bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoselective sequencing: microscopically-guided genomic measurements with subcellular resolution
Authors: Mangiameli Sarah , Chen Haiqi , Earl Andrew S. , Dobkin Julie , Lesman Daniel , Buenrostro Jason , Chen Fei
Published: September 2022

In biological systems, spatial organization is interconnected with genome function and regulation. However, methods that couple high-throughput genomic and epigenomic profiling with spatial information are lacking. Here, we developed Photoselective Sequencing, a spatially-informed DNA sequencing method to assay collections of cells or subcellular regions that share a unifying morphological trait. In Photoselective Sequencing, we prepare a blocked fragment library within a fixed biological specimen. Guided by fluorescence imaging, we remove the block in specific regions of interest using targeted illumination with near-UV light, ultimately allowing high-throughput sequencing of the selected fragments. To validate Photoselective Sequencing, we profile chromatin openness in fluorescently-labeled cell types within the mouse brain and demonstrate strong agreement with published single-cell ATAC-seq data. Using Photoselective Sequencing, we characterize the accessibility profiles of oligodendrocyte-lineage cells within the cortex and corpus-callosum regions of the brain. We develop a new computational strategy for decomposing bulk accessibility profiles by individual cell types, and report a relative enrichment of oligodendrocyte-progenitor-like cells in the cortex. Finally, we leverage Photoselective Sequencing for unbiased profiling of DNA at the nuclear periphery, a key chromatin organizing region. We compare and contrast the Photoselective Sequencing profile with lamin ChIP-seq data, and identify features beyond lamin interaction that are correlated with positioning at the nuclear periphery. These results collectively demonstrate that Photoselective Sequencing is a flexible and generalizable platform for exploring the interplay of spatial structures with genomic and epigenomic properties.
Co-targeting BCL-XL and MCL-1 with DT2216 and AZD8055 synergistically inhibits small-cell lung cancer growth without causing on-target toxicities in mice
Authors: Khan Sajid , Kellish Patrick , Connis Nick , Thummuri Dinesh , Wiegand Janet , Zhang Peiyi , Zhang Xuan , Budamagunta Vivekananda , Hua Nan , Yang Yang , De Umasankar , Jin Lingtao , Zhang Weizhou , Zheng Guangrong , Hromas Robert , Hann Christine , Zajac-Kaye Maria , Kaye Frederic J. , Zhou Daohong
Published: September 2022

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, DT2216+AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied with extended survival in a genetically-engineered mouse (GEM) model of SCLC without causing significant thrombocytopenia or other normal tissue injury. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216+mTOR inhibitor combination in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.
Neural Representations of Post-Decision Choice Confidence and Reward Expectation in the Caudate Nucleus and Frontal Eye Field
Authors: Fan Yunshu , Doi Takahiro , Gold Joshua I. , Ding Long
Published: September 2022

Performance monitoring that supports ongoing behavioral adjustments is often examined in the context of either choice confidence for perceptual decisions (i.e., “did I get it right?”) or reward expectation for reward-based decisions (i.e., “what reward will I receive?”). However, our understanding of how the brain encodes these distinct evaluative signals remains limited because they are easily conflated, particularly in commonly used two-alternative tasks with symmetric rewards for correct choices. Previously we reported behavioral and neural results related to decision formation by monkeys performing a visual motion discrimination task with asymmetric rewards (Doi et al., 2020; Fan et al., 2020). Here we leveraged this task design to partially decouple trial-by-trial estimates of choice confidence and reward expectation and examine their impacts on behavior and their representations in the caudate nucleus (part of the striatum in the basal ganglia) and the frontal eye field (FEF, in prefrontal cortex). We found that these evaluative signals had infrequent, but consistent, effects on the behavior of well-trained monkeys. We also identified distinguishable representations of the two types of signals in FEF and caudate neurons, including different distribution patterns, time courses, and relationships to behavior in the two brain areas. These results suggest that the cortico-striatal decision network may use diverse evaluative signals for performance monitoring and add to our understanding of the different roles of the FEF and caudate nucleus in decision-related computations.
Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex
Authors: Zou Xinle , Shanmugam Sri Karthika , Kanner Scott A. , Sampson Kevin J. , Kass Robert S. , Colecraft Henry M.
Published: September 2022

