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* List gives the number of target gene transcripts that are hit by one or more of the miRNAs in order of decreasing number of miRNAs hitting them. **List gives the number of target gene transcripts that are hit by those miRNAs that are over-expressed in the crypt bottom in addition to hsa-mir-25 and hsa-mir-007

'Legends for Supplementary Figures'

Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial–mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778–90. ©2017 AACR.

List of 83 miRNAs which were differentially expressed in CRC vs normal colon.

Lists of predicted gene targets of miRNAs in the upper crypt. List of predicted gene targets in the crypt bottom. GO term analysis of the top 200 predicted gene targets for both the lists.

'Fig S1. Validation of miRNA chip results by quantitative PCR analysis. Fig S2. Analysis of differentially expressed miRNAs using target predictions. Fig S3. The relative abundance of the common predicted targets in relation to miRNA levels in CRC cell lines. Fig S4. The effect of miRNAs on mRNA expression levels of their predicted targets. Fig S5. Relative expression of four candidate microRNAs in HT29 ALDH+ cells as compared to ALDH- cells. Fig S6. miRNA23b targets 3' UTR of LGR5 and LRIG1 mRNA. Fig S7: miRNA23b affects cell proliferation. Fig S8: CaCo2 data on miRNA23b precursor and antimer effect on sphere forming ability as well as 5FU response.'

Autism spectrum disorder (ASD) has been associated with mitochondrial dysfunction but few studies have examined the relationship between mitochondrial function and ASD symptoms. We measured Complex I and IV and citrate synthase activities in 76 children with ASD who were not receiving vitamin supplementation or medication. We also measured language using the Preschool Language Scales or Clinical Evaluation of Language Fundamentals, adaptive behavior using the Vineland Adaptive Behavioral Scale, social function using the Social Responsiveness Scale and behavior using Aberrant Behavior Checklist, Childhood Behavior Checklist and the Ohio Autism Clinical Impression Scale. Children with ASD demonstrated significantly greater variation in mitochondrial activity compared to controls with more than expected ASD children having enzyme activity outside of the normal range for Citrate Synthase (24%), Complex I (39%) and Complex IV (11%). Poorer adaptive skills were associated with Complex IV activity lower or higher than average and lower Complex I activity. Poorer social function and behavior was associated with relatively higher Citrate Synthase activity. Similar to previous studies we find both mitochondrial underactivity and overactivity in ASD. This study confirms an expanded variation in mitochondrial activity in ASD and demonstrates, for the first time, that such variations are related to ASD symptoms.

Treatment for mitochondrial dysfunction is typically guided by expert opinion with a paucity of empirical evidence of the effect of treatment on mitochondrial activity. We examined citrate synthase and Complex I and IV activities using a validated buccal swab method in 127 children with autism spectrum disorder with and without mitochondrial disease, a portion of which were on common mitochondrial supplements. Mixed-model linear regression determined whether specific supplements altered the absolute mitochondrial activity as well as the relationship between the activities of mitochondrial components. Complex I activity was increased by fatty acid and folate supplementation, but folate only effected those with mitochondrial disease. Citrate synthase activity was increased by antioxidant supplementation but only for the mitochondrial disease subgroup. The relationship between Complex I and IV was modulated by folate while the relationship between Complex I and Citrate Synthase was modulated by both folate and B12. This study provides empirical support for common mitochondrial treatments and demonstrates that the relationship between activities of mitochondrial components might be a marker to follow in addition to absolute activities. Measurements of mitochondrial activity that can be practically repeated over time may be very useful to monitor the biochemical effects of treatments.

