Dissecting RNA biology in hematopoietic stem cells: The long non-coding RNA Meg3 is dispensable for hematopoiesis and alternative polyadenylation orchestrates hematopoietic stem cell function

Hematopoietic stem cells (HSCs) reside at the top of a tightly regulated and hierarchically organized differentiation cascade. They are multipotent and able to self-renew, thereby ensuring life-long replenishment of mature blood cells. In-depth Omics analyses within the hematopoietic hierarchy, including analysis of HSCs and multipotent progenitor (MPP) cells, revealed not only differential expression of protein-coding genes, but also HSC-specific splicing variants and expression of long non-coding RNAs (lncRNAs) upon HSC commitment (Cabezas-Wallscheid et al., 2014; Klimmeck et al., 2014; Luo et al., 2015). Functional analyses revealed that non-coding RNAs and regulation of RNA biogenesis are crucial for HSC function and potency. In this thesis, the role of the lncRNA maternally expressed gene 3 (Meg3) in hematopoiesis is studied. In addition, I introduce the RNA regulatory mechanism alternative polyadenylation (APA) as a mechanism controlling HSC/MPP biology in homeostasis and during replicative stress response by extensive in silico, in vitro and in vivo analyses. Furthermore, I generated an inducible knockout mouse model, targeting the prominent APA regulator poly(A) binding protein 1 (Pabpn1). Thereby, I aimed to dissect the general role of lncRNAs and RNA regulatory mechanisms in hematopoiesis. Project 1: The role of the lncRNA Meg3 in HSCs The tumor suppressor lncRNA Meg3 is encoded in the imprinted Dlk1-Meg3 locus and expressed from the maternally inherited allele (Zhou et al., 2012). We and others found Meg3 to be highly and specifically expressed in the HSC compartment compared to MPP cells. In this thesis, I crossed an inducible Meg3 flox mouse model (Klibanski et al., unpublished) to MxCre mice, generating MxCre Meg3 mat flox/pat wt mice. Cre-induction deleted the maternal allele in the hematopoietic compartment and thereby completely abrogated the expression of Meg3 and its associated miRNA cluster in HSCs. Extensive in vivo and in vitro analyses of adult mice harboring a Meg3-deficient blood system surprisingly did not reveal any impairment of hematopoiesis or stem cell function. In addition, I performed serial transplantation assays to investigate the functional capacity of Meg3-deficient HSCs. Again, knockout cells did not exhibit altered blood contribution, even upon tertiary transplantation. Imprinting of the Dlk1- Meg3 locus has recently been reported to regulate fetal liver HSC function (Qian et al., 2016). To analyze effects of the hematopoiesis-specific Meg3 knockout in the developing embryo, we generated VavCre Meg3 mat flox/pat wt mice. Cre+ offspring were born and developed normally. In- depth analysis of adult animals revealed loss of Meg3 expression in HSCs, but again no hematopoietic impairments were detected. Next, I performed interferon-mediated stimulation in MxCre Meg3 mat flox/pat wt mice. During both activation and recovery phase, Meg3-deficient adult HSCs responded highly similar compared to controls. Taken together, my work shows that the highly expressed, imprinted lncRNA Meg3 is dispensable for the function of HSCs during adulthood and embryonic development. In the adult system, loss of Meg3 does not impair the performance in serial reconstitution assays or response to stress mediators. (Sommerkamp et al., 2019) Project 2: HSC function, differentiation and activation are regulated by APA The majority of mammalian genes have multiple different polyadenylation sites. The RNA editing mechanism APA controls the selection of these sites, thereby altering 3’-UTR length and isoform expression. Thus, APA modulates RNA stability, localization, protein output and even protein localization (Di Giammartino et al., 2011; Tian and Manley, 2017). So far, the role of APA in the regulation of the adult HSC/MPP compartment has not been studied. In this thesis, I show that the APA regulator Pabpn1 is essential for HSCs, as knockdown (KD) of Pabpn1 led to decreased HSC function in vitro and in vivo. To analyze the prevalence of APA at the top of the hematopoietic hierarchy, I established an ultra-low input 3’-Seq approach and performed analysis of HSCs, MPP cells and HSCs activated by inflammation. Bioinformatic analysis revealed dynamic APA patterns in numerous genes between HSCs and MPPs as well as during inflammation-induced HSC stress response. We observed global 3’-UTR shortening both upon HSC differentiation towards MPPs and HSC activation. Further, 3’-Seq analysis of Pabpn1 KD cells revealed that PABPN1 regulates APA in the HSC/MPP compartment. We observed an APA-mediated glutaminase (Gls) isoform switch upon exit of HSCs from quiescence. Gls isoform switching led to enhanced relative expression of the highly active GLS GAC isoform and overall increased GLS protein levels. In line, small molecule-mediated inhibition of glutaminolysis in vitro enhanced HSC maintenance by limiting proliferation. I could show that Gls isoform switching leading to increased glutaminolysis is mediated by the APA regulator NUDT21 and in turn is required for proper HSC function. KD of Nudt21 led to inhibition of Gls isoform switching, impaired HSC function and a partial block in HSC differentiation. In summary, my results install differential employment of APA and associated glutamine metabolism adaptations as novel layers in the regulation of the HSC-controlled hematopoietic hierarchy. (Sommerkamp et al., under revision) Project 3: Generation of Pabpn1flox mice To enable in-depth in vivo analysis of the role of APA in different tissues and disease settings in the future, we generated an inducible Pabpn1flox mouse model. Here, we used the Easi- CRISPR approach (Miura et al., 2018; Quadros et al., 2017) to generate transgenic animals. I performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency in vivo. Two different crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, were injected into one of the pronuclei of zygotes together with a long single-stranded DNA (ssDNA) template. By extensive genotyping, I identified 3 of 17 offspring animals to be correctly targeted. Homozygous offspring mice were successfully bred and can be used in the future to determine functionality of the Pabpn1flox mouse model and to functionally asses the role of APA in different biological settings

