Fit-for-purpose heterodivalent single-domain antibody for gastrointestinal targeting of toxin B from Clostridium difficile.

Single-domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that display extraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross-inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesses high stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications.

Machine Learning Model for Readmission Prediction of Patients With Heart Failure Based on Electronic Health Records: Protocol for a Quasi-Experimental Study for Impact Assessment.

BACKGROUND: Care for patients with heart failure (HF) causes a substantial load on health care systems where a prominent challenge is the elevated rate of readmissions within 30 days following initial discharge. Clinical professionals face high levels of uncertainty and subjectivity in the decision-making process on the optimal timing of discharge. Unwanted hospital stays generate costs and cause stress to patients and potentially have an impact on care outcomes. Recent studies have aimed to mitigate the uncertainty by developing and testing risk assessment tools and predictive models to identify patients at risk of readmission, often using novel methods such as machine learning (ML). OBJECTIVE: This study aims to investigate how a developed clinical decision support (CDS) tool alters the decision-making processes of health care professionals in the specific context of discharging patients with HF, and if so, in which ways. Additionally, the aim is to capture the experiences of health care practitioners as they engage with the system's outputs to analyze usability aspects and obtain insights related to future implementation. METHODS: A quasi-experimental design with randomized crossover assessment will be conducted with health care professionals on HF patients' scenarios in a region located in the South of Sweden. In total, 12 physicians and nurses will be randomized into control and test groups. The groups shall be provided with 20 scenarios of purposefully sampled patients. The clinicians will be asked to take decisions on the next action regarding a patient. The test group will be provided with the 10 scenarios containing patient data from electronic health records and an outcome from an ML-based CDS model on the risk level for readmission of the same patients. The control group will have 10 other scenarios without the CDS model output and containing only the patients' data from electronic medical records. The groups will switch roles for the next 10 scenarios. This study will collect data through interviews and observations. The key outcome measures are decision consistency, decision quality, work efficiency, perceived benefits of using the CDS model, reliability, validity, and confidence in the CDS model outcome, integrability in the routine workflow, ease of use, and intention to use. This study will be carried out in collaboration with Cambio Healthcare Systems. RESULTS: The project is part of the Center for Applied Intelligent Systems Research Health research profile, funded by the Knowledge Foundation (2021-2028). Ethical approval for this study was granted by the Swedish ethical review authority (2022-07287-02). The recruitment process of the clinicians and the patient scenario selection will start in September 2023 and last till March 2024. CONCLUSIONS: This study protocol will contribute to the development of future formative evaluation studies to test ML models with clinical professionals. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/52744.

Interdisciplinary Human-Centered AI for Hospital Readmission Prediction of Heart Failure Patients.

The evolution of clinical decision support (CDS) tools has been improved by usage of new technologies, yet there is an increased need to develop user-friendly, evidence-based, and expert-curated CDS solutions. In this paper, we show with a use-case how interdisciplinary expertise can be combined to develop CDS tool for hospital readmission prediction of heart failure patients. We also discuss how to make the tool integrated in clinical workflow by understanding end-user needs and have clinicians-in-the-loop during the different development stages.

Orally delivered single-domain antibodies against gastrointestinal pathogens.

Single-domain antibodies (sdAbs) are exceptionally stable fragments derived from the antigen-binding domains of immunoglobulins. They can withstand extreme pH, high temperature, and proteolysis, making them suitable for controlling gastrointestinal (GI) infections in humans and animals. sdAbs may function in their native soluble form, although different derived protein formats and the use of delivery vehicles can be useful for improved oral delivery. We discuss selected examples of the use of orally delivered sdAbs for protecting humans and animals against GI infections caused by pathogenic bacteria, viruses, and parasites. We finally provide perspectives on how sdAbs may be applied industrially and what challenges should be overcome for orally delivered sdAbs to reach the market.

Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways.

Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.

C-fiber recovery cycle supernormality depends on ion concentration and ion channel permeability.

