Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B.

Inositol hexakisphosphate kinases (IP6Ks) regulate various biological processes. IP6Ks convert IP6 to pyrophosphates such as diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol tetrakisphosphate (IP8). IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, IP6K1, IP6K2, and IP6K3, which have distinct biological functions. The inositol hexakisphosphate kinase 2 (IP6K2) controls cellular apoptosis. To explore roles for IP6K2 in brain function, we elucidated its protein interactome in mouse brain revealing a robust association of IP6K2 with creatine kinase-B (CK-B), a key enzyme in energy homeostasis. Cerebella of IP6K2-deleted mice (IP6K2-knockout [KO]) produced less phosphocreatine and ATP and generated higher levels of reactive oxygen species and protein oxidative damage. In IP6K2-KO mice, mitochondrial dysfunction was associated with impaired expression of the cytochrome-c1 subunit of complex III of the electron transport chain. We reversed some of these effects by combined treatment with N-acetylcysteine and phosphocreatine. These findings establish a role for IP6K2-CK-B interaction in energy homeostasis associated with neuroprotection.

A functional analysis of mitochondrial respiratory chain cytochrome bc1 complex in Gaeumannomyces tritici by RNA silencing as a possible target of carabrone.

Gaeumannomyces tritici, an ascomycete soilborne fungus, causes a devastating root disease in wheat. Carabrone, a botanical bicyclic sesquiterpenic lactone, is a promising fungicidal agent that can effectively control G. tritici. However, the mechanism of action of carabrone against G. tritici remains largely unclear. Here, we used immunogold for subcellular localization of carabrone and the results showed that carabrone is subcellularly localized in the mitochondria of G. tritici. We then explored the functional analysis of genes GtCytc1 , GtCytb, and GtIsp of the mitochondrial respiratory chain cytochrome bc1 complex in G. tritici by RNA silencing as a possible target of carabrone. The results showed that the silenced mutant ∆GtIsp is less sensitive to carabrone compared to ∆GtCytc1 and ∆GtCytb. Compared with the control, the activities of complex III in all the strains, except ∆GtIsp and carabrone-resistant isolate 24-HN-1, were significantly decreased following treatment with carabrone at EC20 and EC80 in vitro (40%-50% and 70%-80%, respectively). The activities of mitochondrial respiratory chain complex III and the mitochondrial respiration oxygen consumption rates in all the strains, except ∆GtIsp and 24-HN-1, were higher with respect to the control when treated with carabrone at EC20 in vivo. The rates of mitochondrial respiration of all strains, except ∆GtIsp, were significantly inhibited following treatment with carabrone at EC80 (ranging from 57% to 81%). This study reveals that the targeting of the iron-sulphur protein encoded by GtIsp is highly sensitive to carabrone and provides a direction for the research of carabrone's target.

Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.

CYC1, SDHA, UQCRC1, UQCRQ, and SDHB might be important biomarkers in kidney transplant rejection.

BACKGROUND: Kidney transplant rejection is considered as a vital factor of kidney transplant failure. Therefore, it's necessary to search for effective biomarkers for kidney transplant surveillance. METHODS: In this study, we conducted time-series gene expression profiles analysis of samples from kidney transplant patients with different post-transplant days through weighted gene co-expression network analysis (WGCNA). Associations between gene co-expression modules and days post-transplant were determined through spearman rank correlation analysis. Potential kidney transplant rejection-related modules were subjected to gene functional enrichment analysis through clusterProfiler and protein-protein interaction analysis via STRING database. RESULTS: A total of 11 gene co-expression modules were identified, and the pink module which was mainly involved in "energy derivation by oxidation of organic compounds" and "Huntington disease" showed significant correlation with the phenotypic trait "days post-transplant". CYC1, SDHA, UQCRC1, UQCRQ, and SDHB in the pink module exhibited high scores in the protein-protein interaction network analysis. CONCLUSIONS: We reported several potential genes may be associated with the kidney transplant rejection, which should provide novel biomarkers for kidney transplant surveillance.

Morphology and Molecular Characterization of Parabronema smithii (Cobbold, 1882) (Nematoda: Habronematidae) from Wild Asian Elephant (Elephas maximus maximus) of Sri Lanka.

