A comparison between whole transcript and 3' RNA sequencing methods using Kapa and Lexogen library preparation methods.

Background3' RNA sequencing provides an alternative to whole transcript analysis. However, we do not know a priori the relative advantage of each method. Thus, a comprehensive comparison between the whole transcript and the 3' method is needed to determine their relative merits. To this end, we used two commercially available library preparation kits, the KAPA Stranded mRNA-Seq kit (traditional method) and the Lexogen QuantSeq 3' mRNA-Seq kit (3' method), to prepare libraries from mouse liver RNA. We then sequenced and analyzed the libraries to determine the advantages and disadvantages of these two approaches.ResultsWe found that the traditional whole transcript method and the 3' RNA-Seq method had similar levels of reproducibility. As expected, the whole transcript method assigned more reads to longer transcripts, while the 3' method assigned roughly equal numbers of reads to transcripts regardless of their lengths. We found that the 3' RNA-Seq method detected more short transcripts than the whole transcript method. With regard to differential expression analysis, we found that the whole transcript method detected more differentially expressed genes, regardless of the level of sequencing depth.ConclusionsThe 3' RNA-Seq method was better able to detect short transcripts, while the whole transcript RNA-Seq was able to detect more differentially expressed genes. Thus, both approaches have relative advantages and should be selected based on the goals of the experiment

Arquitectura vernácula e identidad cultural en asentamientos rurales en declive

La despoblació i l'abandó de les zones rurals és un fenomen que es repeteix a escala global i de manera transversal a la majoria dels territoris i cultures. Aquest procés de declivi afecta especialment a l'arquitectura vernacla, per la seva modesta materialitat i per tant els valors culturals que en ella resideixen, tant de caràcter material com immaterial. Aquest treball analitza la situació de l'arquitectura vernacla rural en zones en declivi en tres regions d'escala i plantejament cultural tan diferents com les àrees meridionals de la Xina, el Marroc i Espanya, prenent com a referència elements tipològics propis de cada regió. Aprofundeix en experiències en les quals el patrimoni cultural en perill es transforma en força dinamitzadora del territori sobre el qual s'assenta i obre la porta a solucions tant de conservació i valorització dels seus propis elements com de resolució dels problemes socioculturals de fons. La recerca identifica aspectes genèrics i universals del procés de retracció, així com condicions específiques de cadascun dels diversos territoris d'estudi. El treball va trobar com els casos presos en consideració comparteixen algunes condicions en l'enfrontament de les conseqüències de la contracció, com ara dificultats d'adaptació, distància administrativa, fragilitat patrimonial.The depopulation and abandonment of rural areas is a phenomenon that is repeated on a global scale and is transversal to most territories and cultures. This shrinking process particularly affects vernacular architecture, due to its modest materiality and therefore the cultural values that reside in it, both of a material and immaterial nature. This work analyses the situation of rural vernacular architecture in shrinking areas in three regions of different scales and cultural approaches as the southern areas of China, Morocco and Spain, taking as reference typological elements specific to each region. The research delves into experiences in which the endangered cultural heritage is transformed into a dynamizing force for the territory on which it is based and opens the door to different solutions both for the conservation and valorisation of its own elements and for the resolution of the underlying socio-cultural problems. The research identifies generic and universal aspects of the shrinking process, as well as specific conditions of each of the various study territories. The work found how the cases taken into consideration share some conditions in dealing with the consequences of the shrinkage, such as adaptation difficulties, administrative distance, heritage fragility.La despoblación y el abandono de las zonas rurales es un fenómeno que se repite a escala global y de forma transversal a la mayoría de los territorios y culturas. Este proceso de declive afecta especialmente a la arquitectura vernácula, por su modesta materialidad y por tanto los valores culturales que en ella residen, tanto de carácter material como inmaterial. Este trabajo analiza la situación de la arquitectura vernácula rural en zonas en declive en tres regiones de escala y planteamiento cultural tan distintos como las áreas meridionales de China, Marruecos y España, tomando como referencia elementos tipológicos propios de cada región. Profundiza en experiencias en las que el patrimonio cultural en peligro se transforma en fuerza dinamizadora del territorio sobre el que se asienta y abre la puerta a soluciones tanto de conservación y valorización de sus propios elementos como de resolución de los problemas socioculturales de fondo. La investigación identifica aspectos genéricos y universales del proceso de retracción, así como condiciones específicas de cada uno de los diversos territorios de estudio. El trabajo encontró cómo los casos tomados en consideración comparten algunas condiciones en el enfrentamiento de las consecuencias de la contracción, tales como dificultades de adaptación, distancia administrativa, fragilidad patrimonial.Peer Reviewe

Creating New β-Globin-Expressing Lentiviral Vectors by High-Resolution Mapping of Locus Control Region Enhancer Sequences.

