Variable Domain Research Articles

Synthetic translational coupling element for multiplexed signal processing and cellular control

Abstract Repurposing natural systems to develop customized functions in biological systems is one of the main thrusts of synthetic biology. Translational coupling is a common phenomenon in diverse polycistronic operons for efficient allocation of limited genetic space and cellular resources. These beneficial features of translation coupling can provide exciting opportunities for creating novel synthetic biological devices. Here, we introduce a modular synthetic translational coupling element (synTCE) and integrate this design with de novo designed riboregulators, toehold switches. A systematic exploration of sequence domain variants for synTCEs led to the identification of critical design considerations for improving the system performance. Next, this design approach was seamlessly integrated into logic computations and applied to construct multi-output transcripts with well-defined stoichiometric control. This module was further applied to signaling cascades for combined signal transduction and multi-input/multi-output synthetic devices. Further, the synTCEs can precisely manipulate the N-terminal ends of output proteins, facilitating effective protein localization and cellular population control. Therefore, the synTCEs could enhance computational capability and applicability of riboregulators for reprogramming biological systems, leading to future applications in synthetic biology, metabolic engineering and biotechnology.

MAGPIX assay for Influenza A using single domain antibodies

Single domain antibodies (sdAbs) provide versatile binding reagents that offer advantages over conventional antibodies for use in immunoassays. SdAbs consist of the ~15 kDa variable heavy domain from the heavy chain‐only antibodies found in camelids and can be engineered for integration in a variety of immunoassay formats. The Luminex MAGPIX instrument is a high‐throughput platform that uses color‐coded MagPlex beads to enable multiplexed immunoassays and is relatively fast and simple. We developed a MagPlex bead‐based assay for the detection of influenza A virus (IAV), using sdAbs against IAV nucleoprotein (NP). To provide sensitive detection, the sdAbs were formatted into bivalent constructs; and the capture reagent was tailored to provide oriented immobilization on the bead. Using the best capture and reporter pair, we achieved detection of recombinant NP down to 1 ng/mL. Finally, we demonstrated that the developed immunoassay showed promise in detecting IAV in clinical samples.

Effect of a yoga and naturopathy-based lifestyle intervention with nine-month follow-up on heart rate variability in patients with hypertension: A randomized controlled trial

PurposeHypertension is associated with autonomic nervous system (ANS) dysregulation. Heart rate variability (HRV) monitoring is frequently used as a gauge of the ANS balance. The aim of the present study was to assess the effects of yoga and naturopathy on the autonomic variables in patients with hypertension. Methods262 hypertensive patients were randomly allocated to the study group (SG) or control group (CG) in a randomized controlled trial. For ten days, the SG (n = 131) group got received naturopathic treatments and yoga, whereas the CG (n = 131) group did not. HRV which was examined at the beginning and end of the 10-day intervention period and again after 9 months, served as the study's main endpoint. ResultThere was a significant change in the frequency domain of heart rate variability with a significant increase in High frequency (HF) and a significant decrease in low frequency (LF) and LF/HF (all p < 0.05) were observed after follow-up in both SG and CG, but the magnitude of change was more in SG with a significant difference in LF/HF between group. ConclusionResults of this study suggest that 10 days of integrated yoga and naturopathy intervention produces a shift of vagal activity towards parasympathetic activity. Clinical trial registration numberCTRI/2019/01/016883.

Neutralizing VHH Antibodies Targeting the Spike Protein of PEDV

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs’ intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection.

Association between psychological traits and occlusal tactile acuity of healthy individuals.