PURPOSE: The aim of the present study was to carry out a detailed study of morphological features and to determine the phylogenetic position of Parabronema smithii (Cobbold, 1882) found in wild elephants in Sri Lanka. METHODS: Adult worms were collected from stomach ulcers at postmortem examination of wild elephants in the Udawalawe National Park, Sri Lanka. The detailed morphology of P. smithii was studied using light microscopy and, for the first time, scanning electron microscopy. Fifteen morphological characteristics were investigated. The phylogenetic analysis was conducted using the second internal transcribed spacer region (ITS2), and portions of the large subunit ribosomal DNA (28S) and cytochrome c oxidase subunit 1 (cox1). Furthermore, the present study provides a comparison of morphology and morphometrics of Parabronema species that occur in different hosts. CONCLUSION: Parabronema smithii isolated from wild elephants exhibited the key morphological features. Phylogenetic analysis of selected genes revealed that P. smithii is closely associated with P. skrjabini and Habronema spp. Findings of the present study enhance our understanding of the biology and taxonomy of P. smithii in wild elephant in Sri Lanka and will contribute to future phylogeographic studies.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex.

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

Genetic status of indigenous poultry (red jungle fowl) from India.

The global biodiversity of domesticated red jungle fowl (Gallus gallus) is gradually eroding by replacement with commercial poultry breeds and results loss of valuable genetic and physical traits like resistance to disease, extreme environment, etc. posing a threat to the poultry genetic resources. Very fewer reports exist on Indian poultry diversity, especially native chicken of India. Therefore, species identification and inventorying of the poultry genetic resource is indispensable. Thus, the present study aimed to characterize indigenous chicken from bio-diversity hotspot of Sunderban and Northeast India using DNA sequence based barcoding approach. A total of 15 CO1 (Cytochrome c Oxidase-I) DNA barcode of different indigenous chicken were newly sequenced along with 6 previously published sequences from our laboratory and compared with the available data of distinctive genera of Phasianidae as per the standard protocol and are identified as Gallus gallus. About 98.96% of the Phasianid birds were successfully delimitated into the respective species except for 12 congeneric pairs whose minimum interspecific K2P (Kimura 2-parameter) distance overlaps with the maximum intraspecific distance (3.9%). The least genetic divergence is observed between G. gallus and G. varius (0.013%) and highest between G. gallus and G. lafayettei (0.059%). The NJ tree showed a cohesive clustering of indigenous chicken with G. gallus and distinct with respect to all the different species under study, thereby revealing their taxonomic position except for few G. sonneratti that showed mixed clustering with G. gallus. This may be due to the genetic introgression between the species. Nevertheless, the study for the first time provided the molecular identification tag of indigenous poultry from biodiversity hotspot of East and Northeast India and will remain as a potential guide to recognize inimitable and valuable poultry genetic resources for future needs.

A maize cytochrome b-c1 complex subunit protein ZmQCR7 controls variation in the hypersensitive response.

MAIN CONCLUSION: The gene GRMZM2G318346 which encodes a cytochrome b-c1 complex subunit 7 is associated with variation in strength of the hypersensitive response in maize. We previously identified a QTL at 3,545,354 bp (B73 reference genome V2) on maize chromosome 5 associated with variation in the hypersensitive response (HR) conferred by the autoactive R-gene Rp1-D21 (Olukolu et al. in PLoS Genet 10:e1004562 2014). In this study, we show that a gene at this locus, GRMZM2G318346 which encodes a cytochrome b-c1 complex subunit seven (ZmQCR7), an important part of the mitochondrial electron transport chain, can suppress HR mediated by Rp1-D21 in a transient expression assay. ZmQCR7 alleles from two maize lines, W22 and B73 differ for the encoded proteins at just two sites, amino acid 27 (threonine and alanine in B73 and W22, respectively) and amino acid 109 (asparagine and serine), however, the B73 allele is much more effective at suppressing HR. We show that variation at amino acid 27 controlled this variation in HR-suppressing effects. We furthermore demonstrate that the B73 allele of ZmQCR7 can suppress HR induced by RPM1(D505 V), another autoactive R-gene, and that Arabidopsis homologs of ZmQCR7 can also suppress NLR-induced HR. The implications of these findings are discussed.

