Cloning, sequencing, and expression of Brucella abortus heat shock 70 gene

Brucella abortus is a gram negative bacteria that can infect and cause serious disease in many mammals including humans. Any organism, including Brucella, that can survive as an intracellular parasite must be able to survive the stress of a foreign and often hostile environment of the host. Stress usually results in a dramatic change in gene expression including an elevated synthesis of heat shock proteins;We have subcloned, sequenced and expressed the Brucella HSP70 and the upstream sequences. The gene was put under the control of the strong tac promoter. However, when expressed, we saw a high accumulation of a 23 kDa polypeptide. Upon sequencing, this peptide was shown to be from the N-terminus of the protein;Upstream of the initiation codon of the Brucella HSP70 gene is a small ORF complete with its own initiation and termination codon and a good ribosome binding site. Various subclones were made and western blots from these showed that if the Brucella HSP70 gene is expressed without the ORF, the 23 kDa polypeptide accumulates. If the ORF is present, the protein accumulates intact. So, we concluded that this ORF is an important upstream regulatory element. Such a system has not been reported before and seems to be unique to Brucella. However, Brucella is an unusual parasite in that it can survive and multiply in the macrophage--the cell type of the immune system designed to kill it. It\u27s unknown whether this form of regulation has any relationship to survival as an intracellular parasite;To facilitate expression we also constructed seven expression vectors, pJE1-pJE7. All these plasmids were derived from pKK223-3. They all carry the M13 origin of replication to facilitate production of single stranded DNA and an expanded polycloning site to facilitate cloning. pJE1-6 are in vivo constitutive expression vectors with modified promoters such that there is a graded expression with pJE1 carrying the strongest promoter. pJE7 is designed for in vitro regulated expression using the tac promoter. This plasmid carries the lac I[superscript]q gene to prevent over expression from the tac promoter

City Open Data Policies

The capture and analysis of data is transforming the 21st Century. As society becomes more data driven, data has the ability to drive the bottom line for private companies and help the public sector to define where and how services can best be delivered. In City Open Data Policies: Learning by Doing, the National League of Cities identifies how cities can take advantage of the opportunities presented by open data initiatives.SUMMARY OF RECOMMENDATIONSLeadership: Political support stands out as one of the key requirements to implementing a successful open data project.Appropriate Legislation: Enacting legislation or formal policies is a crucial step toward ensuring the growth and sustainability of open data portals. Funding: Open data initiatives do not require high levels of funding. It is, however, important that the programs have their own budget line items where resources are specifically allocated. Technical Approach: Leading U.S. cities rely on commercial platforms that facilitate the implementation of open data initiatives, provide technical expertise, and ensure 24/7 customer support, often at a lower cost than providing these services in-house. Stakeholder Involvement: Open data is a two-way process. It is, therefore, essential to encourage participation and engagement among multiple stakeholders including: community members; non-profits; universities; the press; businesses; city departments; and other levels of government. Many cities adopt a flexible, and usually informal, approach to interact with the stakeholders. Measuring Success: Developing evaluation tools should be an integral part of any future open data policies

Metasomatism of the continental crust and its impact on surface uplift: Insights from reactive‐transport modelling

