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Abstract Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.

We describe a surgical technique to repair medial meniscus posterior root tears through a transtibial pullout repair with a subcortical button for tibial fixation. This technique allows progressive tensioning of the repaired root without losing tension both during suturing of the knots above the button and after the procedure, owing to the specific button configuration.

A rare congenital malformation of the respiratory tract, bronchopulmonary sequestration generally presents in childhood and adolescence with recurrent pneumonia or in adulthood as an incidental finding on thoracic imaging. Manifesting as intrapulmonary or extrapulmonary types, bronchopulmonary sequestration characteristically receives blood supply from the systemic rather than pulmonary circulation. We present a 45-year-old male patient who received a provisional diagnosis of bronchopulmonary sequestration following an incidental finding on routine imaging. This case describes the way in which a provisional diagnosis may be made based upon imaging as well as underscoring the importance of alleviating the burden of additional imaging studies.

Background: Total knee arthroplasty (TKA) has become the gold standard for the definitive treatment of knee osteoarthritis. One crucial aspect in the implantation of a TKA is the precise orientation of the femoral and tibial components. The main purpose of the present study is to assess, in patients undergoing total knee replacement, whether the difference between the planned angulation in the distal femoral cut and the angulation obtained in the postoperative radiological control is low enough to consider the conventional bone-cutting guides reliable. Methods: A retrospective study was designed with a consecutive series of patients who had undergone primary total knee arthroplasty using conventional instrumentation over one year. The authors analysed the main variable (bone cuts) while considering different variables (age, gender, surgeon, prosthesis, laterality, constraint, body mass index and alignment) to identify different patient patterns that justify the results in the main variable. Descriptive variables were analysed using the Mann–Whitney U and Kruskal–Wallis tests. Additionally, the correlation between continuous variables was explored in accordance with the Spearman correlation. Results: A total of 340 patients with a mean age of 75 ± 9.16 years were finally included in the present study. The mean absolute error of the main variable for the femoral coronal bone cut was 1.89° (SD 1.53). For the tibial coronal bone cut, it was 1.31° (SD 2.54). These values correspond to what remains after subtracting the radiological angulation obtained in the postoperative period from the planned intraoperative angulation of the distal femoral cut. No associations were observed between the main variable (the angulation of the proximal tibial cut and distal femoral cut) and the rest of variables for either the femur or tibia. Conclusion: A discrepancy between the planned angulation and the final radiological measure on the coronal plane of the femur and tibia using conventional cutting guides has been demonstrated. The degree of deviation is low enough that it probably does not affect clinical outcomes. Therefore, the use of conventional cutting guides will continue to be an appropriate tool to perform bone cuts in knee replacement surgery.

Human pluripotent stem cells (hPSCs) and mesenchymal stromal/stem cells (hMSCs) are clinically relevant sources for cellular therapies and for modeling human development and disease. Many stem cell-based applications rely on the ability to activate several endogenous genes simultaneously to modify cell fate. However, genetic intervention of these cells remains challenging. Several catalytically dead Cas9 (dCas9) proteins fused to distinct activation domains can modulate gene expression when directed to their regulatory regions by a specific single-guide RNA (sgRNA). In this study, we have compared the ability of the first-generation dCas9-VP64 activator and the second-generation systems, dCas9-SAM and dCas9-SunTag, to induce gene expression in hPSCs and hMSCs. Several stem cell lines were tested for single and multiplexed gene activation. When the activation of several genes was compared, all three systems induced specific and potent gene expression in both single and multiplexed settings, but the dCas9-SAM and dCas9-SunTag systems resulted in the highest and most consistent level of gene expression. Simultaneous targeting of the same gene with multiple sgRNAs did not result in additive levels of gene expression in hPSCs nor hMSCs. We demonstrate the robustness and specificity of second-generation dCas9 activators as tools to simultaneously activate several endogenous genes in clinically relevant human stem cells.

Summary: Chromosomal instability (CIN), a type of genomic instability, favors changes in chromosome number and structure and it is associated with the progression and initiation of multiple diseases, including cancer. Therefore, CIN identification and analysis represents a useful tool for cancer diagnosis and treatment. Here, we report an optimized molecular cytogenetic protocol to detect CIN in formalin-fixed, paraffin-embedded mouse and human tissues, using fluorescent in situ hybridization to visualize and quantify chromosomal alterations such as amplifications, deletions, and translocations.For complete information on the generation and use of this protocol, please refer to Brandt et al. (2018).

