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UniClo technology exploits methylation for universal scarless DNA assembly
No full textSeveral Golden Gate-based DNA assembly techniques have been developed previously with different limitations including the requirement for domestication of sequences with internal sites for the type IIS restriction enzymes used, insertion of persistent scars in assembled DNA and the need for multiple assembly vectors and overhang sequences. We developed UniClo, which overcomes all these problems. Sequences with internal type IIS sites can be assembled, it allows fully scarless hierarchical assembly and requires only three assembly vectors and two universal overhang sequences. This is achieved by three key elements: (i) Recombinant methylases are used in vitro to methylate, and thus inactivate, any sites in fragments to be assembled for the type IIS restriction enzyme used in the assembly as well as the outer sites of the assembly vectors. (ii) A CRISPR-dCas9 molecule is used to protect the type IIS restriction enzyme sites required for the assembly from methylation, thus preserving the activity of these sites. (iii) A set of engineered vectors is used to trim overhangs that would otherwise generate scars. Here, we present a detailed protocol for performing DNA assembly using UniClo and describe the methylation-protection of the fragments to be assembled, methylation of the scarless vectors, the assembly reaction, and analysis of the final assembled DNA molecule. UniClo offers substantial flexibility in the assembly design and enables the assembly of any DNA molecule regardless of its sequence, nature and application
Longitudinal Epidemiology and Variant Dynamics of SARS-CoV-2 in Coastal Kenya (2020–2025): Clinical Features and Wave Patterns
No full textBackground: SARS-CoV-2 is a major cause of outpatient-attended acute respiratory infections (ARIs). Data from Africa are limited on SARS-CoV-2 infection, variants, symptom profile, and longitudinal trends for outpatient presentation. Methods: Starting December 2020, we established ARI surveillance at 5 outpatient clinics in coastal Kenya, recruiting ∼15 participants (any age) per week per clinic for SARS-CoV-2 testing and genome analysis. Participants provided respiratory samples, demographic details, and vaccination and symptom data. We compared SARS-CoV-2 clinical and molecular epidemiology before and during Omicron waves using multivariate logistic regression. Results: By February 2025, we had recruited 14 562 ARI cases, with 1053 (7.2%) testing positive for SARS-CoV-2. The median age of cases was 25 years (IQR, 15–41) and 65.0% were female. Nine infection waves were recorded, with positivity ranging 8.2% to 25.6%. Interwave intervals increased from ≤3 months in 2021 to ≥6 months in 2024. Sixty-eight PANGO lineages were identified from 782 (74.2%) sequenced cases, with 4 predominating local waves (AY.116, BQ.1.8, FY.4.1, LF.7.3.2), which were rare globally (<0.5%) during their detection period. Overall, common symptoms among positive cases were cough (91.5%), nasal discharge (76.7%), and fever (53.1%). Loss of sense of smell was strongly predictive of COVID-19 in the pre–Omicron era, but body malaise, sore throat, joint pain, and nasal discharge were predictive during the Omicron period. Conclusions: SARS-CoV-2 increasingly shows seasonal annual patterns in coastal Kenya, with its clinical features resembling established endemic respiratory viruses. Its case burden is most pronounced in young adults. Locally dominant genetic variants may differ from those globally
Dynamic count models with flexible innovation processes for irregular maritime migration
No full textMotivated by the challenge of analyzing the dynamics of weekly sea border crossings in the Mediterranean (2015–2025) and the English Channel (2018–2025), we develop a Bayesian dynamic framework for modeling heteroskedastic count time series. Building on theoretical considerations and empirical stylized facts, our approach utilizes a Poisson random walk model that allows for heavy-tailed innovations or stochastic volatility dynamics, while incorporating an explicit mechanism to separate structural from sampling zeros. Posterior inference is carried out via a straightforward Markov chain Monte Carlo algorithm. Applying this methodology to the Mediterranean and English Channel data, we compare alternative model specifications through a comprehensive out-of-sample density forecasting exercise. Evaluating each model using log predictive scores and empirical coverage up to the 99th percentile, we find strong evidence for stochastic volatility in the migration innovations, with these models producing well-calibrated forecasts even at extreme quantiles. Our framework can be used to develop risk indicators with direct policy implications for improving governance and preparedness for migration surges. More broadly, the methodology extends to other zero-inflated non-stationary count time series applications, including epidemiological surveillance and public safety incident monitoring