The slow delayed rectifier potassium current, IKs, conducted through pore-forming Q1 and auxiliary E1 ion channel complexes is important for human cardiac action potential repolarization. During exercise or fright, IKs is up-regulated by protein kinase A (PKA)-mediated Q1 phosphorylation to maintain heart rhythm and optimum cardiac performance. Sympathetic upregulation of IKs requires recruitment of PKA holoenzyme (two regulatory- RI or RII- and two catalytic Cα subunits) to Q1 C-terminus by an A kinase anchoring protein (AKAP9). Mutations in Q1 or AKAP9 that abolish their functional interaction result in long QT syndrome type 1 and 11, respectively, which increases the risk of sudden cardiac death during exercise. Here, we investigated the utility of a targeted protein phosphorylation (TPP) approach to reconstitute PKA regulation of IKs in the absence of AKAP9. Targeted recruitment of endogenous Cα to E1-YFP using a GFP/YFP nanobody (nano) fused to RIIα enabled acute cAMP-mediated enhancement of IKs, reconstituting physiological regulation of the channel complex. By contrast, nano-mediated tethering of RIIα or Cα to Q1-YFP constitutively inhibited IKs by retaining the channel intracellularly in the endoplasmic reticulum and Golgi. Proteomic analysis revealed distinct phosphorylation sites are modified by Cα targeted to Q1-YFP compared to free Cα. Thus, functional outcomes of synthetically recruited PKA on IKs regulation is critically dependent on the site of recruitment within the channel complex. The results reveal insights into divergent regulation of IKs by phosphorylation across different spatial and time scales, and suggest a TPP approach to develop new drugs to prevent exercise-induced sudden cardiac death.
Humoral immunogenicity of a Coronavirus Disease 2019 (COVID-19) DNA Vaccine in Rhesus Macaques (Macaca mulatta) Delivered using Needle-free Jet Injection
Authors: Jay Alexandra , Kwilas Steven A , Josleyn Matthew , Ricks Keersten , Hooper Jay
Published: September 2022

A SARS-CoV-2 DNA vaccine targeting the spike protein and delivered by jet injection, nCOV-S(JET), previously shown to protect wild-type and immunosuppressed Syrian hamsters (Mesocricetus auratus), was evaluated via two needle-free delivery methods in rhesus macaques (Macaca mulatta). The methods included intramuscular delivery of 2 mg per vaccination with the PharmaJet Stratis device and intradermal delivery of 0.4 mg per vaccination with the PharmaJet Tropis device. We hypothesized that the nCOV-S(JET) vaccine would mount detectable neutralizing antibody responses when delivered by needle-free jet injection by either the intradermal or intramuscular route. When delivered intramuscularly, the vaccines elicited neutralizing and variant (Beta, Gamma, and Delta) cross-neutralizing antibodies against SARS-CoV-2 in all six animals after three vaccinations. When delivered at a lower dose by the intradermal route, strong neutralizing antibody responses were only detected in two of six animals. This study confirms that a vaccine previously shown to protect in a hamster model can elicit neutralizing and cross-neutralizing antibodies against SARS-CoV-2 in nonhuman primates. We posit that nCOV-S(JET) has the potential for use as booster vaccine in heterologous vaccination strategies against COVID-19.
Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes
Authors: Schulz Lisa Maria , Rothe Patricia , Halbedel Sven , Gründling Angelika , Rismondo Jeanine
Published: September 2022

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.
Ultrahigh-throughput Absorbance Activated Droplet Sorting (UHT-AADS) for enzyme screening at kilohertz frequencies
Authors: Medcalf Elliot J. , Gantz Maximilian , Kaminski Tomasz S. , Hollfelder Florian
Published: September 2022

Droplet microfluidics is a valuable method to ‘beat the odds’ in high throughput screening campaigns such as directed evolution, where valuable hits are infrequent and large library sizes are required. Absorbance-based sorting expands the landscape of range of enzyme families that can be subjected to droplet screening by expanding possible assays beyond fluorescence detection. However, absorbance activated droplet sorting (AADS) is currently ∼10-fold slower than typical fluorescence activated droplet sorting (FADS), meaning that, in comparison, a larger portion of sequence space is inaccessible due to throughput constraints. Here we improve AADS to reach kHz sorting speeds in an order of magnitude increase over previous designs, with close-to-ideal sorting accuracy. This is achieved by a combination of (i) the use of refractive index matching oil that improves signal quality by removal of side scattering (increasing the sensitivity of absorbance measurements); (ii) a sorting algorithm capable of reaching 4 kHz with an Arduino Due; and (iii) a chip design that transmits product detection better into sorting decisions without false positives, namely a single-layered inlet to space droplets further apart and injections of ‘bias oil’ providing a fluidic barrier preventing droplets from entering the incorrect sorting channel. The updated ultrahigh-throughput absorbance activated droplet sorter (UHT-AADS) increases the effective sensitivity of absorbance measurements through better signal quality at a speed that matches the more established fluorescence-activated sorting devices.

