A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes.

Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than can be achieved by studying single TF binding events in isolation. Chromosomal translocations of the lysine methyltransferase 2A (KMT2A) gene produce KMT2A fusion proteins such as KMT2A-AFF1 (previously MLL-AF4), causing poor prognosis acute lymphoblastic leukemias (ALLs) that sometimes relapse as acute myeloid leukemias (AMLs). KMT2A-AFF1 drives leukemogenesis through direct binding and inducing the aberrant overexpression of key genes, such as the anti-apoptotic factor BCL2 and the proto-oncogene MYC However, studying direct binding alone does not incorporate possible network-generated regulatory outputs, including the indirect induction of gene repression. To better understand the KMT2A-AFF1-driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq, and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors such as RUNX1 that regulate target genes downstream of KMT2A-AFF1 using feed-forward loop (FFL) and cascade motifs. A core set of nodes are present in both ALL and AML, and CRISPR screening revealed several factors that help mediate response to the drug venetoclax. Using our GRN, we then identified a KMT2A-AFF1:RUNX1 cascade that represses CASP9, as well as KMT2A-AFF1-driven FFLs that regulate BCL2 and MYC through combinatorial TF activity. This illustrates how our GRN can be used to better connect KMT2A-AFF1 behavior to downstream pathways that contribute to leukemogenesis, and potentially predict shifts in gene expression that mediate drug response.

Ground-Based Mobile Measurements to Track Urban Methane Emissions from Natural Gas in 12 Cities across Eight Countries.

To mitigate methane emission from urban natural gas distribution systems, it is crucial to understand local leak rates and occurrence rates. To explore urban methane emissions in cities outside the U.S., where significant emissions were found previously, mobile measurements were performed in 12 cities across eight countries. The surveyed cities range from medium size, like Groningen, NL, to large size, like Toronto, CA, and London, UK. Furthermore, this survey spanned across European regions from Barcelona, ES, to Bucharest, RO. The joint analysis of all data allows us to focus on general emission behavior for cities with different infrastructure and environmental conditions. We find that all cities have a spectrum of small, medium, and large methane sources in their domain. The emission rates found follow a heavy-tailed distribution, and the top 10% of emitters account for 60-80% of total emissions, which implies that strategic repair planning could help reduce emissions quickly. Furthermore, we compare our findings with inventory estimates for urban natural gas-related methane emissions from this sector in Europe. While cities with larger reported emissions were found to generally also have larger observed emissions, we find clear discrepancies between observation-based and inventory-based emission estimates for our 12 cities.

Successful Control of an Onboard COVID-19 Outbreak Using the Cruise Ship as a Quarantine Facility, Western Australia, Australia.

Onboard quarantining has been only partially effective to control outbreaks of coronavirus disease on cruise ships. We describe the successful use of the ship as a quarantine facility during the response to the outbreak on the MS Artania, which docked in Western Australia, Australia. The health-led 14-day quarantine regime was based on established principles of outbreak management and experiences of coronavirus disease outbreaks on cruise ships elsewhere. The attack rate in the crew was 3.3% (28/832) before quarantine commencement and 4.8% (21/441) during quarantine on board. No crew members became symptomatic after completion of quarantine. Infection surveillance involved telephone correspondence, face-to-face visits, and testing for severe acute respiratory syndrome coronavirus 2. No serious health issues were reported, no response staff became infected, and only 1 quarantine breach occurred among crew. Onboard quarantine could offer financial and operational advantages in outbreak response and provide reassurance to the shore-based wider community regarding risk for infection.

A Gaussian Process Model of Human Electrocorticographic Data.