Long non-coding RNA C5orf66-AS1 prevents oral squamous cell carcinoma through inhibiting cell growth and metastasis.

Oral squamous cell carcinoma (OSCC) is a major cancer type in the head and neck region. Recent studies have reported a marked rise in the incidence of OSCC. The present study was performed to better understand the roles that long non­coding RNAs (lncRNAs) serve in OSCC carcinogenesis. The levels of the lncRNA C5orf66 antisense RNA 1 (C5orf66­AS1) and of cytochrome c1 (CYC1) in OSCC tissues and cells were measured through reverse transcription­quantitative polymerase chain reaction. In addition, the levels of associated proteins were analyzed by western blotting, while MTT assay was used to detect the cell proliferation ability. Wound healing and transwell assays were also used to detect the migration and invasion abilities of OSCC cells in the experimental groups, while flow cytometry was applied to analyze cell apoptosis. The findings revealed that the expression of lncRNA C5orf66­AS1 in OSCC tissues and cells was significantly decreased. Overexpression of lncRNA C5orf66­AS1 significantly inhibited the proliferation, invasion and migration ability of OSCC cells, and promoted cell apoptosis, while lncRNA C5orf66­AS1 downregulation presented the opposite effects. In addition, it was observed that CYC1 was upregulated in OSCC tissues and cells, and was negatively regulated by lncRNA C5orf66­AS1. Notably, CYC1 silencing markedly eliminated the effects of lncRNA C5orf66­AS1 downregulation on OSCC cells. Taken together, these findings indicated that lncRNA C5orf66­AS1 may prevent OSCC progression by inhibiting OSCC cell growth and metastasis via the regulation of CYC1 expression.

The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc1-aa3 supercomplex.

Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa3 has been studied extensively in α-proteobacteria, yeast, and mammals, nothing is known about this process in Actinobacteria. Here, we searched for assembly proteins of the supercomplex by identifying the copper-deprivation stimulon, which might include proteins that insert copper into cytochrome aa3 Using gene expression profiling, we found two copper starvation-induced proteins for supercomplex formation. The Cg2699 protein, named CtiP, contained 16 predicted transmembrane helices, and its sequence was similar to that of the copper importer CopD of Pseudomonas syringae in the N-terminal half and to the cytochrome oxidase maturation protein CtaG of Bacillus subtilis in its C-terminal half. CtiP deletion caused a growth defect similar to that produced by deletion of subunit I of cytochrome aa3, increased copper tolerance, triggered expression of the copper-deprivation stimulon under copper sufficiency, and prevented co-purification of the supercomplex subunits. The secreted Cg1884 protein, named CopC, had a C-terminal transmembrane helix and contained a Cu(II)-binding motif. Its absence caused a conditional growth defect, increased copper tolerance, and also prevented co-purification of the supercomplex subunits. CtiP and CopC are conserved among aerobic Actinobacteria, and we propose a model of their functions in cytochrome aa3 biogenesis. Furthermore, we found that the copper-deprivation response involves additional regulators besides the ECF sigma factor SigC.

Cbp3 and Cbp6 are dispensable for synthesis regulation of cytochrome b in yeast mitochondria.

Cytochrome b (Cytb) is the only mitochondrial encoded subunit from the bc1 complex. Cbp3 and Cbp6 are chaperones necessary for translation of the COB mRNA and Cytb hemylation. Here we demonstrate that their role in translation is dispensable in some laboratory strains, whereas their role in Cytb hemylation seems to be universally conserved. BY4742 yeast requires Cbp3 and Cbp6 for efficient COB mRNA translation, whereas the D273-10b strain synthesizes Cytb at wildtype levels in the absence of Cbp3 and Cbp6. Steady-state levels of Cytb are close to wildtype in mutant D273-10b cells, and Cytb forms non-functional, supercomplex-like species with cytochrome c oxidase, in which at least core 1, cytochrome c1, and Rieske iron-sulfur subunits are present. We demonstrated that Cbp3 interacts with the mitochondrial ribosome and with the COB mRNA in both BY4742 and D273-10b strains. The polymorphism(s) causing the differential function of Cbp3, Cbp6, and the assembly feedback regulation of Cytb synthesis is of nuclear origin rather than mitochondrial, and Smt1, a COB mRNA-binding protein, does not seem to be involved in the observed differential phenotype. Our results indicate that the essential role of Cbp3 and Cbp6 is to assist Cytb hemylation and demonstrate that in the absence of heme b, Cytb can form non-functional supercomplexes with cytochrome c oxidase. Our observations support that an additional protein or proteins are involved in Cytb synthesis in some yeast strains.

