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Spanish Language and Sociolinguistic Analysis -- Editorial page -- Title page -- LCC data -- Dedication page -- Table of contents -- Acknowledgments -- Introduction -- References -- Quantitative analysis in language variation and change -- 1. Distributional analysis -- 1.1 Statistical modeling -- 1.2 The three lines of evidence -- 1.2.1 Statistical significance -- 1.2.2 Constraints -- 1.2.3 Strength -- 2. The case study - variable (that) -- 3. Goldvarb logistic regression -- 4. New statistical tools -- 4.1 The effect of the individual -- 4.2 Independence of observations -- 4.3 How to find interaction in a variable rule analysis -- 4.4 The kitchen sink -- 4.5 Tokens per individual -- 4.6 Tokens per cell -- 4.7 Type/token ratio -- 4.8 The pre-statistical toolkit -- 5. Drawbacks to the variable rule program -- 6. New toolkits for Variationist Sociolinguistics -- 6.1 Rbrul -- 6.2 Basic Rbrul steps -- 6.3. Mixed effects modeling -- 6.4 R -- 6.5 Basic R steps -- 6.5.1 Mixed effects - R -- 6.5.2 Checking for interaction - R -- 6.5.3 Comparison across tools -- 6.5.4 Conditional inference trees -- 7. Practical advice -- 8. Summary -- References -- Combining population genetics (DNA) with historical linguistics -- 1. Introduction -- 2. Palenque: Location and brief description -- 3. Palenque and its African origins -- 3.1 Background -- 3.2 Scholarship into Palenque's African past: Early assumptions and a priori limitations -- 3.3 Palenque's African past: First attempts at narrowing down origins -- 3.4 Palenque's African past: The 1980s to mid-1990s -- 3.5 The growing centrality of Kikongo and connections to Afro-Cuban ritual language -- 3.6 A revelation: It is all Kikongo! -- 4. The state of the discipline from the mid-1990s to 2010 -- 4.1 The monogenetic Kikongo hypothesis around 2010: Lingering doubts

Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. The volume of the flow cell can be controlled by modifying the dimensions of the channel while the reagents are introduced using a syringe pump. In this system, high flow rates disturb the biological system, whereas lower flow rates lead to the generation of a reagent gradient in the flow cell. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. We propose the implementation of multistream laminar microfluidic cells with two inlets and one outlet, which achieve a minimum fluid switching time of 0.25 s. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level. ; European Research Council (ERC) under the European Union Horizon 2020 research and innovation (grant agreement No 681299). The work in the Moreno-Herrero laboratory was also supported by a Spanish Ministry of Economy and Competitiveness Grant, BFU2017-83794-P (AEI/FEDER, UE) to F.M.-H., and Comunidad de Madrid Grants, Tec4Bio – S2018/NMT-4443 and NanoBioCancer – Y2018/BIO-4747 to F.M.-H. J.M.-M. acknowledges support for pre-doctoral fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174). ; Peer reviewed

The mechanical properties of double-stranded RNA (dsRNA) are involved in many of its biological functions and are relevant for future nanotechnology applications. DsRNA must tightly bend to fit inside viral capsids or deform upon the interaction with proteins that regulate gene silencing or the immune response against viral attacks. However, the question of how the nucleotide sequence affects the global mechanical properties of dsRNA has so far remained largely unexplored. Here, we have employed state-of-the-art atomistic molecular dynamics simulations to unveil the mechanical response of different RNA duplexes to an external force. Our results reveal that, similarly to dsDNA, the mechanical properties of dsRNA are highly sequence-dependent. However, we find that the nucleotide sequence affects in a strikingly different manner the stretching and twisting response of RNA and DNA duplexes under force. We find that the elastic response of dsRNA is dominated by the local high flexibility of pyrimidine-purine steps. Moreover, the flexibility of pyrimidine-purine steps is independent of the sequence context, and the global flexibility of the duplex reasonably scales with the number of this kind of base-pair dinucleotides. We conclude that disparities of the mechanical response of dinucleotides are responsible for the differences observed in the mechanical properties of RNA and DNA duplexes. ; Financial support from the Spanish MINECO(projects MDM-2014-0377, MAT2017-83273-R (AEI/FEDER,UE), and BFU2017-83794-P (AEI/FEDER, UE)). F. M.-H.acknowledges support from European Research Council (ERC)under the European Union Horizon 2020 research and inno-vation (grant agreement no. 681299). ; Peer reviewed

