Therapy targeting antigen-specific T cells by a peptide-based tolerizing vaccine against autoimmune arthritis.

A longstanding goal has been to find an antigen-specific preventive therapy, i.e., a vaccine, for autoimmune diseases. It has been difficult to find safe ways to steer the targeting of natural regulatory antigen. Here, we show that the administration of exogenous mouse major histocompatibility complex class II protein bounding a unique galactosylated collagen type II (COL2) peptide (Aq-galCOL2) directly interacts with the antigen-specific TCR through a positively charged tag. This leads to expanding a VISTA-positive nonconventional regulatory T cells, resulting in a potent dominant suppressive effect and protection against arthritis in mice. The therapeutic effect is dominant and tissue specific as the suppression can be transferred with regulatory T cells, which downregulate various autoimmune arthritis models including antibody-induced arthritis. Thus, the tolerogenic approach described here may be a promising dominant antigen-specific therapy for rheumatoid arthritis, and in principle, for autoimmune diseases in general.

Interaction between stabilized droplets of different phases in the same continuous phase of an aqueous three-phase system.

Water-in-water (W/W) emulsions, also called aqueous two-phase systems, are formed by mixing two incompatible polymers in water that phase separate into two distinct phases. They can be stabilized by addition of colloidal particles. Droplets of the dispersed phase can be used to compartmentalize ingredients and induce localized reactions. By mixing more types of incompatible polymers, emulsions containing droplets of different phases can be formed that can potentially capture different ingredients. Here the interaction between dispersed droplets of different types was studied by gently mixing a W/W emulsion containing droplets rich in dextran (DEX) dispersed in a continuous phase rich in polyethylene oxide with an emulsion containing droplets rich in fish gelatin (GEL) dispersed in the same continuous medium. Bis-hydrophilic microgels (MG) consisting of DEX grafted with poly(N-isopropylacrylamide) were added and their effect on the stability of each binary emulsion was investigated. Interestingly, when two very stable emulsions were gently mixed, droplets of different types were observed with confocal scanning laser microscopy to coalesce immediately upon contact. In this manner, Janus-type droplets were formed containing a DEX and a GEL compartment with no MG at the GEL/DEX interface that further associated into strings of alternating droplets. Contact angles between the different phases in emulsions with and without MG were compared and used to determine the effect of the microgels on the interfacial tension between the phases.

Interleukin-22 regulates the complement system to promote resistance against pathobionts after pathogen-induced intestinal damage.

Pathobionts play a critical role in disease development, but the immune mechanisms against pathobionts remain poorly understood. Here, we report a critical role for interleukin-22 (IL-22) in systemic protection against bacterial pathobionts that translocate into the circulation after infection with the pathogen Clostridium difficile. Infection with C. difficile induced IL-22, and infected Il22(-/-) mice harbored high numbers of pathobionts in extraintestinal organs despite comparable pathogen load and intestinal damage in mutant and wild-type mice. Pathobionts exhibited increased resistant against complement-mediated phagocytosis, and their intravenous administration resulted in high animal mortality. Selective removal of translocated commensals rescued Il22(-/-) mice, and IL-22 administration enhanced the elimination of pathobionts. Mechanistically, IL-22 augmented bacterial phagocytosis by increasing the expression and bacterial binding of complement C3. Our study demonstrates an unexpected role for IL-22 in controlling the elimination of pathobionts that enter the systemic circulation through the regulation of the complement system.

Key interactions in the trimolecular complex consisting of the rheumatoid arthritis-associated DRB1*04:01 molecule, the major glycosylated collagen II peptide and the T-cell receptor.

