GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida.

Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse Pseudomonas putida that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.

Has Ghana's Rotavirus Vaccine Switch Met Programmatic Expectations? An Analysis of National Surveillance Data; 2018-2022.

Background: Ghana introduced a 2-dose schedule rotavirus vaccine, Rotarix, into childhood immunization in 2012 but switched to a 3-dose schedule vaccine, Rotavac, in 2020 on account of programmatic advantages offered by the latter, including lower cost per fully immunized child and lower cold chain volume requirement. The objective of the study was to assess the effect of the vaccine switch on the trends of rotavirus vaccine uptake and health facility outpatient department (OPD) attendance due to diarrhea among children aged 1-11 months. Methods: A retrospective analysis was conducted on childhood immunization and diarrhea surveillance data for 2018-2022. The uptake of the different rotavirus vaccine products and the proportion of health facility OPD attendance attributed to diarrhea, respectively, were compared between the pre- and postswitch study periods. Results: The uptake of rotavirus vaccine was sustained following the switch. There were no significant differences in vaccination coverages (rota1, Rotarix coverage [94.3%], vs rota1, Rotavac coverage [95.3%]; P = .757; rota2, Rotarix coverage [91.3%], vs rota2, Rotavac coverage [92.7%]; P = .789). The proportions of health facility OPD attendance due to diarrhea were comparable (preswitch [12.4%] vs postswitch [12.1%]; P = .838). Conclusions: Ghana's rotavirus vaccine switch yielded expected programmatic benefits without any untoward effects. The trends of vaccine uptake and reduction in diarrhea morbidity were sustained. These experiences and lessons from the rotavirus vaccine switch are vital for potential switches for other vaccines in the current immunization schedule to mitigate the annual vaccine expenditure.

Optimization of Exon-Skipping Riboswitches and Their Applications to Control Mammalian Cell Fate.

Mammalian riboswitches that can regulate transgene expression via RNA-small molecule interaction have promising applications in medicine and biotechnology, as they involve no protein factors that can induce immunogenic reactions and are not dependent on specially engineered promoters. However, the lack of cell-permeable and low-toxicity small molecules and cognate aptamers that can be exploited as riboswitches and the modest switching performance of mammalian riboswitches have limited their applications. In this study, we systematically optimized the design of a riboswitch that regulates exon skipping via an RNA aptamer that binds ASP2905. We examined two design strategies to modulate the stability of the aptamer base stem that blocks the 5' splice site to fine-tune the riboswitch characteristics. Furthermore, an optimized riboswitch was used to generate a mouse embryonic stem cell line that can be chemically induced to differentiate into myogenic cells by activating Myod1 expression and a human embryonic kidney cell line that can be induced to trigger apoptosis by activating BAX expression. The results demonstrate the tight chemical regulation of transgenes in mammalian cells to control their phenotype without exogenous protein factors.

Hemodynamic Analysis in Aortic Dilatation after Arterial Switch Operation for Patients with Transposition of Great Arteries Using Computational Fluid Dynamics.

After an arterial switch operation for complete transposition of the great arteries, neo-aortic root dilatation occurs, with unclear hemodynamic effects. This study analyzes three groups (severe dilation, mild dilation, and normal) using computational fluid dynamics (CFD) on cardiac CT scans. Aortic arch angles in severe (median 72.3, range: 68.5-77.2) and mild dilation (76.6, 71.1-85.2) groups are significantly smaller than the normal group (97.3, 87.4-99.0). In the normal and mild dilatation groups, Wall Shear Stress (WSS) exhibits a consistent pattern: it is lowest at the aortic root, gradually increases until just before the bend in the aortic arch, peaks, and then subsequently decreases. However, severe dilation shows disrupted WSS patterns, notably lower in the distal ascending aorta, attributed to local recirculation. This unique WSS pattern observed in severely dilated patients, especially in the transverse aorta. CFD plays an essential role in comprehensively studying the pathophysiology underlying aortic dilation in this population.

[Cardiac arrhythmias in adults with congenital heart disease]. / Herzrhythmusstörungen bei Erwachsenen mit angeborenen Herzfehlern.