Hematopoietic stem cell gene therapy is a promising approach for treating disorders of the hematopoietic system. Identifying combinations of cis-regulatory elements that do not impede packaging or transduction efficiency when included in lentiviral vectors has proven challenging. In this study, we deploy LV-MPRA (lentiviral vector-based, massively parallel reporter assay), an approach that simultaneously analyzes thousands of synthetic DNA fragments in parallel to identify sequence-intrinsic and lineage-specific enhancer function at near-base-pair resolution. We demonstrate the power of LV-MPRA in elucidating the boundaries of previously unknown intrinsic enhancer sequences of the human β-globin locus control region. Our approach facilitated the rapid assembly of novel therapeutic βAS3-globin lentiviral vectors harboring strong lineage-specific recombinant control elements capable of correcting a mouse model of sickle cell disease. LV-MPRA can be used to map any genomic locus for enhancer activity and facilitates the rapid development of therapeutic vectors for treating disorders of the hematopoietic system or other specific tissues and cell types

An autoregressive spatial stochastic frontier analysis for quantifying the sales efficiency of the electric vehicle market: An application to 88 pilot cities in China

This paper proposes the use of an autoregressive spatial stochastic frontier model to measure the sales efficiency of the electric vehicle (EV) market in 88 Chinese cities for the period 2016 to 2021. In contrast to previous research on this topic, the adoption of a stochastic frontier model allows for computing the maximum level of EV sales (i.e., frontier) that each city could have potentially achieved in the timeframe under scrutiny given a certain set of inputs (e.g., central and local purchase subsidies, subsidies for the construction/operation of electric vehicle chargers, average petrol prices, purchase restrictions on conventional vehicles, among others). Further, the spatial-based structure of the model proposed enables the assessment of the impact of similar policy interventions implemented in neighbouring cities on EV sales frontier estimated within the city. The empirical evidence suggests that as the provision of EV charging stations around and within the city increases, so does the maximum number of sellable electric cars. A further interesting finding is that the frontier for EV sales is positively influenced by the electric cars purchased in the previous month in neighbouring areas, revealing the presence of a strong spatial dependency. Finally, this study conducts a simulation exercise wherein three hypothetical scenarios are explored: 1) the implementation of a ten percent tax on petrol, 2) a ten percent increase in the number of public chargers available, and 3) the introduction of policies to improve the air quality of all 88 cities. The results from the simulation analysis suggests that introducing a 10 percent environmental tax on petrol would have resulted in the sales of around 71,000 EVs more across the 88 cities over six years

The crystal structure of Trz1, the long form RNase Z from yeast.

tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3΄ maturation of tRNAs in all domains of life. It is a member of the β-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused β-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two β-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z

Homogeneous Open Quantum Random Walks on a lattice

We study Open Quantum Random Walks for which the underlying graph is a lattice, and the generators of the walk are translation-invariant. We consider the quantum trajectory associated with the OQRW, which is described by a position process and a state process. We obtain a central limit theorem and a large deviation principle for the position process, and an ergodic result for the state process. We study in detail the case of homogeneous OQRWs on a lattice, with internal space $h={\mathbb C}^2$

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background: Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker. Methodology/Principal Findings: K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker. Conclusions/Significance: K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo

MANP Activation Of The cGMP Inhibits Aldosterone Via PDE2 And CYP11B2 In H295R Cells And In Mice

Background: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear. Methods: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice. Results: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased. Conclusions: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment

Barriers and facilitators of the uptake of digital health technology in cardiovascular care: a systematic scoping review.

Digital health technology (DHT) has the potential to revolutionize healthcare delivery but its uptake has been low in clinical and research settings. The factors that contribute to the limited adoption of DHT, particularly in cardiovascular settings, are unclear. The objective of this review was to determine the barriers and facilitators of DHT uptake from the perspective of patients, clinicians, and researchers. We searched MEDLINE, EMBASE, and CINAHL databases for studies published from inception to May 2020 that reported barriers and/or facilitators of DHT adoption in cardiovascular care. We extracted data on study design, setting, cardiovascular condition, and type of DHT. We conducted a thematic analysis to identify barriers and facilitators of DHT uptake. The search identified 3075 unique studies, of which 29 studies met eligibility criteria. Studies employed: qualitative methods (n = 13), which included interviews and focus groups; quantitative methods (n = 5), which included surveys; or a combination of qualitative and quantitative methods (n = 11). Twenty-five studies reported patient-level barriers, most common of which were difficult-to-use technology (n=7) and a poor internet connection (n=7). Six studies reported clinician-level barriers, which included increased workload (n=4) and a lack of integration with electronic medical records (n=3).Twenty-four studies reported patient-level facilitators, which included improved communication with clinicians (n=10) and personalized technology (n=6). Four studies reported clinician-level facilitators, which included approval and organizational support from cardiology departments and/or hospitals (n=3) and technologies that improved efficiency (n=3). No studies reported researcher-level barriers or facilitators. In summary, internet access, user-friendliness, organizational support, workflow efficiency, and data integration were reported as important factors in the uptake of DHT by patients and clinicians. These factors can be considered when selecting and implementing DHTs in cardiovascular clinical settings

Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers

© 2018 Hilfenhaus et al.Through multiple cell–cell and cell–matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.Deutsche Forschungsgemeinschaft (STE 2045/1-1) Fundación Ramón Areces (NO AWARD) Ministerio de Economía y Competitividad (NO AWARD) National Institutes of Health (P40OD018537) Worldwide Cancer Research (13-0170