A new class of teraryl-based AIEgen for highly selective imaging of intracellular lipid droplets and its detection in advanced-stage human cervical cancer tissues.

Lipid droplets (LDs) have drawn much attention in recent years. They serve as the energy reservoir of cells and also play an important role in numerous physiological processes. Furthermore, LDs are found to be associated with several pathological conditions, including cancer and diabetes mellitus. Herein, we report a new class of teraryl-based donor-acceptor-appended aggregation-induced emission luminogen (AIEgen), 6a, for selective staining of intracellular LDs in in vitro live 3T3-L1 preadipocytes and the HeLa cancer cell line. In addition, AIEgen 6a was found to be capable of staining and quantifying the LD accumulation in the tissue sections of advanced-stage human cervical cancer patients. Unlike commercial LD staining dyes Nile Red, BODIPY and LipidTOX, AIEgen 6a showed a high Stokes shift (195 nm), a good fluorescence lifetime decay of 12.7 ns, and LD staining persisting for nearly two weeks.

Disentangling the enigmatic role of ephrin signaling in chronic pain: Moving towards future anti-pain therapeutics.

Chronic pain is a common and debilitating condition with a huge social and economic burden worldwide. Currently, available drugs in clinics are not adequately effective and possess a variety of severe side effects leading to treatment withdrawal and poor quality of life. The ongoing search for new therapeutics with minimal side effects for chronic pain management remains a high research priority. Erythropoietin-producing human hepatocellular carcinoma cell receptor (Eph) is a tyrosine kinase receptor that is involved in neurodegenerative disorders, including pain. The Eph receptor interacts with several molecular switches, such as N methyl d-aspartate receptor (NMDAR), mitogen-activated protein kinase (MAPK), calpain 1, caspase 3, protein kinase a (PKA), and protein kinase Cy (PKCy), which in turn regulates pathophysiology of chronic pain. Here we highlight the emerging evidence of the Ephs/ephrin system as a possible near-future therapeutic target for the treatment of chronic pain and discuss the various mechanism of its involvement. We critically analyse the present status of Eph receptor system and conclude that extrapolating the pharmacological and genetic approaches using a strong therapeutic development framework could serve as next-generation analgesics for the management of chronic pain.

Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.

Picomolar Detection of Lead Ions (Pb2+) by Functionally Modified Fluorescent Carbon Quantum Dots from Watermelon Juice and Their Imaging in Cancer Cells.

Water contamination due to the presence of lead is one of the leading causes of environmental and health hazards because of poor soil and groundwater waste management. Herein we report the synthesis of functionally modified luminescent carbon quantum dots (CQDs) obtained from watermelon juice as potential nanomaterials for the detection of toxic Pb2+ ions in polluted water and cancer cells. By introducing surface passivating ligands such as ethanolamine (EA) and ethylenediamine (ED) in watermelon juice, watermelon-ethanolamine (WMEA)-CQDs and watermelon-ethylenediamine (WMED)-CQDs exhibited a remarkable ~10-fold and ~6-fold increase in fluorescence intensity with respect to non-doped WM-CQDs. The relative fluorescence quantum yields of WMEA-CQDs and WMED-CQDs were found to be 8% and 7%, respectively, in an aqueous medium. Among various functionally-modified CQDs, only WMED-CQDs showed high selectivity towards Pb2+ ions with a remarkably good limit of detection (LoD) of 190 pM, which is less than that of the permissible limit (72 nM) in drinking water. The functionally altered WMED-CQDs detected Pb2+ metal ions in polluted water and in a human cervical cancer cell line (HeLa), thus advocating new vistas for eco-friendly nanomaterials for their use as diagnostic tools in the environment and biomedical research areas.

Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.

Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34+ CD41+ CD235ab+ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers-CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.

Argonaute4 Modulates Resistance to Fusarium brachygibbosum Infection by Regulating Jasmonic Acid Signaling.