In patients with congenital heart disease, cardiac arrhythmias are complex and require a thorough understanding of the anatomy, past surgical and interventional procedures, and the specific electric processes. Supraventricular tachycardias commonly present as emergency situations and should be treated immediately, particularly when there is an underlying complex malformation. Establishing sinus rhythm is usually superior to pure frequency control for hemodynamic reasons. Catheter ablation should be preferred over medical treatment, even though several procedures are often necessary. In addition, bradycardia is seen more frequently in congenital heart defects; this could be aggravated by antiarrhythmic drugs. There are significant differences between the indications and techniques used for pacemaker implantation in patients with congenital heart defects and those without. Patients with complex congenital heart diseases have an increased risk of thromboembolism; therefore, an individual and early indication for low-threshold oral anticoagulation is necessary; direct oral anticoagulants can also be used for this purpose. In risk stratification for sudden cardiac death, the principles of general guidelines are often not applicable, and individualized decisions are required. Recently, a new general risk score for congenital heart disease has been developed. The treatment of cardiac arrhythmias in patients with congenital heart disease should always be performed in close cooperation with specialized centers.

Switch to faricimab after initial treatment with aflibercept in eyes with neovascular age-related macular degeneration.

PURPOSE: To investigate the efficacy and outcomes of switching neovascular age-related macular degeneration (nAMD) patients from aflibercept to faricimab, focusing on visual acuity, retinal fluid management, and treatment intervals. The primary aim was to assess the early outcomes in nAMD patients refractory to aflibercept and explore faricimab's potential as a longer-lasting therapeutic alternative. METHODS: A single-center retrospective study was conducted on 50 refractory nAMD patients at Cleveland Clinic Abu Dhabi from September 2022-May 2023. Patients were switched from aflibercept to faricimab, having met specific criteria for refractory nAMD. The study analyzed best-corrected visual acuity (BCVA), central subfield thickness (CST), and fluid changes post-switch, using Optical Coherence Tomography (OCT). RESULTS: After three faricimab injections, significant reductions in CST were observed, with a notable decrease in retinal fluid. The mean BCVA remained stable throughout the study period. Although there was a decrease in the maximum pigment epithelial detachment (PED) height, it was not statistically significant. Treatment intervals post-switch showed that the majority of patients maintained or extended their treatment intervals, with a significant proportion achieving resolution of intraretinal fluid (IRF) and subretinal fluid (SRF). CONCLUSIONS: Switching to faricimab from aflibercept in refractory nAMD patients led to significant improvements in retinal fluid management and CST, with stable BCVA outcomes. Faricimab presents a promising alternative for patients requiring frequent aflibercept injections, potentially offering a more manageable treatment regimen with extended dosing intervals. This study highlights the need for personalized therapeutic strategies in nAMD treatment, though further research is necessary to optimize treatment switches.

A compact, versatile drug-induced splicing switch system with minimal background expression.

Gene-switch techniques hold promising applications in contemporary genetics research, particularly in disease treatment and genetic engineering. Here, we developed a compact drug-induced splicing system that maintains low background using a human ubiquitin C (hUBC) promoter and optimized drug (LMI070) binding sequences based on the Xon switch system. To ensure precise subcellular localization of the protein of interest (POI), we inserted a 2A self-cleaving peptide between the extra N-terminal peptide and POI. This streamlined and optimized switch system, named miniXon2G, effectively regulated POIs in different subcellular localizations both in vitro and in vivo. Furthermore, miniXon2G could be integrated into endogenous gene loci, resulting in precise, reversible regulation of target genes by both endogenous regulators and drugs. Overall, these findings highlight the performance of miniXon2G in controlling protein expression with great potential for general applicability to diverse biological scenarios requiring precise and delicate regulation.

Angiopoietin-like 4 facilitates human aortic smooth muscle cell phenotype switch and dysfunctions through the PI3K/Akt signaling in aortic dissection.