We present a model-based method for inferring full-brain neural activity at millimeter-scale spatial resolutions and millisecond-scale temporal resolutions using standard human intracranial recordings. Our approach makes the simplifying assumptions that different people's brains exhibit similar correlational structure, and that activity and correlation patterns vary smoothly over space. One can then ask, for an arbitrary individual's brain: given recordings from a limited set of locations in that individual's brain, along with the observed spatial correlations learned from other people's recordings, how much can be inferred about ongoing activity at other locations throughout that individual's brain? We show that our approach generalizes across people and tasks, thereby providing a person- and task-general means of inferring high spatiotemporal resolution full-brain neural dynamics from standard low-density intracranial recordings.

Deubiquitinase Usp8 regulates α-synuclein clearance and modifies its toxicity in Lewy body disease.

In Parkinson's disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. An important outstanding question is whether ubiquitination in Lewy bodies is directly relevant to α-synuclein trafficking or turnover and Parkinson's pathogenesis. By comparative analysis in human postmortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared with the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly colocalized with α-synuclein in endosomal membranes and, both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells, Usp8 knockdown increased the lysosomal degradation of α-synuclein. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss. These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism that reduces its lysosomal degradation in dopaminergic neurons and may contribute to α-synuclein accumulation in Lewy body disease.

BONE QUALITY IN A YOUNG COHORT OF HIV-POSITIVE PATIENTS.

CONTEXT: In HIV+ patients, several factors related to patient and antiretroviral therapy (ART) could determine early onset of bone mineral density (BMD) disturbances. OBJECTIVE: Evaluation of bone quality according to gender in patients from the HIV Romanian cohort. DESIGN: A cross-sectional study in "Prof. Dr. Matei Bals" National Institute for Infectious Diseases, Bucharest between 2016-2018. SUBJECT AND METHODS: We collected data regarding HIV infection, ART history, viral hepatitis co-infections and we calculated patients body mass index (BMI). CD4 cell count, HIV viral load (VL), vitamin-D levels were determined. Dual-energy X-ray absorptiometry (DXA) scans were used to evaluate BMD. RESULTS: We enrolled 97 patients with the median age of 26 years. According to the DXA T-scores, 10 males and 8 females had osteopenia and 4 males and 4 females had osteoporosis. According to Z-scores 2 males and 1 female had osteoporosis. Hip DXA T-scores revealed osteopenia in 6 males and 9 females, whereas T and Z-scores showed osteoporosis in 2 males and 3 females. Lumbar spine (LS) T-score diagnosed osteopenia in 9 males and 6 females, while T and Z-scores revealed osteoporosis in 3 males and females. In males, low T-scores were associated with decreased BMI; low LS DXA Z-scores with low vitamin-D levels; low T and Z-scores and LS-BMD with high VL. CONCLUSIONS: Evaluating bone quality in patients with a long history of HIV infection, multiple factors should be taken into account.

The Drosophila tricellular junction protein Gliotactin regulates its own mRNA levels through BMP-mediated induction of miR-184.

Epithelial bicellular and tricellular junctions are essential for establishing and maintaining permeability barriers. Tricellular junctions are formed by the convergence of three bicellular junctions at the corners of neighbouring epithelia. Gliotactin, a member of the Neuroligin family, is located at theDrosophilatricellular junction, and is crucial for the formation of tricellular and septate junctions, as well as permeability barrier function. Gliotactin protein levels are tightly controlled by phosphorylation at tyrosine residues and endocytosis. Blocking endocytosis or overexpressing Gliotactin results in the spread of Gliotactin from the tricellular junction, resulting in apoptosis, delamination and migration of epithelial cells. We show that Gliotactin levels are also regulated at the mRNA level by micro (mi)RNA-mediated degradation and that miRNAs are targeted to a short region in the 3'UTR that includes a conserved miR-184 target site. miR-184 also targets a suite of septate junction proteins, including NrxIV, coracle and Mcr. miR-184 expression is triggered when Gliotactin is overexpressed, leading to activation of the BMP signalling pathway. Gliotactin specifically interferes with Dad, an inhibitory SMAD, leading to activation of the Tkv type-I receptor and activation of Mad to elevate the biogenesis and expression of miR-184.

