Pharmacokinetics and Absorption, Distribution, Metabolism and Excretion of RGLS4326 in Mouse and Monkey, an Anti-miR-17 Oligonucleotide for the Treatment of Polycystic Kidney Disease.

RGLS4326 is a short oligonucleotide inhibitor of microRNA-17 (miR-17) that preferentially distributes to the kidney and displaces miR-17 from translationally active polysomes. Here, we present pharmacokinetics and absorption, distribution, metabolism, and excretion properties of RGLS4326 from mice and monkeys. RGLS4326 was absorbed rapidly after subcutaneous administration, distributed extensively to the kidney and liver, with preferential distribution to the kidney, and cleared rapidly from plasma by tissue uptake and renal excretion. Plasma exposure increased in a dose-proportional manner with no notable accumulation after repeat doses. Plasma protein binding of RGLS4326 across all species tested was between 79% and 96%. RGLS4326 predominantly distributed to the kidney with a long half-life (t1/2; t1/2 ranged from 8-11 days) and no marked (≤twofold) accumulation in kidney and liver after repeat doses. RGLS4326 was minimally metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species and underwent sequential hydrolysis from both 3' and 5' ends to produce chain-shortened metabolites. There were no human unique metabolites observed. Renal excretion was the major route of elimination of RGLS4326, and a significant fraction (50%-79%) of the dose was recovered intact in the urine of mice and monkeys across all dose levels. RGLS4326 is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, RGLS4326 exhibits low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Pharmacokinetics (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of RGLS4326 were characterized in vivo and in vitro. RGLS4326 shows similar PK and ADME properties across mice and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in preferential exposure of RGLS4326 to the intended target organ (kidney) to drive maximum target engagement. These studies support the interpretation of toxicology and efficacy studies and help characterize the disposition of RGLS4326 in humans.

c-Myc phosphorylation by PKCζ represses prostate tumorigenesis.

Studies showing reduced PKCζ expression or enzymatic activity in different types of human cancers support the clinical relevance of PKCζ as a tumor suppressor. However, the in vivo role of PKCζ and its mechanisms of action in prostate cancer remain unclear. Here we demonstrate that the genetic inactivation of PKCζ in mice results in invasive prostate carcinoma in vivo in the context of phosphatase and tensin homolog deficiency. Bioinformatic analysis of human prostate cancer gene-expression sets revealed increased c-Myc transcriptional activity in PKCζ-inactive cells, which correlated with increased cell growth, invasion, and metastasis. Interestingly, PKCζ knockdown or the overexpression of a kinase-inactive mutant resulted in enhanced cell proliferation and invasion in vitro through increased c-Myc mRNA and protein levels and decreased Ser-373 phosphorylation of c-Myc. Analysis of prostate cancer samples demonstrated increased expression and decreased phosphorylation of c-Myc at Ser-373 in PKCζ knockout tumors. In vivo xenograft studies revealed that c-Myc phosphorylation by PKCζ is a critical event in the control of metastasis. Collectively, these results establish PKCζ as an important tumor suppressor and regulator of c-Myc function in prostate cancer.

First identification and characterization of ovine gammaherpesvirus type 2 in horses and artiodactyla from an outbreak of malignant catarrhal fever in Mexico.