Multiple biological processes involve the stretching of nucleic acids (NAs). Stretching forces induce local changes in the molecule structure, inhibiting or promoting the binding of proteins, which ultimately affects their functionality. Understanding how a force induces changes in the structure of NAs at the atomic level is a challenge. Here, we use all-atom, microsecond-long molecular dynamics to simulate the structure of dsDNA and dsRNA subjected to stretching forces up to 20 pN. We determine all of the elastic constants of dsDNA and dsRNA and provide an explanation for three striking differences in the mechanical response of these two molecules: the threefold softer stretching constant obtained for dsRNA, the opposite twist-stretch coupling, and its nontrivial force dependence. The lower dsRNA stretching resistance is linked to its more open structure, whereas the opposite twist-stretch coupling of both molecules is due to the very different evolution of molecules' interstrand distance with the stretching force. A reduction of this distance leads to overwinding in dsDNA. In contrast, dsRNA is not able to reduce its interstrand distance and can only elongate by unwinding. Interstrand distance is directly correlated with the slide base-pair parameter and its different behavior in dsDNA and dsRNA traced down to changes in the sugar pucker angle of these NAs. ; We thank the Spanish Ministry of Economy and Competitiveness (Projects CSD2010-00024, MAT2014-54484-P, and FIS2014-58328-P) for financial support. F.M.-H. received support from the European Research Council under the European Union's Horizon 2020 Framework Programme for Research and Innovation (Grant 681299). ; Peer reviewed

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA:protein interactions. In particular, combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of the flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation. ; We thank the financial support from the Spanish MINECO (FIS2014-58328-P) and from (ERC) under the European Union's Horizon2020 Research and Innovation programme (grant agreement No 681299). J. M. M. acknowledges a Predoctoral PhD fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174). ). G. L. M. F was supported by the Biotechnology and Biological Sciences Research Council (1363883). M. S. D was supported by the Wellcome Trust (100401 and 077368). ; Peer reviewed

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented. ; Ministerio de Economía y Competitividad (MINECO) [BFU2017-83794-P (AEI/FEDER, UE) to F.M.-H.]; European Research Council (ERC) under the European Union Horizon 2020 research and innovation grant agreemen t[681299 to F.M.-H.]; National Science Foundation [toS.H.L.]. C.C. was supported by a 'Severo Ochoa' postdoc-toral contract from the National Center of Biotechnology (CNB-CSIC); Funding for open access charge: EuropeanResearch Council [681299]. ; Peer reviewed

Bacillus subtilisParB forms multimeric networks involving non-specific DNA bindingleading to DNA condensation. Previously, we found that an excess of the free C-terminal domain(CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assemblednetworks (Fisher et al., 2017). However, interpretation of the molecular basis for this phenomenonwas complicated by our inability to uncouple protein binding from DNA condensation. Here, wehave combined lateral magnetic tweezers with TIRF microscopy to simultaneously control therestrictive force against condensation and to visualise ParB protein binding by fluorescence. Atnon-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA.Our new approach concluded that the free CTD blocks the formation of ParB networks byheterodimerisation with full length DNA-bound ParB. This strongly supports a model in which theCTD acts as a key bridging interface between distal DNA binding loci within ParB networks. ; Financial support from AEI/FEDER, UE (BFU2017-83794-P) and from European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation programme (grant agreement no. 681299). JM-M acknowledges a predoctoral PhD fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref.PRE_2013_11_1174). GLMF was supported by the Biotechnology and Biological Sciences Research Council (1363883). MSD was supported by the Wellcome Trust (100401 and 077368). ; Peer reviewed

Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. Previously, we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks (Fisher et al., 2017). However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualise ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerisation with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks. ; European Research Council (ERC) (Grant agreement no. 681299). ; Basque Country Government Department of Education, Language Policy and Culture.(ref.PRE_2013_11_1174). ; Biotechnology and Biological Sciences Research Council. (1363883). ; Wellcome Trust (100401 and 077368). ; Peer reviewed

Human DNA helicase B (HELB) is a poorly characterized helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single-molecule approaches to characterize the biochemical activities of HELB protein with a particular focus on its interactions with Replication Protein A (RPA) and RPA–single-stranded DNA (ssDNA) filaments. HELB is a monomeric protein that binds tightly to ssDNA with a site size of ∼20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′ to 3′ direction accompanied by the formation of DNA loops. HELB also displays classical helicase activity, but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA, which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from ssDNA. This activity, which can allow other proteins access to ssDNA intermediates despite their shielding by RPA, may underpin the diverse roles of HELB in cellular DNA transactions. ; [Significance] Single-stranded DNA (ssDNA) is a key intermediate in many cellular DNA transactions, including DNA replication, repair, and recombination. Nascent ssDNA is rapidly bound by the Replication Protein A (RPA) complex, forming a nucleoprotein filament that both stabilizes ssDNA and mediates downstream processing events. Paradoxically, however, the very high affinity of RPA for ssDNA may block the recruitment of further factors. In this work, we show that RPA–ssDNA nucleoprotein filaments are specifically targeted by the human HELB helicase. Recruitment of HELB by RPA–ssDNA activates HELB translocation activity, leading to processive removal of upstream RPA complexes. This RPA clearance activity may underpin the diverse roles of HELB in replication and recombination. ; Work in the laboratory of M.S.D. was supported by an Elizabeth Blackwell Early Career Fellowship from the University of Bristol (to O.J.W.) and Wellcome Trust Investigator Grant 100401/Z/12/Z (to M.S.D.). Work in the laboratory of E.A. was supported by NIH Grants GM130746 (to E.A.) and GM133967 (to E.A.). F.M.-H. acknowledges support from the European Research Council under European Union Horizon 2020 Research and Innovation Program Grant Agreement 681299. Work in the laboratory of F.M.-H. was also supported by Spanish Ministry of Science and Innovation Grants BFU2017-83794-P (AEI/FEDER, UE; to F.M.-H.) and PID2020-112998GB-100 (AEI/10.13039/501100011033; to F.M.-H.) and Comunidad de Madrid Grants Tec4-Bio–S2018/NMT-4443 (to F.M.-H.) and NanoBioCancer–Y2018/BIO-4747 (to F.M.-H.).

Double-stranded (ds) RNA mediates the suppression of specific gene expression, it is the genetic material of a number of viruses, and a key activator of the innate immune response against viral infections. The ever increasing list of roles played by dsRNA in the cell and its potential biotechnological applications over the last decade has raised an interest for the characterization of its mechanical properties and structure, and that includes approaches using Atomic Force Microscopy (AFM) and other single-molecule techniques. Recent reports have resolved the structure of dsDNA with AFM at unprecedented resolution. However, an equivalent study with dsRNA is still lacking. Here, we have visualized the double helix of dsRNA under near-physiological conditions and at sufficient resolution to resolve the A-form sub-helical pitch periodicity. We have employed different high-sensitive force-detection methods and obtained images with similar spatial resolution. Therefore, we show here that the limiting factors for high-resolution AFM imaging of soft materials in liquid medium are, rather than the imaging mode, the force between the tip and the sample and the sharpness of the tip apex ; This work was supported by the European Research Council (grant 206117 SM-DNA-REPAIR to F.M.-H.), the Spanish Ministry of Economy and Competitiveness (grants FIS2014-58328-P and FIS2014-51481-ERC to F.M.-H., grant Consolider CSD2010-0024 to J.G.-H. and grant BFU2013-44202 to J.M.V.) and the Madrid Regional Government (grant MAT2013-46753-C2-2 to J.G.-H. and S2013/MIT-2807 to J.M.V.)