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Understanding neuronal connectivity through the post-transcriptional toolkit.

Post-transcriptional regulatory mechanisms have emerged as a critical component underlying the diversification and spatiotemporal control of the proteome during the establishment of precise neuronal connectivity. These mechanisms have been shown to be important for virtually all stages of assembling a neural network, from neurite guidance, branching, and growth to synapse morphogenesis and function. From the moment a gene is transcribed, it undergoes a series of post-transcriptional regulatory modifications in the nucleus and cytoplasm until its final deployment as a functional protein. Initially, a message is subjected to extensive structural regulation through alternative splicing, which is capable of greatly expanding the protein repertoire by generating, in some cases, thousands of functionally distinct isoforms from a single gene locus. Then, RNA packaging into neuronal transport granules and recognition by RNA-binding proteins and/or microRNAs is capable of restricting protein synthesis to selective locations and under specific input conditions. This ability of the post-transcriptional apparatus to expand the informational content of a cell and control the deployment of proteins in both spatial and temporal dimensions is a feature well adapted for the extreme morphological properties of neural cells. In this review, we describe recent advances in understanding how post-transcriptional regulatory mechanisms refine the proteomic complexity required for the assembly of intricate and specific neural networks.

A genome-wide transgenic resource for conditional expression of Drosophila microRNAs.

microRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.

Factors Associated with Discussion of Disasters by Final Year High School Students: An International Cross-sectional Survey.

UNLABELLED: Introduction The effect on behavioral change of educational programs developed to reduce the community's disaster informational vulnerability is not known. This study describes the relationship of disaster education, age, sex, and country-specific characteristics with students discussing disasters with friends and family, a measure of proactive behavioral change in disaster preparedness. METHODS: Three thousand eight hundred twenty-nine final year high school students were enrolled in an international, multi-center prospective, cross-sectional study using a pre-validated written questionnaire. In order to obtain information from different educational systems, from countries with different risk of exposure to disasters, and from countries with varied economic development status, students from Bahrain, Croatia, Cyprus, Egypt, Greece, Italy, Portugal, Romania, and Timor-Leste were surveyed. Logistic regression analyses examined the relationship between the likelihood of discussing disasters with friends and family (dependent variable) and a series of independent variables (age, gender, participation in school lessons about disasters, existence of a national disaster educational program, ability to list pertinent example of disasters, country's economic group, and disaster risk index) captured by the questionnaire or available as published data. RESULTS: There was no statistically significant relationship between age, awareness of one's surroundings, planning for the future, and foreseeing consequences of events with discussions about potential hazards and risks with friends and/or family. The national educational budget did not have a statistically significant influence. Participants who lived in a low disaster risk and high income Organization for Economic Co-operation and Development (OECD) country were more likely to discuss disasters. While either school lessons or a national disaster education program had a unique, significant contribution to the model, neither had a better predictive utility. CONCLUSIONS: The predictors (national disaster program, school lessons, gender, ability to list examples of disasters, country's disaster risk index, and level of economic development), although significant, were not sufficient in predicting disaster discussions amongst teenagers.

Abnormal bundling and accumulation of F-actin mediates tau-induced neuronal degeneration in vivo.

Hyperphosphorylated forms of the microtubule-associated protein (MAP) tau accumulate in Alzheimer's disease and related tauopathies and are thought to have an important role in neurodegeneration. However, the mechanisms through which phosphorylated tau induces neurodegeneration have remained elusive. Here, we show that tau-induced neurodegeneration is associated with accumulation of filamentous actin (F-actin) and the formation of actin-rich rods in Drosophila and mouse models of tauopathy. Importantly, modulating F-actin levels genetically leads to dramatic modification of tau-induced neurodegeneration. The ability of tau to interact with F-actin in vivo and in vitro provides a molecular mechanism for the observed phenotypes. Finally, we show that the Alzheimer's disease-linked human beta-amyloid protein (Abeta) synergistically enhances the ability of wild-type tau to promote alterations in the actin cytoskeleton and neurodegeneration. These findings raise the possibility that a direct interaction between tau and actin may be a critical mediator of tau-induced neurotoxicity in Alzheimer's disease and related disorders.