Following each action potential, C-fiber nociceptors undergo cyclical changes in excitability, including a period of superexcitability, before recovering their basal excitability state. The increase in superexcitability during this recovery cycle depends upon their immediate firing history of the axon, but also determines the instantaneous firing frequency that encodes pain intensity. To explore the mechanistic underpinnings of the recovery cycle phenomenon a biophysical model of a C-fiber has been developed. The model represents the spatial extent of the axon including its passive properties as well as ion channels and the Na/K-ATPase ion pump. Ionic concentrations were represented inside and outside the membrane. The model was able to replicate the typical transitions in excitability from subnormal to supernormal observed empirically following a conducted action potential. In the model, supernormality depended on the degree of conduction slowing which in turn depends upon the frequency of stimulation, in accordance with experimental findings. In particular, we show that activity-dependent conduction slowing is produced by the accumulation of intraaxonal sodium. We further show that the supernormal phase results from a reduced potassium current Kdr as a result of accumulation of periaxonal potassium in concert with a reduced influx of sodium through Nav1.7 relative to Nav1.8 current. This theoretical prediction was supported by data from an in vitro preparation of small rat dorsal root ganglion somata showing a reduction in the magnitude of tetrodotoxin-sensitive relative to tetrodotoxin -resistant whole cell current. Furthermore, our studies provide support for the role of depolarization in supernormality, as previously suggested, but we suggest that the basic mechanism depends on changes in ionic concentrations inside and outside the axon. The understanding of the mechanisms underlying repetitive discharges in recovery cycles may provide insight into mechanisms of spontaneous activity, which recently has been shown to correlate to a perceived level of pain.

Differential axonal conduction patterns of mechano-sensitive and mechano-insensitive nociceptors--a combined experimental and modelling study.

Cutaneous pain sensations are mediated largely by C-nociceptors consisting of both mechano-sensitive (CM) and mechano-insensitive (CMi) fibres that can be distinguished from one another according to their characteristic axonal properties. In healthy skin and relative to CMi fibres, CM fibres show a higher initial conduction velocity, less activity-dependent conduction velocity slowing, and less prominent post-spike supernormality. However, after sensitization with nerve growth factor, the electrical signature of CMi fibres changes towards a profile similar to that of CM fibres. Here we take a combined experimental and modelling approach to examine the molecular basis of such alterations to the excitation thresholds. Changes in electrical activation thresholds and activity-dependent slowing were examined in vivo using single-fibre recordings of CM and CMi fibres in domestic pigs following NGF application. Using computational modelling, we investigated which axonal mechanisms contribute most to the electrophysiological differences between the fibre classes. Simulations of axonal conduction suggest that the differences between CMi and CM fibres are strongly influenced by the densities of the delayed rectifier potassium channel (Kdr), the voltage-gated sodium channels NaV1.7 and NaV1.8, and the Na+/K+-ATPase. Specifically, the CM fibre profile required less Kdr and NaV1.8 in combination with more NaV1.7 and Na+/K+-ATPase. The difference between CM and CMi fibres is thus likely to reflect a relative rather than an absolute difference in protein expression. In support of this, it was possible to replicate the experimental reduction of the ADS pattern of CMi nociceptors towards a CM-like pattern following intradermal injection of nerve growth factor by decreasing the contribution of Kdr (by 50%), increasing the Na+/K+-ATPase (by 10%), and reducing the branch length from 2 cm to 1 cm. The findings highlight key molecules that potentially contribute to the NGF-induced switch in nociceptors phenotype, in particular NaV1.7 which has already been identified clinically as a principal contributor to chronic pain states such as inherited erythromelalgia.

Modeling activity-dependent changes of axonal spike conduction in primary afferent C-nociceptors.

