bluuebunny/crossref_metadata_2025_split huggingface dataset:
CADD from Chlamydia trachomatis is a manganese-dependent oxygenase that employs a self-sacrificing reaction for the synthesis of p-aminobenzoate
Authors: Wooldridge Rowan , Stone Spenser , Pedraza Andrew , Ray W. Keith , Helm Richard F. , Allen Kylie D. 
Published: August 2022

AbstractCADD (chlamydia protein associating with death domains) is a p-aminobenzoate synthase involved in a non-canonical route for tetrahydrofolate biosynthesis in the intracellular bacterial pathogen, Chlamydia trachomatis. The previously solved crystal structure revealed a seven-helix bundle architecture similar to heme oxygenase with a diiron active site, making CADD a founding member of the emerging HDO (heme-oxygenase-like diiron oxidase) superfamily. The CADD-dependent route for pAB biosynthesis was shown to use L-tyrosine as a precursor, however, in vitro reactions were not stimulated by the addition of free L-tyrosine or other tyrosine-derived metabolites, leading to the proposal that the enzyme uses an internal active site tyrosine residue as a precursor to pAB. Here, we perform further biochemical characterization of CADD to clarify the details of the unique self-sacrificing reaction. Surprisingly, the pAB synthase reaction was shown to be dependent on manganese as opposed to iron and the data are most consistent with an active dimanganese cofactor analogous to class Ib and class Id ribonucleotide reductases. Experiments with 18O2 demonstrated the incorporation of two oxygen atoms from molecular oxygen into the pAB product, supporting the proposed mechanism requiring two monooxygenase reactions. Mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, a modification that was increased in reactions that produced pAB in vitro. Additionally, Lys152 was found to be deaminated and oxidized to aminoadipic acid. Taken together, our results support the conclusion that CADD is a manganese-dependent oxygenase that uses Tyr27 and possibly Lys152 as sacrificial substrates for pAB biosynthesis.
The kinase occupancy of T-cell coreceptors reconsidered
Authors: Mørch Alexander M. , Schneider Falk , Jenkins Edward , Santos Ana Mafalda , Fraser Scott E. , Davis Simon J. , Dustin Michael L. 
Published: August 2022

AbstractThe sensitivity of the αβ T-cell receptor (TCR) is enhanced by the coreceptors CD4 and CD8αβ, which are expressed primarily by cells of the helper and cytotoxic T-cell lineages, respectively. The coreceptors bind to major histocompatibility complex (MHC) molecules and associate intracellularly with the Src-family kinase Lck, which catalyzes TCR phosphorylation during receptor triggering. Although coreceptor-kinase occupancy was initially believed to be high, a recent study suggested that most coreceptors exist in an Lck-free state, and that this low occupancy helps to effect TCR antigen discrimination. Here, using the same method, we found instead that the CD4-Lck interaction was stoichiometric (~100%) and that the CD8αβ-Lck interaction was also substantial (~60%). We confirmed our findings in live cells using fluorescence cross-correlation spectroscopy (FCCS) to measure coreceptor-Lck co-diffusionin situ. After introducing structurally guided mutations into the intracellular domain of CD4, we used FCCS to show that stoichiometric Lck coupling required an amphipathic α-helix present in CD4 but not CD8α. In double-positive cells expressing equal numbers of both coreceptors, but limiting amounts of kinase, CD4 out-competed CD8αβ for Lck. In T cells, TCR signaling induced CD4-Lck oligomerization but did not affect the high levels of CD4-Lck occupancy. These findings help settle the question of kinase occupancy and suggest that the binding advantages that CD4 has over CD8 could be important when Lck levels are limiting.Significance statementCD4 and CD8αβ are archetypal coreceptor proteins that potently enhance T-cell antigen sensitivity but how they function is still debated. A fundamental question that remains incompletely resolved is: what fractions of the coreceptors bind the signal-initiating kinase, Lck? Usingin vitroassays and non-invasive fluorescence fluctuation spectroscopy in live cells, we show that most coreceptors are occupied by Lck at the surface of live cells. The structural basis for important differences in the kinase occupancy of CD4 and CD8αβ is also identified. These results provide important context for refining current models of both TCR antigen recognition and cell fate decisions made during thymopoiesis.
Kinesin-5/Cut7 moves bidirectionally on fission-yeast spindles with activity that increases in anaphase
Authors: Gergely Zachary R. , Ansari Saad , Jones Michele H. , McIntosh J. Richard , Betterton Meredith D. 
Published: August 2022

ABSTRACTKinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These tetrameric motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5s typically accumulate more at spindle poles than in the center of the spindle where antiparallel microtubule overlaps are most numerous. While the relevance of this localization has remained unclear, increasing evidence suggests that it occurs due to bidirectional motility or trafficking of kinesin-5 motors. The kinesin-5 motor Cut7p from fission yeast has been shown to move bidirectionally in reconstituted systems. However, bidirectional movement has not been shown in cells and the funtion of the minus-end-directed movement remains unclear. Here, we characterized the motility of kinesin-5/Cut7 on bipolar and monopolar spindles in fission yeast and observed movement both toward plus and minus ends of microtubules. Notably, we found that the activity of the motor increases at the onset on anaphase B. Perturbations to microtubule dynamics did not significantly change the observed Cut7p movement, while Cut7p mutation reduced or abolished observable movement. These results suggest that the directed movement of Cut7p was due to the motility of the motor itself. Mutations of Cut7p that decreased plus-end-directed motility enhanced its spindle-pole localization. In contrast, abolishing Cut7 motility or replacing it with plus-end-directed human Eg5 eliminates the pole localization. Our results suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s in spindle assembly.
SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta
Authors: van Doremalen Neeltje , Singh Manmeet , Saturday Taylor A. , Yinda Claude Kwe , Perez-Perez Lizzette , Bohler W. Forrest , Weishampel Zachary A. , Lewis Matthew , Schulz Jonathan E. , Williamson Brandi N. , Meade-White Kimberly , Gallogly Shane , Okumura Atsushi , Feldmann Friederike , Lovaglio Jamie , Hanley Patrick W. , Shaia Carl , Feldmann Heinz , de Wit Emmie , Munster Vincent J. , Rosenke Kyle 
Published: August 2022

AbstractSince the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Due to confounding factors in the human population, such as pre-existing immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were upregulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggests that in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.
Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform
Authors: Stanhope Bryce J , Peterson Brittany , Knight Brittany , Decadiz Ray Nobles , Pan Roger , Davis Phillip , Fraser Anne , Nuth Manunya , vanWestrienen Jesse , Wendlandt Erik , Goodwin Bruce , Myers Chris , Stone Jennifer , Sozhamannan Shanmuga 
Published: August 2022

ABSTRACTMany SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays overall exhibit high sensitivity and specificity. This variant panel can be used as a Research Use Only screening tool for triaging SARS-CoV-2 positive samples prior to whole genome sequencing.
NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids
Authors: Simoneau Camille R. , Chen Pei-Yi , Xing Galen K. , Khalid Mir M. , Meyers Nathan L. , Hayashi Jennifer M. , Taha Taha Y. , Leon Kristoffer E. , Ashuach Tal , Fontaine Krystal A. , Rodriguez Lauren , Joehnk Bastian , Walcott Keith , Vasudevan Sreelakshmi , Fang Xiaohui , Maishan Mazharul , Schultz Shawn , Roose Jeroen , Matthay Michael A. , Sil Anita , Arjomandi Mehrdad , Yosef Nir , Ott Melanie 
Published: August 2022

AbstractAs SARS-CoV-2 continues to spread worldwide, tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-κB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Confocal microscopy showed more IκBα expression in infected than bystander cells, but found concurrent nuclear translocation of NF-κB that IκBα usually prevents. Overexpressing a nondegradable IκBα mutant reduced NF-κB translocation and increased viral infection. These data demonstrate the functionality of ACE2-OE organoids in SARS-CoV-2 research and identify an incomplete NF-κB feedback loop as a rheostat of viral infection that may promote inflammation and severe disease.
Omicron BA.2 breakthrough infection enhances cross-neutralization of BA.2.12.1 and BA.4/BA.5
Authors: Muik Alexander , Lui Bonny Gaby , Bacher Maren , Wallisch Ann-Kathrin , Toker Aras , Finlayson Andrew , Krüger Kimberly , Ozhelvaci Orkun , Grikscheit Katharina , Hoehl Sebastian , Ciesek Sandra , Türeci Özlem , Sahin Ugur 
Published: August 2022