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The role of evolutionarily metastable oligomeric states in the optimization of catalytic activity
Authors: East Nicholas J. , Clifton Ben E. , Jackson Colin J. , Kaczmarski Joe A.
Published: September 2022

The emergence of oligomers is common during the evolution and diversification of protein families, yet the selective advantage of oligomerization is often cryptic or unclear. Oligomerization can involve the formation of isologous head-to-head interfaces (e.g., in symmetrical dimers) or heterologous head-to-tail interfaces (e.g., in cyclic complexes), the latter of which is less well studied and understood. In this work, we retrace the emergence of the trimeric form of cyclohexadienyl dehydratase from Pseudomonas aeruginosa (PaCDT) by introducing residues that form the PaCDT trimer-interface into AncCDT-5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants (implying evolutionarily metastable oligomeric states) and that trimerization corresponds with a reduction in the KM value of the enzyme from a promiscuous level to the physiologically relevant range. We show that this can be rationalized at the structural and dynamic level by reduced sampling of a non-catalytic conformational substate, and that trimerization was likely followed by a C-terminal extension that further refined the conformational sampling and kinetic properties of the enzyme. This work provides insight into how neutral sampling of metastable oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function.

Importance & Impact Statement

Understanding how and why structural complexity (including homo-oligomerization and sequence insertions) emerges during the evolution and diversification of natural enzymes is a key goal in the study and design of protein function. We show that cyclic homo-oligomeric states can emerge via a small number of substitutions, and that trimerization and a C-terminal extension contributed to the tuning of catalytic properties during the evolution of cyclohexadienyl dehydratase from Pseudomonas aeruginosa.
Metabolic Signatures of Performance in Elite World Tour Professional Cyclists
Authors: Nemkov Travis , Cendali Francesca , Stefanoni Davide , Martinez Janel , Hansen Kirk C , San-Millan Inigo , D'Alessandro Angelo
Published: September 2022

Introduction: Metabolomics studies of recreational and elite athletes have been so far limited to venipuncture-dependent blood sample collection in the setting of controlled training and medical facilities. However, limited to no information is currently available if findings in laboratory settings are translatable to real world scenario in elite competitions. Methods: To characterize molecular profiles of exertion in elite athletes during cycling, we performed metabolomics analyses on blood isolated from twenty-eight international-level elite World Tour professional male athletes from a Union Cycliste Internationale (UCI) World Team taken before and after a graded exercise test (GXT) to volitional exhaustion and before and after a long aerobic training session. Moreover, established signatures were then used to characterize the metabolic physiology of five of these cyclists that were selected to represent the same UCI World Team during a 7-stage elite World Tour race. Results: Using dried blood spot collection to circumvent logistical hurdles associated with field sampling, these studies defined metabolite signatures and fold change ranges of anaerobic or aerobic exertion in elite cyclists, respectively. Blood signatures derived in controlled settings enabled comparison with blood sampled during competition, thus providing insight into fatigue status of the cyclists during the course of the race. Collectively, these studies provide a unique view of alterations in the blood metabolome of elite athletes during competition and at the peak of their performance capabilities.
Chemokine Signaling Caused byMycobacterium aviumBiofilms in the Lung Airway Increases Bacterial Loads by Spatially Diverting Macrophages
Authors: Weathered Catherine , Pennington Kelly , Escalante Patricio , Pienaar Elsje
Published: September 2022

Mycobacterium aviumComplex (MAC) are ubiquitous environmental biofilm-forming microbes that can colonize and infect patient lungs. Incidence and prevalence of MAC infections are increasing globally, and reinfection is common. Thus, MAC infections present a significant public health challenge. MAC infections are notoriously difficult to treat and there is an urgent need for MAC-targeted therapeutics. To identify potential drug targets, we quantify the impact of MAC biofilms and repeated exposure on infection progression using a computational model of MAC infection in lung airways.

MAC biofilms aid epithelial cell invasion, cause premature macrophage apoptosis, and limit antibiotic efficacy. We develop an agent-based model that incorporates the interactions between bacteria, biofilm and immune cells. We perform virtual knockouts to quantify the effects of the sources of biofilm (biofilm simultaneously deposited with bacteria vs. formed in the airway after initial bacterial deposition), and their effects on macrophages (inducing apoptosis and slowing phagocytosis). We also quantify the effects of repeated bacterial exposure to assess the impact of reinfection on infection progression.