Fluctuating β‐sheet secondary structure in DS119 explains the small effects of backbone N‐amination on thermal stability
No full textThe miniprotein DS119 has been used as a model system to probe the effects of introducing residues with backbone N-amination into a region of parallel β-sheet secondary structure. Derivatives featuring backbone N-methylation have also been synthesized for comparison. As expected, N-methylation of Trp9 or Phe33, positioned on the outer edge of the β-strands, led to a reduction in the thermal stability of the protein. However, contrary to predictions, the N-amination of Trp9 or Phe33 did not lead to an increase in thermal stability of DS119. Refinement of the DS119 structure, using NOE restrained molecular dynamics simulations, shows that the β-sheet region is highly fluctuating in nature. Key interstrand hydrogen bonds have populations of 24%–77%, whereas others have populations of less than 10%. In this disordered β-sheet region, the altered conformational preferences arising from backbone N-amination therefore have only minimal effects. This study demonstrates how MD simulation refinement can identify important dynamical features in a protein structure that might be overlooked in standard protein structure determination protocols
Teacher-student relationships and mathematics achievement: the role of socioeconomic status and gender in Chile from a longitudinal and comparative perspective
No full textStudents’ relational experiences with their teachers are central in everyday school interactions, with important consequences for students’ cognitive and socioemotional outcomes. While extensive research explores strategies to enhance school climate, less is understood about the role of teacher-student relationships (TSR), particularly how they vary by student gender and socioeconomic status (SES). Notably, research exploring the differential effects of TSR on mathematics achievement for adolescent students, specifically considering gender and socioeconomic disparities, their evolution over time, and cross-national variations, remains scarce. This study explores gender and socioeconomic differences in students’ perceived relationships with their teachers, their association with mathematics achievement on three measurement occasions in Chile, and their differences with Peru, Paraguay, Spain, Sweden and Denmark in 2022. Drawing on school gender culture and Hoftede’s cultural values theories, we conduct a repeated cross-sectional analysis of data from the Programme for International Student Assessment (PISA) for the years 2009, 2012, and 2022. Using ordinary least square (OLS) regression models, our findings indicate that SES does not moderate the association between gender and TSR in Chile, consistent with Peru, Paraguay, Spain, Sweden, and Denmark. The gender gap in TSR widened in favour of males in PISA 2012 and 2022, with no significant intersectionality between gender and SES in TSR over time in Chile. Boys experience enhanced mathematics achievement with better TSR, especially in Paraguay, Spain, and Denmark, but not in Chile, Peru, or Sweden. In Spain and Sweden, socioeconomically advantaged students benefit more academically from positive TSR, evidenced by higher mathematics achievement, a pattern not observed in Chile, Paraguay, or Denmark. From PISA 2009 to 2022 in Chile, an intensification in the magnitude of the association between TSR and mathematics achievement was observed. The results are discussed considering the recent educational policies implemented in Chile
Ectocervical Hypergranulation Secondary to Transcervical Coil Thread Perforation—An Unusual Cause for Persistent Postcoital Bleeding
No full textCervical perforation by coil threads is an uncommon complication. Predisposing factors may include prior cervical interventions, hypoestrogenic atrophy, thread material, and duration of use. Awareness is essential when evaluating abnormal bleeding or atypical thread presentation despite radiologically correct placement