OBJECTIVES: Rheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T cells. This study characterises the autoantigen-presenting function of DRB1*04:01 (HLA-DRA*01:01/HLA-DRB1*04:01) at a molecular level for prototypic T-cell determinants, focusing on a post-translationally modified collagen type II (Col2)-derived peptide. METHODS: The crystal structures of DRB1*04:01 molecules in complex with the peptides HSP70289-306, citrullinated CILP982-996 and galactosylated Col2259-273 were determined on cocrystallisation. T cells specific for Col2259-273 were investigated in peripheral blood mononuclear cells from patients with DRB1*04:01-positive RA by cytofluorometric detection of the activation marker CD154 on peptide stimulation and binding of fluorescent DRB1*0401/Col2259-273 tetramer complexes. The cDNAs encoding the T-cell receptor (TCR) α-chains and ß-chains were cloned from single-cell sorted tetramer-positive T cells and transferred via a lentiviral vector into TCR-deficient Jurkat 76 cells. RESULTS: The crystal structures identified peptide binding to DRB1*04:01 and potential side chain exposure to T cells. The main TCR recognition sites in Col2259-273 were lysine residues that can be galactosylated. RA T-cell responses to DRB1*04:01-presented Col2259-273 were dependent on peptide galactosylation at lysine 264. Dynamic molecular modelling of a functionally characterised Col2259-273-specific TCR complexed with DRB1*04:01/Col2259-273 provided evidence for differential allosteric T-cell recognition of glycosylated lysine 264. CONCLUSIONS: The MHC-peptide-TCR interactions elucidated in our study provide new molecular insights into recognition of a post-translationally modified RA T-cell determinant with a known dominant role in arthritogenic and tolerogenic responses in murine Col2-induced arthritis.

Impact of Tissue Enolase 1 Protein Overexpression in Esophageal Cancer Progression.

Enolase (ENO) 1 is a key glycolytic enzyme and important player in tumorigenesis. ENO1 overexpression has been correlated with tumor progression and/or worse prognosis in several solid malignancies. However, data concerning the impact of ENO1 in cancer conflict. The study correlated local and circulating ENO1 protein levels in esophageal cancer (EC) with clinicopathological data, to assess its potential clinical value. ENO1 expression was analyzed by immunohistochemistry in paired tumor and non-tumor tissue samples from 40 EC cases and mucosal biopsies from 45 Barrett's esophagus (BE) cases, plus in plasma from these patients and 25 matched healthy controls. ENO1 was abnormally elevated in cancer-cell cytoplasm in both EC types, in esophageal squamous cell carcinoma and in adenocarcinoma (EAC), increasing significantly with tumor stage progression and the transition from BE to EAC. EAC patients exhibited significantly lower ENO1 plasma concentrations than normal subjects. Neither local nor systemic ENO1 expression levels were significantly associated with overall survival. These results indicate ENO1 as potential biomarker, delineating a population of patients with Barrett's esophagus at high risk of cancer, and as new therapeutic opportunity in EC patient management. However, further confirmation might be necessary.

Context-Aware Emotion Recognition in the Wild Using Spatio-Temporal and Temporal-Pyramid Models.

Emotion recognition plays an important role in human-computer interactions. Recent studies have focused on video emotion recognition in the wild and have run into difficulties related to occlusion, illumination, complex behavior over time, and auditory cues. State-of-the-art methods use multiple modalities, such as frame-level, spatiotemporal, and audio approaches. However, such methods have difficulties in exploiting long-term dependencies in temporal information, capturing contextual information, and integrating multi-modal information. In this paper, we introduce a multi-modal flexible system for video-based emotion recognition in the wild. Our system tracks and votes on significant faces corresponding to persons of interest in a video to classify seven basic emotions. The key contribution of this study is that it proposes the use of face feature extraction with context-aware and statistical information for emotion recognition. We also build two model architectures to effectively exploit long-term dependencies in temporal information with a temporal-pyramid model and a spatiotemporal model with "Conv2D+LSTM+3DCNN+Classify" architecture. Finally, we propose the best selection ensemble to improve the accuracy of multi-modal fusion. The best selection ensemble selects the best combination from spatiotemporal and temporal-pyramid models to achieve the best accuracy for classifying the seven basic emotions. In our experiment, we take benchmark measurement on the AFEW dataset with high accuracy.

Myeloid Cell-Restricted STAT3 Signaling Controls a Cell-Autonomous Antifibrotic Repair Program.

