Strand Separation Research Articles

Managerial talent and managerial practices: Are they complements?

We examine the role of managerial talent and its interaction with managerial practices in determining firm performance. We build a matched firm-director panel dataset for the universe of limited liability companies in Italy, tracking directors across different firms over time. We define managerial talent as the individual contribution to the variation of the firms’ total factor productivity, estimated using a two-way fixed effects model. Combining the data with survey information on a representative sample of firms, we then document that our measure of talent correlates with ex-ante and ex-post indicators of ability, i.e. managers’ educational attainment and their forecast precision with respect to the firm’s future performance. Most important, we leverage information on the adoption of managerial practices within the firm to examine potential synergies between managerial talent and structured managerial practices, thus bridging two separate strands of the literature. While talent and structured practices are positively associated with firm productivity on their own, there is evidence of complementarities between the two. These findings hold both in a cross-sectional setting and in a panel analysis that accounts for time-invariant firm heterogeneity. Overall, our results indicate that the effectiveness of managerial practices varies with managers’ ability to implement them.

Probing telomeric-like G4 structures with full or partial 2′-deoxy-5-hydroxyuridine substitutions

Guanine quadruplexes (G4s) are stable four-stranded secondary DNA structures held together by noncanonical G-G base tetrads. We synthesised the nucleoside analogue 2′-deoxy-5-hydroxyuridine (H) and inserted its phosphoramidite into telomeric repeat-type model oligonucleotides. Full and partial substitutions were made, replacing all guanines in all the three tetrads of a three-tier G4 structure, or only in the putative upper, central, or lower tetrads. We characterised these modified structures using CD, UV absorbance spectroscopy, native gel studies, and a capture oligo-based G4 disruption kinetic assay. The strand separation activity of BLM helicase on these substituted structures was also investigated. Two of the partially H-substituted constructs adopted G4-like structures, but displayed lower thermal stabilities compared to unsubstituted G4. The construct modified in its central tetrad remained mostly denatured, but the possibility of a special structure for the fully replaced variant remained open. H substitutions did not interfere with the G4-resolving activity of BLM helicase, but its efficiency was highly influenced by construct topology and even more by the G4 ligand PhenDC3. Our results suggest that the H modification can be incorporated into G quadruplexes, but only at certain positions to maintain G4 stability. The destabilizing effect observed for 2′-deoxy-5-hydroxyuridine indicates that the cytosine deamination product 5-hydroxyuracil and its nucleoside counterpart in RNA (5-hydroxyuridine), might also be destabilizing in cellular DNA and RNA quadruplexes. The kinetic assay employed in this study can be generally employed for a fast comparison of the stabilities of various G4s either in their free or ligand-bound states.

Retention behavior of short double-stranded oligonucleotide and its potential impurities by anion-exchange chromatography under non-denaturing conditions

Small interfering RNA (siRNA), consisting of two complementary single-stranded RNAs with overhanging bases, is being adopted as a potent and specific inhibitor of target gene expression. However, non-duplexed single strands and undesired double strands composed of impurities (e.g., n-1 mer) could be produced in addition to the target double strand in the siRNA manufacturing process. Compared to the liquid chromatography analysis of single strands, the analysis of the duplexes under non-denaturing conditions is challenging, since restricted chromatographic conditions are required to maintain the Watson-Crick hydrogen bonds. This study reports the analysis of double-stranded oligomers having approximately 20 base pairs with some overhanging bases as non-denatured forms by anion-exchange chromatography (AEX). Optimization of the chromatographic conditions could potentially achieve the adequate separation of excess single strands from the double strand. Non-optimal duplexes, such as duplexes with long overhangs or bulge structures, were prepared by intentionally deleting terminal or middle nucleotide(s) of either the sense or the antisense strand, and these non-optimal duplexes were eluted at different retention times in most of the cases. Interestingly, under alkaline chromatographic conditions (pH 9.0), non-optimal duplexes containing a shortmer tended to exhibit a stronger retention than their parent duplexes, although they possessed a less negative charge. This study demonstrated some retention behavior of double strands with overhangs by AEX under non-denaturing conditions.