Argonautes (AGOs) associate with noncoding RNAs to regulate gene expression during development and stress adaptation. Their role in plant immunity against hemibiotrophic fungal infection remains poorly understood. Here, we explore the function of AGOs in the interaction of wild tobacco (Nicotiana attenuata) with a naturally occurring hemibiotrophic pathogen, Fusarium brachygibbosum Among all AGOs, only transcripts of AGO4 were elicited after fungal infection. The disease progressed more rapidly in AGO4-silenced (irAGO4) plants than in wild type, and small RNA (smRNA) profiling revealed that 24-nucleotide smRNA accumulation was severely abrogated in irAGO4 plants. Unique microRNAs (miRNAs: 130 conserved and 208 novel, including 11 canonical miRNA sequence variants known as "isomiRs") were identified in infected plants; silencing of AGO4 strongly changed miRNA accumulation dynamics. Time-course studies revealed that infection increased accumulation of abscisic acid, jasmonates, and salicylic acid in wild type; in irAGO4 plants, infection accumulated lower jasmonate levels and lower transcripts of jasmonic acid (JA) biosynthesis genes. Treating irAGO4 plants with JA, methyl jasmonate, or cis-(+)-12-oxo-phytodienoic acid restored wild-type levels of resistance. Silencing expression of RNA-directed RNA polymerases RdR1 and RdR2 (but not RdR3) and Dicer-like3 (DCL3, but not DCL2 or DCL4) increased susceptibility to F brachygibbosum The relevance of AGO4, RdR1, RdR2, and DCL3 in a natural setting was revealed when plants individually silenced in their expression (and their binary combinations) were planted in a diseased field plot in the Great Basin Desert of Utah. These plants were more susceptible to infection and accumulated lower JA levels than wild type. We infer that AGO4-dependent smRNAs play a central role in modulating JA biogenesis and signaling during hemibiotrophic fungal infections.

Complex regulation of microRNAs in roots of competitively-grown isogenic Nicotiana attenuata plants with different capacities to interact with arbuscular mycorrhizal fungi.

BACKGROUND: Nicotiana attenuata is an ecological model plant whose 2.57 Gb genome has recently been sequenced and assembled and for which miRNAs and their genomic locations have been identified. To understand how this plant's miRNAs are reconfigured during plant-arbuscular mycorrhizal fungal (AMF) interactions and whether hostplant calcium- and calmodulin dependent protein kinase (CCaMK) expression which regulates the AMF interaction also modulates miRNAs levels and regulation, we performed a large-scale miRNA analysis of this plant-AMF interaction. RESULTS: Next generation sequencing of miRNAs in roots of empty vector (EV) N. attenuata plants and an isogenic line silenced in CCaMK expression (irCCaMK) impaired in AMF-interactions grown under competitive conditions with and without AMF inoculum revealed a total of 149 unique miRNAs: 67 conserved and 82 novel ones. The majority of the miRNAs had a length of 21 nucleotides. MiRNA abundances were highly variable ranging from 400 to more than 25,000 reads per million. The miRNA profile of irCCaMK plants impaired in AMF colonization was distinct from fully AMF-functional EV plants grown in the same pot. Six conserved miRNAs were present in all conditions and accumulated differentially depending on treatment and genotype; five (miR6153, miR403a-3p, miR7122a, miR167-5p and miR482d, but not miR399a-3p) showed the highest accumulation in AMF inoculated EV plants compared to inoculated irCCaMK plants. Furthermore, the accumulation patterns of sequence variants of selected conserved miRNAs showed a very distinct pattern related to AMF colonization - one variant of miR473-5p specifically accumulated in AMF-inoculated plants. Also abundances of miR403a-3p, miR171a-3p and one of the sequence variants of miR172a-3p increased in AMF-inoculated EV compared to inoculated irCCaMK plants and to non-inoculated EV plants, while miR399a-3p was most strongly enriched in AMF inoculated irCCaMK plants grown in competition with EV. The analysis of putative targets of selected miRNAs revealed an involvement in P starvation (miR399), phytohormone signaling (Nat-R-PN59, miR172, miR393) and defense (e.g. miR482, miR8667, Nat-R-PN-47). CONCLUSIONS: Our study demonstrates (1) a large-scale reprograming of miRNAs induced by AMF colonization and (2) that the impaired AMF signaling due to CCaMK silencing and the resulting reduced competitive ability of irCCaMK plants play a role in modulating signal-dependent miRNA accumulation.