PURPOSE: Vascular smooth muscle cell (VSMC) phenotype switch and dysfunctions have been reported to participate in aortic dissection (AD) progression. This study was aimed to investigate the role of angiopoietin-like 4 (ANGPTL4) in regulating VSMCs phenotype switch. MATERIALS AND METHODS: Key genes were analyzed in AD using public datasets, and it was found that the central differential gene ANGPTL4 was up-regulated in AD. The KEGG signaling pathway annotation was performed to validate the associated pathways, and the expression of ANGPTL4 was verified using multiple datasets and clinical samples. Furthermore, the specific functions of ANGPTL4 on platelet-derived growth factor-BB (PDGF-BB)-treated human aortic smooth muscle cell (HASMC) phenotypes were investigated. The dynamic effects of ANGPTL4 and core signaling antagonists on HASMC phenotypes were examined. RESULTS: Hub gene ANGPTL4 was significantly up-regulated in AD. ANGPTL4 was linked to the PI3K/Akt signaling, angiogenesis, and neovascularization and remodeling. ANGPTL4 overexpression further enhanced PDGF-BB effects on HASMC phenotypes, including promoted cell viability and migration, decreased contractile VSMC markers α-SMA and SM22α, elevated ECM degradation markers MMP-2 and MMP-9, and promoted phosphorylation of PI3K and Akt. ANGPTL4 knockdown partially abolished PDGF-BB-induced contractile/synthetic VSMCs imbalance and HASMC dysfunctions. Furthermore, in ANGPTL4-overexpressing HASMCs pre-treated with PDGF-BB, the PI3K/Akt signaling inhibitor LY294002 also partially eliminated the effects caused by the PDGF-BB treatment and ANGPTL4 overexpression. CONCLUSIONS: ANGPTL4 is significantly up-regulated in AD. ANGPTL4 overexpression further enhanced PDGF-BB effects on HASMC phenotype switch and dysfunctions, which might be involved in the PI3K/Akt signaling.

The many facets of RuII(dppe)2 acetylide compounds.

In this contribution, we describe the various research domains in which RuII alkynyl derivatives are involved. Their peculiar molecular properties stem from a strong and intimate overlap between the metal centered d orbitals and the p system of the acetylide ligands, resulting in plethora of fascinating properties such as strong and tunable visible light absorption with a strong MLCT character  essential for sensing, photovoltaics, light-harvesting applications or non-linear optical properties. Likewise, the d/p mixing results in tunable redox properties at low potential due to the raising of the HOMO level, and making those compounds particularly suited to achieve redox switching of various properties associated to the acetylide conjugated ligand, such as photochromism, luminescence or magnetism, for charge transport at the molecular level and in field effect transistor devices, or charge storage for memory devices. Altogether, we show in this review the potential of RuII acetylide compounds, insisting on the molecular design and suggesting further research developments for this class of organometallic dyes, including supramolecular chemistry.

Delayed Presentation of Baffle Obstruction in an Adult Post-Mustard Repair of Transposition: Computed Tomography Demonstration.

Atrial switch surgery is performed in patients with transposition of the great arteries. One of the complications of this surgery is obstruction of the baffle created. We describe the computed tomography findings of one such case where there was delayed presentation of recurrent Mustard baffle obstruction in addition to pulmonary venous drainage obstruction in an adult previously operated on for intra-atrial repair of transposition of the great arteries.

A multi-network model of Parkinson's disease tremor: exploring the finger-dimmer-switch theory and role of dopamine in thalamic self-inhibition.

BACKGROUND: Tremor is a cardinal symptom of Parkinson's disease (PD) that manifests itself through complex oscillatory activity across multiple neuronal populations. According to the finger-dimmer-switch (FDS) theory, tremor is triggered by transient pathological activity in the basal ganglia-thalamo-cortical (BTC) network (the finger) and transitions into an oscillatory form within the inner circuitry of the thalamus (the switch). The cerebello-thalamo-cortical (CTC) network (the dimmer) is then involved in sustaining and amplifying tremor amplitude. In this study, we aimed to investigate the generation and progression dynamics of PD tremor oscillations by developing a comprehensive and interacting FDS model that transitions sequentially from healthy to PD to tremor and then to tremor-off state. Methods: We constructed a computational model consisting of 700 neurons in 11 regions of BTC, CTC, and thalamic networks. Transition from healthy to PD state was simulated through modulating dopaminergic synaptic connections; and further from PD to tremor and tremor-off by modulating projections between the thalamic reticular nucleus (TRN), anterior ventrolateral nucleus (VLa), and posterior ventrolateral nucleus (VLp). Results: Sustained oscillations in the frequency range of PD tremor emerged in thalamic VLp (5 Hz) and cerebellar dentate nucleus (3 Hz). Increasing self-inhibition in the thalamus through dopaminergic modulation significantly decreased tremor amplitude. Conclusion/Significance: Our results confirm the mechanistic power of the FDS theory in describing the PD tremor phenomenon and emphasize the role of dopaminergic modulation on thalamic self-inhibition. These insights pave the way for novel therapeutic strategies aimed at reducing the tremor by strengthening thalamic self-inhibition, particularly in dopamine-resistant patients.