Lipodystrophy syndrome in HIV treatment-multiexperienced patients: implication of resistin.

BACKGROUND: Impaired production of adipocytokines is a major factor incriminated in the occurrence of lipodystrophy (LD). OBJECTIVE: To evaluate LD prevalence and subtypes in HIV treatment-multiexperienced patients, and to determine the correlations between adipocytokines and LD subtypes. METHODS: Cross-sectional study in a Romanian tertiary care hospital, between 2008 and 2010, in HIV-positive patients, undergoing cART for ≥6 months. LD diagnosis, based on clinical and anthropometric data, was classified into lipoatrophy (LA), lipohypertrophy (LH) and mixed fat redistribution (MFR). Blood samples were collected for leptin, adiponectin and resistin assessments. RESULTS: We included 100 patients, 44 % with LD, among which LA had 63 %. LA patients had sex ratio, median age, treatment duration and median number of ARV regimens of 1, 20, 93 and 3.5 compared to non-LD patients: 1.65, 31, 44 and 1. LH and MFR patients were older and had higher total and LDL cholesterol versus non-LD patients. For both overall group and female group, LA was associated in univariate and multivariate analysis with increased resistin (p = 0.02 and 0.04) and number of ARV regimens (p < 0.001). Determination coefficient (Nagelkerke R (2)) of increased resistin and the number of ARV combinations in the presence of LA was 33 % in overall group and 47 % in female patients. CONCLUSIONS: In our young HIV-positive population, LD had high prevalence with predominance of LA subtype. LA was associated with high resistin levels and greater number of ARV regimens in overall group and female subgroup. Resistin could be used as a marker of peripheral adipose tissue loss and might be used as a target for new anti-LD therapeutic strategies.

Does disaster education of teenagers translate into better survival knowledge, knowledge of skills, and adaptive behavioral change? A systematic literature review.

An increasing number of people are affected worldwide by the effects of disasters, and the United Nations International Strategy for Disaster Reduction (UNISDR) has recognized the need for a radical paradigm shift in the preparedness and combat of the effects of disasters through the implementation of specific actions. At the governmental level, these actions translate into disaster and risk reduction education and activities at school. Fifteen years after the UNISDR declaration, there is a need to know if the current methods of disaster education of the teenage population enhance their knowledge, knowledge of skills in disasters, and whether there is a behavioral change which would improve their chances for survival post disaster. This multidisciplinary systematic literature review showed that the published evidence regarding enhancing the disaster-related knowledge of teenagers and the related problem solving skills and behavior is piecemeal in design, approach, and execution in spite of consensus on the detrimental effects on injury rates and survival. There is some evidence that isolated school-based intervention enhances the theoretical disaster knowledge which may also extend to practical skills; however, disaster behavioral change is not forthcoming. It seems that the best results are obtained by combining theoretical and practical activities in school, family, community, and self-education programs. There is a still a pressing need for a concerted educational drive to achieve disaster preparedness behavioral change. School leavers' lack of knowledge, knowledge of skills, and adaptive behavioral change are detrimental to their chances of survival.

Water and power reserve capacity of health facilities in the Greek islands.