Accurate recapitulation of human disease in animal models requires generation of complex and heterogeneous genetic variation. Here the authors combine RCAS-TVA with CRISPR-Cas9 to generate mouse models of cancer.

Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.

Background and study aims The aim of the current study was to review the outcomes of a large-scale international registry on endoscopic ultrasound-guided gallbladder drainage (EGBD) that encompasses different stent systems in patients who are at high-risk for cholecystectomy. Patients and methods This was a retrospective international multicenter registry on EGBD created by 13 institutions around the world. Consecutive patients who received EGBD for several indications were included. Outcomes include technical and clinical success, unplanned procedural events (UPE), adverse events (AEs), mortality, recurrent cholecystitis and learning curve of the procedure. Results Between June 2011 and November 2017, 379 patients were recruited to the study. Technical and clinical success were achieved in 95.3 % and 90.8 % of the patients, respectively. The 30-day AE rate was 15.3 % and 30-day mortality was 9.2 %. UPEs were significantly more common in patients with EGBD performed for conversion of cholecystostomy and symptomatic gallstones (P

The t(4;11)(q21;q23) translocation is associated with high-risk infant pro-B-cell acute lymphoblastic leukemia and arises prenatally during embryonic/fetal hematopoiesis. The developmental/pathogenic contribution of the t(4;11)-resulting MLL-AF4 (MA4) and AF4-MLL (A4M) fusions remains unclear; MA4 is always expressed in patients with t(4;11)+ B-cell acute lymphoblastic leukemia, but the reciprocal fusion A4M is expressed in only half of the patients. Because prenatal leukemogenesis manifests as impaired early hematopoietic differentiation, we took advantage of well-established human embryonic stem cell-based hematopoietic differentiation models to study whether the A4M fusion cooperates with MA4 during early human hematopoietic development. Co-expression of A4M and MA4 strongly promoted the emergence of hemato-endothelial precursors, both endothelial- and hemogenic-primed. Double fusion-expressing hemato-endothelial precursors specified into significantly higher numbers of both hematopoietic and endothelial-committed cells, irrespective of the differentiation protocol used and without hijacking survival/proliferation. Functional analysis of differentially expressed genes and differentially enriched H3K79me3 genomic regions by RNA-sequencing and H3K79me3 chromatin immunoprecipitation-sequencing, respectively, confirmed a hematopoietic/endothelial cell differentiation signature in double fusion-expressing hemato-endothelial precursors. Importantly, chromatin immunoprecipitation-sequencing analysis revealed a significant enrichment of H3K79 methylated regions specifically associated with HOX-A cluster genes in double fusion-expressing differentiating hematopoietic cells. Overall, these results establish a functional and molecular cooperation between MA4 and A4M fusions during human hematopoietic development.

[This corrects the article DOI: 10.1371/journal.pgen.1007387.].

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.

The Consortium on Asthma among African-ancestry Populations in the Americas (CAAPA) aims to better understand population genetics of the African diaspora. Here, it uses deeply sequenced whole-genomes to describe the impact of admixture and potential disease burden of deleterious variants.

Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency.

Condensin is a conserved chromosomal complex necessary to promote mitotic chromosome condensation and sister chromatid resolution during anaphase. Here, we report that yeast condensin binds to replicated centromere regions. We show that centromeric condensin relocalizes to chromosome arms as cells undergo anaphase segregation. We find that condensin relocalization is initiated immediately after the bipolar attachment of sister kinetochores to spindles and requires Polo kinase activity. Moreover, condensin localization during anaphase involves a higher binding rate on DNA and temporally overlaps with condensin’s DNA overwinding activity. Finally, we demonstrate that topoisomerase 2 (Top2) is also recruited to chromosome arms during anaphase in a condensin-dependent manner. Our results uncover a functional relation between condensin and Top2 during anaphase to mediate chromosome segregation.

