targeting of smaller and stiffer tissues calls for increases in actuation frequency and refinement of mechanical driver positioning. Furthermore, the anisotropic nature of soft tissues results in driver position related changes in observed displacement wave patterns. These challenges motivate the investigation and development of the concept of active MRE driver positioning through visual servoing under MR imaging. OBJECTIVE: This work demonstrates the initial prototype of an MRE driver positioning system, allowing capture of displacement wave patterns from various mechanical vibration loading angles under different vibration frequencies through MR imaging. METHODS: Three different configurations of the MRE driver positioning robot are tested with an intervertebral disc (IVD) shaped gel phantom. RESULTS: Both the octahedral shear stress signal to noise ratio (OSS-SNR) and estimated stiffness show statistically significant dependence on driver configuration in each of the three phantom IVD regions. CONCLUSION: This dependence demonstrates that driver configuration is a critical factor in MRE, and that the developed robot is capable of producing a range of configurations. SIGNIFICANCE: This work presents the first demonstration of an active, imaging guided MRE driver positioning system, with significance for the future application of MRE to a wider range of human tissues.

Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway.

BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an important technique in male infertility treatment. Currently, sperm selection for ICSI in human assisted reproductive technology (ART) is subjective, based on a visual assessment by the operator. Therefore, it is desirable to develop methods that can objectively provide an accurate assessment of the shape and size of sperm heads that use low-magnification microscopy available in most standard fertility clinics. Recent studies have shown a correlation between sperm head size and shape and chromosomal abnormalities, and fertilization rate, and various attempts have been made to establish automated computer-based measurement of the sperm head itself. For example, a dictionary-learning technique and a deep-learning-based method have both been developed. Recently, an automatic algorithm was reported that detects sperm head malformations in real time for selection of the best sperm for ICSI. These data suggest that a real-time sperm selection system for use in ICSI is necessary. Moreover, these systems should incorporate inverted microscopes (×400-600 magnification) but not the fluorescence microscopy techniques often used for a dictionary-learning technique and a deep-learning-based method. These advances are expected to improve future success rates of ARTs. In this review, we summarize recent reports on the assessment of sperm head shape, size, and acrosome status in relation to fertility, and propose further improvements that can be made to the ARTs used in infertility treatments.

α-Synuclein Propagation Mouse Models of Parkinson's Disease.

Parkinson's disease (PD) is pathologically characterized by intraneuronal α-synuclein (α-Syn) inclusions called Lewy bodies (LBs) and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Autopsy studies have suggested that Lewy pathology initially occurs in the olfactory bulb and enteric nervous system, subsequently spreading in the brain stereotypically. Recent studies have demonstrated that templated fibrillization and intercellular dissemination of misfolded α-Syn underlie this pathological progression. Injection of animals with α-Syn preformed fibrils (PFFs) can recapitulate LB-like inclusions and the subsequent intercellular transmission of α-Syn pathology. Moreover, targeting specific brain regions or body parts enables the generation of unique models depending on the injection sites. These features of α-Syn PFF-injected animal models provide a platform to explore disease mechanisms and to test disease modifying therapies in PD research. Here, we describe a methodology for the generation of α-Syn PFFs and the surgery on mice.

A Smart Tendon Hammer System for Remote Neurological Examination.

The deep tendon reflex exam is an important part of neurological assessment of patients consisting of two components, reflex elicitation and reflex grading. While this exam has traditionally been performed in person, with trained clinicians both eliciting and grading the reflex, this work seeks to enable the exam by novices. The COVID-19 pandemic has motivated greater utilization of telemedicine and other remote healthcare delivery tools. A smart tendon hammer capable of streaming acceleration measurements wirelessly allows differentiation of correct and incorrect tapping locations with 91.5% accuracy to provide feedback to users about the appropriateness of stimulation, enabling reflex elicitation by laypeople, while survey results demonstrate that novices are reasonably able to grade reflex responses. Novice reflex grading demonstrates adequate performance with a mean error of 0.2 points on a five point scale. This work shows that by assisting in the reflex elicitation component of the reflex exam via a smart hammer and feedback application, novices should be able to complete the reflex exam remotely, filling a critical gap in neurological care during the COVID-19 pandemic.