Scanning Switch-off Microscopy for Super-Resolution Fluorescence Imaging.

Super-resolution (SR) microscopy provides a revolutionary optical imaging approach by breaking the diffraction limit of light, while the commonly required special instrumentation with complex optical setup hampers its popularity. Here, we present a scanning switch-off microscopy (SSM) concept that exploits the omnipresent switch-off response of fluorophores to enable super-resolution imaging using a commercial confocal microscope. We validated the SSM model with theoretical calculations and experiments. An imaging resolution of ∼100 nm was obtained for DNA origami nanostructures and cellular cytoskeletons using fluorescent labels of Alexa 405, Alexa 488, Cy3, and Atto 488. Notably, super-resolution imaging of live cells was realized with SSM, by employing a dronpa fluorescent protein as the fluorescent label. In principle, this SSM method can be applied to any excitation laser scanning-based microscope.

F-53B stimulated vascular smooth muscle cell phenotypic switch and vascular remodeling via ferroptosis-related pathway.

The compound 6:2 chlorinated polyfluorinated ether sulfonate (F53B), an alternative to perfluorooctane sulfonate (PFOS), has been widely utilized in China. Although the connection between the exposure and toxicity of F53B is established, the role and mechanisms of the compound in promoting vascular remodeling are yet to be elucidated. Thus, the present study investigated the impact of F53B on the function of vascular smooth muscle cells (VSMCs) and vascular remodeling. The data exhibited that F53B stimulates vascular morphological alterations in vivo, and exposure to the compound caused excessive VSMCs ferroptosis and phenotype switching, as determined using phenotype and molecular assays. Moreover, Fer-1 reversed F-53B-induced VSMC dysfunction and vascular remodeling. Furthermore, F53B activated the ferroptosis-related pathway, encompassing ATR expression and LOC101929922/miR-542-3p/ACSL4 pathway. Thus, the current results elaborated on the multifaceted toxicities of F53B that induce vascular remodeling, thereby necessitating the assessment of vasotoxicity risks associated with the compound.

Tilted Spins in Chains of Molecular Switches on Pb(100).

A complex based on a Ni(II) porphyrin exhibiting spin crossover on Ag(111) is studied on Pb(100) by scanning tunneling microscopy at 0.3 K. Strong molecular interactions between the phenyl and pentafluorophenyl moieties lead to the formation of molecular chains and cause a faceting of the substrate surface. The chains are located along double and multiple substrate steps that deviate from high-symmetry directions. Tunneling spectroscopy reveals spin-flip excitations of an S = 1 system. Measurements in high magnetic fields are used to identify a tilt of the complex and its hard anisotropy axis with respect to the surface normal. Electron injection into the substrate near the molecular rows induces a transition to a state with larger inelastic cross section, leaving the spin state unchanged.

Activation induces shift in nutrient utilization that differentially impacts cell functions in human neutrophils.

Neutrophils utilize a variety of metabolic sources to support their crucial functions as the first responders in innate immunity. Here, through in vivo and ex vivo isotopic tracing, we examined the contributions of different nutrients to neutrophil metabolism under specific conditions. Human peripheral blood neutrophils, in contrast to a neutrophil-like cell line, rely on glycogen storage as a major metabolic source under resting state but rapidly switch to primarily using extracellular glucose upon activation with various stimuli. This shift is driven by a substantial increase in glucose uptake, enabled by rapidly increased GLUT1 on cell membrane, that dominates the simultaneous increase in gross glycogen cycling capacity. Shifts in nutrient utilization impact neutrophil functions in a function-specific manner: oxidative burst depends on glucose utilization, whereas NETosis and phagocytosis can be flexibly supported by either glucose or glycogen, and neutrophil migration and fungal control are enhanced by the shift from glycogen utilization to glucose utilization. This work provides a quantitative and dynamic understanding of fundamental features in neutrophil metabolism and elucidates how metabolic remodeling shapes neutrophil functions, which has broad health relevance.

A molten globule ensemble primes Arf1-GDP for the nucleotide switch.