Pre-operative progesterone benefits operable breast cancer patients by modulating surgical stress.

PURPOSE: We have reported a survival benefit of single injection of hydroxyprogesterone prior to surgery for primary tumour in patients with node-positive operable breast cancer. Hydroxyprogesterone was meant to recapitulate the luteal phase of menstrual cycle in these women. We wanted to understand the molecular basis of action of hydroxyprogesterone on primary breast tumours in a peri-operative setting. METHODS: We performed whole transcriptome sequencing (RNA-Seq) of primary breast tumour samples collected from patients before and after hydroxyprogesterone exposure and controls. Paired breast cancer samples were obtained from patients who were given hydroxyprogesterone before surgery and a group of patients who were subjected to only surgery. RESULTS: A test of significance between the two groups revealed 207 significantly altered genes, after correction for multiple hypothesis testing. We found significantly contrasting gene expression patterns in exposed versus unexposed groups; 142 genes were up-regulated post-surgery among exposed patients, and down-regulated post-surgery among unexposed patients. Significantly enriched pathways included genes that respond to progesterone, cellular stress, nonsense-mediated decay of proteins and negative regulation of inflammatory response. These results suggest that cellular stress is modulated by hydroxyprogesterone. Network analysis revealed that UBC, a mediator of stress response, to be a major node to which many of the significantly altered genes connect. CONCLUSIONS: Our study suggests that pre-operative exposure to progesterone favourably modulates the effect of surgical stress, and this might underlie its beneficial effect when administered prior to surgery.

Argonaute 8 (AGO8) Mediates the Elicitation of Direct Defenses against Herbivory.

In Nicotiana attenuata, specific RNA-directed RNA polymerase (RdR1) and the Dicer-like (DCL3 and DCL4) proteins are recruited during herbivore attack to mediate the regulation of defense responses. However, the identity and role(s) of Argonautes (AGOs) involved in herbivory remain unknown. Of the 11 AGOs in the N. attenuata genome, we silenced the expression of 10. Plants silenced in NaAGO8 expression grew normally but were highly susceptible to herbivore attack. Larvae of Manduca sexta grew faster when consuming inverted-repeat stable transformants (irAGO8) plants but did not differ from the wild type when consuming plants silenced in AGO1 (a, b, and c), AGO2, AGO4 (a and b), AGO7, or AGO10 expression. irAGO8 plants were significantly compromised in herbivore-induced levels of defense metabolites such as nicotine, phenolamides, and diterpenoid glycosides. Time-course analyses revealed extensively altered microRNA profiles and the reduced accumulation of MYB8 transcripts and of the associated genes of the phenolamide and phenylpropanoid pathways as well as the nicotine biosynthetic pathway. A possible AGO8-modulated microRNA-messenger RNA target network was inferred. Furthermore, comparative analysis of domains revealed the diversity of AGO conformations, particularly in the small RNA-binding pocket, which may influence substrate recognition/binding and functional specificity. We infer that AGO8 plays a central role in the induction of direct defenses by modulating several regulatory nodes in the defense signaling network during herbivore response. Thus, our study identifies the effector AGO of the herbivore-induced small RNA machinery, which in N. attenuata now comprises RdR1, DCL3/4, and AGO8.

Profiling conserved biological pathways in Autosomal Dominant Polycystic Kidney Disorder (ADPKD) to elucidate key transcriptomic alterations regulating cystogenesis: A cross-species meta-analysis approach.