Perampanel Inhibits α-Synuclein Transmission in Parkinson's Disease Models.

BACKGROUND: The intercellular transmission of pathogenic proteins plays a key role in the clinicopathological progression of neurodegenerative diseases. Previous studies have demonstrated that this uptake and release process is regulated by neuronal activity. OBJECTIVE: The objective of this study was to examine the effect of perampanel, an antiepileptic drug, on α-synuclein transmission in cultured cells and mouse models of Parkinson's disease. METHODS: Mouse primary hippocampal neurons were transduced with α-synuclein preformed fibrils to examine the effect of perampanel on the development of α-synuclein pathology and its mechanisms of action. An α-synuclein preformed fibril-injected mouse model was used to validate the effect of oral administration of perampanel on the α-synuclein pathology in vivo. RESULTS: Perampanel inhibited the development of α-synuclein pathology in mouse hippocampal neurons transduced with α-synuclein preformed fibrils. Interestingly, perampanel blocked the neuronal uptake of α-synuclein preformed fibrils by inhibiting macropinocytosis in a neuronal activity-dependent manner. We confirmed that oral administration of perampanel ameliorated the development of α-synuclein pathology in wild-type mice inoculated with α-synuclein preformed fibrils. CONCLUSION: Modulation of neuronal activity could be a promising therapeutic target for Parkinson's disease, and perampanel could be a novel disease-modifying drug for Parkinson's disease. © 2021 International Parkinson and Movement Disorder Society.

Effects of CREG1 on Age-Associated Metabolic Phenotypes and Renal Senescence in Mice.

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.

α-Synuclein Spread from Olfactory Bulb Causes Hyposmia, Anxiety, and Memory Loss in BAC-SNCA Mice.

BACKGROUND: Patients with Parkinson's disease (PD) show motor symptoms as well as various non-motor symptoms. Postmortem studies of PD have suggested that initial alpha-synuclein (α-Syn) pathology develops independently in the olfactory bulb and lower brainstem, spreading from there stereotypically. However, it remains unclear how these two pathological pathways contribute to the clinicopathological progression of PD. OBJECTIVE: The objective of this study was to examine the clinicopathological contribution of α-Syn spread from the olfactory bulb. METHODS: We conducted pathological and behavioral analyses of human α-Syn bacterial artificial chromosome transgenic mice injected with α-Syn preformed fibrils into the bilateral olfactory bulb up to 10 months postinjection. RESULTS: α-Syn preformed fibril injections induced more widespread α-Syn pathology in the transgenic mice than that in wild-type mice. Severe α-Syn pathology in the transgenic mice injected with α-Syn preformed fibrils was initially observed along the olfactory pathway and later in the brain regions that are included in the limbic system and have connections with it. The α-Syn pathology was accompanied by regional atrophy, neuron loss, reactive astrogliosis, and microglial activation, which were remarkable in the hippocampus. Behavioral analyses revealed hyposmia, followed by anxiety-like behavior and memory impairment, but not motor dysfunction, depression-like behavior, or circadian rhythm disturbance. CONCLUSION: Our data suggest that α-Syn spread from the olfactory bulb mainly affects the olfactory pathway and limbic system as well as its related regions, leading to the development of hyposmia, anxiety, and memory loss in PD. © 2021 International Parkinson and Movement Disorder Society.

A critical role of STING-triggered tumor-migrating neutrophils for anti-tumor effect of intratumoral cGAMP treatment.

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-ß1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

A Direct Drive Parallel Plane Piezoelectric Needle Positioning Robot for MRI Guided Intraspinal Injection.

Recent developments in the field of cellular therapeutics have indicated the potential of stem cell injections directly to the spinal cord. Injections require either open surgery or a Magnetic Resonance Imaging (MRI) guided injection. Needle positioning during MRI imaging is a significant hurdle to direct spinal injection, as the small target region and interlaminar space require high positioning accuracy. OBJECTIVE: To improve both the procedure time and positioning accuracy, an MRI guided robotic needle positioning system is developed. METHODS: The robot uses linear piezoelectric motors to directly drive a parallel plane positioning mechanism. Feedback is provided through MRI during the orientation procedure. Both accuracy and repeatability of the robot are characterized. RESULTS: This system is found to be capable of repeatability below 51 µm. Needle endpoint error is limited by imaging modality, but is validated to 156 µm. CONCLUSION: The reported robot and MRI image feedback system is capable of repeatable and accurate needle guide positioning. SIGNIFICANCE: This high accuracy will result in a significant improvement to the workflow of spinal injection procedures.