Tactile acuity is a somatosensory measure of the extent to which humans can discern tactile stimuli. It is influenced by how peripheral signals are processed centrally. In the oral cavity, Occlusal Tactile Acuity (OTA) is the ability to perceive minimal thicknesses between antagonist teeth. The aim of the current study was to assess the association between psychological traits and OTA of otherwise healthy individuals. Sixty-three volunteers (32 males; mean age ± SD: 24.6 ± 2.7 years) participated in this study. Somatosensory amplification, anxiety, depression, physical symptoms and pain catastrophizing were scored using questionnaires, and subgroups of severity were created per variable based on cut-offs. OTA was measured using 9 aluminium foils with thickness (ranging from 8 to 72 μm) and one sham test (without foil). Each thickness was tested 10 times in random order, the participants were instructed to report whether they felt the foil between their molars and the mean percentage of correct answers was computed. A linear mixed model was used with OTA as a dependent variable and psychological domain as an independent variable. Significantly different OTA was observed among the anxiety subgroups (p = .003), supporting a decreased perception of thicknesses 24 and 32 μm (p = .018 and p < .001, respectively) in participants with moderate/severe anxiety compared to those with no/mild anxiety. Significantly different OTA was also observed among the pain catastrophizing subgroups (p = .008), showing decreased perception of thicknesses 32 and 40 μm (p < .001 and p = .007, respectively) in severe catastrophizing levels, compared to no/mild catastrophizing levels. No significant differences were observed for the other variables. Healthy adults with increased anxiety or pain catastrophizing levels show decreased interdental acuity as compared to participants with minor or no psychological impairment.

Premise‐Variable‐Domain‐Partition‐Dependent Output Feedback Switched Controllers Design for Switched IT2 Fuzzy Systems With Switching Signal Transmission Delays and Local Nonlinear Model

ABSTRACTThis article studies the guaranteed cost extended dissipative stabilization problem of switched IT2 fuzzy systems (SIT2FSs) with switching signal transmission delays and local nonlinear model. The guaranteed cost performance, guaranteed cost strict dissipative performance, guaranteed cost − performance and guaranteed cost passivity performance are considered for SIT2FSs in a unified form. First, a novel asynchronous control scheme is proposed for SIT2FSs with local nonlinear model. The controller gains in mismatched interval are designed separately. By using a premise‐variable‐domain‐partition‐dependent switched fuzzy Lyapunov functions (PDSFLFs) approach, the membership‐function‐shape‐independent (MFSI) existence conditions of a class of IT2 fuzzy switched controllers, which can ensure the guaranteed cost extended dissipative performance of SIT2FSs, are established. Then, by utilizing the shape information of membership functions and dividing the premise variable domain into multiple partitions, the membership‐function‐shape‐dependent (MFSD) existence conditions of a class of premise‐variable‐domain‐partition‐dependent (PVDPD) IT2 fuzzy switched controllers are presented. The results indicate that less conservative results can be achieved by using the PVDPD IT2 fuzzy switched controllers, and better system performance can be gotten compared with the Type‐1 fuzzy switched controllers. Finally, the effectiveness of the results is demonstrated through a robot arm model.

WDR45 variants as a major cause for a clinically variable intellectual disability syndrome from early infancy in females.

Pathogenic variants of WD repeat domain 45 (WDR45) cause neurodegeneration with brain iron accumulation 5 (NBIA5), which is characterised by progressive neurological regression and brain iron accumulation in adulthood. Early diagnosis of NBIA5 patients is difficult because they often show only a non-specific developmental delay in childhood, but it is essential for lifelong medical management. We investigated 32 females with developmental delays for coding variants of WDR45 using Sanger sequencing. Whole-genome sequencing (WGS) and X chromosome inactivation (XCI) analysis were also performed. We identified two disease-causing variants, one of which was a novel stop-loss variant, c.1051delG p.(Val351CysfsTer60), in a female with severe developmental delay from early infancy with epileptic spasms. The XCI analysis (which we originally developed) suggested a random pattern in white blood cells. WGS did not reveal any other pathogenic variants, including those in two iron transporter genes. Together with our previous findings in the WGS study, WDR45 variants accounted for 12% (6/51) of the females with developmental delay, suggesting that WDR45 is a major gene in females with developmental delay. Pathogenic variants of WDR45 result in various phenotypes that do not necessarily correlate with variant types or XCI skewing patterns.

Addressing the antibody germline bias and its effect on language models for improved antibody design.