PURPOSE: Initiation and progression of fluid filled cysts mark Autosomal Dominant Polycystic Kidney Disease (ADPKD). Thus, improved therapeutics targeting cystogenesis remains a constant challenge. Microarray studies in single ADPKD animal models species with limited sample sizes tend to provide scattered views on underlying ADPKD pathogenesis. Thus we aim to perform a cross species meta-analysis to profile conserved biological pathways that might be key targets for therapy. METHODS: Nine ADPKD microarray datasets on rat, mice and human fulfilled our study criteria and were chosen. Intra-species combined analysis was performed after considering removal of batch effect. Significantly enriched GO biological processes and KEGG pathways were computed and their overlap was observed. For the conserved pathways, biological modules and gene regulatory networks were observed. Additionally, Gene Set Enrichment Analysis (GSEA) using Molecular Signature Database (MSigDB) was performed for genes found in conserved pathways. RESULTS: We obtained 28 modules of significantly enriched GO processes and 5 major functional categories from significantly enriched KEGG pathways conserved in human, mice and rats that in turn suggest a global transcriptomic perturbation affecting cyst - formation, growth and progression. Significantly enriched pathways obtained from up-regulated genes such as Genomic instability, Protein localization in ER and Insulin Resistance were found to regulate cyst formation and growth whereas cyst progression due to increased cell adhesion and inflammation was suggested by perturbations in Angiogenesis, TGF-beta, CAMs, and Infection related pathways. Additionally, networks revealed shared genes among pathways e.g. SMAD2 and SMAD7 in Endocytosis and TGF-beta. CONCLUSION: Our study suggests cyst formation and progression to be an outcome of interplay between a set of several key deregulated pathways. Thus, further translational research is warranted focusing on developing a combinatorial therapeutic approach for ADPKD redressal.

Wild tobacco genomes reveal the evolution of nicotine biosynthesis.

Nicotine, the signature alkaloid of Nicotiana species responsible for the addictive properties of human tobacco smoking, functions as a defensive neurotoxin against attacking herbivores. However, the evolution of the genetic features that contributed to the assembly of the nicotine biosynthetic pathway remains unknown. We sequenced and assembled genomes of two wild tobaccos, Nicotiana attenuata (2.5 Gb) and Nicotiana obtusifolia (1.5 Gb), two ecological models for investigating adaptive traits in nature. We show that after the Solanaceae whole-genome triplication event, a repertoire of rapidly expanding transposable elements (TEs) bloated these Nicotiana genomes, promoted expression divergences among duplicated genes, and contributed to the evolution of herbivory-induced signaling and defenses, including nicotine biosynthesis. The biosynthetic machinery that allows for nicotine synthesis in the roots evolved from the stepwise duplications of two ancient primary metabolic pathways: the polyamine and nicotinamide adenine dinucleotide (NAD) pathways. In contrast to the duplication of the polyamine pathway that is shared among several solanaceous genera producing polyamine-derived tropane alkaloids, we found that lineage-specific duplications within the NAD pathway and the evolution of root-specific expression of the duplicated Solanaceae-specific ethylene response factor that activates the expression of all nicotine biosynthetic genes resulted in the innovative and efficient production of nicotine in the genus Nicotiana Transcription factor binding motifs derived from TEs may have contributed to the coexpression of nicotine biosynthetic pathway genes and coordinated the metabolic flux. Together, these results provide evidence that TEs and gene duplications facilitated the emergence of a key metabolic innovation relevant to plant fitness.

Elevated blood plasma levels of epinephrine, norepinephrine, tyrosine hydroxylase, TGFß1, and TNFα associated with high-altitude pulmonary edema in an Indian population.