Our results show that chemokines released by biofilm-induced apoptosis bias macrophage chemotaxis towards pockets of infected and apoptosed macrophages. This bias results in fewer macrophages finding extracellular bacteria, allowing the extracellular planktonic bacteria to replicate freely. These spatial macrophage trends are further exacerbated with repeated deposition of bacteria.

Our model indicates that interventions to either abrogate macrophages’ apoptotic responses to bacterial biofilms and/or reduce frequency of patient exposure to bacteria will lower bacterial load, and likely overall risk of infection.
Working strokes produced by curling protofilaments at disassembling microtubule tips can be biochemically tuned and vary with species
Authors: Murray Lucas E. , Kim Haein , Rice Luke M. , Asbury Charles L.
Published: September 2022

The disassembly of microtubules can generate force and drive intracellular motility. During mitosis, chromosomes remain persistently attached via kinetochores to the tips of disassembling microtubules, which pull the sister chromatids apart. According to the conformational wave hypothesis, such force generation requires that protofilaments curl outward from the disassembling tips to exert pulling force directly on kinetochores. Rigorously testing this idea will require modifying the mechanical and energetic properties of curling protofilaments, but no way to do so has yet been described. Here, by direct measurement of working strokes generated in vitro by curling protofilaments, we show that their mechanical energy output can be increased by adding magnesium, and that yeast microtubules generate larger and more energetic working strokes than bovine microtubules. Both the magnesium and species-dependent increases in work output can be explained by a lengthening of protofilament curls, without any change in their bending stiffness or intrinsic curvature. These observations demonstrate how work output from curling protofilaments can be tuned and suggest evolutionary conservation of the amount of curvature strain energy stored in the microtubule lattice.
Recycling upstream redox enzymes expands the regioselectivity of cycloaddition in pseudo-aspidosperma alkaloid biosynthesis
Authors: Kamileen Mohamed O. , DeMars Matthew D. , Hong Benke , Nakamura Yoko , Paetz Christian , Lichman Benjamin R. , Sonawane Prashant D. , Caputi Lorenzo , O’Connor Sarah E.
Published: September 2022

Nature uses cycloaddition reactions to generate complex natural product scaffolds. Dehydrosecodine is a highly reactive biosynthetic intermediate that undergoes cycloaddition to generate several alkaloid scaffolds that are the precursors to pharmacologically important compounds such as vinblastine and ibogaine. Here we report how dehydrosecodine can be subjected to redox chemistry, which in turn allows cycloaddition reactions with alternative regioselectivity. By incubating dehydrosecodine with reductase and oxidase biosynthetic enzymes that act upstream in the pathway, we can access the rare pseudo-aspidosperma alkaloids, pseudo-tabersonine and pseudo-vincadifformine, both in vitro and by reconstitution in the plant Nicotiana benthamiana from an upstream intermediate. We propose a stepwise mechanism to explain the formation of the pseudo-tabersonine scaffold by structurally characterizing enzyme intermediates, and by monitoring the incorporation of deuterium labels. This discovery highlights how plants use redox enzymes to enantioselectively generate new scaffolds from common precursors.
Microcosm cultures of a complex synthetic community reveal ecology and genetics of gut microbial organization
Authors: Jin Xiaofan , Yu Feiqiao B. , Yan Jia , Weakley Allison , Pollard Katherine S.
Published: September 2022

The behavior of microbial communities depends on both taxonomic composition and physical structure. Metagenomic sequencing of fecal samples has revealed the composition of human gut microbiomes, but we remain less familiar with the spatial organization of microbes between regions such as lumen and mucosa, as well as the microbial genes that regulate this organization. To discover the determinants of spatial organization in the gut, we simulate mucosal colonization over time using an in vitro culture approach incorporating mucin hydrogel microcosms with a complex yet defined community of 123 human strains for which we generated high-quality genome assemblies. Tracking strain abundance longitudinally using shotgun metagenomic measurements, we observe distinct and strain-specific spatial organization in our cultures with strains enriched on mucin microcosms versus in supernatant, reminiscent of mucosa versus lumen enrichment in vivo. Our high taxonomic resolution data enables a comprehensive search for microbial genes that underlie this spatial organization. We identify gene families positively associated with microcosm-enrichment, including several known for biofilm and adhesion functions such as efflux pumps, gene expression regulation, and membrane proteases, as well as a novel link between a coenzyme F420 hydrogenase gene family and lipo/exopolysaccharide biosynthesis. Our strain-resolved abundance measurements also demonstrate that incorporation of microcosms yields a more diverse community than liquid-only culture by allowing co-existence of closely related strains. Altogether these findings demonstrate that microcosm culture with synthetic communities can effectively simulate lumen versus mucosal regions in the gut, providing measurements of microbial organization with high taxonomic resolution to enable identification of specific bacterial genes and functions associated with spatial structure.
Bacterial Argonaute proteins aid cell division in the presence of topoisomerase inhibitors in Escherichia coli
Authors: Olina Anna , Agapov Aleksei , Yudin Denis , Kuzmenko Anton , Aravin Alexei A. , Kulbachinskiy Andrey
Published: September 2022