Retracing the Response of Rangifer to Postglacial Climate Change in Arctic Islands
No full textRangifer tarandus L. plays a key role in Arctic ecosystems as the most numerous and widespread large herbivore. Sea ice is vital for maintaining genetic connectivity in Arctic islands, yet the historical role of sea ice in shaping R. tarandus biogeography is unknown. We studied the role of sea ice changes and ice sheet retreat since the last glacial period in the timing of island dispersal. We compiled published datasets of mitochondrial control region sequences that informed population history scenarios, which were evaluated in a coalescent‐based approximate Bayesian computation (ABC) modelling framework to test hypotheses of island (re)colonisation and to estimate divergence and admixture. Population events were compared with modelled and proxy‐based paleo‐sea ice cover and published ice sheet chronologies. Our analysis supports Holocene dispersal onto deglaciated Arctic islands, rather than High Arctic glacial refugia. The degree of population admixture and the effect of sea ice were dependent on regional geography and climate history. North American initial island population divergence occurred as sea ice cover was declining. A lack of strong genetic structure and the occurrence of late Holocene admixture suggest that Canadian Arctic Archipelago populations were somewhat connected by sea ice during the Holocene. The Svalbard, Franz Josef land, and West Greenland colonisations arose through long‐distance dispersal. Here, divergence times occurred post‐deglaciation but broadly align with subfossil‐based colonisation estimates, suggesting dispersal limitation due to sea ice conditions, potentially requiring appropriate ocean currents and sea ice drift directionality and speeds. Our study sheds light on the Late Quaternary (~60 ka—present) history of Arctic island Rangifer and suggests that ice sheet retreat, sea ice, and ocean currents were important in shaping present‐day genetic patterns. Regional differences in postglacial dynamics suggest that dispersal during contemporary climate change may vary regionally and depend upon diminishing connectivity provided by sea ice
GJA5 and ATP1A1 perturbations recapitulate inflammation-related beat irregularities in iPSC-based atrial myocardium tissue model
No full textAtrial fibrillation (AF) is the most common cardiac arrhythmia, linked to greater risk of heart failure, stroke and death. Inflammation has been connected to AF emergence, however mechanisms of inflammation-caused AF remain thus far elusive, leading to a lack of mechanism-based treatments. An isogenic, 3D tissue model containing hiPSC-derived atrial-like cardiomyocytes (aCM), cardiac fibroblasts (cfb), and cardiac macrophages was engineered using custom injection-molded pillar devices. Electrophysiological changes were examined via sharp electrode recordings, calcium imaging, and multi-electrode assays. Gene function was interrogated using siRNA knock-down, lentiviral overexpression, and pharmacological modulation. In silico tissue and whole-heart models validated findings under simulated stress and heterogeneous conditions. Activation of M1 macrophages led to a 50% reduction in contraction amplitude, action potential spike amplitude (aCM+cfb+M1: 61.3 mV ±13.9 vs control: 71.6 mV ±14.5, p < 0.01) and increased beat irregularity (M1: 150.7% ± 388.9 vs control, p < 0.001). Calcium transient amplitude was reduced (12.3 a.u. ± 14.7, p < 0.05) and upstroke velocity slowed. SCN5A knock-down reduced contraction amplitude (−51.9% ± 37.2, p < 0.01) without inducing arrhythmias, whereas combined GJA5 and ATP1A1 knock-down induced significant irregularity (403% ± 371.3, p < 0.001), increased conduction heterogeneity (+18%), and reduced velocity (−52.4%). In silico modeling confirmed that paired 50% downregulation of sodium-potassium pump and tissue conductivity induced AF under tachycardia even without ectopic activity. This work reveals a novel, inflammation-driven mechanism for AF initiation. Combined downregulation of GJA5 (connexin 40) and ATP1A1 (NaK ATPase) disrupted intercellular connectivity and ion flux, establishing a substrate for arrhythmogenesis. These results were robust across in vitro, genetic/pharmacological, and in silico models, defining new avenues for translational intervention