Biomarkers are essential to unravel the locked pathophysiology of any disease. This study investigated the role of biomarkers and their interactions with each other and with the clinical parameters to study the physiology of high-altitude pulmonary edema (HAPE) in HAPE-patients (HAPE-p) against adapted highlanders (HLs) and healthy sojourners, HAPE-controls (HAPE-c). For this, seven circulatory biomarkers, namely, epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor beta 1, tumor necrosis factor alpha (TNFα), platelet-derived growth factor beta beta, and C-reactive protein (CRP), were measured in blood plasma of the three study groups. All the subjects were recruited at ~3,500 m, and clinical features such as arterial oxygen saturation (SaO2), body mass index, and mean arterial pressure were measured. Increased levels of epinephrine, norepinephrine, tyrosine hydroxylase, transforming growth factor-beta 1, and TNFα were observed in HAPE-p against the healthy groups, HAPE-c, and HLs (P<0.0001). CRP levels were decreased in HAPE-p against HAPE-c and HLs (P<0.0001). There was no significant difference or very marginal difference in the levels of these biomarkers in HAPE-c and HLs (P>0.01). Correlation analysis revealed a negative correlation between epinephrine and norepinephrine (P=4.6E-06) in HAPE-p and positive correlation in HAPE-c (P=0.004) and HLs (P=9.78E-07). A positive correlation was observed between TNFα and CRP (P=0.004) in HAPE-p and a negative correlation in HAPE-c (P=4.6E-06). SaO2 correlated negatively with platelet-derived growth factor beta beta (HAPE-p; P=0.05), norepinephrine (P=0.01), and TNFα (P=0.005) and positively with CRP (HAPE-c; P=0.02) and norepinephrine (HLs; P=0.04). Body mass index correlated negatively with epinephrine (HAPE-p; P=0.001) and positively with norepinephrine and tyrosine hydroxylase in HAPE-c (P<0.05). Mean arterial pressure correlated positively with TNFα in HAPE-p and norepinephrine in HLs (P<0.05). Receiver operating characteristic curve analysis yielded a positive predictive value for these biomarkers with HAPE (area under the curve >0.70, P<0.05). The results clearly suggest that increased plasma levels of these circulatory biomarkers associated with HAPE.

Tumor heterogeneity and cancer stem cell paradigm: updates in concept, controversies and clinical relevance.

Although tumor heterogeneity is widely accepted, the existence of cancer stem cells (CSCs) and their proposed role in tumor maintenance has always been challenged and remains a matter of debate. Recently, a path-breaking chapter was added to this saga when three independent groups reported the in vivo existence of CSCs in brain, skin and intestinal tumors using lineage-tracing and thus strengthens the CSC concept; even though certain fundamental caveats are always associated with lineage-tracing approach. In principle, the CSC hypothesis proposes that similar to normal stem cells, CSCs maintain self renewal and multilineage differentiation property and are found at the central echelon of cellular hierarchy present within tumors. However, these cells differ from their normal counterpart by maintaining their malignant potential, alteration of genomic integrity, epigenetic identity and the expression of specific surface protein profiles. As CSCs are highly resistant to chemotherapeutics, they are thought to be a crucial factor involved in tumor relapse and superficially appear as the ultimate therapeutic target. However, even that is not the end; further complication is attributed by reports of bidirectional regeneration mechanism for CSCs, one from their self-renewal capability and another from the recently proposed concept of dynamic equilibrium between CSCs and non-CSCs via their interconversion. This phenomenon has currently added a new layer of complexity in understanding the biology of tumor heterogeneity. In-spite of its associated controversies, this area has rapidly emerged as the center of attention for researchers and clinicians, because of the conceptual framework it provides towards devising new therapies.

Ameliorative action of curcumin in cisplatin-mediated hepatotoxicity: an in vivo study in Wistar rats.

