SOX9 modulates cancer biomarker and cilia genes in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with high mortality. The cellular origins of PDAC are largely unknown; however, ductal cells, especially centroacinar cells (CACs), have several characteristics in common with PDAC, such as expression of SOX9 and components of the Notch-signaling pathway. Mutations in KRAS and alterations to Notch signaling are common in PDAC, and both these pathways regulate the transcription factor SOX9. To identify genes regulated by SOX9, we performed siRNA knockdown of SOX9 followed by RNA-seq in PANC-1s, a human PDAC cell line. We report 93 differentially expressed (DE) genes, with convergence on alterations to Notch-signaling pathways and ciliogenesis. These results point to SOX9 and Notch activity being in a positive feedback loop and SOX9 regulating cilia production in PDAC. We additionally performed ChIP-seq in PANC-1s to identify direct targets of SOX9 binding and integrated these results with our DE gene list. Nine of the top 10 downregulated genes have evidence of direct SOX9 binding at their promoter regions. One of these targets was the cancer stem cell marker EpCAM. Using whole-mount in situ hybridization to detect epcam transcript in zebrafish larvae, we demonstrated that epcam is a CAC marker and that Sox9 regulation of epcam expression is conserved in zebrafish. Additionally, we generated an epcam null mutant and observed pronounced defects in ciliogenesis during development. Our results provide a link between SOX9, EpCAM and ciliary repression that can be exploited in improving our understanding of the cellular origins and mechanisms of PDAC.

Age-dependent electrical and morphological remodeling of the Drosophila heart caused by hERG/seizure mutations.

Understanding the cellular-molecular substrates of heart disease is key to the development of cardiac specific therapies and to the prevention of off-target effects by non-cardiac targeted drugs. One of the primary targets for therapeutic intervention has been the human ether a go-go (hERG) K+ channel that, together with the KCNQ channel, controls the rate and efficiency of repolarization in human myocardial cells. Neither of these channels plays a major role in adult mouse heart function; however, we show here that the hERG homolog seizure (sei), along with KCNQ, both contribute significantly to adult heart function as they do in humans. In Drosophila, mutations in or cardiac knockdown of sei channels cause arrhythmias that become progressively more severe with age. Intracellular recordings of semi-intact heart preparations revealed that these perturbations also cause electrical remodeling that is reminiscent of the early afterdepolarizations seen in human myocardial cells defective in these channels. In contrast to KCNQ, however, mutations in sei also cause extensive structural remodeling of the myofibrillar organization, which suggests that hERG channel function has a novel link to sarcomeric and myofibrillar integrity. We conclude that deficiency of ion channels with similar electrical functions in cardiomyocytes can lead to different types or extents of electrical and/or structural remodeling impacting cardiac output.

Detection of Genes Related to Antibiotic Resistance in Leptospira.

Leptospirosis is a disease caused by the bacteria of the Leptospira genus, which can usually be acquired by humans through contact with urine from infected animals; it is also possible for this urine to contaminate soils and bodies of water. The disease can have deadly consequences in some extreme cases. Fortunately, until now, patients with leptospirosis have responded adequately to treatment with doxycycline and azithromycin, and no cases of antibiotic resistance have been reported. However, with the extensive use of such medications, more bacteria, such as Staphylococci and Enterococci, are becoming resistant. The purpose of this study is to determine the presence of genes related to antibiotic resistance in the Leptospira genus using bioinformatic tools, which have not been undertaken in the past. Whole genomes from the 69 described Leptospira species were downloaded from NCBI's GeneBank and analyzed using CARD (The Comprehensive Antibiotic Resistant Database) and RAST (Rapid Annotations using Subsystem Technology). After a detailed genomic search, 12 genes associated with four mechanisms were found: resistance to beta-lactamases, vancomycin, aminoglycoside adenylyltransferases, as well as multiple drug efflux pumps. Some of these genes are highly polymorphic among different species, and some of them are present in multiple copies in the same species. In conclusion, this study provides evidence of the presence of genes related to antibiotic resistance in the genomes of some species of the genus Leptospira, and it is the starting point for future experimental evaluation to determine whether these genes are transcriptionally active in some species and serovars.

CD11b suppresses TLR7-driven inflammatory signaling to protect against lupus nephritis.