Brown hagfish from the northwest and east coasts of Honshu, Japan are genetically different.

The brown hagfish (Eptatretus atami) is one of several known hagfish species occurring in Japanese coastal waters. To date, there has been no research studying genetic polymorphisms in the species. In the present study, we analyzed differences in nucleotide sequences between two populations: one from Suruga Bay on the Pacific coast of Honshu, Japan, and the other from the Sea of Japan, off Akita on the northwest coast of Honshu. We sequenced part of the cytochrome oxidase subunit 1 gene (COX1) from the mitochondrial genome, and three G protein-coupled receptor genes from the nuclear genome. Phylogenetic networks of all four genes showed divergence between the two populations. Further, comparison of the COX1 data using a phylogenetic tree for a range of hagfish species indicated clear differences between the populations, suggesting that they differ at the species level. The numbers of their teeth, in particular of fused cusps (anterior/posterior multicusps), also supported these findings. Individuals of the Suruga Bay population had 3/3 fused cusps, as described for E. atami, whereas individuals of the Akita population had 3/2 fused cusps. These results suggest that the brown hagfish from the Sea of Japan, off the northwest coast of Honshu, is a distinct species from E. atami.

Four Human Cases of Diphyllobothrium nihonkaiense (Eucestoda: Diphyllobothriidae) in China with a Brief Review of Chinese Cases.

We described 4 human infection cases of zoonotic fish-tapeworm, Diphyllobothrium nihonkaiense, identified with morphological and molecular characters and briefly reviewed Chinese cases in consideration of it as an emerging parasitic disease in China. The scolex and mature and gravid proglottids of some cases were seen, a rosette-shaped uterus was observed in the middle of the mature and gravid proglottids, and the diphyllobothriid eggs were yellowish-brown in color and displayed a small knob or abopercular protuberance on the opposite end of a lid-like opening. The average size of the eggs was recorded as 62-67×42-45 µm. The parasitic materials gathered from 4 human cases were morphologically identified as belonging to the genera Diphyllobothrium and Adenocephalus. The phylogenetic analysis based on the nucleotide sequences of cytochrome c oxidase subunit 1 gene of the etiologic agents confirmed that the 4 cases were D. nihonkaiense infection. The finding of 4 additional D. nihonkaiense cases suggests that D. nihonkaiense might be a major causative species of human diphyllobothriasis in China. A combined morphological and molecular analysis is the main method to confirm D. nihonkaiense infection.

MicroRNA-661 Enhances TRAIL or STS Induced Osteosarcoma Cell Apoptosis by Modulating the Expression of Cytochrome c1.

AIM: Osteosarcoma (OS) is an aggressive bone malignancy that affects rapidly growing bones and is associated with a poor prognosis. Our previous study showed that cytochrome c1 (CYC1), a subunit of the cytochrome bc1 complex (complex III) of the mitochondrial electron chain, is overexpressed in human OS tissues and cell lines and its silencing induces apoptosis in vitro and inhibits tumor growth in vivo. Here, we investigated the mechanism underlying the modulation of CYC1 expression in OS and its role in the resistance of OS to apoptosis. METHODS: qRT-PCR, luciferase reporter assay, western blotting, fow cytometry, and animal experiments were performed in this study. RESULTS: MicroRNA (miR)-661 was identified as a downregulated miRNA in OS tissues and cells and shown to directly target CYC1. Ectopically expressed miR-661 inhibited OS cell growth, promoted apoptosis, and reduced the activity of mitochondrial complex III. miR-661 overexpression enhanced TRAIL or STS induced apoptosis and promoted the release of cytochrome c into the cytosol, which induced caspase-9 activation, and these effects were abolished by a caspase-3 inhibitor. Overexpression of CYC1 rescued the effects of miR-661 on sensitizing OS cells to TRAIL or STS induced apoptosis, indicating that the antitumor effect of miR-661 is mediated by the downregulation of CYC1. In vivo, miR-661 overexpression sensitized tumors to TRAIL or STS induced apoptosis in a xenograft mouse model, and these effects were attenuated by co-expression of CYC1. CONCLUSION: Taken together, our results indicate that miR-661 plays a tumor suppressor role in OS mediated by the downregulation of CYC1, suggesting a potential mechanism underlying cell death resistance in OS.