The phagocytic integrins and complement receptors αMβ2/CR3 and αXβ2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of β2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

Natural populations often grow, shrink, and migrate over time. Such demographic processes can affect genome-wide levels of genetic diversity. Additionally, genetic variation in functional regions of the genome can be altered by natural selection, which drives adaptive mutations to higher frequencies or purges deleterious ones. Such selective processes affect not only the sites directly under selection but also nearby neutral variation through genetic linkage via processes referred to as genetic hitchhiking in the context of positive selection and background selection (BGS) in the context of purifying selection. While there is extensive literature examining the consequences of selection at linked sites at demographic equilibrium, less is known about how non-equilibrium demographic processes influence the effects of hitchhiking and BGS. Utilizing a global sample of human whole-genome sequences from the Thousand Genomes Project and extensive simulations, we investigate how non-equilibrium demographic processes magnify and dampen the consequences of selection at linked sites across the human genome. When binning the genome by inferred strength of BGS, we observe that, compared to Africans, non-African populations have experienced larger proportional decreases in neutral genetic diversity in strong BGS regions. We replicate these findings in admixed populations by showing that non-African ancestral components of the genome have also been affected more severely in these regions. We attribute these differences to the strong, sustained/recurrent population bottlenecks that non-Africans experienced as they migrated out of Africa and throughout the globe. Furthermore, we observe a strong correlation between FST and the inferred strength of BGS, suggesting a stronger rate of genetic drift. Forward simulations of human demographic history with a model of BGS support these observations. Our results show that non-equilibrium demography significantly alters the consequences of selection at linked sites and support the need for more work investigating the dynamic process of multiple evolutionary forces operating in concert.

Femoroacetabular impingement is uncommonly associated to a rim fracture. Complete resection of the fragment might result in iatrogenic instability or poor femoral head coverage. In this report, we describe the step-by-step surgical technique of arthroscopic partial resection of a rim fracture, reduction and internal fixation of the remaining fragment to correct the impingement, and preserve the adequate acetabular coverage.

En este trabajo se implementó una funciónde contorno de un árbol para establecermedidas de similitud molecular.El estudio se realizó con 73 moléculasorgánicas divididas en 8 grupos funcionalesoptimizadas con un nivel de teoríaDFT//B3LYP/6-31G(d,p) a las cualesse les calculó el Potencial ElectrostáticoMolecular y el Laplaciano de la DensidadElectrónica en una rejilla tridimensional.A partir de los valores de estaspropiedades, caracterizando y codificandosegún su topología, se generarongrafos (árboles) que se compararon a travésde la función propuesta. La caracterizacióny clasificación de las moléculasorgánicas con el Potencial ElectrostáticoMolecular muestra una separacióncorrespondiente a moléculas que en suestructura poseen heteroátomos con funcionesquímicas por lo menos estructuralmentesimilares, y con el Laplacianode la Densidad Electrónica se obtuvocomo resultado una clasificación acordecon el número de pares de electrones libresasociados a los heteroátomos en lasmoléculas y a la naturaleza de los átomosque los aportan. Lo anterior evidenciaque las funciones de contorno de árbolpropuestas en el estudio son una alternativarápida para clasificar a grosso modomoléculas orgánicas.

Cancer is the second leading cause of death globally and remains a major economic and social burden. Although our understanding of cancer at the molecular level continues to improve, more effort is needed to develop new therapeutic tools and approaches exploiting these advances. Because of its high efficiency and accuracy, the CRISPR-Cas9 genome editing technique has recently emerged as a potentially powerful tool in the arsenal of cancer therapy. Among its many applications, CRISPR-Cas9 has shown an unprecedented clinical potential to discover novel targets for cancer therapy and to dissect chemical-genetic interactions, providing insight into how tumours respond to drug treatment. Moreover, CRISPR-Cas9 can be employed to rapidly engineer immune cells and oncolytic viruses for cancer immunotherapeutic applications. Perhaps more importantly, the ability of CRISPR-Cas9 to accurately edit genes, not only in cell culture models and model organisms but also in humans, allows its use in therapeutic explorations. In this review, we discuss important considerations for the use of CRISPR/Cas9 in therapeutic settings and major challenges that will need to be addressed prior to its clinical translation for a complex and polygenic disease such as cancer.