INTRODUCTION: Geographically isolated islands are vulnerable during natural or technological disasters. During disasters, island health facilities should be able to secure power and water in order to continue operations. OBJECTIVE: This study sought to determine the existence of Greek island health facility backup systems for water and power. When such systems existed, reserve capacity was quantified and compared to the Pan American Health Organization (PAHO) Hospital Safety Index standards. METHODS: A standardized, self-administered questionnaire was sent to major health care facilities belonging to the national health system in all Greek islands. The biggest facility available in each island was included (hospital, health center, or health post). For Crete and Euboea, all hospitals were included. RESULTS: Fifty-four of 85 facilities queried (27 hospitals, 17 health centers and 41 health posts) responded, for a response rate of 64%. Responding to the survey were 16 hospitals, 12 health centers and 26 health posts. In 70% of responding facilities (all 16 hospitals, 10 health centers, and 12 health posts) a backup water tank was available, while 72% (all 16 hospitals, 11 health centers, and 12 health posts) had a backup power supply system. Twenty-seven facilities provided data on water reserve, with 15 (56%) reporting a reserve for three or more days. Twenty facilities provided data on fuel stock and power consumption; six (30%) had energy reserves for more than 72 hours, and eight (40%) had reserves for 24-72 hours. CONCLUSIONS: Greek state-supported island health facilities responding to the questionnaire had water and power reserves for use in an emergency. Health centers and health posts were less prepared than hospitals. Of the responding health facilities, half had a water backup system and approximately one-third had power backup systems with reserves that would last for at least 72 hours.

Semmaphorin 3 A causes immune suppression by inducing cytoskeletal paralysis in tumour-specific CD8+ T cells.

Semaphorin-3A (SEMA3A) functions as a chemorepulsive signal during development and can affect T cells by altering their filamentous actin (F-actin) cytoskeleton. The exact extent of these effects on tumour-specific T cells are not completely understood. Here we demonstrate that Neuropilin-1 (NRP1) and Plexin-A1 and Plexin-A4 are upregulated on stimulated CD8+ T cells, allowing tumour-derived SEMA3A to inhibit T cell migration and assembly of the immunological synapse. Deletion of NRP1 in both CD4+ and CD8+ T cells enhance CD8+ T-cell infiltration into tumours and restricted tumour growth in animal models. Conversely, over-expression of SEMA3A inhibit CD8+ T-cell infiltration. We further show that SEMA3A affects CD8+ T cell F-actin, leading to inhibition of immune synapse formation and motility. Examining a clear cell renal cell carcinoma patient cohort, we find that SEMA3A expression is associated with reduced survival, and that T-cells appear trapped in SEMA3A rich regions. Our study establishes SEMA3A as an inhibitor of effector CD8+ T cell tumour infiltration, suggesting that blocking NRP1 could improve T cell function in tumours.

Two distinct modes of guidance signalling during collective migration of border cells.

Although directed migration is a feature of both individual cells and cell groups, guided migration has been studied most extensively for single cells in simple environments. Collective guidance of cell groups remains poorly understood, despite its relevance for development and metastasis. Neural crest cells and neuronal precursors migrate as loosely organized streams of individual cells, whereas cells of the fish lateral line, Drosophila tracheal tubes and border-cell clusters migrate as more coherent groups. Here we use Drosophila border cells to examine how collective guidance is performed. We report that border cells migrate in two phases using distinct mechanisms. Genetic analysis combined with live imaging shows that polarized cell behaviour is critical for the initial phase of migration, whereas dynamic collective behaviour dominates later. PDGF- and VEGF-related receptor and epidermal growth factor receptor act in both phases, but use different effector pathways in each. The myoblast city (Mbc, also known as DOCK180) and engulfment and cell motility (ELMO, also known as Ced-12) pathway is required for the early phase, in which guidance depends on subcellular localization of signalling within a leading cell. During the later phase, mitogen-activated protein kinase and phospholipase Cgamma are used redundantly, and we find that the cluster makes use of the difference in signal levels between cells to guide migration. Thus, information processing at the multicellular level is used to guide collective behaviour of a cell group.

Lysosomal dysfunction promotes cleavage and neurotoxicity of tau in vivo.