High-elevation, low-relief continental plateaus are major topographic features and profoundly influence atmospheric circulation, sediment transport and storage, and biodiversity. Although orogenic surface-uplift mechanisms for modern continental plateaus near known plate margins like Tibet are well-characterized, they cannot account for examples in intracontinental settings like the Colorado Plateau. In contrast to canonical plate-tectonic uplift mechanisms, broad-scale hydration-induced metasomatism of the lower crust has been suggested to reduce its density and increase its buoyancy sufficiently to contribute to isostatic uplift. However, the relationships between key petrophysical properties in these environments are not fully quantified, which limits application of this model. Here, we develop a series of petrological models that describe the petrological and topographic effects of fluid–rock interaction in non-deforming continental crust of varying composition. We apply an open-system petrological modelling framework that utilizes reactive-transport calculations to determine the spatial and temporal scales over which mineralogic transformations take place compared with the magnitude of infiltration of aqueous fluids derived from devolatilization of subducting oceanic lithosphere. The buoyancy effect of hydration-induced de-densification is most significant for metabasic lower crust, intermediate for metapelitic crust, and minimal for granodioritic crust. We apply these results to a case study of the ~2 km-high Colorado Plateau and demonstrate that under ideal conditions, hydration of its lower–middle crust by infiltrating aqueous fluids released by the Farallon slab during Cenozoic low-angle subduction could have uplifted the plateau surface by a maximum of ~1 km over 16 Myr. However, realistically, although hydration likely has a measurable effect on surface tectonics, the uplift of orogenic plateaus is likely dominantly controlled by other factors, such as lithospheric delamination

exo-FISH: protocol for detecting DNA breaks in repetitive regions of mammalian genomes

Detecting DNA breaks in defined regions of the genome is critical to advancing our understanding of genome stability maintenance. Here, we present exo-FISH, a protocol to label exposed single-stranded DNA in defined repetitive regions of mammalian genomes by combining in vitro restriction enzyme digestion on fixed cells with fluorescence in situ hybridization (FISH). We describe steps for cell harvesting and fixation, slide treatments, and FISH probe hybridization. We then detail procedures for imaging and analysis. For complete details on the use and execution of this protocol, please refer to Saayman et al. (2023)

Study of extraction processes and their impact on bioactivity of botanicals

Ph.DDOCTOR OF PHILOSOPH

Factors affecting language proficiency of English language learners at language institutes in the United States

The purpose of this study was to investigate the perspectives of English language\ud learners (ELLs) about the importance of the five factors, learning environment, learning\ud strategies, motivation, beliefs, and linguistic distance related to learning English and to\ud determine the relationship between these factors with ELLs' language learning and their\ud language proficiency. Two questions guided this study: (l) What do ELLs perceive to\ud be the important factors contributing to their language learning? (2) What are the\ud relationships between these factors and ELLs' language proficiency?\ud A total of 606 ELLs who studied at English language institutes in the United\ud States constituted the final sample. The 43 item survey was used to explore ELLs'\ud perspectives about English language learning. Employing a series of statistical\ud techniques, descriptive analyses, confirmatory factor analyses, and multiple regression,\ud this study investigated the important factor contributing to language learning from ELLs'\ud perspectives.\ud Findings revealed that ELLs' perceive that their beliefs about language learning\ud were the important factor contributing to their language learning, followed by the\ud learning environment, learning strategies, motivation, and linguistic distance. The\ud linguistic distance subscale had the less importance ratings. In addition, the relationship\ud between five factors and ELLs' language proficiency, the results showed that two of the\ud five factors (learning strategies and linguistic distance) contributed significantly to the\ud prediction of ELLs' language proficiency, and motivation is marginally significantly to\ud the prediction of ELLs' language proficiency. That is, learning strategies was the most\ud important effect on ELLs' language proficiency, followed by linguistic distance, and\ud motivation.\ud Learning a language is a complex process. In order to broaden students' desire to\ud learn in school, teachers may need to consider the activities carefully to increase ELLs'\ud interest to learn more and provide the opportunities for them to express their opinion in\ud the classroom. This learning process involves teachers, the learning environment in the\ud classroom and outside the classroom as well as the learners' cooperation. This research\ud has implications for further study of relationship among factors that ELLs perceive to be\ud important to language learning and to instruction in language institutes.Simpson, Anne Dr.\ud Compton-Hall, Margaret Dr.\ud Patterson, Leslie Dr.\ud Dam, Phap Dr.\ud Yang, Philip Dr

Does regular walking improve lipid levels in adults?