AbstractRecently, we reported that BNT162b2-vaccinated individuals after Omicron BA.1 breakthrough infection have strong serum neutralizing activity against Omicron BA.1, BA.2, and previous SARS-CoV-2 variants of concern (VOCs), yet less against the highly contagious Omicron sublineages BA.4 and BA.5 that have displaced previous variants. As the latter sublineages are derived from Omicron BA.2, we characterized serum neutralizing activity of COVID-19 mRNA vaccine triple-immunized individuals who experienced BA.2 breakthrough infection. We demonstrate that sera of these individuals have broadly neutralizing activity against previous VOCs as well as all tested Omicron sublineages, including BA.2 derived variants BA.2.12.1, BA.4/BA.5. Furthermore, applying antibody depletion we showed that neutralization of BA.2 and BA.4/BA.5 sublineages by BA.2 convalescent sera is driven to a significant extent by antibodies targeting the N-terminal domain (NTD) of the spike glycoprotein, whereas their neutralization by Omicron BA.1 convalescent sera depends exclusively on antibodies targeting the receptor binding domain (RBD). These findings suggest that exposure to Omicron BA.2, in contrast to BA.1 spike glycoprotein, triggers significant NTD specific recall responses in vaccinated individuals and thereby enhances the neutralization of BA.4/BA.5 sublineages. Given the current epidemiology with a predominance of BA.2 derived sublineages like BA.4/BA.5 and rapidly ongoing evolution, these findings are of high relevance for the development of Omicron adapted vaccines.
Potential anti-monkeypox virus activity of atovaquone, mefloquine, and molnupiravir, and their potential use as treatments
Authors: Akazawa Daisuke , Ohashi Hirofumi , Hishiki Takayuki , Morita Takeshi , Iwanami Shoya , Kim Kwang Su , Jeong Yong Dam , Park Eun-Sil , Kataoka Michiyo , Shionoya Kaho , Mifune Junki , Tsuchimoto Kana , Ojima Shinjiro , Azam Aa Haeruman , Nakajima Shogo , Park Hyeongki , Yoshikawa Tomoki , Shimojima Masayuki , Kiga Kotaro , Iwami Shingo , Maeda Ken , Suzuki Tadaki , Ebihara Hideki , Takahashi Yoshimasa , Watashi Koichi 
Published: August 2022

AbstractMonkeypox virus (MPXV) is a zoonotic orthopoxvirus that causes smallpox-like symptoms in humans and caused an outbreak in May 2022 that led the WHO to declare global health emergency. In this study, from a screening of approved-drug libraries using an MPXV infection cell system, atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 μM, which is more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted post-entry process to impair intracellular virion accumulation. Inhibitors of dihydroorotate dehydrogenase, an atovaquone’s target enzyme, showed conserved anti-MPXV activities. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by seven days at clinically relevant drug concentrations. Moreover, atovaquone and molnupiravir exhibited pan-Orthopoxvirus activity against vaccinia and cowpox viruses. These data suggest that atovaquone would be potential candidates for treating monkeypox.
Brand repositioning: a case of Ufone, Pakistan
Authors: Taimoor Sadaf , Wasti Fizah , Adil Qurat Ul Ain , Raees Sikander , Arshad Umair 
Published: August 2022

Learning outcomes
In the light of the case and the accompanying case questions, the students should understand:1. The theoretical underpinnings of the brand positioning and brand repositioning.2. Critical evaluation of marketing communication material in the light of theoretical underpinnings.3. The nuances of operating in emerging markets in technology-driven sectors.4. The intricate link between the business goals and communication goals5. The application of the attention-interest-desire-action model and the brand media wheel when translating business strategies into communication strategies.

Case overview/synopsis
It was in June 2016, when Asad Haroon, the young head of brands at Ufone, a Pakistani originated telecom operating company, was posed with a challenge of dwindling subscriber identity module card sales, deteriorating average revenue per user and an exponential increase in customer churn. The telecom industry itself was in a flux due to various factors such as changes in the regulatory frameworks and technological shifts.Asad felt that the reason for the brand’s decline might be the lack of synergy between the business strategy and the brand’s communication strategy.Asad knew that he would have to make some unpopular choices and review his brand’s communication strategy which had not yet proved to create a harmony between communication goals and business goals. However, he was unsure about how and if at all he should go about changing a marketing legacy and the brand’s positioning which had reigned the minds of his peers and his customers for so long.

Complexity academic level
This case is aimed toward undergraduate students enrolled in courses of principles of marketing, marketing communications and corporate media strategy.

Supplementary materials
Teaching notes are available for educators only.