Identification of Triplophysa species from the Qinghai-Tibetan Plateau (QTP) and its adjacent regions through DNA barcodes.

The genus Triplophysa is the largest and most difficult to identity morphologically fish group of superfamily Cobitoidea with 140 currently valid species, and is mainly distributed in the Qinghai-Tibetan Plateau (QTP) and adjacent regions. Most species within this genus possess highly similar morphological characteristics for adaption to the highland environment and are very difficult to be identified only based on morphology. The traditional species identification, mainly based on external morphological diagnostic characters, leads to inconsistent results in many cases. Herein, we provided a molecular method based on mitochondrial cytochrome c subunit I (COI) for the identification of Triplophysa fishes. Thirty-three Triplophysa species, 244 individuals, were used to determine whether barcoding was effective in discriminating species for this genus. The mean intraspecific and interspecific K2P distances ranged from 0 to 14.9% (mean, 2.9%) and 0 to 23.4% (mean, 9.7%), respectively. The tree-based analysis displayed most of species formed discrete clusters with strong bootstrap support values (>90%). The results showed that most of Triplophysa species could be identified by DNA barcode and indicated DNA barcode could be used as a molecular marker for these species.

NapB in excess inhibits growth of Shewanella oneidensis by dissipating electrons of the quinol pool.

Shewanella, a group of ubiquitous bacteria renowned for respiratory versatility, thrive in environments where various electron acceptors (EAs) of different chemical and physiological characteristics coexist. Despite being extensively studied, we still know surprisingly little about strategies by which multiple EAs and their interaction define ecophysiology of these bacteria. Previously, we showed that nitrite inhibits growth of the genus representative Shewanella oneidensis on fumarate and presumably some other CymA (quinol dehydrogenase)-dependent EAs by reducing cAMP production, which in turn leads to lowered expression of nitrite and fumarate reductases. In this study, we demonstrated that inhibition of fumarate growth by nitrite is also attributable to overproduction of NapB, the cytochrome c subunit of nitrate reductase. Further investigations revealed that excessive NapB per se inhibits growth on all EAs tested, including oxygen. When overproduced, NapB acts as an electron shuttle to dissipate electrons of the quinol pool, likely to extracellullar EAs, because the Mtr system, the major electron transport pathway for extracellular electron transport, is implicated. The study not only sheds light on mechanisms by which certain EAs, especially toxic ones, impact the bacterial ecophysiology, but also provides new insights into how electron shuttle c-type cytochromes regulate multi-branched respiratory networks.

In meso crystal structure of a novel membrane-associated octaheme cytochrome c from the Crenarchaeon Ignicoccus hospitalis.

The Crenarchaeon Ignicoccus hospitalis lives in symbiosis with Nanoarchaeum equitans providing essential cell components and nutrients to its symbiont. Ignicoccus hospitalis shows an intriguing morphology that points toward an evolutionary role in driving compartmentalization. Therefore, the bioenergetics of this archaeal host-symbiont system remains a pressing question. To date, the only electron acceptor described for I. hospitalis is elemental sulfur, but the organism comprises genes that encode for enzymes involved in nitrogen metabolism, e.g., one nitrate reductase and two octaheme cytochrome c, Igni_0955 (IhOCC) and Igni_1359. Herein, we detail functional and structural studies of the highly abundant IhOCC, including an X-ray crystal structure at 1.7 Å resolution, the first three-dimensional structure of an archaeal OCC. The trimeric IhOCC is membrane associated and exhibits significant structural and functional differences to previously characterized homologs within the hydroxylamine oxidoreductases (HAOs) and octaheme cytochrome c nitrite reductases (ONRs). The positions and spatial arrangement of the eight hemes are highly conserved, but the axial ligands of the individual hemes 3, 6 and 7 and the protein environment of the active site show significant differences. Most notably, the active site heme 4 lacks porphyrin-tyrosine cross-links present in the HAO family. We show that IhOCC efficiently reduces nitrite and hydroxylamine, with possible relevance to detoxification or energy conservation. DATABASE: Structural data are available in the Protein Data Bank under the accession number 4QO5.