Q: Evidence-based answer: Minimally. Regular moderateintensity walking for a period of 4 or more weeks minimally decreased total cholesterol (TC) and low-density lipoprotein (LDL) levels by about 7 mg/dL in women with overweight or obesity (strength of recommendation [SOR]: C, systematic review and meta-analysis on disease-oriented evidence). For adults ages 40 to 65 years, regular walking for 3 or more months inconsistently affected cholesterol and triglyceride levels (SOR: C, based on 3 randomized controlled trials [RCTs] with disease-oriented evidence).Kayla Hatchell, MD; Emily Chin, DO; Brian Vukelic, MD; Katherine Fortenberry, PhD; Dominick Ose, DrPH; Eliza Taylor, MPH, BS, CHES; Rachel Goossen, MD (University of Utah), Rick Guthmann, MD, MPH (Advocate Health Care Illinois Masonic Medical Center Program)Includes bibliographical reference

Using Environmental DNA to Detect Whales and Dolphins in the New York Bight

Determining how cetaceans and other threatened marine animals use coastal habitats is critical to the effective conservation of these species. Environmental DNA (eDNA) is an emerging tool that can potentially be used to detect cetaceans over broad spatial and temporal scales. In particular, eDNA may present a useful complementary method for monitoring their presence during visual surveys in nearshore areas, and for co-detecting prey. In conjunction with ongoing visual surveys, we tested the ability of eDNA metabarcoding to detect the presence and identity of cetaceans in the New York Bight (NYB), and to identify fish species (potential prey) present in the area. In almost all cases in which humpback whales and dolphins were visually observed, DNA from these species was also detected in water samples. To assess eDNA degradation over time, we took samples in the same location 15 and 30min after a sighting in seven instances, and found that eDNA often, but not always, dropped to low levels after 30min. Atlantic menhaden were detected in all samples and comprised the majority of fish sequences in most samples, in agreement with observations of large aggregations of this important prey species in the NYB. While additional data are needed to better understand how factors such as behavior and oceanographic conditions contribute to the longevity of eDNA signals, these results add to a growing body of work indicating that eDNA is a promising tool to complement visual and acoustic surveys of marine megafauna

Microstructural and geochemical constraints on the evolution of deep arc lithosphere

Mantle xenoliths from the Sierra Nevada, California, USA, sampled a vertical column (60–120 km) of lithosphere that formed during Mesozoic continental arc magmatism. This lithosphere experienced an anticlockwise P-T-t path resulting in rapid cooling that effectively “quenched in” features inherited from earlier high-temperature conditions. Here we combine new mineral chemistry data (water, trace element, and major element concentrations) with mineral crystallographic preferred orientations (CPOs) to investigate the relationship between melt infiltration and deformation. The peridotites record a refertilization trend with increasing depth, starting from shallow, coarse-protogranular, less-melt-infiltrated spinel peridotite with strong, orthorhombic olivine CPO to deep, fine-porphyroclastic, highly melt-infiltrated garnet peridotite with weak, axial-[010] olivine CPO. In contrast to the observed axial-[010] CPOs, subgrain boundary orientations and misorientation axes suggest the dominant activation of the (001)[100] slip system, suggesting deformation under moderately hydrous conditions. After accounting for effects of subsolidus cooling, we see coherent trends between mineral trace element abundance and water content, indicating that melt infiltration led to an increase in water content of the peridotites. However, measured olivine and pyroxene water contents in all peridotites (5–10 and 30–500 wt ppm, respectively) are lower than that required to promote dominant (001)[100] slip system observed in both natural and experimental samples. These results suggest that deformation occurred earlier along the P-T path, probably during or shortly after hydrous melt infiltration. Subsequent rapid cooling at 90 Ma led to water loss from olivine (owing to decreased solubility at low temperature), leaving behind a deep arc lithosphere that remained viscously coupled to the Farallon slab until the opening of the slab window in the late Cenozoic