Myeloid cells can be beneficial as well as harmful in tissue regenerative responses. The molecular mechanisms by which myeloid cells control this critical decision of the immune system are not well understood. Using two different models of physiological acute or pathological chronic skin damage, in this study we identified myeloid cell-restricted STAT3 signaling as important and an injury context-dependent regulator of skin fibrosis. Targeted disruption of STAT3 signaling in myeloid cells significantly accelerated development of pathological skin fibrosis in a model of chronic bleomycin-induced tissue injury, whereas the impact on wound closure dynamics and quality of healing after acute excision skin injury was minor. Chronic bleomycin-mediated tissue damage in control mice provoked an antifibrotic gene signature in macrophages that was characterized by upregulated expression of IL-10, SOCS3, and decorin. In contrast, in STAT3-deficient macrophages this antifibrotic repair program was abolished whereas TGF-ß1 expression was increased. Notably, TGF-ß1 synthesis in cultured control bone marrow-derived macrophages (BMDMs) was suppressed after IL-10 exposure, and this suppressive effect was alleviated by STAT3 deficiency. Accordingly, coculture of IL-10-stimulated control BMDMs with fibroblasts suppressed expression of the TGF-ß1 downstream target connective tissue growth factor in fibroblasts, whereas this suppressive effect was lost by STAT3 deficiency in BMDMs. Our findings highlight a previously unrecognized protective role of myeloid cell-specific STAT3 signaling in immune cell-mediated skin fibrosis, and its regulatory pathway could be a potential target for therapy.

A common variant near TGFBR3 is associated with primary open angle glaucoma.

Primary open angle glaucoma (POAG), a major cause of blindness worldwide, is a complex disease with a significant genetic contribution. We performed Exome Array (Illumina) analysis on 3504 POAG cases and 9746 controls with replication of the most significant findings in 9173 POAG cases and 26 780 controls across 18 collections of Asian, African and European descent. Apart from confirming strong evidence of association at CDKN2B-AS1 (rs2157719 [G], odds ratio [OR] = 0.71, P = 2.81 × 10(-33)), we observed one SNP showing significant association to POAG (CDC7-TGFBR3 rs1192415, ORG-allele = 1.13, Pmeta = 1.60 × 10(-8)). This particular SNP has previously been shown to be strongly associated with optic disc area and vertical cup-to-disc ratio, which are regarded as glaucoma-related quantitative traits. Our study now extends this by directly implicating it in POAG disease pathogenesis.

Performance Analysis of Physical Layer Security of Opportunistic Scheduling in Multiuser Multirelay Cooperative Networks.

In this paper, we study the physical layer security (PLS) of opportunistic scheduling for uplink scenarios of multiuser multirelay cooperative networks. To this end, we propose a low-complexity, yet comparable secrecy performance source relay selection scheme, called the proposed source relay selection (PSRS) scheme. Specifically, the PSRS scheme first selects the least vulnerable source and then selects the relay that maximizes the system secrecy capacity for the given selected source. Additionally, the maximal ratio combining (MRC) technique and the selection combining (SC) technique are considered at the eavesdropper, respectively. Investigating the system performance in terms of secrecy outage probability (SOP), closed-form expressions of the SOP are derived. The developed analysis is corroborated through Monte Carlo simulation. Numerical results show that the PSRS scheme significantly improves the secure ability of the system compared to that of the random source relay selection scheme, but does not outperform the optimal joint source relay selection (OJSRS) scheme. However, the PSRS scheme drastically reduces the required amount of channel state information (CSI) estimations compared to that required by the OJSRS scheme, specially in dense cooperative networks.

Outage Performance Analysis of Relay Selection Schemes in Wireless Energy Harvesting Cooperative Networks over Non-Identical Rayleigh Fading Channels.

In this paper, we study relay selection in decode-and-forward wireless energy harvesting cooperative networks. In contrast to conventional cooperative networks, the relays harvest energy from the source's radio-frequency radiation and then use that energy to forward the source information. Considering power splitting receiver architecture used at relays to harvest energy, we are concerned with the performance of two popular relay selection schemes, namely, partial relay selection (PRS) scheme and optimal relay selection (ORS) scheme. In particular, we analyze the system performance in terms of outage probability (OP) over independent and non-identical (i.n.i.d.) Rayleigh fading channels. We derive the closed-form approximations for the system outage probabilities of both schemes and validate the analysis by the Monte-Carlo simulation. The numerical results provide comprehensive performance comparison between the PRS and ORS schemes and reveal the effect of wireless energy harvesting on the outage performances of both schemes. Additionally, we also show the advantages and drawbacks of the wireless energy harvesting cooperative networks and compare to the conventional cooperative networks.

