Preclinical evaluation of ADVM-062, a novel intravitreal gene therapy vector for the treatment of blue cone monochromacy.

Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 1010 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM.

Seroprevalence and Risk Factors Possibly Associated with Emerging Zoonotic Vaccinia Virus in a Farming Community, Colombia.

In 2014, vaccinia virus (VACV) infections were identified among farmworkers in Caquetá Department, Colombia; additional cases were identified in Cundinamarca Department in 2015. VACV, an orthopoxvirus (OPXV) used in the smallpox vaccine, has caused sporadic bovine and human outbreaks in countries such as Brazil and India. In response to the emergence of this disease in Colombia, we surveyed and collected blood from 134 farmworkers and household members from 56 farms in Cundinamarca Department. We tested serum samples for OPXV antibodies and correlated risk factors with seropositivity by using multivariate analyses. Fifty-two percent of farmworkers had OPXV antibodies; this percentage decreased to 31% when we excluded persons who would have been eligible for smallpox vaccination. The major risk factors for seropositivity were municipality, age, smallpox vaccination scar, duration of time working on a farm, and animals having vaccinia-like lesions. This investigation provides evidence for possible emergence of VACV as a zoonosis in South America.

A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases.

RATIONALE: Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome. OBJECTIVE: The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro. METHODS AND RESULTS: By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development. CONCLUSIONS: Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

SCF(Fbxw7/hCdc4) targets cyclin E2 for ubiquitin-dependent proteolysis.

E-type cyclins (E1 and E2) regulate the S phase program in the mammalian cell division cycle. Expression of cyclin E1 and E2 is frequently deregulated in a variety of cancer types and a wealth of experimental evidence supports an oncogenic role of these proteins in human tumorigenesis. Although the molecular mechanisms responsible for cyclin E1 deregulation in cancer are well defined, little is known regarding cyclin E2. Here we report that cyclin E2 is targeted for ubiquitin-dependent proteolysis by the ubiquitin ligase SCF(Fbxw7/hCdc4). Ubiquitylation is triggered by phosphorylation of cyclin E2 on residues Thr392 and Ser396, and to a lesser extent Thr74, contained in two consensus Cdc4-phosphodegrons. Furthermore, we found that ectopic expression of cyclin E1 enhances the ubiquitin-dependent proteolysis of cyclin E2 in vivo, suggesting a potential cross-talk in the regulation of E-type cyclin activity. Since SCF(Fbxw7/hCdc4) is functionally inactivated in several human cancer types, alteration of this molecular pathway could contribute to the deregulation of cyclin E2 in tumorigenesis.

The RBCC gene RFP2 (Leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD.

RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.

FBXW7/hCDC4 is a general tumor suppressor in human cancer.

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of approximately 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.

Factores de riesgo de leptospirosis y sus métodos diagnósticos / Risk Factors for Leptospirosis and Its Diagnostic Methods / Fatores de risco da leptospirose e seus métodos de diagnóstico

Resumen: la leptospirosis es una enfermedad transmitida de animales a humanos, provocada por especies patógenas de Leptospira spp. Este microorganismo sobrevive en el ambiente varias semanas tanto en agua como en el suelo. Además de esto, los reservorios principales son los roedores, caninos, porcinos y bovinos. Los brotes son frecuentes en países tropicales y subtropicales. El objetivo inicial es identificar los factores de riesgo de leptospirosis y los métodos diagnósticos por medio de una revisión narrativa. Se realizó una búsqueda de la literatura de artículos en español y en las bases de datos Google Scholar, Lilacs, Redalyc, Medigraphic, Scielo, ProQuest, Publimed y ScienceDirect, entre 2016 y 2021, haciendo uso de palabras clave como leptospirosis, factores de riesgo, diagnóstico, epidemiología y zoonosis. Teniendo en cuenta la revisión narrativa de la literatura en los 50 artículos consultados se puede determinar que los trabajadores con mayor predisposición a contagio de leptospirosis son los agricultores, trabajadores de mataderos, piscícolas, recolectores de basura, veterinarios auxiliares, ordeñadores de granja, industria pecuaria. Dentro los factores determinantes se encontró el uso inapropiado de elementos de protección personal (EPP), desconocimiento de la enfermedad, consumo de aguas contaminadas, comer en plantas de sacrificio, entre otros. Una de las conclusiones es que se identificó la similitud en los diferentes artículos consultados en cuanto al contagio, que se presenta más en hombres que en mujeres debido a las diferentes prácticas que realizan, también se evidenció que la zona rural tiene poca atención médica y representa un alto índice de transmisión de leptospirosis.