Drug concentration estimation using contrast-enhanced MRI in intra-arterial chemotherapy for head and neck cancers.

OBJECTIVE: In cases of head and neck cancer treated with intra-arterial chemotherapy, no objective indices are available for determining the distribution of anticancer drugs administered to multiple arteries. To establish such indices, noninvasive measurements of drug concentrations are required in the arterial perfusion area of each artery. In MRI, changes in 1/T1 (Δ1/T1) are correlated with the contrast agent concentration. We focused on these properties and investigated whether it is possible to estimate anticancer drug concentrations within tissue based on Δ1/T1. METHODS: We employed the fast spin echo (FSE) sequence to determine optimum imaging parameters using a phantom. Subsequently, contrast agent was administered via the lingual and external carotid arteries for seven cases of tongue cancer. Δ1/T1 were then measured in tumor and nontumor tissues. The results of this study were compared with those of a previous study in which intratumor concentrations of anticancer agent were measured in excised specimens. RESULTS: The optimum imaging parameters for the FSE was two repetition times (TR, 500 and 1000 ms). When compared with the external carotid artery administration, the lingual artery administration of contrast agent resulted in significantly higher Δ1/T1 in both tumor and nontumor tissues (2.13 and 2.62 times, respectively). The multiplying factor for the nontumor tissue and high homogeneity of the contrast agent concentration were reasonably consistent with the results of the previous study. CONCLUSION: This method can be applied to estimating intratissue concentrations of intra-arterially administered anticancer drugs, thus possibly providing useful information in determining the distribution of anticancer drugs.

Structural basis for histone variant H3tK27me3 recognition by PHF1 and PHF19.

The Polycomb repressive complex 2 (PRC2) is a multicomponent histone H3K27 methyltransferase complex, best known for silencing the Hox genes during embryonic development. The Polycomb-like proteins PHF1, MTF2, and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3t in testis and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.

Digital PCR-based plasma cell-free DNA mutation analysis for early-stage pancreatic tumor diagnosis and surveillance.

BACKGROUND: Cell-free DNA (cfDNA) shed from tumors into the circulation offers a tool for cancer detection. Here, we evaluated the feasibility of cfDNA measurement and utility of digital PCR (dPCR)-based assays, which reduce subsampling error, for diagnosing pancreatic ductal adenocarcinoma (PDA) and surveillance of intraductal papillary mucinous neoplasm (IPMN). METHODS: We collected plasma from seven institutions for cfDNA measurements. Hot-spot mutations in KRAS and GNAS in the cfDNA from patients with PDA (n = 96), undergoing surveillance for IPMN (n = 112), and normal controls (n = 76) were evaluated using pre-amplification dPCR. RESULTS: Upon Qubit measurement and copy number assessment of hemoglobin-subunit (HBB) and mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) in plasma cfDNA, HBB offered the best resolution between patients with PDA relative to healthy subjects [area under the curve (AUC) 0.862], whereas MT-ND1 revealed significant differences between IPMN and controls (AUC 0.851). DPCR utilizing pre-amplification cfDNA afforded accurate tumor-derived mutant KRAS detection in plasma in resectable PDA (AUC 0.861-0.876) and improved post-resection recurrence prediction [hazard ratio (HR) 3.179, 95% confidence interval (CI) 1.025-9.859] over that for the marker CA19-9 (HR 1.464; 95% CI 0.674-3.181). Capturing KRAS and GNAS could also provide genetic evidence in patients with IPMN-associated PDA and undergoing pancreatic surveillance. CONCLUSIONS: Plasma cfDNA quantification by distinct measurements is useful to predict tumor burden. Through appropriate methods, dPCR-mediated mutation detection in patients with localized PDA and IPMN likely to progress to invasive carcinoma is feasible and complements conventional biomarkers.