The versatile binding properties of antibodies have made them an extremely important class of biotherapeutics. However, therapeutic antibody development is a complex, expensive and time-consuming task, with the final antibody needing to not only have strong and specific binding, but also be minimally impacted by developability issues. The success of transformer-based language models in protein sequence space and the availability of vast amounts of antibody sequences, has led to the development of many antibody-specific language models to help guide antibody design. Antibody diversity primarily arises from V(D)J recombination, mutations within the CDRs, and/or from a few non-germline mutations outside the CDRs. Consequently, a significant portion of the variable domain of all natural antibody sequences remains germline. This affects the pre-training of antibody-specific language models, where this facet of the sequence data introduces a prevailing bias towards germline residues. This poses a challenge, as mutations away from the germline are often vital for generating specific and potent binding to a target, meaning that language models need be able to suggest key mutations away from germline. In this study, we explore the implications of the germline bias, examining its impact on both general-protein and antibody-specific language models. We develop and train a series of new antibody-specific language models optimised for predicting non-germline residues. We then compare our final model, AbLang-2, with current models and show how it suggests a diverse set of valid mutations with high cumulative probability. AbLang-2 is trained on both unpaired and paired data, and is freely available at https://github.com/oxpig/AbLang2.git. Supplementary data are available at Journal Name online.

Preparation of divalent camelid single-domain antibody and its application in immunoassays for Salmonella detection in food.

Salmonella-related foodborne infections are commonly caused by the serovars of S. Typhimurium, which can be detected using antibody-based immunoassays. The monovalent variable domain of thecamelid heavy chain antibody (VHH) performs excellently in constructing multivalent VHH variants, which generally exhibit higher affinities with antigens and consequently enhance the assay sensitivity. In this study, the divalent variants of VHHs (diVHHs) targeting S. Typhimurium were generated by encoding the monovalent VHH genes assembled in tandem with a flexible linker peptide (G4S)2. Soluble diVHHs were produced in a prokaryotic expression system and purified with a yield of 4.22 mg/L. Benefiting from their stability and antigen-binding abilities towards tested Salmonella serovars, diVHH-based immunoassays were developed. The diVHH-based sandwich immunoassay, using diVHH as capture antibody, exhibited a detection limit of 1.04×102 CFU/mL and enabled as low as 10 CFU/mL S. Typhimurium to be detectedafter 6 h of enrichment in lettuce. Furthermore, this assay can be applied to spiked lettuce, chicken, and pork samples, showing acceptable recoveries ranging from 83 to 106%. This study presented feasible strategies for VHH multivalence and established a superior sensitivity VHH-based immunoassay for monitoring and analyzing Salmonella contamination in food.

Variable Domain First-Order First-Degree Entailment and Some of Its Children

Variable Domain First-Order First-Degree Entailment and Some of Its Children

Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

Molecular Modeling and In Vitro Functional Analysis of the RGS12 PDZ Domain Variant Associated with High-Penetrance Familial Bipolar Disorder

Variable frequency interharmonic domain methodology for transient analysis and simulation of distributed generation systems

As the integration of renewable energy resources into the power grid increases, accurate modeling of variable frequency distributed generation systems becomes essential for ensuring grid stability, reliability, and optimal operation. Existing models often fail in effectively capturing the frequency dynamics of these systems, particularly when dealing with variable frequency sources. This gap hinders comprehensive analysis and the development of effective planning, control, and mitigation strategies.To address this challenge, this paper introduces a novel modeling methodology named here as the variable frequency interharmonic domain (VFID), which is based on the flexible extended harmonic domain (FEHD) approach. VFID dynamically adjusts the set of pre-selected frequencies within the state-space system to reflect changes in variable frequency sources. This method eliminates the need for post-processing routines, accurately representing both instantaneous values and frequency evolution of time-varying harmonics and interharmonics.Key results demonstrate that VFID offers significant improvements in simulation accuracy over conventional FEHD and PSCAD methods, making it a valuable tool for analyzing and simulating modern distributed generation systems. This advancement not only fills a critical research gap but also contributes to the development of more resilient and sustainable energy infrastructures.

On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.