BACKGROUND AND AIMS: Cisplatin (CP) is regarded as a major anticancer drug against a wide spectrum of leukemia and other malignancies. The mode of action of CP on cancer cells is attributed to its property of releasing free radicals which, at the same time, has the potential to damage liver and kidney cells. The tissue-specific toxicity of CP to the kidneys is well documented. However, there is a paucity of literature on systemic toxicity and also the amelioration of such effect. The aim of the present study was to evaluate the damage caused by CP and its modulation by using the antioxidant capacity of curcumin (CMN, a natural polyphenolic compound) in Wistar rats. METHODS: We evaluated the ameliorative effect of CMN (200 mg/kg body weight, b.w.) on the toxicity of CP in rat liver. Oxidative stress biomarkers, enzymatic and nonenzymatic antioxidants were evaluated for assessing the mitigatory potential of CMN against CP-induced toxicity. RESULTS: A single dose of CP (7 mg/kg b.w.) caused marked hepatic damage. CP caused a significant increase in lipid peroxidation (LPO) level and protein carbonyl (PC) content. Pretreatment of rat with CMN significantly restored the LPO levels and PC content. It also replenished the CP-induced modulatory effects on altered enzymatic and nonenzymatic antioxidants in liver. CONCLUSION: Our results clearly demonstrated that the role of oxidative stress is detrimental in systemic toxicity induced by CP and suggest a mitigatory effect of CMN on CP-induced hepatotoxicity in rat.

Inhibition of Comt with tolcapone slows progression of polycystic kidney disease in the more severely affected PKD/Mhm (cy/+) substrain of the Hannover Sprague-Dawley rat.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. METHODS: Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. RESULTS: Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. CONCLUSIONS: Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.

Parallel analysis of mRNA and microRNA microarray profiles to explore functional regulatory patterns in polycystic kidney disease: using PKD/Mhm rat model.

Autosomal polycystic kidney disease (ADPKD) is a frequent monogenic renal disease, characterised by fluid-filled cysts that are thought to result from multiple deregulated pathways such as cell proliferation and apoptosis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of many genes associated with such biological processes and human pathologies. To explore the possible regulatory role of miRNAs in PKD, the PKD/Mhm (cy/+) rat, served as a model to study human ADPKD. A parallel microarray-based approach was conducted to profile the expression changes of mRNAs and miRNAs in PKD/Mhm rats. 1,573 up- and 1,760 down-regulated genes were differentially expressed in PKD/Mhm. These genes are associated with 17 pathways (such as focal adhesion, cell cycle, ECM-receptor interaction, DNA replication and metabolic pathways) and 47 (e.g., cell proliferation, Wnt and Tgfß signaling) Gene Ontologies. Furthermore, we found the similar expression patterns of deregulated genes between PKD/Mhm (cy/+) rat and human ADPKD, PKD1(L3/L3), PKD1(-/-), Hnf1α-deficient, and Glis2(lacZ/lacZ) models. Additionally, several differentially regulated genes were noted to be target hubs for miRNAs. We also obtained 8 significantly up-regulated miRNAs (rno-miR-199a-5p, -214, -146b, -21, -34a, -132, -31 and -503) in diseased kidneys of PKD/Mhm rats. Additionally, the binding site overrepresentation and pathway enrichment analyses were accomplished on the putative targets of these 8 miRNAs. 7 out of these 8 miRNAs and their possible interactions have not been previously described in ADPKD. We have shown a strong overlap of functional patterns (pathways) between deregulated miRNAs and mRNAs in the PKD/Mhm (cy/+) rat model. Our findings suggest that several miRNAs may be associated in regulating pathways in ADPKD. We further describe novel miRNAs and their possible targets in ADPKD, which will open new avenues to understand the pathogenesis of human ADPKD. Furthermore they could serve as a useful resource for anti-fibrotic therapeutics.

The pro-apoptotic protein Bim is a microRNA target in kidney progenitors.

Understanding the mechanisms that regulate nephron progenitors during kidney development should aid development of therapies for renal failure. MicroRNAs, which modulate gene expression through post-transcriptional repression of specific target mRNAs, contribute to the differentiation of stem cells, but their role in nephrogenesis is incompletely understood. Here, we found that the loss of miRNAs in nephron progenitors results in a premature depletion of this population during kidney development. Increased apoptosis and expression of the pro-apoptotic protein Bim accompanied this depletion. Profiling of miRNA expression during nephrogenesis identified several highly expressed miRNAs (miR-10a, miR-106b, miR-17-5p) in nephron progenitors that are either known or predicted to target Bim. We propose that modulation of apoptosis by miRNAs may determine congenital nephron endowment. Furthermore, our data implicate the pro-apoptotic protein Bim as a miRNA target in nephron progenitors.

