bluuebunny/crossref_metadata_2025_split huggingface dataset:
ENKD1 is a centrosomal and ciliary microtubule-associated protein important for primary cilium assembly and Hedgehog signaling
Authors: Tiryaki Fatmanur , Deretic Jovana , Firat-Karalar Elif Nur 
Published: July 2021

AbstractCentrioles and cilia are conserved, microtubule-based structures critical for cell function and development. Their structural and functional defects cause cancer and developmental disorders. How microtubules are organized into ordered structures by microtubule-associated proteins (MAPs) and tubulin modifications is best understood during mitosis but is largely unexplored for the centrioles and the ciliary axoneme, which are composed of remarkably stable microtubules that maintain their length at steady state. In particular, we know little about the identity of the centriolar and ciliary MAPs and how they work together during the assembly and maintenance of the cilium and centriole. Here, we identified Enkurin domain containing 1 (ENKD1) as a component of the centriole wall and the axoneme in mammalian cells, and showed that it has extensive proximity interactions with these compartments and MAPs. Using in vitro and cellular assays, we found that ENKD1 is a new MAP that promotes microtubule polymerization and regulates microtubule organization and stability. Consistently, overexpression of ENKD1 increased tubulin polymerization and acetylation and disrupted microtubule organization. Cells depleted for ENKD1 were defective in ciliary length and content regulation and failed to respond to Hedgehog pathway activation. Together, our results establish ENKD1 as a new centriolar and ciliary MAP that regulate primary cilium structure and function, and advances our understanding of the functional and regulatory relationship between MAPs and the primary cilium.
Structural insight into the stabilization of microtubules by taxanes
Authors: Prota Andrea E. , Lucena-Agell Daniel , Ma Yuntao , Estévez-Gallego Juan , Li Shuo , Bargsten Katja , Josa-Prado Fernando , Altmann Karl-Heinz , Gaillard Natacha , Kamimura Shinji , Mühlethaler Tobias , Gago Federico , Oliva María A. , Steinmetz Michel O. , Fang Wei-Shuo , Díaz J. Fernando 
Published: July 2021

AbstractPaclitaxel (Taxol®) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drug’s mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 Å resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the βM loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, binding of the taxane core to the taxane site displaces the S9-S10 loop of β-tubulin resulting in microtubule expansion. Our results provide detailed new insights into the mechanism of microtubule-stabilization by taxanes.
Lipid-induced polymorphic amyloid fibrils formation by α-synuclein
Authors: Singh Bhanu Pratap , Morris Ryan J , Horrocks Mathew H , Kunath Tilo , MacPhee Cait E 
Published: July 2021

ABSTRACTMany proteins that self-assemble into amyloid and amyloid-like fibres can adopt polymorphic forms. These forms have been observed both in vitro and in vivo and can arise through variations in the steric-zipper interactions between β-sheets, variations in the arrangements between protofilaments, and differences in the number of protofilaments that make up a given fibre class. Different polymorphs which arise from the same precursor molecule not only exhibit different levels of toxicity, but importantly can contribute to different disease conditions. In this work, we show that in the presence of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, a highly abundant lipid in the plasma membrane of neurons, the aggregation of α-synuclein is markedly accelerated and yields a diversity of polymorphic forms under identical experimental conditions. This morphological diversity includes thin and curly amyloid fibrils, helical and twisted ribbons, nanotubes and flat sheets. TEM analysis of fibrils sampled from the early stage of the growth phase shows the presence of helical and twisted ribbons, indicating that these morphological variants form at the early stages of aggregation. Total internal reflection fluorescence microscopy (TIRFM) indicated the presence of lipids collocated with the mature fibrils. This finding has important implication as the presence of α-synuclein with co-localized high lipid content has been reported in Lewy bodies, the pathological hallmark of Parkinson’s disease and other synucleinopathies. Thus, the present study demonstrates that an interface, such as that provided by a lipid membrane, can not only modulate the kinetics of α-synuclein amyloid aggregation but also plays an important role in the formation of morphological variants by incorporating lipid molecules in the process of amyloid fibril formation.
Targeted Bacteriophage T4 Nanoparticles Reverse HIV-1 Latency in CD4+ Human T Cells
Authors: Batra Himanshu , Zhu Jingen , Jain Swati , Ananthaswamy Neeti , Mahalingam Marthandan , Tao Pan , Lange Camille , Hill Shawn , Zhong Chaojie , Kearney Mary F. , Hu Haitao , Maldarelli Frank , Rao Venigalla B. 
Published: July 2021