Ovine gammaherpesvirus 2 (OvHV-2), a member of the genus Macavirus, causes sheep-associated malignant catarrhal fever (SA-MCF), a fatal lymphoproliferative disease affecting a wide variety of ungulates in addition to horses. This study described an outbreak of SA-MCF in Mexico and the identification of the OvHV-2 virus in primary rabbit testis cultures through the generation of intranuclear inclusion bodies, syncytia, immunofluorescence (IF), immunocytochemistry (ICC), immunohistochemistry (IHC), endpoint polymerase chain reaction (PCR), and partial sequencing of the ORF75 gene. The animals involved in this outbreak showed mucogingival ulcers in the vestibule of the mouth and tongue, hypersalivation, corneal opacity, reduced food consumption, and weight loss of variable severity. These clinical signs and the histopathological findings suggested the diagnosis of SA-MCF. Buffy coat fractions from the anticoagulated blood samples of ill animals were collected and analyzed by PCR. Positive buffy coats were used to inoculate the primary cell cultures of rabbit testis to identify the virus. Small clusters of refractile cytomegalic cells, characteristic of viral cytopathic effects, were observed between 48 and 72 h post-infection. Furthermore, intranuclear acidophilic inclusion bodies (IBs) were identified in the inoculated primary culture cells, and the cytoplasm showed immunoreactivity with hyperimmune rabbit serum against OvHV-2. Moreover, in the liver histological sections from sick deer, immunoreactive juxtanuclear IBs were identified with the same rabbit hyperimmune serum. The obtained sequences were aligned with the OvHV-2 sequences reported in GenBank and revealed a nucleotide identity higher than 98%. Based on the evidence provided in this study, we conclude that the outbreak of SA-MCF in the municipality of Tequisquiapan in the state of Queretaro, Mexico, was caused by OvHV-2. This is the second study reporting that horses are susceptible to OvHV-2 infection and can develop SA-MCF. We identified for the first time in Mexico, the presence of OvHV-2 in buffy coats from horses and Artiodactyla.

PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression.

Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation occurs when PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are repressed via their 3'-UTR miR-17 binding element. Eliminating this motif (Pkd1∆17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also inhibited via its 3'-UTR miR-17 motif, and Pkd2∆17-induced Polycystin-2 derepression retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, modeling PKD1∆17 or PKD2∆17 alleles in patient-derived primary ADPKD cultures leads to smaller cysts, reduced proliferation, lower pCreb1 expression, and improved mitochondrial membrane potential. Thus, evading 3'-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.

Assessment of the hemagglutinating activity of the Porcine orthorubulavirus.

Blue eye disease (BED) in pigs is caused by Porcine orthorubulavirus (PRV) of the Paramyxoviridae family. It is an endemic disease in swine production in the central region of Mexico and causes nervous signs and high mortality in suckling pigs, pneumonia in growing pigs, orchitis in boars and mummification during gestation. PRV hemagglutinates most red blood cells (RBCs) of domestic species. For serological diagnosis, the hemagglutination inhibition test is used, and in this test, guinea pig, bovine and chicken RBCs have been commonly used. In this investigation, hemagglutination with PRV was evaluated using the RBCs of seven domestic species (chicken, bovine, horse, pig, dog, guinea pig and rabbit). In the hemagglutination test, the following parameters were evaluated: temperature (25 °C and 37 °C), bottoms of the wells (V and U), erythrocyte concentration (0.5%, 0.75%, and 1%), and reading time (15, 30, 45, 60 and 90 min). Significant differences (P < 0.001) were found in most of the evaluated treatments. The best hemagglutination results were obtained with chicken, bovine and horse RBCs. The hemagglutination titer is higher (2 dilutions) when using chicken RBCs than when using bovine or horse RBCs. If chicken RBCs are used in the inhibition of hemagglutination, the test will be more sensitive, while it is more specific when bovine or horse RBCs are used. The hemagglutination readings are imprecise when using RBCs from dogs, pigs, guinea pigs and rabbits. RBCs from these species should not be used for the diagnosis or investigation of PRV.

Role and expression of FRS2 and FRS3 in prostate cancer.