A soft-hard combination-based cooperative spectrum sensing scheme for cognitive radio networks.

In this paper we propose a soft-hard combination scheme, called SHC scheme, for cooperative spectrum sensing in cognitive radio networks. The SHC scheme deploys a cluster based network in which Likelihood Ratio Test (LRT)-based soft combination is applied at each cluster, and weighted decision fusion rule-based hard combination is utilized at the fusion center. The novelties of the SHC scheme are as follows: the structure of the SHC scheme reduces the complexity of cooperative detection which is an inherent limitation of soft combination schemes. By using the LRT, we can detect primary signals in a low signal-to-noise ratio regime (around an average of -15 dB). In addition, the computational complexity of the LRT is reduced since we derive the closed-form expression of the probability density function of LRT value. The SHC scheme also takes into account the different effects of large scale fading on different users in the wide area network. The simulation results show that the SHC scheme not only provides the better sensing performance compared to the conventional hard combination schemes, but also reduces sensing overhead in terms of reporting time compared to the conventional soft combination scheme using the LRT.

Structural basis of the green-blue color switching in proteorhodopsin as determined by NMR spectroscopy.

Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green-blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14-C15. Upon mutation, a significantly stretched C14-C15 bond, deshielding of C15, and a slight alteration of the retinal chain's out-of-plane twist was observed. The L105Q blue switch therefore acts locally on the retinal itself and induces a conjugation defect between the isomerization region and the imine linkage. Consequently, the S0-S1 energy gap increases, resulting in the observed blue shift. The distortion of the chromophore structure also offers an explanation for the elongated primary reaction detected by pump-probe spectroscopy, while chemical shift perturbations within the protein can be linked to the elongation of late-photocycle intermediates studied by flash photolysis. Besides resolving a long-standing problem, this study also demonstrates that the combination of data obtained from high-field and DNP-enhanced MAS NMR spectroscopy together with time-resolved optical spectroscopy enables powerful synergies for in-depth functional studies of membrane proteins.

Reconstructing transient pressures in pipe networks from local observations by using physics-informed neural networks.

Reconstructing transient states presents significant challenges, particularly within complex pipe networks. These challenges arise due to nonlinear behaviours, inherent uncertainties in the system, and limitations in data availability. This work proposed a novel approach employing Physics-Informed Neural Networks (PINN) to reconstruct transient states in pipe networks, even with limited sensor data. To integrate the complex topology of pipe network systems into neural networks, the method integrates the PINN framework with an efficient elastic water column (EWC) model which can be simply formulated across diverse pipe network configurations. The results showed the proposed PINN method can accurately reconstruct the pressure and flow variation at unmonitored locations, even provided with noisy data at a limited number of locations. One of its advantages lies in its ability to effectively capture extreme values that hold potential significance for pipe infrastructure, providing a promising avenue for pipe failure analysis and enhanced safety management. Laboratory experiments have also been conducted to validate the efficacy and reliability of this method, thus further underlining its potential for real-world applications.

Human MHC Class II and Invariant Chain Knock-in Mice Mimic Rheumatoid Arthritis with Allele Restriction in Immune Response and Arthritis Association.

Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.

Effect of charge on the stabilization of water-in-water emulsions by thermosensitive bis-hydrophilic microgels.