Morphometric analysis of potential osteochondral autografts for resurfacing unicondylar defects of the proximal phalanx in PIP joint injuries.

PURPOSE: This study was designed to morphometrically assess the base of the little and ring finger metacarpals as potential osteochondral donors to resurface distal condylar defects of the proximal phalanx. METHODS: The proximal phalanges were dissected from all 4 fingers in 10 cadaveric hands and the following measurements were obtained from the distal condylar surface: anteroposterior height, radial-ulnar width, and radius of curvature. Measurements were obtained from posteroanterior and lateral radiographic views, which were digitized and analyzed using digital imaging software. Comparable measurements were obtained from the base of the small and ring metacarpals. RESULTS: The anteroposterior dimension of both potential donor metacarpals was large enough to resurface the distal condyles of each of the proximal phalanges; however, this was not true for the radial-ulnar dimensions. The distal ulnar condyle of the long finger proximal phalanx was largest, measuring 4.9 (+/- 0.) mm dorsally and 6.2 (+/- 0.5) mm volarly in the radial-ulnar dimension. Only the small metacarpal base had sufficient stock in the radial-ulnar dimension (9.4 [+/- 1.7]) mm dorsally and 10.6 [+/- 2.0] mm volarly) to resurface this condyle. With respect to radius of curvature (ROC), the donor-to-recipient ROC ratio was 1.43 for the small metacarpal base versus 2.12 for the ring metacarpal base. Linear regression analysis revealed a stronger relationship in ROC between donor and recipient condyle when the small metacarpal base served as the donor (R = 0.96 vs R = 0.60). CONCLUSIONS: As determined from morphometric measurements of the 2 potential donor sites tested, the base of the small metacarpal provides the best match for resurfacing distal condylar defects of the proximal phalanges

Detección e identificación de los hongos comestibles y tóxicos en Guatemala

Esta primera fase del proyecto consiste en profundizar en el trabajo llevado a cabo en años anteriores. Alcanzando los objetivos propuestos será posible ensamblar una estructura que permite continuar con la segunda fase del proyecto, que en sí será la identificación de todos los hongos comestibles tóxicos posibles. Los objetivos principales son: Ordenar y adecuar el material recolectado desde 1983, tanto el bibliográfico como el herborizado.Estandarizar el herbario con las normas internacionales para hacer de él un útil instrumento de referencia, hasta el momento, único en el país.Adquirir documentación relevante sobre macromicetos y preparar material suficiente para proseguir con la segunda fase del estudio.Crear las condiciones necesarias para que el Laboratorio de Micología sea tomado como un Centro de Referencia sobre macromicetos a nivel nacional y preparado para responder las consultas que se originen

El compromiso laboral y la calidad de servicio de la empresa Mongroup E.I.R.L Surco 2021

La siguiente investigación se refirió al compromiso laboral y la calidad de servicio en la empresa MONGROUP EIRL, Surco 2021, el objetivo general fue determinar el nivel de correlación que existía entre las dos variables: compromiso laboral y calidad de servicio, para ello se utilizó como instrumento de recolección de datos a la encuesta, para lo cual se elaboró un cuestionario con 22 items, que se aplicó a los colaboradores de la empresa Mongroup ETRL, los cuales fueron elegido de manera probabilística en un número de 52. La investigación fue de enfoque cuantitativo, se utilizó la escala de Likert y los resultados fueron procesados de manera numéricas en el SPSS V.25. el diseño fue no experimental, se describió el problema tal y como se presentó, sin manipular los datos recogidos. Para determinar si existía la correlación se utilizó la prueba no paramétrica de Rho de Spearman el cual arrojó un valor de 0.47 y una significación bilateral de 0.000 el cual es menor que 0.05 lo cual nos permitió validar la hipótesis alterna y rechazar la hipótesis nula. Las conclusiones del trabajo permitieron demostrar el nivel de correlación que existe entre las variables compromiso laboral y calidad de servicio, recomendando a la administración que trabaje en estrategias que permitan que los colaboradores se identifiquen con la institución porque esa manera podrá mejorar la calidad de servicio que ofrece la empresa