Expansion of the lysosomal system, including cathepsin D upregulation, is an early and prominent finding in Alzheimer's disease brain. Cell culture studies, however, have provided differing perspectives on the lysosomal connection to Alzheimer's disease, including both protective and detrimental influences. We sought to clarify and molecularly define the connection in vivo in a genetically tractable model organism. Cathepsin D is upregulated with age in a Drosophila model of Alzheimer's disease and related tauopathies. Genetic analysis reveals that cathepsin D plays a neuroprotective role because genetic ablation of cathepsin D markedly potentiates tau-induced neurotoxicity. Further, generation of a C-terminally truncated form of tau found in Alzheimer's disease patients is significantly increased in the absence of cathepsin D. We show that truncated tau has markedly increased neurotoxicity, while solubility of truncated tau is decreased. Importantly, the toxicity of truncated tau is not affected by removal of cathepsin D, providing genetic evidence that modulation of neurotoxicity by cathepsin D is mediated through C-terminal cleavage of tau. We demonstrate that removing cathepsin D in adult postmitotic neurons leads to aberrant lysosomal expansion and caspase activation in vivo, suggesting a mechanism for C-terminal truncation of tau. We also demonstrate that both cathepsin D knockout mice and cathepsin D-deficient sheep show abnormal C-terminal truncation of tau and accompanying caspase activation. Thus, caspase cleavage of tau may be a molecular mechanism through which lysosomal dysfunction and neurodegeneration are causally linked in Alzheimer's disease.

Ileo-ceco-descendento-colic intussusception in adult - a case report.

INTRODUCTION: Intestinal intussusception in adult represents a rare cause of intestinal obstruction, therefore the diagnostic and therapeutic methods must be adapted to each case. MATERIALS AND METHODS: We present the case of a 30 year old female admitted in our service suffering from a subocclusive symptomatology. After preoperative tests were completed in another medical unit, we presumed the possibility of intestinal intussusception. The intraoperative exploration revealed the presence of a terminal ileum tumor (4x5x5 cm) with the invaginated segment progressed on ileo-ceco-transversodescendento-colic trajectory. After partial desinvagination, we performed right hemicolectomy with ileo-transverse end-to-endanastomosis. RESULTS: The patient was discharged healthy on the 7th postoperative day. The histopathological findings revealed submucosal ileal lipoma. CONCLUSIONS: Tumors of the terminal ileum, cecum or right colon could have an important role in the etiology of mechanical intestinal obstructions. These tumors can be a starting point for intestinal intussusception, which can advance to the left colon.

Interrogation of Functional miRNA-Target Interactions by CRISPR/Cas9 Genome Engineering.

Posttranscriptional silencing by microRNAs (miRNAs) is a critical constituent of eukaryotic gene regulation. miRNAs are short (~22 nt) noncoding RNAs capable of specifically targeting the miRNA-induced silencing complex (miRISC) to transcripts bearing a complementary miRNA response element (MRE). Although recent methodological advances have greatly improved our understanding of miRNA biogenesis and the mechanisms by which miRNAs repress their cognate targets, exploring the physiological relevance of direct miRNA-target interactions in vivo has remained an outstanding challenge. Here we describe the experimental protocol underlying a novel approach, which allows direct in situ interrogation of specific miRNA-MRE interactions by CRISPR/Cas9-mediated genome engineering (Bassett G et al., Nat Commun 5, 4640, 2014). In this instance, the CRISPR/Cas9 system is first used to catalyze homology-directed replacement of candidate MREs with molecular barcodes at endogenous loci. Subsequently, the effect of MRE mutation on transcript abundance (i.e., MRE activity) can be rapidly evaluated by routine quantitative PCR. This strategy enables functional investigation of a putative miRNA-target pair in a pool of transiently transfected cells, obviating the need for generation of clonal cell lines or transgenic animals. This protocol can be implemented in any cell line in less than 2 weeks and can readily be scaled up for multiplex studies. To facilitate the conceptual workflow underlying this strategy, we also describe a genome-wide resource for automated design and computational evaluation of CRISPR/Cas9 guide RNAs targeting all predicted MREs in various species (miR-CRISPR).