SUMMARYA major barrier for HIV-1 eradication is the latent virus reservoir containing stably integrated and silent proviruses in CD4+ T-cells. Targeted reactivation and removal of this latent reservoir is a potential strategy for HIV-1 cure but remains a major challenge. Here, we investigated whether CD4-targeted bacteriophage T4 capsid nanoparticles that mimic HIV envelope can reactivate HIV-1 latency. The nanoparticles were arrayed with CD4-binding CD4-DARPin, or HIV-1 gp140 envelope trimer. When exposed to J-Lat T-cell model of HIV-1 latency or primary T-lymphocytes from human PBMCs, these nanoparticles activated CD4+ T-cells without causing global T-cell activation, which led to activation of HIV-1 proviral transcription, viral protein production and release. Intriguingly, the observed T-cell activation and HIV-1 latency reversal do not involve the classic PKC or NFAT pathways and did not lead to cytokine storm. These studies indicate that engineered non-infectious bacteriophages can be exploited for HIV-1 cure and targeted T-cell therapies.Graphical AbstractHighlightsEngineered Bacteriophage T4 nanoparticles target CD4+ T Cells.CD4-targeted T4 nanoparticles activate and reverse HIV-1 latency in CD4+ T cells.The T4 nanoparticles target CD4+ T cells in human PBMCs and activate resting CD4+ T cells.T4 nanoparticle-mediated activation is independent of the classic PKC and NF-AT pathways.
Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons
Authors: Chua Jason P. , Bedi Karan , Paulsen Michelle T. , Ljungman Mats , Tank Elizabeth M. H. , Kim Erin S. , Colón-Mercado Jennifer M. , Ward Michael E. , Weisman Lois S. , Barmada Sami J. 
Published: July 2021

ABSTRACTAutophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously-encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Accordingly, our findings establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis, but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.
Integrin activation is an essential component of SARS-CoV-2 infection
Authors: Simons Peter , Rinaldi Derek A. , Bondu Virginie , Kell Alison M. , Bradfute Steven , Lidke Diane , Buranda Tione 
Published: July 2021

AbstractCellular entry of coronaviruses depends on binding of the viral spike (S) protein to a specific cellular receptor, the angiotensin-converting enzyme 2 (ACE2). Furthermore, the viral spike protein expresses an RGD motif, suggesting that cell surface integrins may be attachment co-receptors. However, using infectious SARS-CoV-2 requires a biosafety level 3 laboratory (BSL-3), which limits the techniques that can be used to study the mechanism of cell entry. Here, we UV-inactivated SARS-CoV-2 and fluorescently labeled the envelope membrane with octadecyl rhodamine B (R18) to explore the role of integrin activation in mediating both cell entry and productive infection. We used flow cytometry and confocal fluorescence microscopy to show that fluorescently labeled SARS-CoV-2R18particles engage basal-state integrins. Furthermore, we demonstrate that Mn2+, which activates integrins and induces integrin extension, enhances cell binding and entry of SARS-CoV-2R18in proportion to the fraction of integrins activated. We also show that one class of integrin antagonist, which binds to the αI MIDAS site and stabilizes the inactive, closed conformation, selectively inhibits the engagement of SARS-CoV-2R18with basal state integrins, but is ineffective against Mn2+-activated integrins. At the same time, RGD-integrin antagonists inhibited SARS-CoV-2R18binding regardless of integrin activity state. Integrins transmit signals bidirectionally: ‘inside-out’ signaling primes the ligand binding function of integrins via a talin dependent mechanism and ‘outside-in’ signaling occurs downstream of integrin binding to macromolecular ligands. Outside-in signaling is mediated by Gα13and induces cell spreading, retraction, migration, and proliferation. Using cell-permeable peptide inhibitors of talin, and Gα13binding to the cytoplasmic tail of an integrin’s β subunit, we further demonstrate that talin-mediated signaling is essential for productive infection by SARS-CoV-2.
Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases
Authors: Hobbs Helen T , Shah Neel H , Badroos Jean M , Gee Christine L , Marqusee Susan , Kuriyan John 
Published: July 2021

ABSTRACTThe catalytic activity of Syk-family tyrosine kinases is regulated by a tandem-SH2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation which stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to doubly-phosphorylated tyrosine-containing motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the auto-inhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.
Regulation of stem cell identity by miR-200a during spinal cord regeneration
Authors: Walker Sarah E. , Sabin Keith Z. , Gearhart Micah D. , Yamamoto Kenta , Echeverri Karen 
Published: July 2021

AbstractAxolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal markerbrachyuryin spinal cord cells after injury. However, these cells still express the neural stem cell markersox2.In vivolineage tracing allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulatebrachyuryvia a seed sequence in the 3’UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.Summary StatementAfter spinal cord injury, miR-200 fine-tunes expression levelsbrachyuryandβ-cateninto direct spinal cord stem into cells of the mesodermal or ectodermal lineage.
Distinct shifts in site-specific glycosylation pattern of SARS-CoV-2 spike proteins associated with arising mutations in the D614G and Alpha variants
Authors: Kuo Chu-Wei , Yang Tzu-Jing , Chien Yu-Chun , Yu Pei-Yu , Hsu Shang-Te Danny , Khoo Kay-Hooi 
Published: July 2021