Experimental Control of Macrophage Pro-Inflammatory Dynamics Using Predictive Models.

Macrophage activity is a major component of the healthy response to infection and injury that consists of tightly regulated early pro-inflammatory activation followed by anti-inflammatory and regenerative activity. In numerous diseases, however, macrophage polarization becomes dysregulated and can not only impair recovery, but can promote further injury and pathogenesis, e.g., after trauma or in diabetic ulcers. Dysregulated macrophages may either fail to polarize or become chronically polarized, resulting in increased production of cytotoxic factors, diminished capacity to clear pathogens, or failure to promote tissue regeneration. In these cases, a method of predicting and dynamically controlling macrophage polarization will enable a new strategy for treating diverse inflammatory diseases. In this work, we developed a model-predictive control framework to temporally regulate macrophage polarization. Using RAW 264.7 macrophages as a model system, we enabled temporal control by identifying transfer function models relating the polarization marker iNOS to exogenous pro- and anti-inflammatory stimuli. These stimuli-to-iNOS response models were identified using linear autoregressive with exogenous input terms (ARX) equations and were coupled with non-linear elements to account for experimentally identified supra-additive and hysteretic effects. Using this model architecture, we were able to reproduce experimentally observed temporal iNOS dynamics induced by lipopolysaccharides (LPS) and interferon gamma (IFN-γ). Moreover, the identified model enabled the design of time-varying input trajectories to experimentally sustain the duration and magnitude of iNOS expression. By designing transfer function models with the intent to predict cell behavior, we were able to predict and experimentally obtain temporal regulation of iNOS expression using LPS and IFN-γ from both naïve and non-naïve initial states. Moreover, our data driven models revealed decaying magnitude of iNOS response to LPS stimulation over time that could be recovered using combined treatment with both LPS and IFN-γ. Given the importance of dynamic tissue macrophage polarization and overall inflammatory regulation to a broad number of diseases, the temporal control methodology presented here will have numerous applications for regulating immune activity dynamics in chronic inflammatory diseases.

Forced expression of miR-143 and -145 in cardiomyocytes induces cardiomyopathy with a reductive redox shift.

BACKGROUND: Animal model studies show that reductive stress is involved in cardiomyopathy and myopathy, but the exact physiological relevance remains unknown. In addition, the microRNAs miR-143 and miR-145 have been shown to be upregulated in cardiac diseases, but the underlying mechanisms associated with these regulators have yet to be explored. METHODS: We developed transgenic mouse lines expressing exogenous miR-143 and miR-145 under the control of the alpha-myosin heavy chain (αMHC) promoter/enhancer. RESULTS: The two transgenic lines showed dilated cardiomyopathy-like characteristics and early lethality with markedly increased expression of miR-143. The expression of hexokinase 2 (HK2), a cardioprotective gene that is a target of miR-143, was strongly suppressed in the transgenic hearts, but the in vitro HK activity and adenosine triphosphate (ATP) content were comparable to those observed in wild-type mice. In addition, transgenic complementation of HK2 expression did not reduce mortality rates. Although HK2 is crucial for the pentose phosphate pathway (PPP) and glycolysis, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) was unexpectedly higher in the hearts of transgenic mice. The expression of gamma-glutamylcysteine synthetase heavy subunit (γ-GCSc) and the in vitro activity of glutathione reductase (GR) were also higher, suggesting that the recycling of GSH and its de novo biosynthesis were augmented in transgenic hearts. Furthermore, the expression levels of glucose-6-phosphate dehydrogenase (G6PD, a rate-limiting enzyme for the PPP) and p62/SQSTM1 (a potent inducer of glycolysis and glutathione production) were elevated, while p62/SQSTM1 was upregulated at the mRNA level rather than as a result of autophagy inhibition. Consistent with this observation, nuclear factor erythroid-2 related factor 2 (Nrf2), Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) were activated, all of which are known to induce p62/SQSTM1 expression. CONCLUSIONS: Overexpression of miR-143 and miR-145 leads to a unique dilated cardiomyopathy phenotype with a reductive redox shift despite marked downregulation of HK2 expression. Reductive stress may be involved in a wider range of cardiomyopathies than previously thought.