Fee-for-service as a business model of growing importance: the academic biobank experience

Biorepositories offer tremendous scientific value to a wide variety of customer groups (academic, commercial, industrial) in their ability to deliver a centralized, standardized service model, encompassing both biospecimen storage and related laboratory services. Generally, the scientific expertise and economies of scale that are offered in centralized, properly resourced research biobanks has yielded value that has been well-recognized by universities, pharmaceutical companies, and other sponsoring institutions. However, like many facets of the economy, biobanks have been under increasing cost pressure in recent years. This has been a particular problem in the academic arena, where direct support from grant sources (both governmental and philanthropic) typically now is more difficult to secure, or provides reduced financial support, relative to previous years. One way to address this challenge is to establish or enhance a well-defined fee-for-service model which is properly calibrated to cover operational costs while still offering competitive value to users. In this model, customers are never charged for the biospecimens themselves, but rather for the laboratory services associated with them. Good communication practices, proper assessment of value, implementation of best practices, and a sound business plan are all needed for this initiative to succeed. Here we summarize our experiences at Washington University School of Medicine in the expectation they will be useful to others

Different methods of external fixation for treating distal radial fractures in adults

BACKGROUND: Fracture of the distal radius is a common injury. A surgical treatment is external fixation, where metal pins inserted into bone on either side of the fracture are then fixed to an external frame. OBJECTIVES: To evaluate the evidence from randomised controlled trials comparing different methods of external fixation for distal radial fractures in adults. SEARCH METHODS: We searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised Register (June 2007), the Cochrane Central Register of Controlled Trials, MEDLINE, EMBASE and other databases, conference proceedings and reference lists of articles. No language restrictions were applied. SELECTION CRITERIA: Randomised or quasi‐randomised controlled clinical trials which compared different methods of external fixation in adults with a distal radial fracture. DATA COLLECTION AND ANALYSIS: All review authors independently performed study selection. Two authors independently assessed the included trials and performed data extraction. MAIN RESULTS: Nine small trials involving 510 adults with potentially or evidently unstable fractures, were grouped into five comparisons. The interventional, clinical and methodological heterogeneity of trials precluded data pooling. Only one trial had secure allocation concealment. Two trials comparing a bridging (of the wrist) external fixator versus pins and plaster external fixation found no significant differences in function or deformity. One trial found tendencies for more serious complications but less subsequent discomfort and deformity in the fixator group. Three trials compared non‐bridging versus bridging fixation. Of the two trials testing uni‐planar non‐bridging fixation, one found no significant differences in functional or clinical outcomes; the other found non‐bridging fixation significantly improved grip strength, wrist flexion and anatomical outcome. The third trial found no significant findings in favour of multi‐planar non‐bridging fixation of complex intra‐articular fractures. One trial using a bridging external fixator found that deploying an extra external fixator pin to fix the 'floating' distal fragment gave superior functional and anatomical results. One trial found no evidence of differences in clinical outcomes for hydroxyapatite coated pins compared with standard uncoated pins. Two trials compared dynamic versus static external fixation. One trial found no significant effects from early dynamism of an external fixator. The poor quality of the other trial undermines its findings of poorer functional and anatomical outcomes for dynamic fixation. AUTHORS' CONCLUSIONS: There is insufficient robust evidence to determine the relative effects of different methods of external fixation. Adequately powered studies could provide better evidence

SARS-COV-2 variants: epidemiology, pathophysiology and the importance of vaccines

El SARS-CoV-2 es un virus ARN monocatenario de la familia de los coronavirus, causante de la COVID-19 (Coronavirus Disease 2019). Este virus es responsable de la pandemia actual que, desde su aparición a finales de 2019, ha provocado la muerte de millones de personas y ha tenido un impacto global no solo a nivel sanitario sino también económico y social. Por ello, el presente artículo tiene como objetivo revisar la información más actualizada sobre el SARS-CoV-2, empezando por describir los mecanismos de transmisión del virus, su fisiopatología y filogenética. Asimismo, presentará a las variantes emergentes del SARS-CoV-2, su relevancia para la salud pública local y global, su epidemiología en Perú, y finalmente, el rol y la importancia de las vacunas en este contexto.SARS-CoV-2 is a single-stranded RNA virus of the coronavirus family, which causes COVID-19 (Coronavirus Disease 2019). This virus is responsible for the current pandemic, which, since its emergence in late 2019, has caused millions of deaths and has had a global impact not only on public health but also on social and economic areas. Therefore, this article aims to review the most up-to-date information on SARS-CoV-2, beginning with the description of the pathophysiology and phylogenetics of the virus. Also, we will present the emerging SARS-CoV-2 variants, their relevance for local and global public health, their epidemiology in Peru, and finally, the role and importance of vaccines in this context