Malignant transformation of tissue stem cells (SC) may be the root of most cancer. Accordingly, we identified miRNA expression patterns in the normal human colonic SC niche to understand how cancer stem cells (CSC) may arise. In profiling miRNA expression in SC-enriched crypt subsections isolated from fresh, normal surgical specimens, we identified 16 miRNAs that were differentially expressed in the crypt bottom, creating an SC signature for normal colonic epithelia (NCE). A parallel analysis of colorectal cancer tissues showed differential expression of 83 miRNAs relative to NCE. Within the 16 miRNA signature for the normal SC niche, we found that miR-206, miR-007-3, and miR-23b individually could distinguish colorectal cancer from NCE. Notably, miR-23b, which was increased in colorectal cancer, was predicted to target the SC-expressed G protein-coupled receptor LGR5. Cell biology investigations showed that miR-23b regulated CSC phenotypes globally at the level of proliferation, cell cycle, self-renewal, epithelial–mesenchymal transition, invasion, and resistance to the colorectal cancer chemotherapeutic agent 5-fluorouracil. In mechanistic experiments, we found that miR-23b decreased LGR5 expression and increased ALDH+ CSCs. CSC analyses confirmed that levels of LGR5 and miR-23b are inversely correlated in ALDH+ CSCs and that distinct subpopulations of LGR5+ and ALDH+ CSCs exist. Overall, our results define a critical function for miR-23b, which, by targeting LGR5, contributes to overpopulation of ALDH+ CSCs and colorectal cancer. Cancer Res; 77(14); 3778–90. ©2017 AACR.

Myocardial ischemia results in early and progressive damage to mitochondrial structure and function, but the molecular events leading to these changes have not been clearly established. We hypothesized that mitochondrial dysfunction and a coordinated expression of nuclear and mitochondrial genes occur in a time-dependent manner by relating the time courses of changes in parameters of mitochondrial bioenergetics after ischemia-reperfusion. Using a Langendorff rat heart model, mitochondrial bioenergetics and protein levels were assessed at different times of ischemia and ischemia/reperfusion. Mitochondrial and nuclear gene expression (super array analysis) and mitochondrial DNA levels were evaluated after late ischemia. Ischemia induced progressive and marked decreases in complex I, III, and V activities. Reperfusion (15, 30, and 60 min) after 45 min of ischemia had little further effect on enzyme activities or respiration. Super array analysis after 45 min ischemia revealed increased levels of the proteins with more pronounced increases in the corresponding mRNAs. Expression of mitochondrial and nuclear genes involved in oxidative phosphorylation increased after 45 min of ischemia but not after reperfusion. Myocardial ischemia induces mitochondrial dysfunction and differential but coordinated expression of nuclear and mitochondrial genes in a time-dependent manner. Our observations are pertinent to the search for molecular stimuli that generate mitochondrial defects and alter mitochondrial and nuclear transcriptional responses that may impact ischemic preconditioning and cardioprotection.

Background Specific myocardial mitochondrial enzymatic dysfunction and apoptotic remodeling occur in pacing-induced heart failure. We sought to define their regional distribution and molecular basis in the failing heart. Methods and Results Enzyme dysfunction was assessed in mitochondrial subpopulations and immunoblot analysis was performed using homogenate proteins from the left atria (LA) and left ventricle (LV) of paced and control mongrel dogs. A greater range of enzymatic defects (complex I, III, and V) was found in mitochondria subpopulations from the LV as compared with the LA (where only complex V was defective). Analysis of paced LV proteins demonstrated a downregulated expression of both mitochondrial genes (eg, cytochrome b ) and nuclear genes (eg, ATP synthase β subunit, mitochondrial creatine kinase). Protease-activated products of both mitochondrial (eg, apoptosis inducing factor) and cytosolic (eg, caspase-3) apoptogenic proteins were increased in both the LA and LV. Nuclear-localized apoptotic markers (eg, p53, p21) were also significantly increased in the LV of paced dogs. Conclusion Abnormal activity of several mitochondrial enzymes and increased apoptogenic pathway appear to be mediated, at least in part, by an orchestrated shift in expression (both nuclear and mitochondrial DNA) of respiratory chain subunits (eg, cyt b , ATP-β), mitochondrial bioenergetic enzymes (eg, mitochondrial creatine kinase), global transcription factor (eg, PGC-1), and apoptotic proteins (eg, p53, p21) with distinct differences in their regional distribution and in the subpopulations of mitochondria affected.