Cytochrome c and c1 heme lyases are essential in Plasmodium berghei.

Malaria parasites possess a de novo heme synthetic pathway. Interestingly, this pathway is dispensable during the blood stages of development in mammalian hosts. The assembly of the two most important hemeproteins, cytochromes c and c1, is mediated by cytochrome heme lyase enzymes. Plasmodium spp. possess two cytochrome heme lyases encoded by separate genes. Given the redundancy of heme synthesis, we sought to determine if heme lyase function also exhibits redundancy. To answer this question, we performed gene knockout experiments. We found that the PBANKA_143950 and PBANKA_0602600 Plasmodium berghei genes encoding cytochrome c (Pbcchl) and cytochrome c1 (Pbcc1hl) heme lyases, respectively, can only be disrupted when a complementary gene is present. In contrast, four genes in the de novo heme synthesis pathway can be disrupted without complementation. This work provides evidence that Pbcchl and Pbcc1hl are both essential and thus may be antimalarial targets.

CYC1 Predicts Poor Prognosis in Patients with Breast Cancer.

Cytochrome c-1 (CYC1) is an important subunit of mitochondrial complex III. However, its role in tumor progression is unclear. We found that CYC1 was upregulated in breast tumor tissues, especially in tissues with lymph node metastasis. And higher expression of CYC1 correlates with poor prognosis in breast cancer patients using online databases and tools. Then we confirmed that CYC1 contributed to metastasis and proliferation in two highly metastatic human breast cancer cell lines. Digging into the biological function of CYC1, we found the activity of mitochondrial complex III decreased due to silencing CYC1. Then the ratio of AMP to ATP increased and AMPK was activated. Analyzing units of other mitochondrial complexes, we did not find knockdown of CYC1 expression reduced expression of any other unit of OXPHOS. We concluded that CYC1 promoted tumor metastasis via suppressing activation of AMPK and contributed to tumor growth via facilitating production of ATP. Our results indicated that CYC1 plays crucial roles in breast cancer progression and might be a predictive factor assisting future patient diagnosis.

Limited predictability of postmortem human brain tissue quality by RNA integrity numbers.

The RNA integrity number (RIN) is often considered to be a critical measure of the quality of postmortem human brains. However, it has been suggested that RINs do not necessarily reflect the availability of intact mRNA. Using the Agilent bioanalyzer and qRT-PCR, we explored whether RINs provide a meaningful way of assessing mRNA degradation and integrity in human brain samples by evaluating the expression of 3'-5' mRNA sequences of the cytochrome C-1 (CYC1) gene. Analysis of electropherograms showed that RINs were not consistently correlated with RNA or cDNA profiles and appeared to be poor predictors of overall cDNA quality. Cycle thresholds from qRT-PCR analysis to quantify the amount of CYC1 mRNA revealed positive correlations of RINs with amplification of full-length transcripts, despite the variable degree of linear degradation along the 3'-5' sequence. These data demonstrate that in postmortem human brain tissue the RIN is an indicator of mRNA quantity independent of degradation, but does not predict mRNA integrity, suggesting that RINs provide an incomplete measure of brain tissue quality. Quality assessment of postmortem human brains by RNA integrity numbers (RINs) may be misleading, as they do not measure intact mRNAs. We show that the RIN is an indicator of mRNA quantity independent of degradation, but does not predict mRNA integrity, suggesting that RINs provide an incomplete measure of brain tissue quality. Our results resolve controversial assumption on interpreting quality assessments of human postmortem brains by RINs.