The adenosine di-phosphate (ADP) ribosylation factor (Arf) small guanosine tri-phosphate (GTP)ases function as molecular switches to activate signaling cascades that control membrane organization in eukaryotic cells. In Arf1, the GDP/GTP switch does not occur spontaneously but requires guanine nucleotide exchange factors (GEFs) and membranes. Exchange involves massive conformational changes, including disruption of the core ß-sheet. The mechanisms by which this energetically costly switch occurs remain to be elucidated. To probe the switch mechanism, we coupled pressure perturbation with nuclear magnetic resonance (NMR), Fourier Transform infra-red spectroscopy (FTIR), small-angle X-ray scattering (SAXS), fluorescence, and computation. Pressure induced the formation of a classical molten globule (MG) ensemble. Pressure also favored the GDP to GTP transition, providing strong support for the notion that the MG ensemble plays a functional role in the nucleotide switch. We propose that the MG ensemble allows for switching without the requirement for complete unfolding and may be recognized by GEFs. An MG-based switching mechanism could constitute a pervasive feature in Arfs and Arf-like GTPases, and more generally, the evolutionarily related (Ras-like small GTPases) Rags and Gα GTPases.

Customizable OpenGUS immunoassay: A homogeneous detection system using ß-glucuronidase switch and label-free antibody.

We developed a customizable OpenGUS immunoassay that enables rapid and sensitive detection of analytes without requiring antibody modification. This immunoassay employs label-free whole antibodies, an antibody-binding Z domain (ZD) derived from Staphylococcal protein A, and a ß-glucuronidase (GUS) switch mutant, allowing for easy replacement of antibodies to tailor the immunoassays for various targeted antigens. The working principle is that the OpenGUS probe, the fusion protein of ZD and a GUS switch, converts the antibody-antigen interaction into GUS activation in a one-pot reaction. To enhance the signal-to-background ratio of the immunoassay, a GUS switch mutant that exhibits reduced background activation was developed by screening several additional mutations at the diagonal interface residue H514. Moreover, we optimized the composition of the reaction buffer, including organic solvents, salt, and surfactant. Under optimal conditions, we customized OpenGUS immunoassays for Cry j 1, human C-reactive protein, and human lactoferrin, achieving around 10-20-fold maximum fluorescence (15 min) or colorimetric (2 h) responses with picomolar to low nanomolar level detection limit, simply by using commercially available IgGs. Additionally, the three analytes were successfully detected in complex matrices similar to those used in practical applications. We believe that this customizable OpenGUS immunoassay will pave the way for the prompt development of rapid and sensitive homogeneous immunoassays for point-of-care diagnostics, high-throughput testing, and onsite environmental assessments.

Cooperative Folding as a Molecular Switch in an Evolved Antibody Binder.

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody-binding. Hence, these variants contained molecular "on/off" switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.

Visible-Photo-Assisted Phase Switching of Antiferroelectric-to-Ferroelectric Orders in an I3--Intercalated 2D Perovskite.

Antiferroelectric (AFE) has emerged as a promising branch of electroactive materials, due to their intriguing physical attributes stemming from the electric field-induced antipolar-to-polar phase transformation. However, the requirement of an extremely high electric field strength to switch adjacent sublattice polarization poses great challenges for exploiting molecular AFE system. Although photoirradiation is striking as a noncontact and nondestructive manipulation tool to optimize physical properties, the optical control of antiferroelectricity is still unexplored. Here, by adopting light-sensitive triiodide I3- anion into the 2D perovskite family, we have designed the first I3--intercalated molecular AFE of (t-ACH)2EA2Pb3I10(I3)0.5·((H3O)(H2O))0.5 (1, t-ACH = trans-4-aminomethyl-1-cyclohexanecarboxylate, EA = ethylammonium). The I3--intercalating gives an ultra-narrow bandgap of 1.65 eV with strong absorption. In terms of AFE structure, the anti-parallel alignment of electric dipoles results in a spontaneous polarization of 4.3 µC/cm2. Strikingly, 1 merely shows AFE behaviour in the dark even under ultrahigh voltage, while the field-induced ferroelectric state can be facilely obtained upon visible illumination. Such unprecedented photo-assisted phase switching ascribes to the incorporation of photoactive I3- anions, which reduce the AFE-to-ferroelectric switching barrier for 1. This pioneering work on the photo-assisting transformation of ferroic orders paves a new way to develop future photoactive materials with significant potential applications.