Prokaryotic Argonaute (pAgo) proteins are guide-dependent nucleases that function in host defense against invaders. Recently, it was shown that TtAgo from Thermus thermophilus also participates in the completion of DNA replication by decatenating chromosomal DNA. Here, we show that two pAgos from cyanobacteria Synechococcus elongatus (SeAgo) and Limnothrix roseae (LrAgo) act as DNA-guided DNA nucleases in Escherichia coli and aid cell division in the presence of the gyrase inhibitor ciprofloxacin. Both pAgos are preferentially loaded with small DNA guides derived from the sites of replication termination. The amount of pAgo-associated small DNAs (smDNAs) from the termination sites is increased in the presence ciprofloxacin, suggesting that smDNA biogenesis depends on DNA replication and is stimulated by gyrase inhibition. Ciprofloxacin also enhances asymmetry in the distribution of smDNAs around Chi-sites, indicating that it induces double-strand breaks that serve as a source of smDNA during their processing by RecBCD. While active in E. coli, SeAgo does not protect its native host S. elongatus from ciprofloxacin. These results suggest that pAgo nucleases help to complete replication of chromosomal DNA by targeting the sites of termination, and may switch their functional activities when expressed in different host species.
Development of human lateral prefrontal sulcal morphology and its relation to reasoning performance
Authors: Willbrand Ethan H. , Ferrer Emilio , Bunge Silvia A. , Weiner Kevin S.
Published: September 2022

Previous findings show that the morphology of folds (sulci) of the human cerebral cortex flatten during postnatal development. However, previous studies did not consider the relationship between sulcal morphology and cognitive development in individual participants. Here, we fill this gap in knowledge by leveraging cross-sectional morphological neuroimaging data in the lateral prefrontal cortex (LPFC) from individual human participants (6-36 years old, males and females; N = 108; 3672 sulci), as well as longitudinal morphological and behavioral data from a subset of child and adolescent participants scanned at two timepoints (6-18 years old; N = 44; 2992 sulci). Manually defining thousands of sulci revealed that LPFC sulcal morphology (depth, surface area, gray matter thickness, and local gyrification index) differed between children (6-11 years old)/adolescents (11-18 years old) and young adults (22-36 years old) cross-sectionally, but only cortical thickness showed both cross-sectional differences between children and adolescents and presented longitudinal changes during childhood and adolescence. Furthermore, a data-driven approach relating morphology and cognition identified that longitudinal changes in cortical thickness of four rostral LPFC sulci predicted longitudinal changes in reasoning performance, a higher-level cognitive ability that relies on LPFC. Contrary to previous findings, these results suggest that sulci may flatten either after this time frame or over a longer longitudinal period of time than previously presented. Crucially, these results also suggest that longitudinal changes in the cortex within specific LPFC sulci are behaviorally meaningful—providing targeted structures, and areas of the cortex, for future neuroimaging studies examining the development of cognitive abilities.

Significance Statement

Recent work has shown that individual differences in neuroanatomical structures (indentations, or sulci) within the lateral prefrontal cortex (LPFC) are behaviorally meaningful during childhood and adolescence. Here, we describe how specific LPFC sulci develop at the level of individual participants for the first time—from both cross-sectional and longitudinal perspectives. Further, we show, also for the first time, that the longitudinal morphological changes in these structures are behaviorally relevant. These findings lay the foundation for a future avenue to precisely study the development of the cortex and highlight the importance of studying the development of sulci in other cortical expanses and charting how these changes relate to the cognitive abilities those areas support at the level of individual participants.
Nanobody engineering for SARS-CoV-2 neutralization and detection
Authors: Hannula Liina , Kuivanen Suvi , Lasham Jonathan , Kant Ravi , Kareinen Lauri , Bogacheva Mariia , Strandin Tomas , Sironen Tarja , Sharma Vivek , Saviranta Petri , Kipar Anja , Vapalahti Olli , Huiskonen Juha T. , Rissanen Ilona
Published: September 2022