Aim: Mitochondrial function studies in autism spectrum disorders (ASD) have detected skeletal muscle mitochondrial enzyme deficiencies in respiratory complex (RC) activities. As a muscle biopsy is expensive and invasive, we assessed RC-I and RC-IV activities in buccal swabs. Methods: 92 children with ASD and 68 controls were studied with immunocapture for RC-I and microspectrophotometry for RC-IV. Results: Significant RC activity deficiencies were found in 39 (42%) ASD patients (p < 0.01) and more prevalent in more severe cases. Aberrant RC overactivity was seen in 9 children. RC-I/RC-IV activity ratio was significantly increased in 64% of the entire ASD cohort including 76% of those more severely affected (p < 0.05). Conclusion: Buccal swab analysis revealed extensive RC abnormalities in ASD providing a noninvasive biomarker to assess mitochondrial function in ASD patients.

Phelan-McDermid Syndrome (PMS), which is defined by a deletion within 22q13, demonstrates significant phenotypic variation. Given that six mitochondrial genes are located within 22q13, including complex I and IV genes, we hypothesize that mitochondrial complex activity abnormalities may explain phenotypic variation in PMS symptoms. Complex I, II, II + III and IV activity was measured in 51 PMS participants. Caretakers completed questionnaires and provided genetic information through the PMS foundation registry. Complex activity was abnormal in 59% of PMS participants. Abnormalities were found in complex I and IV but not complex II + III and II activity, consistent with disruption of genes within the 22q13 region. However, complex activity abnormalities were not related to specific gene deletions suggesting a “neighboring effect” of regional deletions on adjacent gene expression. A specific combination of symptoms (autism spectrum disorder, developmental regression, failure-to-thrive, exercise intolerance/fatigue) was associated with complex activity abnormalities. 64% of 106 individuals in the PMS foundation registry who did not have complex activity measured also endorsed this pattern of symptoms. These data suggest that mitochondrial abnormalities, specifically abnormalities in complex I and IV activity, may explain some phenotypic variation in PMS individuals. These results point to novel pathophysiology mechanisms and treatment targets for PMS patients.

Normal human colon crypt protein extract was resolved by two-dimensional gel electrophoresis using pH 6-11 immobilized pH gradient strips in the first dimension. The optimized isoelectric focusing protocol includes cup-loading sample application at the anode and 1.2% hydroxyethyl disulfide (DeStreak), 15% 2-propanol and 5% glycerol in the rehydration buffer. Spots were well resolved across the entire pH range up to 11. A total of 311 protein spots were identified by mass spectrometry and peptide mass mapping. After combining isoforms, 231 nonredundant proteins were grouped into 16 categories according to their subcellular locations, and 17 categories according to their physiological functions. Histone proteins, ribosomal proteins and mitochondrial proteins were among the well-resolved highest p/ proteins. Application of this protocol to the analysis of normal and neoplastic colon crypts will contribute to the proteomic study of colorectal tumorigenesis since a significant portion of the human proteins is in basic pH range.