Cycling as work: mobility and informality in Indian cities

Set against the marginalization of cycling in Kolkata and Delhi, this article shows how cargo-cyclists and cycle rickshaw pullers make productive contributions to urban economies and negotiate constraints to their mobility in Indian cities. As cheap vehicles, bicycles and cycle rickshaws not only provide opportunities for social and economic mobility, but also contribute to, generate, and sustain vital urban economies. Cycle workers ensure the smooth transportation of goods, people, and services in Indian cities. Situating cycle work at the crossroads of anthropology and urban planning, this article demonstrates how the interdependence of urban economies, regulation of space, and constraints to everyday mobility advances knowledge on contemporary Indian cities. Instead of seeing these spheres as separate strands of investigation and analysis, we suggest that cycling as work draws them together.

Genome wide hypomethylation and youth-associated DNA gap reduction promoting DNA damage and senescence-associated pathogenesis

Introduction: The United States currently faces two opioid crises, an evolved crisis currently manifesting as widespread abuse of illicit opioids, and a crisis in pain management largely manufactured by the Centers for Disease Control and Prevention 2016 Guideline. Our goal in this paper is to identify root causes, trace the trajectory of forces unleashed over time, and define potential solutions to these crises. Methods: Analytic review of the scientific, socioeconomic, and historical literature. Background: Age-associated epigenetic alteration is the underlying cause of DNA damage in aging cells. Two types of youth-associated DNA-protection epigenetic marks, global methylation, and youth-associated genomic stabilization DNA gap (youth-DNA-gap) reduce when cell ages. The epigenomic mark reduction promotes DNA damage and accelerates aging hallmarks. While DNA hypomethylation destabilizes DNA by several mechanisms, the DNA sequence around the youth-DNA-gap is hypermethylated. Therefore, the genomic instability mechanisms underlying DNA hypomethylation and youth-DNA-gap reduction are linked. Results: DNA gap prevents DNA damage by relieving the torsion forces caused by a twisted wave during DNA strand separation by transcription or replication. When the cells begin to age, hypomethylation and youth-DNA-gap reduction can occur as consequences of the efflux of intranuclear HMGB1. The methylated DNA gaps are formed by several proteins. Box A of HMGB1 possesses a molecular scissor role in producing youth-DNA-gaps. So the lack of a gap-producing role of HMGB1 results in a youth-DNA-gap reduction. The histone deacetylation role of SIRT1, an aging prevention protein, prevents DNA ends of youth-DNA-gaps from being recognized as pathologic DNA breaks. Youth-DNA-gaps are methylated and determined genome distribution by AGO4, an effector protein in RNA-directed DNA methylation. The lack of intranuclear HMGB1 promotes global hypomethylation due to two subsequent mechanisms. First is the loss of AGO4-methylating DNA. The other is the accumulation of DNA damage due to lacking HMGB1-produced DNA gap promoting DNA demethylation while undergoing DNA repair. DNA torsion due to youth-DNA-gap reduction increases DNA damage and, consequently, the DNA damage response (DDR). Persistent DDR promotes cellular senescence. Accumulating senescent cells leads to the deterioration of the structure and function of the human body. Rejuvenating DNA (RED) by adding DNA protection epigenetic marks using genomic stability molecule (GEM) such as box A of HMGB1 increases DNA durability, limits DNA damage, rejuvenates senescence cells, and improves organ structure and function deterioration due to aging. Conclusion: Reducing youth-associated epigenetic marks is degenerative diseases' primary molecular pathogenesis mechanism. REDGEM is a new therapeutic strategy inhibiting the upstream molecular aging process that will revolutionize the treatment of DNA damage or age-associated diseases and conditions.

Comparative analysis of chlorambucil-induced DNA lesion formation and repair in a spectrum of different human cell systems.

