New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi

We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.National Institutes of Health (U.S.) (Grant GM035010)National Institutes of Health (U.S.) (Grant GM118135)National Institutes of Health (U.S.) (Grant R15AI130950

Evidence for a Structural Role for Acid-Fast Lipids in Oocyst Walls of Cryptosporidium, Toxoplasma, and Eimeria

Coccidia are protozoan parasites that cause significant human disease and are of major agricultural importance. Cryptosporidium spp. cause diarrhea in humans and animals, while Toxoplasma causes disseminated infections in fetuses and untreated AIDS patients. Eimeria is a major pathogen of commercial chickens. Oocysts, which are the infectious form of Cryptosporidium and Eimeria and one of two infectious forms of Toxoplasma (the other is tissue cysts in undercooked meat), have a multilayered wall. Recently we showed that the inner layer of the oocyst walls of Toxoplasma and Eimeria is a porous scaffold of fibers of β-1,3-glucan, which are also present in fungal walls but are absent from Cryptosporidium oocyst walls. Here we present evidence for a structural role for lipids in the oocyst walls of Cryptosporidium, Toxoplasma, and Eimeria. Briefly, oocyst walls of each organism label with acid-fast stains that bind to lipids in the walls of mycobacteria. Polyketide synthases similar to those that make mycobacterial wall lipids are abundant in oocysts of Toxoplasma and Eimeria and are predicted in Cryptosporidium. The outer layer of oocyst wall of Eimeria and the entire oocyst wall of Cryptosporidium are dissolved by organic solvents. Oocyst wall lipids are complex mixtures of triglycerides, some of which contain polyhydroxy fatty acyl chains like those present in plant cutin or elongated fatty acyl chains like mycolic acids. We propose a two-layered model of the oocyst wall (glucan and acid-fast lipids) that resembles the two-layered walls of mycobacteria (peptidoglycan and acid-fast lipids) and plants (cellulose and cutin). IMPORTANCE Oocysts, which are essential for the fecal-oral spread of coccidia, have a wall that is thought responsible for their survival in the environment and for their transit through the stomach and small intestine. While oocyst walls of Toxoplasma and Eimeria are strengthened by a porous scaffold of fibrils of β-1,3-glucan and by proteins cross-linked by dityrosines, both are absent from walls of Cryptosporidium. We show here that all oocyst walls are acid fast, have a rigid bilayer, dissolve in organic solvents, and contain a complex set of triglycerides rich in polyhydroxy and long fatty acyl chains that might be synthesized by an abundant polyketide synthase. These results suggest the possibility that coccidia build a waxy coat of acid-fast lipids in the oocyst wall that makes them resistant to environmental stress.National Institutes of Health (U.S.) (Grant GM31318

Evaluating the usefulness of Protection Motivation Theory for predicting climate change mitigation behavioral intentions among a US sample of climate change deniers and acknowledgers.

BACKGROUND: This paper summarizes data from 7 studies that used Protection Motivation Theory (PMT) to guide climate messaging with the goal of increasing climate-mitigating behavioral intentions. Together, the studies address 5 research questions. 1) Does PMT predict behavioral intentions in the context of climate change mitigation? 2) Does PMT work similarly for climate change deniers vs acknowledgers? 3) Are the effects of threat and efficacy additive or multiplicative? 4) Does adding measures of collective threat and efficacy improve the model accuracy for a collective problem like climate change? 5) Can threat and efficacy appraisals - and ultimately behavioral intentions - be shifted through climate messaging? METHODS: Seven online experiments were conducted on US adults (N = 3,761) between 2020 and 2022. Participants were randomly assigned to a control condition or to one of several experimental conditions designed to influence threat, efficacy, or both. Participants indicated their belief in climate change, ethnicity, gender, and political orientation. They completed measures of personal threat and efficacy, collective threat and efficacy, and behavioral intentions. RESULTS: Multiple regressions, ANCOVAs, and effect sizes were used to evaluate our research questions. Consistent with PMT, threat and efficacy appraisals predicted climate mitigation behavioral intentions, even among those who denied climate change. Different interactions emerged for climate deniers and acknowledgers, suggesting that in this context threat and efficacy are not just additive in their effects (but these effects were small). Including measures of collective threat and efficacy only modestly improved the model. Finally, evidence that threat and efficacy appraisals can be shifted was weak and inconsistent; mitigation behavioral intentions were not reliably influenced by the messages tested. CONCLUSIONS: PMT effectively predicts climate change mitigation behavioral intentions among US adults, whether they deny climate change or acknowledge it. Threat appraisals may be more impactful for deniers, while efficacy appraisals may be more impactful for acknowledgers. Including collective-level measures of threat and efficacy modestly improves model fit. Contrary to PMT research in other domains, threat and efficacy appraisals were not easily shifted under the conditions tested here, and increases did not reliably lead to increases in behavioral intentions