The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

Generation of Antibody Libraries for Phage Display: Human Fab Format.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains VH and VL (Vκ or Vλ), fused to the human constant domains CH1 of IgG1 and CL (Cκ or Cλ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (VH, Vκ, and Vλ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.

Generation of Antibody Libraries for Phage Display: Chimeric Rabbit/Human Fab Format.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (VH, Vκ, and Vλ) fused to human constant domains. The human constant domains, CH1 of IgG1 and CL (Cκ or Cλ), not only provide established purification and detection handles but also facilitate higher expression in Escherichia coli compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 108-1011 independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.

Utilizing Bayesian generalization network for reliable fault diagnosis of machinery with limited data

To address the issues of overfitting, domain generalization challenges, and lack of credibility brought by limited data samples in mechanical fault diagnosis in practical engineering, this paper proposes a reliable Bayesian generalization network (BGNet). A Bayesian convolutional layer is constructed based on variational inference, treating all parameters in the convolutional layer as random variables. This approach makes a single model function similar to an ensemble of an infinite number of models, and thus enhancing the model's capability of overfitting resistance and domain generalization. The parameters of the variational distribution are updated to approximate the posterior distribution by local reparametrization and Monte Carlo sampling to optimize the evidence lower bound (ELBO) loss. Confidence information is extracted from the model results and, uncertainty estimation and decomposition schemes are designed to provide interpretability. The proposed method is applied to analyze the experimental data of bearing and gearbox faults. The results show that in a multi-source domain scenario with limited samples, the proposed method demonstrates high diagnostic accuracy, effectively describes the relationship between domain variability and uncertainty, and significantly outperforms several benchmark and state-of-the-art models.

PLAbDab-nano: a database of camelid and shark nanobodies from patents and literature.

Nanobodies are essential proteins of the adaptive immune systems of camelid and shark species, complementing conventional antibodies. Properties such as their relatively small size, solubility and high thermostability makeVHH (variable heavy domain of theheavy chain) and VNAR (variable new antigen receptor) modalities a promising therapeutic format and a valuable resource for a wide range of biological applications. The volume of academic literature and patents related to nanobodies has risen significantly over the past decade. Here, we present PLAbDab-nano, a nanobody complement to the Patent and Literature Antibody Database (PLAbDab). PLAbDab-nano is a self-updating, searchable repository containing ∼5000 annotated VHH and VNAR sequences. We describe the methods used to curate the entries in PLAbDab-nano, and highlight how PLAbDab-nano could be used to design diverse libraries, as well as find sequences similar to known patented or therapeutic entries. PLAbDab-nano is freely available as a searchable web server (https://opig.stats.ox.ac.uk/webapps/plabdab-nano/).

The development of single-domain VHH nanobodies that target the Candida albicans cell surface.

Candida albicans causes life-threatening invasive infections that are hard to diagnose and treat, with drug resistance leading to treatment failure. The goal of this study was to develop VHH (single variable domain on a heavy chain) nanobodies to detect drug-resistant infections. Llamas were immunized with a mixture of heat killed and fixed C. albicans cells of different morphologies. Llama lymphocyte RNA was used to generate phage display libraries that were tested for binding to C. albicans cells or cell wall fractions, and single antibody domains were isolated. The libraries were panned against echinocandin-resistant C. albicans isolates and counter-selected against echinocandin-susceptible isolates with the aim of isolating binding domains specific for antigens on drug-resistant cells. Thirty diverse VHH nanobodies were selected, and binding characteristics were assessed via dose-response ELISA. Binding was tested against a variety of C. albicans isolates and other Candida species, indicating that the VHHs were specific for C. albicans. The VHH nanobodies were sorted into four distinct groups based on their binding patterns. Two of the groups bound preferentially to the yeast cell poles and hyphae, respectively. Nanobody binding to C. albicans deletion mutants was tested by fluorescence microscopy and ELISA to identify the antigen targets. VHH19 nanobody, belonging to the largest group, recognized the Als4 adhesin. VHH14 antibody in the hyphae-specific group recognized Als3. None of the isolated VHH nanobodies was selective for drug-resistant clinical isolates. Our data indicate that this approach can generate valuable single-domain antibodies specific to C. albicans proteins.IMPORTANCEThe human fungal pathogen Candida albicans causes a range of diseases from superficial mucosal infections such as oral and vaginal thrush to life-threatening, systemic infections. Accurate and rapid diagnosis of these infections remains challenging, and currently, there are no rapid ways to diagnose drug-resistant infections without performing drug susceptibility testing from blood culture, which can take several days. In this proof-of-concept study, we have generated a diverse set of single domain VHH antibodies (nanobodies) from llamas that recognize and bind specifically to C. albicans cell surface. The nanobodies were classified into four groups based on their binding patterns, for example, cell poles or hyphae. Specific nanobodies were verified as recognizing the important adhesin Als4 or the hyphae associated invasin Als3, respectively. The data validate the approach that small VHH antibody domains hold future promise for diagnostic applications and as probes to study the fungal cell surface.