HYPOTHESIS: Molecular surfactants are not able to stabilize water-in-water (W/W) emulsions, unlike nano or micro-particles, which can achieve this in some cases. However, the effect of electrostatic interactions between particles on the emulsion stability has rarely been investigated. We hypothesize that introducing charges modifies the stabilization capacity of particles and renders it both pH- and ionic strength-dependent. EXPERIMENTS: Charge was introduced into bis-hydrophilic and thermoresponsive dextran/polyN-isopropylacrylamide microgels by replacing a small fraction of polyN-isopropylacrylamide with acrylic acid groups. The size of the microgels was obtained by dynamic light scattering. The stability and microstructure of dextran/poly(ethyleneoxide)-based W/W emulsions, was studied as a function of pH, NaCl concentration and temperature using confocal microscopy and by analytical centrifugation. FINDINGS: The swelling degree of charged microgels depends on the pH, ionic strength and the temperature. In the absence of salt, charged microgels do not adsorb at the interface and have little stabilizing effect even after neutralization. However, the interfacial coverage and the stability increase with rising concentration of NaCl. Saltinduced stabilization of these emulsions was also observed at 50 °C. Increasing the temperature strongly influences the emulsion stability at low pH.

Antigen-presenting autoreactive B cells activate regulatory T cells and suppress autoimmune arthritis in mice.

B cells undergo several rounds of selection to eliminate potentially pathogenic autoreactive clones, but in contrast to T cells, evidence of positive selection of autoreactive B cells remains moot. Using unique tetramers, we traced natural autoreactive B cells (C1-B) specific for a defined triple-helical epitope on collagen type-II (COL2), constituting a sizeable fraction of the physiological B cell repertoire in mice, rats, and humans. Adoptive transfer of C1-B suppressed arthritis independently of IL10, separating them from IL10-secreting regulatory B cells. Single-cell sequencing revealed an antigen processing and presentation signature, including induced expression of CD72 and CCR7 as surface markers. C1-B presented COL2 to T cells and induced the expansion of regulatory T cells in a contact-dependent manner. CD72 blockade impeded this effect suggesting a new downstream suppressor mechanism that regulates antigen-specific T cell tolerization. Thus, our results indicate that autoreactive antigen-specific naïve B cells tolerize infiltrating T cells against self-antigens to impede the development of tissue-specific autoimmune inflammation.

A study implementing real-time PCR to identify Strongyloides species of third-stage larvae in human stool samples from Southern Vietnam.

Background: Strongyloidiasis, a neglected disease caused by intestinal nematodes of the genus, is endemic to tropical and subtropical areas such as Vietnam. Morphological methods only identify the genus, while DNA-molecular techniques are susceptible in Strongyloides spp. detection. The study aims to determine the prevalence of dominant Strongyloides species among the population in Duc Hoa district, Long An, Vietnam. Methods: A cross-sectional study used 1190 stool specimens collected from July 2017 to November 2018. All samples were transported within 2 h, stored at 2-8°C, and processed within 48 h for microscopy smear and culture at the Laboratory of Medical Parasitology, Pham Ngoc Thach University of Medicine (PNT). Then all positive samples with the above 2 methods were verified by real-time PCR technique. Real-time PCR amplification was conducted at the Laboratory of Molecular Biology, PNT. Results: Direct microscopy and modified Harada-Mori culture detected Strongyloides spp. larvae in 79/1190 samples (6.6%). About 94.2% of the DNA samples were Strongyloides stercoralis, 2.9% were co-infections with Strongyloides ratti and S. stercoralis, and 2.9% were patients with S. ratti. The identity of 12/14 sequences was confirmed as S. stercoralis with a high level of similarity (91.3%-100%) and over 98% for S. ratti. Conclusion: DNA-molecular techniques and sequence analysis are highly suitable for identifying Strongyloides species isolated from stool samples. It is remarkable evidence of the presence of zoonosis S. ratti disease in human, not just the known S. stercoralis. It is likely to result in a certain proportion of people being infected by this animal-borne infectious pathogen.

Prevalence and causes of blindness in children in Vietnam.