Lupus Nephritis (LN) is a severe complication of systemic lupus erythematosus (SLE) that affects kidney function. Here, we investigated the role of CD11b, a protein encoded by the ITGAM gene, in the development of LN and its functional activation as a therapeutic strategy. Genetic coding variants of ITGAM significantly increase the risk for SLE and LN by producing a less active CD11b and leading to elevated levels of type I interferon (IFN I). However, a molecular mechanism for how these variants increase LN risk has been unclear. Here, we determined that these variants also significantly associate with elevations in soluble urokinase plasminogen activator receptor (suPAR), a known biomarker linked to kidney disease, suggesting a novel molecular connection. Pharmacologic activation of CD11b with a novel, clinical-stage agonist ONT01 significantly suppressed suPAR production in myeloid cells and reduced systemic inflammation and kidney damage in multiple experimental models of LN. Importantly, delaying treatment with ONT01 until after disease onset also significantly reduced serum suPAR and inflammatory cytokines, and decreased immune complex deposition in the glomerulus, glomerulonephritis and albuminuria, suggesting that CD11b activation is therapeutic for LN. Genetic activation of CD11b via a gain-of-function CD11b mutation also showed complete protection from LN, whereas genetic deletion of CD11b worsened the disease in mice, providing further evidence of the role of CD11b activation in regulating LN. Finally, transfer of human LN PBMCs generated human LN like disease in mice that was significantly reduced by ONT01. Together, these data provide strong evidence that ONT01 mediated CD11b activation can therapeutically modulate TLR7-driven inflammation and protect against LN. These findings support clinical development of CD11b agonists as novel therapeutics for treating lupus nephritis in human patients.

Seed morphology and germination of Willd. ex Schult. emulating environmental conditions within ant nest.

Damiana (Turnera diffusa Willd. ex Schult.) is a species of plant used in traditional Mexican medicine for its aphrodisiac properties. Although it has a high commercial demand, both nationally and internationally, its sexual propagation is not usual due to the low percentage of seed germination. It has been proposed that ants play an important role in germination, due to the presence of elaiosomes. Therefore, the objectives of this study were to characterize the seed morphology of T. diffusa for agronomic purposes, analyze their viability, and evaluate their germination by simulating environmental conditions of an ant nest. For the morphological characterization, 30 seeds were selected and evaluated for the variables of color, size, and weight. Viability was evaluated with a tetrazolium test using two lots of seeds collected in 2016 and 2017, with different concentrations and three exposure times at 40°C. The germination of T. diffusa was evaluated under three pre-germination treatments and nine germination treatments. The results of the study showed that the seeds of T. diffusa have an average size of 0.725 mm long and 0.182 mm wide; the color of the seeds varies from brown to black when ripe and yellowish white when immature. There are no significant differences in the viability percentage (60%) for seeds collected in 2016 and 2017 (p = 0.20). On the other hand, there are significant differences between all the pre-germination and germination tests analyzed. Seeds of T. diffusa have the highest percentage of germination (36%) with the presence of elaiosome and 500 ppm of GA3. The germination interval of the seeds occurs over a period of six to 39 days. The application of GA3 in the germination of the seeds indicates that they present a physiological latency which was inhibited at concentrations of 500 and 300 ppm.

Photorhabdus and Xenorhabdus as Symbiotic Bacteria for Bio-Control Housefly (Musca domestica L.).

<b>Background and Objective:</b> The housefly poses a threat to the public health of humans and domestic animals since it can carry and transmit pathogens. Despite there are many attempts to control this insect, most of them depend on conventional pesticides. Thus, the current study aimed to evaluate the efficacy of whole-cell suspension, cell-free supernatant and crude cells of the symbiotic bacteria <i>Photorhabdus</i> sp. and <i>Xenorhabdus</i> sp., as bio-control agents for housefly stages. <b>Materials and Methods:</b> The <i>Photorhabdus</i> sp. and <i>Xenorhabdus</i> sp., were isolated from the entomopathogenic nematodes, <i>Heterorhabditis indica</i> and <i>Steinernema feltiae</i>, respectively. The phenotypic, as well as the enzymatic characterizations of both bacteria, were determined. In addition, histopathological changes of the alimentary canal of <i>M. domestica</i> adults treated with whole-cell suspensions (at 3×10⁸cells mL¹) of both bacteria were carefully examined using transmission electron microscopy. <b>Results:</b> The results showed that both symbiotic bacteria significantly suppressed larvae, pupae and adults of <i>M. domestica</i>, particularly when they were applied as whole-cell suspensions. For example, the highest concentration of whole-cell suspension, cell-free supernatant and crude cells of <i>Photorhabdus</i> sp., induced larval mortalities by 94.7, 64.0 and 45.3%, while those of <i>Xenorhabdus</i> sp., induced larval mortalities by 58.7, 46.7 and 30.7% at 96 hrs, respectively. The results also showed that whole-cell suspensions of both symbiotic bacteria caused severe histopathological changes in the ultrastructure of the treated adults' alimentary canal. <b>Conclusion:</b> Both symbiotic bacteria can be effectively used, particularly the whole-cell suspension, as bio-control agents against the housefly either in the larval or adult stage.