BACKGROUND: FGF receptor substrates (FRS2 and FRS3) are key adaptor proteins that mediate FGF-FGFR signalling in benign as well as malignant tissue. Here we investigated FRS2 and FRS3 as a means of disrupting global FGF signalling in prostate cancer. METHODS: FRS2 and FRS3 manipulation was investigated in vitro using over-expression, knockdown and functional assays. FRS2 and FRS3 expression was profiled in cell lines and clinical tumors of different grades. RESULTS: In a panel of cell lines we observed ubiquitous FRS2 and FRS3 transcript and protein expression in both benign and malignant cells. We next tested functional redundancy of FRS2 and FRS3 in prostate cancer cells. In DU145 cells, specific FRS2 suppression inhibited FGF induced signalling. This effect was not apparent in cells stably over-expressing FRS3. Indeed FRS3 over-expression resulted in enhanced proliferation (p = 0.005) compared to control cells. Given this functional redundancy, we tested the therapeutic principle of dual targeting of FRS2 and FRS3 in prostate cancer. Co-suppression of FRS2 and FRS3 significantly inhibited ERK activation with a concomitant reduction in cell proliferation (p < 0.05), migration and invasion (p < 0.05). Synchronous knockdown of FRS2 and FRS3 with exposure to cytotoxic irradiation resulted in a significant reduction in prostate cancer cell survival compared to irradiation alone (p < 0.05). Importantly, this synergistic effect was not observed in benign cells. Finally, we investigated expression of FRS2 and FRS3 transcript in a cohort of micro-dissected tumors of different grades as well as by immunohistochemistry in clinical biopsies. Here, we did not observe any difference in expression between benign and malignant biopsies. CONCLUSIONS: These results suggest functional overlap of FRS2 and FRS3 in mediating mitogenic FGF signalling in the prostate. FRS2 and FRS3 are not over-expressed in tumours but targeted dual inhibition may selectively adversely affect malignant but not benign prostate cells.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.

Adipocyte p62/SQSTM1 Suppresses Tumorigenesis through Opposite Regulations of Metabolism in Adipose Tissue and Tumor.

Obesity is a leading risk factor for cancer. However, understanding the crosstalk between adipocytes and tumor cells in vivo, independently of dietary contributions, is a major gap in the field. Here we used a prostate cancer (PCa) mouse model in which the signaling adaptor p62/Sqstm1 is selectively inactivated in adipocytes. p62 loss in adipocytes results in increased osteopontin secretion, which mediates tumor fatty acid oxidation and invasion, leading to aggressive metastatic PCa in vivo. Furthermore, p62 deficiency triggers in adipocytes a general shutdown of energy-utilizing pathways through mTORC1 inhibition, which supports nutrient availability for cancer cells. This reveals a central role of adipocyte's p62 in the symbiotic adipose tissue-tumor collaboration that enables cancer metabolic fitness.

On-line MSPD-SPE-HPLC/FLD analysis of polycyclic aromatic hydrocarbons in bovine tissues.

A fast method was optimized and validated for simultaneous trace determination of four polycyclic aromatic hydrocarbons: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in bovine tissues. The determination was performed by matrix solid-phase dispersion (MSPD) coupled on-line to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with fluorescence detection (FLD). The sample was dispersed on C18 silica sorbent and then the on-line MSPD-SPE-HPLC/FLD method was applied. Several parameters were optimized: cleaning and elution sequences applied to the MSPD cartridge, the flow rate and dilution of extract used for SPE loading. The on-line method was validated over a concentration range of 0.1-0.6ngg-1 obtaining good linearity (r⩾0.998) and precision (RSD)⩽10%. Recovery ranged from 96 to 99% and the limits of detection were 0.012ngg-1. This methodology was applied to liver samples from unhealthy animals. The results demonstrate that MSDP-SPE-HPLC/FLD method provides reliable, sensitive, accurate and fast data to the food control.

ATF4-Induced Metabolic Reprograming Is a Synthetic Vulnerability of the p62-Deficient Tumor Stroma.