The effects of physiologically relevant hypoxia on the catalytic activity of cytochrome c oxidase (CytOX), mitochondrial gene expression, and both nuclear and mitochondrial encoded CytOX mRNA levels were investigated in murine monocyte macrophages, mouse C2C12 skeletal myocytes and rat adrenal pheochromocytoma PC12 cells. Our results suggest a coordinated down regulation of mitochondrial genome-coded CytOX I and II and nuclear genome-coded CytOX IV and Vb mRNAs during hypoxia. Hypoxia also caused a severe decrease in mitochondrial transcription rates, and associated decrease in mitochondrial transcription factor A. The enzyme from hypoxia exposed cells exhibited altered subunit content as revealed by blue native gel electrophoresis. There was a generalized decline in mitochondrial function that led to a decrease in total cellular heme and ATP pools. We also observed a decrease in mitochondrial heme aa3 content and decreased levels of CytOX subunit I, IV and Vb, though the catalytic efficiency of the enzyme (TN for cytochrome c oxidase) remained nearly the same. Increased glycolytic flux and alterations in the kinetic characteristics of the CytOX might be the two mechanisms by which hypoxic cells maintain adequate ATP levels to sustain life processes. Reoxygenation nearly completely reversed hypoxia-mediated changes in CytOX mRNA contents, rate of mitochondrial transcription, and the catalytic activity of CytOX enzyme. Our results show adaptive changes in CytOX structure and activity during physiological hypoxia.

The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue-specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by approximately 50% reduced, mitochondrial genome-encoded COX I and II mRNAs and nuclear genome-encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50-80% reduction in mitochondrial transcription and translation rates in heme-depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme-depleted tissues contained a 30-50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme-depleted tissues. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.

Making a diagnosis of mitochondrial disease (MD) is extremely challenging and often employs the analysis of respiratory complex (RC) activities in biopsied skeletal muscle. Given both the invasive nature and expense of biopsied-muscle based testing for mitochondrial defects, buccal swab enzyme analysis has been explored as an alternative approach to the more invasive muscle biopsy. Case studies have recently suggested that buccal swabs from patients can be used to accurately assess mitochondrial enzyme activities including RC I and RC IV using a dipstick methodology combined with spectrophotometric analysis. In this study, forty patients with suspected MD who have previously been found to have significant defects in either RC I or RC IV in skeletal muscle were assessed by buccal swab analysis and compared to enzyme values obtained with unaffected controls (n=106) in the same age range. Buccal citrate synthase was used as an indicator of overall mitochondrial content, correlating well with overall buccal mitochondrial frataxin levels and was found to be elevated above control levels in 28% of the patients in this cohort. Of 26 cases with significant muscle RC I deficiency, 20 displayed significantly reduced levels of buccal RC I activity. All 7 of the patients with muscle RC IV deficiency showed significant buccal RC IV defect and 6 of the 7 patients with combined defects in muscle RC I and IV activity levels also exhibited analogous deficiencies in both buccal RC I and RC IV activities. In conclusion, the relatively high correlation (over 82%) of buccal and muscle RC deficiencies further supports the validity of this non-invasive approach as a potentially useful tool in the diagnosis of MD.

Ageing reduces the ability of cardiac myocytes to respond to inotropic agents. We hypothesized that hypoxia-inducible factor-1 (HIF-1) would improve the functional and Ca(2+) transient responses of ageing myocytes to the inotropic agents and this would act, in part, through altered mitochondrial activity. Young (3-4 months) and older Fischer 344 rats (18-20 months) were used. Hypoxia-inducible factor-1alpha was upregulated with ciclopirox olamine (CPX, 50 mg kg(1) on 2 days). Hypoxia-inducible factor-1 upregulation was detected by Western blot. Cardiomyocyte contraction and Ca(2+) transients were measured at baseline and after forskolin and ouabain. We also measured mitochondrial complex activities and production of reactive oxygen species (ROS). In the young group, forskolin (31%) and ouabain (31%) significantly increased percentage shortening. Similar changes were observed in the young + CPX group. Calcium transients also responded in a similar manner. However, in the older group, forskolin (12%) and ouabain (6%) did not significantly increase myocyte contractility or Ca(2+) transients. In the older + CPX group, the effects of forskolin (34%) and ouabain (29%) were restored. In the young + CPX group, there was increased ROS production and mitochondrial complex I and III activity compared with the young group. These differences were not observed in older groups. These data demonstrate an impaired functional and Ca(2+) effect of positive inotropic agents in older myocytes. Upregulation of HIF-1 restored this blunted response, but this was not related to changed mitochondrial activity induced by HIF-1. Thus, we found that HIF-1 improved inotropy in older myocytes without requiring mitochondrial activity changes.