AbstractExtensive glycosylation of the spike protein of SARS-CoV-2 virus not only shields the major part of it from host immune responses, but glycans at specific sites also act on its conformation dynamics and contribute to efficient host receptor binding, and hence infectivity. As variants of concern arise during the course of the COVID-19 pandemic, it is unclear if mutations accumulated within the spike protein would affect its site-specific glycosylation pattern. The Alpha variant derived from the D614G lineage is distinguished from others by having deletion mutations located right within an immunogenic supersite of the spike N-terminal domain that make it refractory to most neutralizing antibodies directed against this domain. Despite maintaining an overall similar structural conformation, our mass spectrometry-based site-specific glycosylation analyses of similarly produced spike proteins with and without the D614G and Alpha variant mutations reveal a significant shift in the processing state of N-glycans on one specific N-terminal domain site. Its conversion to a higher proportion of complex type structures is indicative of altered spatial accessibility attributable to mutations specific to the Alpha variant that may impact its transmissibility. This and other more subtle changes in glycosylation features detected at other sites provide crucial missing information otherwise not apparent in the available cryogenic electron microscopy-derived structures of the spike protein variants.
Life situations and lived experiences of young people who are not in education, employment, or training in South Africa
Authors: Matli Walter , Ngoepe Mpho 
Published: July 2021

PurposeIn recent years, South Africa has continued to experience a high rate of young people who are not in employment, education, or training (NEET). The emergence of the concept ‘NEET’ affords researchers an opportunity to more closely examine the life situations and experiences of this population sub-group. The purpose of this paper is thus to further explore the life situations and lived experiences of people in South Africa who are NEET. Structuration theory is adopted to better understand how social structures play a role in the lives of people who are NEET.Design/methodology/approachSemi-structured interviews were carried out with 24 NEET people over a period of two months in South Africa. This paper presents a section of the findings from a broader study that used self-administered interviews to collect data in three metropolitan municipalities of Gauteng Province, the economic heartland of South Africa.FindingsThe findings indicate that poverty remains an obstacle for many young people in furthering their studies or developing their ideas into businesses. Also evident is that there are NEETs who are taking action to disconnect from the NEET sub-group by means of seeking work, funding, or volunteering for initiatives, among other activities. Lack of financial support affects most NEETs perpetuating their vulnerability and their efforts to detach themselves from their NEET status. It is evident that most NEETs who took part in this study are still seeking opportunities to become economically active, regardless of their current financial situations.Originality/valueThere remains a lack of adequate exploration and understanding of the experiences and life situations of people who are NEET. The relevance of this study is in its contribution towards extending the applicability of structuration theory to understanding the life situations of people who are NEET in South Africa. This study contributes to the literature by providing the experiences and life situations of people who are NEET in a country with already high levels of inequalities. It is hoped that this study may provide the basis for developing more specific policy solutions able to address the NEET issues in South Africa and in society more generally.
Discovering the relationship among knowledge management, sustainability marketing and service improvement: the moderating role of consumer interest
Authors: Liu Chih-Hsing Sam , Dong Tse-Ping 
Published: July 2021

PurposeThe purpose of this study was to demonstrate “what” critical attributes are actually associated with the development of blockchain technology and “how” it influences service improvement in the context of tourism and hospitality operations.Design/methodology/approachThis paper aims to provide a new and novel viewpoint by using the blockchain perspective to discover the relationships between knowledge management, sustainability marketing and service improvement using 311 samples collected from tourism and hospitality firms in Taiwan.FindingsThe results show that knowledge management may influence service improvement through sustainability marketing commitment. Furthermore, the study found that consumer interest may strengthen the relationships between knowledge management, sustainability marketing and service improvement. Additionally, the sub-dimension of knowledge management is also discussed in the integrated mediation–moderation model.Practical implicationsThe results provide sufficient support for employees or managers undertaking new technology or development to facilitate business-to-consumer trade models in their present and future operations.Originality/valueThe study advances the existing tourism and hospitality literature by identifying consumer interest as an important effective characteristic that may positively strengthen the relationship among critical attributes of knowledge management and sustainability marketing commitment during the service improvement process.
Managing emergency situations with lean and advanced manufacturing technologies: an empirical study on the Rumbia typhoon disaster
Authors: Pu Xiaodie , Chen Meng , Cai Zhao , Chong Alain Yee-Loong , Tan Kim Hua 
Published: July 2021

PurposeThis study aims to examine the impact of lean manufacturing (LM) on the financial performance of companies affected by emergency situations. It additionally explores the role of advanced manufacturing technologies (AMTs) in complementing LM to enhance financial performance in emergency and non-emergency situations.Design/methodology/approachBoth survey and archival data were collected from 219 manufacturing companies in China. With longitudinal data collected before and after an emergency situation (i.e. Typhoon Rumbia), regression analysis was conducted to investigate the effects of LM and AMTs on financial performance in different contexts.FindingsOur results reveal an inverted U-shaped relationship between LM and financial performance in the context of emergency. We also found that AMTs exerted a positive moderation effect on the inverted U-shaped relationship, indicating high levels of AMTs that mitigated the inefficiency of LM in coping with supply chain emergencies.Research limitations/implicationsThrough simultaneous investigation of LM and AMTs as bundles of practices and their fit with different contexts, this study takes a systems approach to fit that advances the application of contingency theory in the Operations Management literature to more complex patterns of fit.Originality/valueThis study illuminates how AMTs support LM practices in facilitating organizational performance in different contexts. Specifically, this study unravels the interaction mechanisms between AMTs and LM in influencing financial performance in emergency and non-emergency situations.