Chlorambucil (CLB) belongs to the class of nitrogen mustards (NMs), which are highly reactive bifunctional alkylating agents and were the first chemotherapeutic agents developed. They form DNA interstrand crosslinks (ICLs), which cause a blockage of DNA strand separation, inhibiting essential processes in DNA metabolism like replication and transcription. In fast replicating cells, e.g., tumor cells, this can induce cell death. The upregulation of ICL repair is thought to be a key factor for the resistance of tumor cells to ICL-inducing cytostatic agents including NMs. To monitor induction and repair of CLB-induced ICLs, we adjusted the automated reversed fluorometric analysis of alkaline DNA unwinding assay (rFADU) for the detection of ICLs in adherent cells. For the detection of monoalkylated DNA bases we established an LC-MS/MS method. We performed a comparative analysis of adduct formation and removal in five human cell lines and in peripheral blood mononuclear cells (PBMCs) after treatment with CLB. Dose-dependent increases in adduct formation were observed, and suitable treatment concentrations were identified for each cell line, which were then used for monitoring the kinetics of adduct formation. We observed significant differences in the repair kinetics of the cell lines tested. For example, in A2780 cells, hTERT immortalized VH10 cells, and in PBMCs a time-dependent repair of the two main monoalkylated DNA-adducts was confirmed. Regarding ICLs, repair was observed in all cell systems except for PBMCs. In conclusion, LC-MS/MS analyses combined with the rFADU technique are powerful tools to study the molecular mechanisms of NM-induced DNA damage and repair. By applying these methods to a spectrum of human cell systems of different origin and transformation status, we obtained insight into the cell-type specific repair of different CLB-induced DNA lesions, which may help identify novel resistance mechanisms of tumors and define molecular targets for therapeutic interventions.

Pressure-less spark plasma sintering of 3D-plotted titanium porous structures

Additive manufacturing of titanium porous structures by direct ink writing involves the removal of the binder needed for powder extrusion and subsequent sintering to consolidate the 3D-plotted body. In this work, pressure-less spark plasma sintering (PL-SPS) was systematically studied for fast consolidation of titanium porous structures. Furthermore, poloxamer 407 was used as the binder and the lowest temperature possible was identified for its thermal elimination. The results show for the first time that PL-SPS generated sintering conditions similar to those generated by conventional pressure-less sintering, producing hierarchical titanium porous structures with equivalent densification, shrinkage, and surface roughness, but with minimal grain growth. The thermal responses of the die and material showed efficient radiation heat transfer, allowing fast heating (100 °C/min) of one sample per run, promoting the formation of sintering necks and powder densification in 10 min, which is much faster than conventional sintering that requires at least 2 h of dwell time. However, the process operates at a sintering temperature 200–300 °C above the conventional sintering temperature, and at the expense of high consumption of electrical energy to achieve such a high heating rate. The mechanical strength of the resulting titanium structures increases with increasing strand densification at nearly constant strand separation, resulting in strong and plastic porous structures.

Development of a visual multiplex fluorescent LAMP assay for the detection of foot-and-mouth disease, vesicular stomatitis and bluetongue viruses.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that can be used to amplify target genes at a constant temperature, and it has several advantages, including convenience, specificity and sensitivity. However, due to the special interpretation methods of this technology for reaction results, all the previously reported LAMP detection methods have been restricted to identifying a single target, which limits the application of this technology. In this study, we modified conventional LAMP to include a quencher-fluorophore composite probe complementary to the F1c segment of the inner primer FIP; upon strand separation, a gain in the visible fluorescent signal was observed. The probes could be labeled with different fluorophores, showing different colors at the corresponding wavelengths. Therefore, this multiplex LAMP (mLAMP) assay can simultaneously detect 1-3 target sequences in a single LAMP reaction tube, and the results are more accurate and intuitive. In this study, we comprehensively demonstrated a single-reaction mLAMP assay for the robust detection of three cattle viruses without nonspecific amplification of other related pathogenic cattle viruses. The detection limit of this mLAMP assay was as low as 526-2477 copies/reaction for the recombinant plasmids. It is expected that this mLAMP assay can be widely used in clinical diagnosis.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

Sequence-specific remodeling of a topologically complex RNP substrate by Spb4.

DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-specific remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize Spb4 in a post-catalytic, high-energy intermediate that drives the organization of the three-way junction at the base of rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.

Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging.