Tumors undergo nutrient stress and need to reprogram their metabolism to survive. The stroma may play a critical role in this process by providing nutrients to support the epithelial compartment of the tumor. Here we show that p62 deficiency in stromal fibroblasts promotes resistance to glutamine deprivation by the direct control of ATF4 stability through its p62-mediated polyubiquitination. ATF4 upregulation by p62 deficiency in the stroma activates glucose carbon flux through a pyruvate carboxylase-asparagine synthase cascade that results in asparagine generation as a source of nitrogen for stroma and tumor epithelial proliferation. Thus, p62 directly targets nuclear transcription factors to control metabolic reprogramming in the microenvironment and repress tumorigenesis, and identifies ATF4 as a synthetic vulnerability in p62-deficient tumor stroma.

Metabolic reprogramming of stromal fibroblasts through p62-mTORC1 signaling promotes inflammation and tumorigenesis.

The tumor microenvironment plays a critical role in cancer progression, but the precise mechanisms by which stromal cells influence the epithelium are poorly understood. Here we show that p62 levels were reduced in the stroma of several tumors and that its loss in the tumor microenvironment or stromal fibroblasts resulted in increased tumorigenesis of epithelial prostate cancer cells. The mechanism involves the regulation of cellular redox through an mTORC1/c-Myc pathway of stromal glucose and amino acid metabolism, resulting in increased stromal IL-6 production, which is required for tumor promotion in the epithelial compartment. Thus, p62 is an anti-inflammatory tumor suppressor that acts through the modulation of metabolism in the tumor stroma.

Determination of organophosphorus pesticides in bovine tissue by an on-line coupled matrix solid-phase dispersion-solid phase extraction-high performance liquid chromatography with diode array detection method.

A miniaturized method based on matrix solid-phase dispersion coupled to solid phase extraction and high performance liquid chromatography with diode array detection (MSPD-SPE-HPLC/DAD) was developed for the trace simultaneous determination of the following organophosphorus pesticides (OPPs) in bovine tissue: parathion-methyl, fenitrothion, parathion, chlorfenvinphos, diazinon, ethion, fenchlorphos, chlorpyrifos and carbophenothion. To perform the coupling between MSPD and SPE, 0.05 g of sample was dispersed with 0.2 g of C(18) silica sorbent and packed into a stainless steel cartridge containing 0.05 g of silica gel in the bottom. After a clean-up of high and medium polarity interferences with water and an acetonitrile:water mixture, the OPPs were desorbed from the MSPD cartridge with pure acetonitrile and directly transferred to a dynamic mixing chamber for dilution with water and preconcentration into an SPE 20 mm × 2.0 mm I.D. C(18) silica column. Subsequently, the OPPs were eluted on-line with the chromatographic mobile phase to the analytical column and the diode array detector for their separation and detection, respectively. The method was validated and yielded recovery values between 91% and 101% and precision values, expressed as relative standard deviations (RSD), which were less than or equal to 12%. Linearity was good and ranged from 0.5 to 10 µg g(-1), and the limits of detection of the OPPs were in the range of 0.04-0.25 µg g(-1). The method was satisfactorily applied to the analysis of real samples and is recommended for food control, research efforts when sample amounts are limited, and laboratories that have ordinary chromatographic instrumentation.

Factores de riesgo para la obesidad en la infancia / Risck factors for childhood obesity

La obesidad infantil constituye un importante problema de salud, debido a su asociación con enfermedades de tipo crónico y/o degenerativo que pueden presentarse desde la infancia hasta la edad adulta. En la actualidad, se conocen los factores de riesgo asociados a este padecimiento en países desarrollados, más poco se conoce de éstos en los países en desarrollo. Este artículo presenta información relevante sobre los determinantes de la obesidad de la infancia. Por su origen, y tomando en cuenta la posibilidada de prevención, estos factores se han dividido en dos grupos. Por un lado, se realiza una síntesis de los estudios sobre los determinantes biológicos de la obesidad infantil, y por el otro, se revisan los aspectos sociales y ambientales asociados con este padecimiento. Además, se señalan aspectos de interés sobre la prevalencia de la enfermedad tanto en países en vías de desarrollo como en desarrollados, y se presentan recomendaciones sobre el tratamiento correspondiente