The paper gives an extension to the existing approach to hardware/software codesign of embedded systems by including the missed out network element commonly seen in the today's embedded systems. The approach tries to provide a verification platform for networked embedded system. Virtual system modeling of networks & their interaction with hardware-software is the key issue in the heterogeneous co-simulation of networked embedded system.

This work proposes cascaded Fuzzy Logic Controllers as new hardware architecture so that it works in an accurate and fast manner. The MAX-MIN interface can be simplified as lookup table operations. Both membership function values and index numbers of input MF's are pre-stored in the lookup table and the observed crisp inputs serve as the addresses for reading them. Each output MF is treated as a singleton located at the center of the MF. So, the computational complexity of COG defuzzification is significantly reduced. As a case study for a single-phase converter, the fuzzy method of Δ∝ angle compensation is used to linearize the converter transfer characteristics in discontinuous conduction mode. On the basis of the information collected from the graphs; the fine tuning of the output of FLC1 is suggested to achieve better linearization. The required fine-tuning is achieved by using another fuzzy controller FLC2 cascaded with first fuzzy controller FLC1. All components of the FLC are simulated in VHDL using the concept of moment of equilibrium principle for MAX and MIN modules. The VHDL analysis shows faster response to conclude that dedicated cascaded hardware processor will give very accurate linear control for the converter under study.

The effects of physiologically relevant hypoxia on the catalytic activity of cytochrome c oxidase (CytOX), mitochondrial gene expression, and both nuclear and mitochondrial encoded CytOX mRNA levels were investigated in murine monocyte macrophages, mouse C2C12 skeletal myocytes and rat adrenal pheochromocytoma PC12 cells. Our results suggest a coordinated down regulation of mitochondrial genome-coded CytOX I and II and nuclear genome-coded CytOX IV and Vb mRNAs during hypoxia. Hypoxia also caused a severe decrease in mitochondrial transcription rates, and associated decrease in mitochondrial transcription factor A. The enzyme from hypoxia exposed cells exhibited altered subunit content as revealed by blue native gel electrophoresis. There was a generalized decline in mitochondrial function that led to a decrease in total cellular heme and ATP pools. We also observed a decrease in mitochondrial heme aa3 content and decreased levels of CytOX subunit I, IV and Vb, though the catalytic efficiency of the enzyme (TN for cytochrome c oxidase) remained nearly the same. Increased glycolytic flux and alterations in the kinetic characteristics of the CytOX might be the two mechanisms by which hypoxic cells maintain adequate ATP levels to sustain life processes. Reoxygenation nearly completely reversed hypoxia-mediated changes in CytOX mRNA contents, rate of mitochondrial transcription, and the catalytic activity of CytOX enzyme. Our results show adaptive changes in CytOX structure and activity during physiological hypoxia.

Ezekiel J. J. Kruglick, Shirish Dainle, Kristofer S. J. PisterUniversity of California at Los AngelesDept. ofElectrical EngineeringLos Angeles, CA 90024, USAABSTRACTA design is presented and tested to build and actuate reflection based three dimensional micro-opto-mechanicalsystems in standard CMOS using only a single maskiess post-processing step. We have built two generations of structuresand actuated them at various frequencies. The initial design uses the various components of CMOS to make a rigid boxstructure with a deflecting mirror. The second generation design uses a bimodal compliant mirror which becomes flat uponbeing pulled against the substrate. Results and photos are presented to show the actuation. Possible developments whichtake advantage ofthe CMOS underlying the structure are discussed.Keywords: micromechanics, optics, CMOS, xenon difluoride, compliant structures, electrostatic actuation1. MICRO-OPTICS AND CMOS