A psychobiography of Viktor Emil Frankl

The first study conducted in South Africa of a prominent figure traces back as far as 1939. Since then, extensive research has been done in the field of psychobiography, resulting in a growing interest in this field and evolving into an established research genre in South Africa. Despite the increase in the use of psychobiographies as a research approach internationally and specifically in South Africa, its use is still considered relatively under-utilised and, therefore, the pursuit of further development of psychobiographies in South Africa is required. In light of South Africa’s effort to promote and advance the use of psychobiographies, South African researchers may be motivated to continue studying significant and exceptional lives in South Africa and abroad. Viktor Frankl was selected for this study based on his uniqueness, significance and his interesting life. He is also considered an exceptional individual who has shaped modern psychological thinking. Frankl had written over 40 books on his theory and many studies have been conducted based on his existential theory. Frankl’s contribution to the academic world has been recognised and acknowledged by significant institutions through his own and others’ research. Although much has been written on the life and work of Frankl, none of the literature utilises specific psychological focus and no psychobiographical study of the life of Frankl exists. Therefore, the researcher selected Frankl as the subject for this psychobiography through purposive sampling. The aim of the study was to provide a psychological exploration and description of Frankl’s life against the backdrop of his socio-cultural context. In order to achieve this aim, the researcher employed two psychological frameworks to guide in the description and exploration of his life. The psychological frameworks included Levinson’s life structure theory of adult development and Frankl’s existential theory. The study aimed to describe Frankl’s development with the use of Levinson’s theory while describing how he attained meaning in his life with the use of his own existential theory. Due to the exploratory-descriptive nature of this study, the objective fell within the inductive research approach. The researcher utilised a systematic coding method to arrange the data. The theoretical framework of Levinson’s life structure theory guided the coding system, as well as the key concepts of Frankl’s existential theory. In addition, the researcher utilised Alexander’s guidelines for the extraction of salient data, together with McAdam’s methods and recommendations for managing the data. Lastly, the researcher followed the 12-step method of conducting a psychobiography, as proposed by Du Plessis. Findings from this study found a correlation between Frankl’s life and the eras and transitional periods as theorised by Levinson’s theory. The findings of this study, therefore, support the use of Levinson’s theory with regards to the time frames and its use in understanding Frankl’s development. Also, the researcher found that Levinson’s theory was useful in understanding Frankl’s personality development within his context throughout his lifespan. With regards to Frankl’s existential theory, the researcher found that the use of the theory was suitable in understanding Frankl’s search for meaning. Frankl’s concepts of freedom of will, will to meaning, meaning of life, existential vacuum, supra-meaning, dimensional ontology, the three triads and noö-dynamics assisted in explaining Frankl’s drive to find meaning in his life, take responsibility to attain such meaning and to strive for transcendence beyond his suffering. The researcher found Frankl’s theory useful in explaining his drive, motives, needs and patterns of behaviour within this context. The holistic and integrative approach of the study allowed for an in-depth exploration and description of Frankl’s life and development within his socio-historical context. In addition to contributing to the existing body of knowledge on Frankl, his life and his theory, the study also contributed to the growing field of psychobiographical research. The study acknowledged and highlighted that further in-depth examination of the lives of extraordinary personalities could significantly contribute to psychobiographical studies. Based on the psychological frameworks used to explore and describe Frankl’s life, recommendations are made for future research

There\u27ll Be A Hot Time In The Town of Berlin

Photograph of Frank Sinatrahttps://scholarsjunction.msstate.edu/cht-sheet-music/10592/thumbnail.jp

The disruption of GDP-fucose de novo biosynthesis suggests the presence of a novel fucose-containing glycoconjugate in <i>Plasmodium</i> asexual blood stages