SMC complexes play key roles in genome maintenance, where they ensure efficient genome replication and segregation. The SMC complex Smc5/6 is a crucial player in DNA replication and repair, yet many molecular features that determine its roles are unclear. Here, we use single-molecule microscopy to investigate Smc5/6's interaction with DNA. We find that Smc5/6 forms oligomers that dynamically redistribute on dsDNA by 1D diffusion and statically bind to ssDNA. Using combined force manipulation and single-molecule microscopy, we generate ssDNA-dsDNA junctions that mimic structures present in DNA repair intermediates or replication forks. We show that Smc5/6 accumulates at these junction sites, stabilizes the fork, and promotes the retention of RPA. Our observations provide a model for the complex's enrichment at sites of replication stress and DNA lesions from where it coordinates the recruitment and activation of downstream repair proteins.

Religious accommodation, agonism, agnosticism in healthcare: A commentary on Joshua Hordern, 'Accommodating religion and belief in healthcare: Political threats, agonistic democracy and established religion'.

Religious accommodation, agonism, agnosticism in healthcare: A commentary on Joshua Hordern, 'Accommodating religion and belief in healthcare: Political threats, agonistic democracy and established religion'.

Scale Compromise: Three Challenges of Large-Scale but Citizen-Driven Urban Planning

Amongst various urban crises, some fundamental ones require long-term policies and large-scale developments to be effectively mitigated. Since multiple government-led large-scale projects raised more and more public opposition, the question whether such grand endeavors can be approached by citizen-driven initiatives became urgent. However, large-scale urban planning issues are still believed to be too big, too complex, and too difficult to be solved from the bottom up. This paper examines such recurring belief—here termed the “scale compromise”—and conceptualizes it across two essential dimensions of urban planning: spatial and political. The scale compromise is presented twofold, as each side of the compromise is embedded within a separate strand of urban planning practice: large-scale urban planning as traditionally associated with the authority-led approach, and citizen-driven urbanism as traditionally linked with locality. Regarding each approach, respectively, the causes, consequences, and difficulties of the scale compromise are discussed from both perspectives. Finally, by breaking down the resulting compromise across three integral aspects of planning—the socio-economic, organizational, and technical—the “scale compromise” conceptual framework indicates three scale-related challenges. Accordingly, this paper argues why all the three challenges must be met concurrently, as failing at one squanders the efforts made for the others. Finally, some recent experience in the development of urban greenway projects in the U.S. are discussed as promising points of reference for dealing with the scale compromise and for seeking new solutions to fundamental urban crisis.

Proton transfer during DNA strand separation as a source of mutagenic guanine-cytosine tautomers

Proton transfer between the DNA bases can lead to mutagenic Guanine-Cytosine tautomers. Over the past several decades, a heated debate has emerged over the biological impact of tautomeric forms. Here, we determine that the energy required for generating tautomers radically changes during the separation of double-stranded DNA. Density Functional Theory calculations indicate that the double proton transfer in Guanine-Cytosine follows a sequential, step-like mechanism where the reaction barrier increases quasi-linearly with strand separation. These results point to increased stability of the tautomer when the DNA strands unzip as they enter the helicase, effectively trapping the tautomer population. In addition, molecular dynamics simulations indicate that the relevant strand separation time is two orders of magnitude quicker than previously thought. Our results demonstrate that the unwinding of DNA by the helicase could simultaneously slow the formation but significantly enhance the stability of tautomeric base pairs and provide a feasible pathway for spontaneous DNA mutations.

The effects of health shocks on risk preferences: Do personality traits matter?

Older individuals hold a disproportionate amount of total wealth, and are particularly vulnerable to shocks to health. Accordingly, there is a interest in understanding the extent to which health detriments influence financial choices and portfolio holdings within this group of society. A separate strand of literature has recently focused on the role of non-cognitive skills, and in particular personality traits, in shaping attitudes towards risk. We combine these literatures to explore the extent to which unantici pated shocks to health display heterogeneous impacts on preferences towards financial risk via portfolio investments and stock market participation. We find that health shocks have a negative effect on the level of risk at the household level when men, but not women, experience the shock. Moreover, there appears to be heterogeneity in the response by personality trait. Households where men display dominant traits for neuroticism, extraversion and openness to experience tend to be most affected in investment decisions following a health shock. The household becomes less risk tolerant when the level of neuroticism or openness increases, and more risk tolerant when the level of extroversion of the man increases.