In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, ‘nanobodies’. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for viral evolution in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel detection assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.
A miniature airlock system to aid the cryo-cooling of protein crystals grown under anoxic conditions
Authors: van der Linden Peter , Engilberge Sylvain , Atta Mohamed , Carpentier Philippe
Published: September 2022

Crystallographic studies of structures and mechanisms of dioxygen-employing enzymes or oxygen-sensitive proteins usually require these molecules to be prepared and crystallized under anoxic conditions in gloveboxes. Handling, cryo-cooling and extraction of these crystals from gloveboxes for X-ray data collection can be a tricky operation, during which samples might be ruined if they are accidentally exposed to atmospheric oxygen. This work reports the design of a new miniature airlock system and describes the associated method in order to simplify these tasks. The system allows flash-cooling of crystals harvested in a glovebox directly into an external liquid nitrogen container via a miniature airlock while maintaining strict anoxic conditions. The method was assessed using hen egg-white lysozyme crystals soaked in dihydroresorufin (an O2 indicator) and with the reduced tRNA-hydroxylase enzyme MiaE.
Evaluation of failure risks for manual tightening operations in automotive assembly lines
Authors: Altinisik Armagan , Yildirim Utku , Topcu Y. Ilker
Published: September 2022

Purpose

The tightening operations are one of the most critical operations in automotive assembly lines because of its direct impact on customer safety. This study aims to evaluate the major complexity drivers for manual tightening operations, correlate with real tightening failure data and propose mitigations to improve the complexity.

Design/methodology/approach

In the first stage, the complexity drivers for manual tightening operations were identified. Then, the relative importance of the risk attributes was defined by using pairwise comparisons questionnaire. Further, failure mode effect analysis–analytic hierarchy process (FMEA–AHP) and AHP ratings methods were applied to 20 manual tightening operations in automotive assembly lines. Finally, the similarities between the revealed results and the real failure rates of a Turkish automotive factory were examined and a sensitivity analysis was conducted.

Findings

The correlation between the proposed methods and manual tightening failure data was calculated as 83%–86%. On the other hand, the correlation between FMEA–AHP and AHP ratings was found as 92%. Poor ergonomics, operator competency and training, operator concentration-loose attention fatigue, manual mouthing before the tightening operation, frequent task changes, critical tightening sequence, positioning of the part and/or directional assembly were found relatively critical for the selected 20 tightening operations.

Originality/value

This is a unique study for the evaluation of the attributes for manual tightening complexity in automotive assembly lines. The output of this study can be used to improve manual tightening failures in manual assembly lines and to create low complexity assembly lines in new model launches.
Analysing the risk-return relationship in privately held firms: the contingent effect of being a family firm
Authors: Rojo Ramírez Alfonso Andrés , Martínez-Victoria MCarmen , Martínez-Romero María J.
Published: September 2022

Purpose

The relationship between risk and return has been widely analysed in the scope of listed companies. However the present literature leaves uncovered an important study area with regards to privately held firms. In order to cover this gap, this study analyses the risk-return trade-off in the context of private enterprises. Furthermore, the authors incorporate the contingent effect of being a family firm on the abovementioned relationship.

Design/methodology/approach

Using information from the SABI (Sistema de Análisis de Balances Ibéricos) database, a sample of 2,297 private manufacturing firms were analysed for the period of 2009–2016. So as to ascertain the proposed hypotheses, dynamic panel data methodology was applied. Specifically, the authors estimated the two-step general method of moments (GMM).

Findings

The obtained findings reveal that, according to prospect theory arguments, privately held firms adopt a conservative attitude toward risk when results are higher than a target level, while becoming risk seeking when results are lower than a target level. Moreover, the fact of being a family firm softens the risk-return relationship both when performance is above the target level and also when firms find themselves in the lowest performing case.

Originality/value

This article is, to the best of the authors' knowledge, one of the first studies dealing with the risk-return relationship in a privately held firm context. Moreover, the inclusion of being a family firm as a contingent factor in the abovementioned link is a complete novelty.
Why do good soldiers in good organizations behave wrongly? The vicarious licensing effect of perceived corporate social responsibility
Authors: Dadaboyev Sherzodbek Murodilla Ugli , Choi Sungwon , Paek Soyon
Published: September 2022

Purpose

While most corporate social responsibility (CSR) research has focused on its positive effects, the potential “dark side” of CSR has received scant attention. Grounded in vicarious moral licensing theory and insights from related literature, the current study examines how employees' perceptions of external CSR could result in unintentional negative consequences like unethical pro-organizational behavior via psychological entitlement. The study also investigates the direct and conditional effects of private self-awareness.