Glycosylation is an important posttranslational protein modification in all eukaryotes. Besides glycosylphosphatidylinositol (GPI) anchors and N-glycosylation, O-fucosylation has been recently reported in key sporozoite proteins of the malaria parasite. Previous analyses showed the presence of GDP-fucose (GDP-Fuc), the precursor for all fucosylation reactions, in the blood stages of Plasmodium falciparum. The GDP-Fuc de novo pathway, which requires the action of GDP-mannose 4,6-dehydratase (GMD) and GDP-L-fucose synthase (FS), is conserved in the parasite genome, but the importance of fucose metabolism for the parasite is unknown. To functionally characterize the pathway we generated a PfGMD mutant and analyzed its phenotype. Although the labelling by the fucose-binding Ulex europaeus agglutinin I (UEA-I) was completely abrogated, GDP-Fuc was still detected in the mutant. This unexpected result suggests the presence of an alternative mechanism for maintaining GDP-Fuc in the parasite. Furthermore, PfGMD null mutant exhibited normal growth and invasion rates, revealing that the GDP-Fuc de novo metabolic pathway is not essential for the development in culture of the malaria parasite during the asexual blood stages. Nonetheless, the function of this metabolic route and the GDP-Fuc pool that is generated during this stage may be important for gametocytogenesis and sporogonic development in the mosquito

Potential hazards of air pollutant emissions from unconventional oil and natural gas operations on the respiratory health of children and infants

Abstract: Research on air pollutant emissions associated with unconventional oil and gas (UOG) development has grown significantly in recent years. Empirical investigations have focused on the identification and measurement of oil and gas air pollutants [e.g. volatile organic compounds (VOCs), particulate matter (PM), methane] and the influence of UOG on local and regional ambient air quality (e.g. tropospheric ozone). While more studies to better characterize spatial and temporal trends in exposure among children and newborns near UOG sites are needed, existing research suggests that exposure to air pollutants emitted during lifecycle operations can potentially lead to adverse respiratory outcomes in this population. Children are known to be at a greater risk from exposure to air pollutants, which can impair lung function and neurodevelopment, or exacerbate existing conditions, such as asthma, because the respiratory system is particularly vulnerable during development inutero, the postnatal period, and early childhood. In this article, we review the literature relevant to respiratory risks of UOG on infants and children. Existing epidemiology studies document the impact of air pollutant exposure on children in other contexts and suggest impacts near UOG. Research is sparse on long-term health risks associated with frequent acute exposures -especially in children -hence our interpretation of these findings may be conservative. Many data gaps remain, but existing data support precautionary measures to protect the health of infants and children

Approachability in Stackelberg Stochastic Games with Vector Costs

The notion of approachability was introduced by Blackwell [1] in the context of vector-valued repeated games. The famous Blackwell's approachability theorem prescribes a strategy for approachability, i.e., for `steering' the average cost of a given agent towards a given target set, irrespective of the strategies of the other agents. In this paper, motivated by the multi-objective optimization/decision making problems in dynamically changing environments, we address the approachability problem in Stackelberg stochastic games with vector valued cost functions. We make two main contributions. Firstly, we give a simple and computationally tractable strategy for approachability for Stackelberg stochastic games along the lines of Blackwell's. Secondly, we give a reinforcement learning algorithm for learning the approachable strategy when the transition kernel is unknown. We also recover as a by-product Blackwell's necessary and sufficient condition for approachability for convex sets in this set up and thus a complete characterization. We also give sufficient conditions for non-convex sets.Comment: 18 Pages, Submitted to Dynamic Games and Application

Overlooked post-translational modifications of proteins in Plasmodium falciparum: N- and O-glycosylation - A Review

Human malignant malaria is caused by Plasmodium falciparum and accounts for almost 900,000 deaths per year, the majority of which are children and pregnant women in developing countries. There has been significant effort to understand the biology of P. falciparum and its interactions with the host. However, these studies are hindered because several aspects of parasite biology remain controversial, such as N- and O-glycosylation. This review describes work that has been done to elucidate protein glycosylation in P. falciparum and it focuses on describing biochemical evidence for N- and O-glycosylation. Although there has been significant work in this field, these aspects of parasite biochemistry need to be explored further