Se evaluó la capacidad estacional de utilizar un evento de precipitación en tres especies arbustivas con diferentes sistemas radiculares (dimórficos: Balbisia peduncularis, Senna cumingii; profundo: Haplopappus parvifolius) en la Quebrada El Romeral, norte-centro de Chile. El sitio posee un clima tipo mediterráneo árido con influencia de neblinas costeras y una precipitación promedio anual de 78 mm en los últimos 30 años. La utilización de precipitación artificial (i.e., 25 mm en otoño, invierno y primavera) por parte de las plantas se estimó por la composición de isótopos de hidrógeno (delta²H) en el agua del xilema y los potenciales hídricos de pre-alba (psipa) antes y después del riego. Los resultados indican que las tres especies utilizan una mezcla de dos fuentes de agua (agua superficial proveniente de las precipitaciones, y en mayor proporción, agua subterránea). A excepción de invierno, sólo las especies de sistemas radiculares dimórficos son capaces de reducir su déficit hídrico después de la aplicación de la precipitación artificial. La reducción en las precipitaciones observada en los últimos 100 años afectaría diferencialmente la productividad de las especies con sistema radicular dimórfico, y en especial al arbusto forrajero Balbisia peduncularis.Seasonal ability to use precipitation by three shrub species with different root systems (dimorphic: Balbisia peduncularis and Senna cummingii; deep: Haplopappus parvifolius) was evaluated in Quebrada El Romeral, north-central Chile. The site has an arid Mediterranean climate with coastal fog influence and an annual mean precipitation of 78 mm during the last 30 years. The use of artificial rain (i.e., 25 mm in fall, winter and spring) by plants was estimated by the hydrogen isotope composition (delta²H) in the xylem water and the pre-dawn water potentials (psipa) before and after watering. The results show that the three species use a mixture of two water sources (shallow water coming from rainfall and a greater proportion of subterranean water). Except in winter, the species with dimorphic roots are able to reduce their water deficits after the artificial watering. The decrease in rainfall observed for the last 100 years would affect the productivity of the species with dimorphic root systems, especially the forage shrub Balbisia peduncularis.

Recombinase mediated cassette exchange (RMCE) is a two-step process leading to genetic modification in a specific genomic target sequence. The process involves insertion of a docking genetic cassette in the genome followed by DNA transfer of a second cassette flanked by compatible recombination signals and expression of the recombinase. Major technical drawbacks are cell viability upon transfection, toxicity of the enzyme, and the ability to target efficiently cell types of different origins. To overcome such drawbacks, we developed an RMCE assay that uses an integrase-deficient lentivirus (IDLV) vector in the second step combined with promoterless trapping of double selectable markers. Additionally, recombinase expression is self-limiting as a result of the exchangeable reaction, thus avoiding toxicity. Our approach provides proof-of-principle of a simple and novel strategy with expected wide applicability modelled on a human cell line with randomly integrated copies of a genetic landing pad. This strategy does not present foreseeable limitations for application to other cell systems modified by homologous recombination. Safety, efficiency, and simplicity are the major advantages of our system, which can be applied in low-to-medium throughput strategies for screening of cDNAs, non-coding RNAs during functional genomic studies, and drug screening.

Chemokines are extracellular mediators of complex regulatory circuits involved principally in cell-to-cell communication. Most studies to date of the essential chemokine Cxcl12 (Sdf-1) have focused on the ubiquitously expressed secreted isoforms alpha and beta. Here we show that, unlike these isoforms and all other known chemokines, the alternatively transcribed gamma isoform is an intracellular protein that localizes to the nucleolus in differentiated mouse Cardiac tissue. Our results demonstrate that nucleolar transportation is encoded by a nucleolar-localization signal in the unique carboxy-terminal region of Sdf-1gamma, and is competent both in vivo and in vitro. The molecular mechanism underlying these unusual chemokine properties involves cardiac-specific transcription of an mRNA containing a unique short-leader sequence lacking the signal peptide and translation from a non-canonical CUG codon. Our results provide an example of genome economy even for essential and highly conserved genes such as Cxcl12, and suggest that chemokines can exert tissue specific functions unrelated to cell-to-cell communication.