Subject code
CSS 8: Marketing
Queer and trans youth (not) knowing: experiences of epistemic (in)justice in the context of an LGBTQ+-inclusive secondary curriculum
Authors: Schey Ryan 
Published: August 2022

PurposeCurrent legislative, policy and cultural efforts to censor and illegalize classroom discussions and curricular representations of LGBTQ+ people reflect longstanding challenges in English education. In an effort to explore what curricular inclusion can (not) accomplish – especially what and how current struggles over inclusion, censorship, illegalization and ultimately representation in English education might (not) contribute to queer and trans liberation – the purpose of this article is to feature the experiences of queer and trans youth as knowers in classroom lessons with LGBTQ+-inclusive curriculum.Design/methodology/approachDrawing from a yearlong literacy ethnography at a Midwestern high school in which the author explored youth and adults reading, writing and talking about sexual and gender diversity, in this article the author focuses on one literacy learning context at the high school, a co-taught sophomore humanities that combined English language arts and social studies.FindingsEngaging theories of epistemic (in) justice, the findings of this article highlight the experiences of queer and trans youth – especially two queer youth of Color, Camden and Imani – as knowers in the context of an LGBTQ+-inclusive classroom curriculum. The author describes epistemic harms with respect to distortions of credibility and homonormative assimilationist requirements and reflects on alternative possibilities that youth gestured toward through their small resistances.Originality/valueBy centering the experiences of LGBTQ+ youth, this article contributes to research about LGBTQ+-inclusive curriculum in English teaching. Previous research, when empirical rather than conceptual, has tended to focus on the perspectives of teachers.
Factors influencing adoption of construction technologies in Vietnam's residential construction projects
Authors: Momade Mohammed Hamza , Durdyev Serdar , Van Tam Nguyen , Shahid Shamsuddin , Mbachu Jasper , Momade Yusra 
Published: August 2022

PurposeVietnam's construction technology (CT) adoption is low when compared to other countries with similar gross domestic product (GDP) per capita resulting in lesser productivity. The research objectives are: (1) To undertake an extensive literature review on CT adoption challenges; (2) To investigate CT adoption challenges unique to Vietnam's construction sector; and (3) To propose data-driven solutions for a greater rate of CT adoption.Design/methodology/approachA two-stage descriptive survey method was adopted in alignment with the research aim and objectives. Based on the literature review of 215 articles, a questionnaire was designed and administered to experienced construction managers (CM) to identify whether CT has been adopted, barriers to adoption, drivers, and the most popular CT tools. Descriptive statistics were used to summarize the characteristics of interest in the empirical dataset and SPSS-based inferential statistics to estimate the means, frequency counts, variance and test hypotheses that informed the drawing of conclusions concerning the research objectives.FindingsThe popular CT tools identified were Autodesk, Microsoft Office and Primavera. The most influential CT adoption barriers: (1) Unknow`n impact on productivity, (2) Late implementation of software in construction projects, (3) Lack of understanding of importance and needs in the construction industry (4) Lack of funds during budget planning for technological advances and implementation (5) Lack of experts required for technological change, and insufficient skills in the industry.Practical implicationsIt is expected that the findings could inform data-driven regulatory and practice reforms targeted at increasing greater uptake of CT in Vietnam with potential for replication in countries facing similar adoption challenges.Originality/valueThe findings are intended to support data-driven regulatory and practice improvements aimed at improving CT adoption in Vietnam, with the possibility for replication in other countries facing comparable problems.
Is trade, energy consumption and economic growth threat to environmental quality in Bahrain–evidence from VECM and ARDL bound test approach
Authors: Alam Md Shabbir 
Published: August 2022

PurposeThe Kingdom of Bahrain has had tremendous development in various areas in the last decade. As a result of this, increasing energy consumption in Bahrain puts a strain on the country's energy supplies and increased CO2 emissions. This study investigates the determinants of carbon emissions in Bahrain.Design/methodology/approachThis study employs the autoregressive distributed lag (ARDL) bounds test and vector error correction model (VECM) Granger causality cointegration methods for empirical analysis during 1980-2020. The unit root test and residual diagnosis have been applied to see the stationarity and normality of the model.FindingsThe analysis suggests no short run causality amid carbon emission, international trade, capital formation, economic development and energy consumption, but a long-run association jointly exist from the exogenous variables toward endogenous variables. The results of the study also revealed that trade and economic growth in Bahrain react negatively to environmental deterioration.Practical implicationsThis research study’s outcome will help the policymakers to build sound external and environmental policies to sustain economic growth and suggested policymakers to emphasize on sustainable usage of energy, alternatives of energy supply, and creation of renewable energy to mitigate the impact of CO2 emission.Social implicationsThe alternatives of energy supply and creation of renewable energy can positively influence the socio-economic state of the nation, like new job opportunities, revenue generation.Originality/valueThis study is unique as no other study till now has covered this period. The findings are also different as the past studies found short-run causality with the control variables, but the study found a long-run causality jointly.