Action potential initiation and conduction along peripheral axons is a dynamic process that displays pronounced activity dependence. In patients with neuropathic pain, differences in the modulation of axonal conduction velocity by activity suggest that this property may provide insight into some of the pathomechanisms. To date, direct recordings of axonal membrane potential have been hampered by the small diameter of the fibers. We have therefore adopted an alternative approach to examine the basis of activity-dependent changes in axonal conduction by constructing a comprehensive mathematical model of human cutaneous C-fibers. Our model reproduced axonal spike propagation at a velocity of 0.69 m/s commensurate with recordings from human C-nociceptors. Activity-dependent slowing (ADS) of axonal propagation velocity was adequately simulated by the model. Interestingly, the property most readily associated with ADS was an increase in the concentration of intra-axonal sodium. This affected the driving potential of sodium currents, thereby producing latency changes comparable to those observed for experimental ADS. The model also adequately reproduced post-action potential excitability changes (i.e., recovery cycles) observed in vivo. We performed a series of control experiments replicating blockade of particular ion channels as well as changing temperature and extracellular ion concentrations. In the absence of direct experimental approaches, the model allows specific hypotheses to be formulated regarding the mechanisms underlying activity-dependent changes in C-fiber conduction. Because ADS might functionally act as a negative feedback to limit trains of nociceptor activity, we envisage that identifying its mechanisms may also direct efforts aimed at alleviating neuronal hyperexcitability in pain patients.

Long-lasting small-amplitude TRP-mediated dendritic depolarizations in CA1 pyramidal neurons are intrinsically stable and originate from distal tuft regions.

In several regions of the nervous system, neurons display bi- or multistable intrinsic properties. Such stable states may be subthreshold and long-lasting, and can appear as a sustained afterdepolarization. In hippocampal CA1 pyramidal neurons, small-amplitude (1 mV) long-lasting (seconds) afterdepolarizations have been reported and are thought to depend on calcium-activated nonselective (CAN) currents recently identified as transient receptor potential (TRP) channels. Continuing our previous experimental and computational work on synaptically metabotropic glutamate receptor (mGluR)-activated TRP currents, we here explore small-amplitude long-lasting depolarizations in a detailed multicompartmental model of a CA1 pyramidal neuron. We confirm a previous hypothesis suggesting that the depolarization results from an interplay of TRP and voltage-gated calcium channels, and contribute to the understanding of the depolarization in several ways. Specifically, we show that: (i) the long-lasting depolarization may be intrinsically stable to weak excitatory and inhibitory input, (ii) the phenomenon is essentially located in distal apical dendrites, (iii) induction is facilitated if simultaneous input arrives at several dendritic branches, and if calcium- and/or mGluR-evoked signals undergo summation, suggesting that both spatial and temporal synaptic summation might be required for the depolarization to occur and (iv) we also show that the integration of inputs to oblique dendrites is strongly modulated by the presence of small-amplitude long-lasting depolarizations in distal tuft dendrites. To conclude, we suggest that small-amplitude long-lasting dendritic depolarizations may contribute to sustaining neural information during behavioural tasks in cases where information is separated in time, as in trace conditioning and delay tasks.

Low-frequency summation of synaptically activated transient receptor potential channel-mediated depolarizations.

Neurons sum their input by spatial and temporal integration. Temporally, presynaptic firing rates are converted to dendritic membrane depolarizations by postsynaptic receptors and ion channels. In several regions of the brain, including higher association areas, the majority of firing rates are low. For rates below 20 Hz, the ionotropic receptors α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and N-methyl-d-aspartate (NMDA) receptor will not produce effective temporal summation. We hypothesized that depolarization mediated by transient receptor potential (TRP) channels activated by metabotropic glutamate receptors would be more effective, owing to their slow kinetics. On the basis of voltage-clamp and current-clamp recordings from a rat slice preparation, we constructed a computational model of the TRP channel and its intracellular activation pathway, including the metabotropic glutamate receptor. We show that synaptic input frequencies down to 3-4 Hz and inputs consisting of as few as three to five pulses can be effectively summed. We further show that the time constant of integration increases with increasing stimulation frequency and duration. We suggest that the temporal summation characteristics of TRP channels may be important at distal dendritic arbors, where spatial summation is limited by the number of concurrently active synapses. It may be particularly important in regions characterized by low and irregular rates.