Systems biology approach to identify transcriptome reprogramming and candidate microRNA targets during the progression of polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation throughout the kidney parenchyma. It is caused by mutations in either of two genes, PKD1 and PKD2. Mice that lack functional Pkd1 (Pkd1⁻/⁻), develop rapidly progressive cystic disease during embryogenesis, and serve as a model to study human ADPKD. Genome wide transcriptome reprogramming and the possible roles of micro-RNAs (miRNAs) that affect the initiation and progression of cyst formation in the Pkd1⁻/⁻ have yet to be studied. miRNAs are small, regulatory non-coding RNAs, implicated in a wide spectrum of biological processes. Their expression levels are altered in several diseases including kidney cancer, diabetic nephropathy and PKD. RESULTS: We examined the molecular pathways that modulate renal cyst formation and growth in the Pkd1⁻/⁻ model by performing global gene-expression profiling in embryonic kidneys at days 14.5 and 17.5. Gene Ontology and gene set enrichment analysis were used to identify overrepresented signaling pathways in Pkd1⁻/⁻ kidneys. We found dysregulation of developmental, metabolic, and signaling pathways (e.g. Wnt, calcium, TGF-ß and MAPK) in Pkd1⁻/⁻ kidneys. Using a comparative transcriptomics approach, we determined similarities and differences with human ADPKD: ~50% overlap at the pathway level among the mis-regulated pathways was observed. By using computational approaches (TargetScan, miRanda, microT and miRDB), we then predicted miRNAs that were suggested to target the differentially expressed mRNAs. Differential expressions of 9 candidate miRNAs, miRs-10a, -30a-5p, -96, -126-5p, -182, -200a, -204, -429 and -488, and 16 genes were confirmed by qPCR. In addition, 14 candidate miRNA:mRNA reciprocal interactions were predicted. Several of the highly regulated genes and pathways were predicted as targets of miRNAs. CONCLUSIONS: We have described global transcriptional reprogramming during the progression of PKD in the Pkd1⁻/⁻ model. We propose a model for the cascade of signaling events involved in cyst formation and growth. Our results suggest that several miRNAs may be involved in regulating signaling pathways in ADPKD. We further describe novel putative miRNA:mRNA signatures in ADPKD, which will provide additional insights into the pathogenesis of this common genetic disease in humans.

Genomic characterization of Wilms' tumor suppressor 1 targets in nephron progenitor cells during kidney development.

The Wilms' tumor suppressor 1 (WT1) gene encodes a DNA- and RNA-binding protein that plays an essential role in nephron progenitor differentiation during renal development. To identify WT1 target genes that might regulate nephron progenitor differentiation in vivo, we performed chromatin immunoprecipitation (ChIP) coupled to mouse promoter microarray (ChIP-chip) using chromatin prepared from embryonic mouse kidney tissue. We identified 1663 genes bound by WT1, 86% of which contain a previously identified, conserved, high-affinity WT1 binding site. To investigate functional interactions between WT1 and candidate target genes in nephron progenitors, we used a novel, modified WT1 morpholino loss-of-function model in embryonic mouse kidney explants to knock down WT1 expression in nephron progenitors ex vivo. Low doses of WT1 morpholino resulted in reduced WT1 target gene expression specifically in nephron progenitors, whereas high doses of WT1 morpholino arrested kidney explant development and were associated with increased nephron progenitor cell apoptosis, reminiscent of the phenotype observed in Wt1(-/-) embryos. Collectively, our results provide a comprehensive description of endogenous WT1 target genes in nephron progenitor cells in vivo, as well as insights into the transcriptional signaling networks controlled by WT1 that might direct nephron progenitor fate during renal development.

Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease.

BACKGROUND: MicroRNAs (miRNAs) play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD). Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly. RESULTS: Given the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs. CONCLUSION: We find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.