Comparative Analysis of the NDVI and NGBVI as Indicators of the Protective Effect of Beneficial Bacteria in Conditions of Biotic Stress.

Precision agriculture has the objective of improving agricultural yields and minimizing costs by assisting management with the use of sensors, remote sensing, and information technologies. There are several approaches to improving crop yields where remote sensing has proven to be an important methodology to determine agricultural maps to show surface differences which may be associated with many phenomena. Remote sensing utilizes a wide variety of image sensors that range from common RGB cameras to sophisticated, hyper-spectral image cameras which acquire images from outside the visible electromagnetic spectrum. The NDVI and NGBVI are computer vision vegetation index algorithms that perform operations from color masks such as red, green, and blue from RGB cameras and hyper-spectral masks such as near-infrared (NIR) to highlight surface differences in the image to detect crop anomalies. The aim of the present study was to determine the relationship of NDVI and NGBVI as plant health indicators in tomato plants (Solanum lycopersicum) treated with the beneficial bacteria Bacillus cereus-Amazcala (B. c-A) as a protective agent to cope with Clavibacter michiganensis subsp. michiganensis (Cmm) infections. The results showed that in the presence of B. c-A after infection with Cmm, NDVI and NGBVI can be used as markers of plant weight and the activation of the enzymatic activities related to plant defense induction.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Silencing of CCR4-NOT complex subunits affects heart structure and function.

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function.This article has an associated First Person interview with the first author of the paper.

Prolonged Exposure to Microgravity Reduces Cardiac Contractility and Initiates Remodeling in Drosophila.

Understanding the effects of microgravity on human organs is crucial to exploration of low-earth orbit, the moon, and beyond. Drosophila can be sent to space in large numbers to examine the effects of microgravity on heart structure and function, which is fundamentally conserved from flies to humans. Flies reared in microgravity exhibit cardiac constriction with myofibrillar remodeling and diminished output. RNA sequencing (RNA-seq) in isolated hearts revealed reduced expression of sarcomeric/extracellular matrix (ECM) genes and dramatically increased proteasomal gene expression, consistent with the observed compromised, smaller hearts and suggesting abnormal proteostasis. This was examined further on a second flight in which we found dramatically elevated proteasome aggregates co-localizing with increased amyloid and polyQ deposits. Remarkably, in long-QT causing sei/hERG mutants, proteasomal gene expression at 1g, although less than the wild-type expression, was nevertheless increased in microgravity. Therefore, cardiac remodeling and proteostatic stress may be a fundamental response of heart muscle to microgravity.

The HAND1 frameshift A126FS mutation does not cause hypoplastic left heart syndrome in mice.

AIMS: To test if a human Hand1 frame shift mutation identified in human samples is causative of hypoplastic left heart syndrome (HLHS). METHODS AND RESULTS: HLHS is a poorly understood single ventricle congenital heart defect that affects two to three infants in every 10 000 live births. The aetiologies of HLHS are largely unknown. The basic helix-loop-helix transcription factor HAND1 is required for normal heart development. Interrogation of HAND1 sequence from fixed HLHS tissues identified a somatic frame-shift mutation at Alanine 126 (NP_004812.1 p.Ala126Profs13X defined as Hand1A126fs). Hand1A126fs creates a truncated HAND1 protein that predictively functions as dominant negative. To determine if this mutation is causative of HLHS, we engineered a conditional Hand1A126fs mouse allele. Activation of this allele with Nkx2.5Cre results in E14.5 lethality accompanied by cardiac outflow tract and intraventricular septum abnormalities. Using αMHC-Cre or Mef2CAHF-Cre to activate Hand1A126fs results in reduced phenotype and limited viability. Left ventricles of Hand1A126FS mutant mice are not hypoplastic. CONCLUSIONS: Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.