Thermal performance of a novel hybrid heat pipe with composite heat transfer characteristics

Considering the capillary threshold of oscillating heat pipes (OHP), a novel hybrid heat pipe (HHP) was designed with channels with hydraulic diameters that alternately exceed and fall below this threshold, parallelly arrayed within a unified copper substrate. This technology attains a comprehensive heat transfer performance within the HHP, seamlessly transitioning from pure phase change heat transfer to hybrid pulsating flow heat transfer to effectively accommodate the escalating demands of heat load. Performance and visualization are used to analyze the operational characteristics of HHP. The results indicate that the HHP mainly transfers heat through the phase change in the large channels in lower heat input, while the working fluid in the small channels remains calm. Once the heat input is sufficient, the geyser boiling in the large channels and the liquid slug pulsating in the small channels jointly carry out heat transfer. Furthermore, the liquid content in the large and small channels can be adaptively adjusted to each other. The high-speed liquid in the small channels can be squeezed into the large channels to ensure the sufficient pulsating space and achieve efficient heat transfer under high heat input. In addition, the operation of the HHP can be significantly impacted by the filling ratio (FR). In the phase change stage, the FR of 47.6 % can be considered as the turning point. After transitioning to the hybrid pulsating flow stage, the HHP with low FR can get a better performance. This new type of heat pipe with gradually changing operation characteristics may be critical to solve thermal problems in variable heat load domain.

Conformational Differences in the Light Chain Constant Domain of Immunoglobulin G and Free Light Chain May Influence Proteolysis in AL Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is a life-threatening disease caused by the deposition of light chain (LC) and its fragments containing variable (VL) and portions of constant (CL) domains. AL patients feature either monoclonal free LCs (FLCs) circulating as covalent and noncovalent homodimers, or monoclonal immunoglobulin (Ig) wherein the LC and heavy chain (HC) form disulfide-linked heterodimers, or both. The role of full-length Ig in AL amyloidosis is unclear as prior studies focused on FLC or VL domain. We used a mammalian cell-based expression system to generate four AL patient-derived full-length IgGs, two non-AL IgG controls, and six corresponding FLC proteins derived from an IGLV6-57 germline precursor. Comparison of proteins’ secondary structure, thermal stability, proteolytic susceptibility, and disulfide link reduction suggested the importance of local vs. global conformational stability. Analysis of IgGs vs. corresponding FLCs using hydrogen–deuterium exchange mass spectrometry revealed major differences in the local conformation/dynamics of the CL domain. In all IgGs vs. FLCs, segments containing β-strand and α-helix βAC-αACBC were more dynamic/exposed while segment βDC-βEC was less dynamic/exposed. Notably, these segments overlapped proteolysis-prone regions whose in vivo cleavage generates LC fragments found in AL deposits. Altogether, the results suggest that preferential cleavage in segments βAC-αACBC of FLC or βDC-βEC of LC in IgG helps generate amyloid protein precursors. We propose that protecting these segments using small-molecule stabilizers, which bind to the interfacial cavities CL-CL in FLC and/or CL-CH1 in IgG, is a potential therapeutic strategy to complement current approaches targeting VL-VL or VL-CL stabilization of LC dimer.