Abstract: Leptospirosis is a disease transmitted from animals to humans, caused by pathogenic species of Leptospira spp. This microorganism can survive in the environment for several weeks in both water and soil. The main reservoirs include rodents, dogs, pigs, and cattle. Outbreaks are common in tropical and subtropical countries. The initial objective is to identify the risk factors for leptospirosis and the diagnostic methods through a narrative review. A literature search was conducted for articles in Spanish and English, using databases such as Google Scholar, Lilacs, Redalyc, Medigraphic, Scielo, ProQuest, Publimed, and ScienceDirect, from 2016 to 2021. Keywords used included leptospirosis, risk factors, diagnosis, epidemiology, and zoonosis. Based on the narrative review of 50 consulted articles, it was determined that workers most predisposed to leptospirosis are farmers, slaughterhouse workers, fishery workers, garbage collectors, veterinary assistants, farm milkers, and livestock industry workers. Determining factors included inappropriate use of personal protective equipment (PPE), lack of knowledge about the disease, consumption of contaminated water, and eating in slaughterhouses, among others. One of the conclusions drawn is the consistency among the different articles consulted regarding infection, which occurs more frequently in men than in women due to their various practices. It was also evident that rural areas have limited medical attention, leading to a high transmission rate of leptospirosis.

Resumo: a leptospirose é uma doença transmitida de animais para humanos, causada por espécies patogênicas de Leptospira spp. Esse microrganismo sobrevive no ambiente por várias semanas, tanto na água quanto no solo. Além disso, os principais reservatórios incluem roedores, cães, suínos e bovinos. Surtos de leptospirose são comuns em países tropicais e subtropicais. O objetivo inicial deste estudo é identificar os fatores de risco associados à leptospirose e os métodos de diagnóstico por meio de uma revisão narrativa. Realizou-se uma busca na literatura em espanhol e inglês, bem como em várias bases de dados, como o Google Scholar, Lilacs, Redalyc, Medigraphic, Scielo, ProQuest, Publimed e ScienceDirect, no período de 2016 a 2021, utilizando palavras-chave como leptospirose, fatores de risco, diagnóstico, epidemiologia e zoonoses. Considerando a revisão narrativa da literatura em 50 artigos consultados, foi possível determinar que os trabalhadores com maior predisposição à leptospirose são aqueles envolvidos na agricultura, trabalhadores de matadouros, piscicultura, coletores de lixo, veterinários auxiliares, ordenhadores de fazendas e trabalhadores da indústria pecuária. Foram encontrados fatores determinantes como uso inadequado de equipamentos de proteção pessoal (EPP), falta de conhecimento sobre a doença, consumo de água contaminada, consumo de alimentos em locais de abate, entre outros. Uma das conclusões é que houve semelhança nos diferentes artigos consultados em relação à taxa de infecção, que é mais comum em homens do que em mulheres devido às diferentes práticas que realizam. Além disso, foi evidenciado que as áreas rurais têm acesso limitado à assistência médica e representam um alto índice de transmissão de leptospirose.

The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming.

Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

CDK-mediated activation of the SCF(FBXO) (28) ubiquitin ligase promotes MYC-driven transcription and tumourigenesis and predicts poor survival in breast cancer.

SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCF(FBXO28) activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCF(FBXO28) plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.

Both SCF(Cdc4alpha) and SCF(Cdc4gamma) are required for cyclin E turnover in cell lines that do not overexpress cyclin E.

The ubiquitin-mediated turnover of cyclin E is regulated by phosphorylation and the activity of the ubiquitin ligase SCF(Cdc4) (also known as SCF(Fbw7)). In 293A cells, SCF complexes containing two different Cdc4 isoforms, alpha and gamma, are required for efficient cyclin E ubiquitylation. Whereas SCF(Cdc4gamma) ubiquitylates cyclin E directly, SCF(Cdc4alpha) serves as a cofactor for Pin1-mediated prolyl isomerization of the cyclin E phosphodegron, essential to potentiate ubiquitylation. In the current study, we show that the requirement for both Cdc4alpha and gamma is general, except in cell lines where cyclin E is expressed at an elevated level. Under these circumstances, Cdc4alpha is sufficient for cyclin E turnover. Furthermore, the requirement for Cdc4gamma can be bypassed by ectopic overexpression of cyclin E.