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics

Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

Precarious Employment during the COVID-19 Pandemic, Disability-Related Discrimination, and Mental Health

Drawing on separate strands of research documenting the psychological consequences of (a) precarious employment and other challenges associated with the COVID-19 pandemic and (b) ableism, this study incorporates both into an examination of disability-based differences in the joint significance of discrimination and work precarity during the pandemic for mental health. Analyses utilizing data from a regional survey of people with and without disabilities in the Intermountain West (N = 2,012) provide evidence that precarious employment, greater discrimination, and disability independently predict depressive symptoms. Further, in the context of greater discrimination, more precarious employment is found to have greater significance for people with disabilities compared to those who are not currently disabled. These findings challenge us to think about how we engage in research concerning ableism and macro-level stressors, and underscore the role of power structures and positionality in shaping the psychological impact of employment challenges experienced during the pandemic.

Evolutionary Invariant of the Structure of DNA Double Helix in RNAP II Core Promoters.

Eukaryotic and archaeal RNA polymerase II (POL II) machinery is highly conserved, regardless of the extreme changes in promoter sequences in different organisms. The goal of our work is to find the cause of this conservatism. The representative sets of aligned promoter sequences of fifteen organisms belonging to different evolutional stages were studied. Their textual profiles, as well as profiles of the indexes that characterize the secondary structure and the mechanical and physicochemical properties, were analyzed. The evolutionarily stable, extremely heterogeneous special secondary structure of POL II core promoters was revealed, which includes two singular regions—hexanucleotide “INR” around TSS and octanucleotide “TATA element” of about −28 bp upstream. Such structures may have developed at some stage of evolution. It turned out to be so well matched for the pre-initiation complex formation and the subsequent initiation of transcription for POL II machinery that in the course of evolution there were selected only those nucleotide sequences that were able to reproduce these structural properties. The individual features of specific sequences representing the singular region of the promoter of each gene can affect the kinetics of DNA-protein complex formation and facilitate strand separation in double-stranded DNA at the TSS position.

Metal-Organic Framework-Loaded Engineering DNAzyme for the Self-Powered Amplified Detection of MicroRNA.

DNAzyme shows great promise in designing a highly sensitive and specific sensing platform; however, the low cellular uptake efficiency, instability, and especially the insufficient cofactor supply inhibit the intracellular molecule sensor applications. Herein, we demonstrate a novel type of DNAzyme-based self-driven intracellular sensor for microRNA (miRNA) detection in living cells. The sensor consists of a metal-organic framework [zeolite imidazole framework (ZIF-8)] core loaded with a shell consisting of a rationally designed DNAzyme, where the substrate strand is modified with FAM and BHQ-1 nearby both the sides of the restriction site, respectively, while the enzyme strand consists of two separate strands with a complementary fragment to the substrate strand and the targeting miRNA, respectively. The ZIF-8 nanoparticles enable the efficient delivery of DNAzyme into the cell and protect the DNAzyme from degradation. The pH-responsive ZIF-8 degradation is accompanied with the release of the DNAzyme and Zn2+ cofactors, and the intracellular target miRNAs recognize and activate the DNAzyme driven by the Zn2+ cofactors to cleave the substrate strand, resulting in the release of the FAM-labeled shorter product strand and increased fluorescence for miRNA detection. The self-driven approach can be generally applied to various miRNAs' detection through DNAzyme engineering.

More-than photography and sculpture: a diffractive reading

In historical and theoretical writings on photography and sculpture, the two media are mostly addressed separately, with some points of overlap. The aim of this article is to examine artworks by contemporary artists Rachel de Joode and artist duo Nerhol from the hypothesis that they are not either photography or sculpture, but both and even more. They occupy new spaces, both material/physical and conceptual, reaching dimensions that are more-than: more than photography and more than sculpture. I aim for a renewed understanding of these media through diffractive reading, an approach with roots in feminist and new materialist discourse. Diffractive reading is about understanding things through each other, rather than finding meaning through preconceived static oppositions. As the artworks analyzed are understood as material-discursive practices, the theoretical and historical backgrounds of both photography and sculpture become increasingly relevant. How can and how should photography and sculpture be theorized as expanding fields? How do their ways of making meaning differ from each other and how do they converge? Through diffractive reading, photography and sculpture are not understood as two separate strands of practice coming together, but rather as manifestations of hybrid identities within the same artwork.