Design/methodology/approach

A two-wave survey of 609 full-time employees from various occupations was conducted to empirically test the hypotheses. Several techniques and remedies were applied to control the quality of the sample data and mitigate the effects of potential common method bias.

Findings

The results demonstrate that unethical pro-organizational behavior can be an unintentional negative outcome of perceived external CSR, and psychological entitlement mediates the relationship.

Research limitations/implications

This work contributes to the moral licensing literature by examining vicarious moral licensing in the work domain. It offers several new and significant implications for research on CSR, psychological entitlement, and unethical pro-organizational behavior. The results suggest that managers should be mindful of unethical pro-organizational behavior as a potential negative consequence of external CSR engagement.

Originality/value

This study is among the first attempts to examine vicarious moral licensing in the work domain and investigates a largely neglected research area – the negative aspect of external CSR.
The cross-country evaluation of nuclear science and technology publications
Authors: Loan Fayaz Ahmad , Shah Ufaira Yaseen
Published: September 2022

Purpose

The purpose of this study is to measure the quality of nuclear science and technology research using the scientometric indicators, like activity index, attractivity index and publication efficiency index.

Design/methodology/approach

The present scientometric study is based on the bibliographic data extracted from the core collection of the Web of Science database from 1989 to 2019. The data was collected using the advanced search feature, with a search string of 28 related terms. The bibliographic data for all the journal articles, excluding all other formats, was downloaded for statistical analysis.

Findings

The present study highlights the growth pattern and the research efforts devoted to the field of nuclear science and technology worldwide, as reflected through the Core Collection of the Web of Science database, for 31 years from 1989 to 2019. The USA has emerged as the topmost producer, with a total share of almost 29% with respect to the world literature followed by Japan, Germany, France and UK. However, the relative citation impact has been the highest for Netherlands followed by the USA, Germany, France and UK, respectively. The study depicted that the activity index, also called as the relative performance indicator, has been the highest for the USA, whereas the attractivity index has been the highest for China, indicating its dominance in attracting citations to its publications in the field of nuclear science and technology. The publication efficiency index, the value has been the maximum for Iran, for which the value has been greater than 1 (1.618). The measure suggests that Iran’s research effectiveness in the field of nuclear science and technology has been more than the research effort devoted to the field.

Research limitations/implications

The paper was based on the data retrieved from a single database, and the results may vary across other databases. Hence, caution should be taken while generalizing the findings.

Originality/value

Several scientometric studies have taken place in the field of nuclear science and technology, particularly in its allied branches, but none of the studies has focused on evaluating the research efforts carried out by the countries in terms of activity index, attractivity index and publication efficiency index, and the present study is an attempt to fill the gap.
Knowledge management practices on innovation performance in the hotel industry: mediated by organizational learning and organizational creativity
Authors: Patwary Ataul Karim , Alwi Mohamad Khairi , Rehman Shafique Ur , Rabiul Md Karim , Babatunde Adeneye Yusuf , Alam Mirza Mohammad Didarul
Published: September 2022

Purpose

The purpose of this study is to investigate the role of knowledge management (KM) practices on innovation performance. It also examines whether organisational creativity and organizational learning mediate the relationship between KM practices and innovation performance.

Design/methodology/approach

The study uses a quantitative method by surveying (n = 291) hotel employees from Malaysia using self-administered questionnaires. Data collected were analysed by using partial least squares structural equation modelling.

Findings

Findings reveal that KM positively impacts innovation performance among Malaysian hospitality workers. This study further reveals that organisational learning and organisational creativity significantly mediate the relationship between KM and innovation performance.

Originality/value

Theoretically, this study establishes how KM practices influence innovation performance. Organizations that practice organizational learning and creativity are more likely to benefit from increased innovation outcomes through KM practices.
Teaching vocational pupils in their pyjamas: a socio-material perspective on challenges in the age of Covid-19
Authors: Carlsson Sandra , K Flensner Karin , Svensson Lars , Willermark Sara
Published: September 2022

Purpose

Due to the global outbreak of Covid-19, Swedish teachers in upper secondary education were forced to conduct emergency remote teaching. As of today, there is a stream of research that addresses digitalization in education in light of the pandemic. Previous studies show that the challenges with the sudden intensification of digitalization have been particularly challenging in practical and aesthetic subjects. The research question is as follows: What challenges did vocational teachers experience during the emergency remote teaching caused by Covid-19 and what emergent tactics can be identified in vocational teaching practice?