OBJECTIVE: To estimate the prevalence of blindness in children in Vietnam and to assess the major causes. DESIGN: A population-based study sampled children from 16 provinces across Vietnam. The second study examined children attending all blind schools in Vietnam. PARTICIPANTS: In 16 provinces, 28 800 children aged 0-15 were sampled. In 28 blind schools, 569 children aged 0-15 were examined. INTERVENTION: In children not seeing well according to the parents, presenting visual acuity (PVA) was assessed. If PVA was <3/60 in one or both eyes, the child was examined by an ophthalmologist. All children in blind schools were examined by a pediatric ophthalmologist. MAIN OUTCOME MEASURES: Blindness was defined as PVA <3/60 in the better eye. Causes of visual loss were classified using the World Health Organization classification. RESULTS: In the population-based study, 22 children had a PVA <3/60 in the better eye, a prevalence of 7.6/10 000 children (95% confidence interval [CI], 4.9-11.8/10 000). Fourteen children had a pinhole visual acuity <3/60 in the better eye, a prevalence of 4.9/10 000 (95% CI, 2.8-8.4/10 000). An estimated 16 400 (95% CI, 10 500-25 300), children were blind from all causes, with 36.4% from uncorrected refractive errors. In the blind schools, 411 children had a PVA <3/60 in the better eye and 55.5% were male. Conditions of the retina (24.6%) and cornea (24.0%) predominated. Retinopathy of prematurity (ROP) caused blindness in 32.6% of children younger than 10 years, but in only 6% of older children. The converse was true for corneal scarring and phthisis (14.0% and 27.3%, respectively). All other causes were similar between age groups (53.5% and 66.7%, respectively). More than half of all causes were avoidable. CONCLUSIONS: Vietnam is developing very rapidly, and this is impacting health indices. The mortality rate of those younger than 5 years declined from 65/100 live births in 1980 to 14/100 in 2008. The findings of this study show these changes, because the childhood blindness prevalence was relatively low, and the causes show improved control of measles and vitamin A deficiency, as well as increased services for premature babies. Eye care services for children should now focus on refractive errors, cataract, and control of ROP.

Physics-informed neural networks for hydraulic transient analysis in pipeline systems.

In water pipeline systems, monitoring and predicting hydraulic transient events are important to ensure the proper operation of pressure control devices (e.g., pressure reducing valves) and prevent potential damages to the network infrastructure. Simulating transient pressures using traditional numerical methods, however, require a complete model with known boundary and initial conditions, which is rarely able to obtain in a real system. This paper proposes a new physics-based and data-driven method for targeted transient pressure reconstruction without the need of having a complete pipe system model. The new method formulates a physics-informed neural network (PINN) by incorporating both measured data and physical laws of the transient flow in the training process. This enables the PINN to learn and explore hidden information of the hydraulic transient (e.g., boundary conditions and wave damping characteristics) that is embedded in the measured data. The trained PINN can then be used to predict transient pressures at any location of the pipeline. Results from two numerical and one experimental case studies showed a high accuracy of the pressure reconstruction using the proposed approach. In addition, a series of sensitivity analyses have been conducted to determine the optimal hyperparameters in the PINN and to understand the effects of the sensor configuration on the model performance.

Structural dynamics determine voltage and pH gating in human voltage-gated proton channel.

Voltage-gated proton (Hv) channels are standalone voltage sensors without separate ion conductive pores. They are gated by both voltage and transmembrane proton gradient (i.e., ∆pH), serving as acid extruders in most cells. Like the canonical voltage sensors, Hv channels are a bundle of four helices (named S1 -S4), with the S4 segment carrying three positively charged Arg residues. Extensive structural and electrophysiological studies on voltage-gated ion channels, in general, agree on an outwards movement of the S4 segment upon activating voltage, but the real-time conformational transitions are still unattainable. With purified human voltage-gated proton (hHv1) channels reconstituted in liposomes, we have examined its conformational dynamics, including the S4 segment at different voltage and pHs using single-molecule fluorescence resonance energy transfer (smFRET). Here, we provide the first glimpse of real-time conformational trajectories of the hHv1 voltage sensor and show that both voltage and pH gradient shift the conformational dynamics of the S4 segment to control channel gating. Our results indicate that the S4 segment transits among three major conformational states and only the transitions between the inward and outward conformations are highly dependent on voltage and pH. Altogether, we propose a kinetic model that explains the mechanisms underlying voltage and pH gating in Hv channels, which may also serve as a general framework for understanding the voltage sensing and gating in other voltage-gated ion channels.