Design/methodology/approach

The empirical data consists of (1) interviews with two vocational teachers and, (2) workshops with 25 teacher students from different vocational programmes that addressed vocational teaching during the Covid-19 crisis.

Findings

Emergency remote teaching meant challenges due to the changed socio-material environment that cannot easily be transformed to a vocational teaching setting. The challenges were related to authentic situations and material, problem solving and dexterity. Tactics that emerged as a response to the challenges were mainly connected to attempts to mimic vocational practices.

Originality/value

Contributions include explaining specific challenges and possibilities in developing vocational competence when teaching is digitalised. Furthermore, it increases the understanding of the relationship between theory and practice in vocational education. By adopting a socio-material perspective on vocational competence, the authors enhance the understanding of the importance of a shared socio-material environment.
Quality of digital learning experiences – effective, efficient, and appealing designs?
Authors: Jahnke Isa
Published: September 2022

Purpose

Fischeret al.(2022) present a framework for rethinking education, including broad design components such as learning-on-demand or learning takes place in the context of authentic problems. How can we bring those design components into practice? I argue that the design of innovative learning approaches for the digital age requires rigor in design and evaluation methods to understand what works and how and why it works.

Design/methodology/approach

This case study applies the research to improve approach with iterative steps of design, development, and formative evaluation. Methods to be used are borrowed from user and learning experience fields, such as usability studies and pre/posttests for learning growth.

Findings

The design of digital learning experiences rests on the three dimensions of how learners interact with (a) the digital tool, space, or service; (b) the pedagogical elements of goals, activities, and assessments (e.g. sense-making); and (c) other peers or instructors.

Originality/value

This study provides useful insights on how to conduct research to improve versus research to prove and indicates the importance of data analysis related to the effectiveness, efficiency, and attractiveness of digital learning experiences. Learning experience design and research provides sufficient rigor and could play an important role in new ways of learning in the digital age with the goal “making learning a part of life” as pointed out by Fischeret al.(2022).
Application of multilevel confirmatory factor analysis to compositional organizational constructs
Authors: Ganesh Pavithra , Srivastava Kailash B.L.
Published: September 2022

Purpose

This study aims to offer insight into the applications of multilevel confirmatory factor analysis (MCFA) within organizational behavior (OB) studies, specifically when using macro-level variables. The authors used the case of a scale measuring collective organizational engagement (COE) to demonstrate the advantage of using MCFA over single-level confirmatory factor analysis (CFA).

Design/methodology/approach

The authors used nine referent-shift items to measure the dimensions of physical, cognitive and emotional COE. MCFA and aggregated single-level CFA were conducted on the same data using Mplus V. 8.7, and the authors compared standardized parameters from the two techniques.

Findings

The results indicate a three-factor model of collective engagement with CFA and MCFA. Stronger item loadings, factor correlations and composite reliability were found in the MCFA within-level model compared to aggregated CFA model. MCFA also supported an alternate one-factor-between-three-factors-within model of COE.

Originality/value

This study demonstrates the application of MCFA with a compositional construct and compares MCFA to mean-aggregated single-level CFA. It presents a case for OB researchers to apply MCFA to compositional constructs as a best methodological practice.
The variation of family business branding across stakeholders: a social identity perspective
Authors: Zhang Zhibiao , Rowan Peter
Published: September 2022

Purpose

Family business brand communication depends on the effect of the family on stakeholders' perception, and the family influences stakeholders differently, raising the question of whether family business branding varies across stakeholders. Drawing on social identity theory, this research classifies a family firm's stakeholders into family (in-group) and non-family (out-group) stakeholders and explores the communication of family business brands to these two groups of stakeholders.

Design/methodology/approach

Data for this research were gathered from a questionnaire survey of 327 Chinese family firms.

Findings

The results show that family business brand communication differs between family and non-family stakeholders. Additionally, family harmony has a positive relationship with family business branding to family stakeholders and an inverse U-shaped relationship with family business branding to non-family stakeholders.

Originality/value

This research is the first to demonstrate that family business brand communication varies across stakeholders and that the effect of family characteristics (family harmony in this research) on family business branding differs between stakeholders. In addition, it expands the scope of the out-group in family firms to embrace all non-family stakeholders and suggests an intergroup opposition between family and non-family stakeholders, which is important for advancing family firm theory.