Tension Threshold Research Articles

Knockout of EPO gene in blunt snout bream (Megalobrama amblycephala) by CRISPR/Cas9 reveals its roles in hypoxia-tolerance

Erythropoietin (EPO) is a glycoprotein hormone that plays a crucial role as a downstream regulator in the Hypoxia-inducible Factor (HIF) signaling pathway, mainly involved in the regulation of erythropoiesis. The open reading frame (ORF) of EPO in blunt snout bream (Megalobrama amblycephala, BSB) was 561 bp, encoding 186 amino acids. Phylogenetic analysis revealed it clusters with homologs in others teleost. We designed two target sites (targets 1, 2) to obtain gene knockout mutants, with the mature mRNA sequence of EPO that were located in exon 2, exon 3, respectively. The mutation efficiency of targets 1, 2 was 19.60%, 24.22%, respectively, both containing 6 mutation types. F1EPO+/− mutants with the same mutation type were screened, and select F2EPO−/− mutants after mating. At the age of five months, EPO−/− fish demonstrated a significant (p < 0.05) increase in the average oxygen tension threshold for the loss of equilibrium (LOEcrit) and the shedding degree of interlamellar cell masses (ILCM), whereas a significant (p < 0.05) decrease was observed in the number of red blood cells (RBC) and the concentration of hemoglobin (Hb) under hypoxia conditions, in comparison to the control group. Notably, injection of EPO mRNA rescued the downregulation of EPO expression in EPO−/− mutant, and overexpression of the EPO gene did not disturb the normal development of BSB embryos. These findings provide new insights into the potential for applying CRISPR/Cas9 in other aquaculture fish species and the breeding of hypoxia-tolerant BSB.

Effective cell membrane tension protects red blood cells against malaria invasion.

A critical step in how malaria parasites invade red blood cells (RBCs) is the wrapping of the membrane around the egg-shaped merozoites. Recent experiments have revealed that RBCs can be protected from malaria invasion by high membrane tension. While cellular and biochemical aspects of parasite actomyosin motor forces during the malaria invasion have been well studied, the important role of the biophysical forces induced by the RBC membrane-cytoskeleton composite has not yet been fully understood. In this study, we use a theoretical model for lipid bilayer mechanics, cytoskeleton deformation, and membrane-merozoite interactions to systematically investigate the influence of effective RBC membrane tension, which includes contributions from the lipid bilayer tension, spontaneous tension, interfacial tension, and the resistance of cytoskeleton against shear deformation on the progression of membrane wrapping during the process of malaria invasion. Our model reveals that this effective membrane tension creates a wrapping energy barrier for a complete merozoite entry. We calculate the tension threshold required to impede the malaria invasion. We find that the tension threshold is a nonmonotonic function of spontaneous tension and undergoes a sharp transition from large to small values as the magnitude of interfacial tension increases. We also predict that the physical properties of the RBC cytoskeleton layer-particularly the resting length of the cytoskeleton-play key roles in specifying the degree of the membrane wrapping. We also found that the shear energy of cytoskeleton deformation diverges at the full wrapping state, suggesting the local disassembly of the cytoskeleton is required to complete the merozoite entry. Additionally, using our theoretical framework, we predict the landscape of myosin-mediated forces and the physical properties of the RBC membrane in regulating successful malaria invasion. Our findings on the crucial role of RBC membrane tension in inhibiting malaria invasion can have implications for developing novel antimalarial therapeutic or vaccine-based strategies.

Evaluation of hypoxia tolerance in F3 hybrids of blunt snout bream (Megalobrama amblycephala♀) × topmouth culter (Culter alburnus♂)

To compare gill hypoxia adaptation mechanisms, the hypoxia-related physiological indices were measured in F3 self-crossing population of blunt snout bream (Megalobrama amblycephala)♀ × topmouth culter (Culter alburnus)♂ hybrids. The critical oxygen pressure (Pcrit) of F3 population was 28.33 ± 1.58 Torr, which is higher than topmouth culter (21.69 ± 0.85 Torr) and lower than blunt snout bream (33.00 ± 0.97 Torr). Moreover, the lower oxygen tension threshold for loss of equilibrium (LOEcrit) of F3 hybrids was 1.09 mg·L−1 higher than topmouth culter (0.91 mg·L−1) and lower than blunt snout bream (1.30 mg·L−1). These results indicated that the hypoxia tolerance of F3 hybrid fish was significantly higher than that of blunt snout bream. In the hypoxic environment, gill remodeling reactions increased the mass-specific lamellar area. In this study, gill remodeling was observed in blunt snout bream, topmouth culter and F3 hybrids. However, a particularly intriguing finding was that the gill recovery ability of F3 population was better than that of blunt snout bream after reoxygenation treatment. This might be related to the capability of physiological and biochemical regulation and lactic acid excretion of F3 hybrids. Together, these results showed that the F3 population inherited the gill remodeling mechanism of their parents, which had better hypoxia tolerance than blunt snout bream, and they could change the morphology and physiological and biochemical levels of gill tissue to cope with hypoxia.

Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response.

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, ∆mscL, ∆mscS, a double knockout ∆mscS ∆mscK, and a triple knockout ∆mscL ∆mscS ∆mscK, in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains, MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the ∆mscL strain, suggesting either a crosstalk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants.

Functional exploration of SNP mutations in HIF2αb gene correlated with hypoxia tolerance in blunt snout bream (Megalobrama amblycephala).

Blunt snout bream (Megalobrama amblycephala) is sensitive to hypoxia environment. Hypoxia-inducible factor (HIF) is the most critical factor in the HIF pathway, which strictly regulates the hypoxia stress process of fish. In this study, we found six hifα genes in blunt snout bream that demonstrated different expressions under hypoxia conditions. In HEK293T cells, all six hifαs were detected to activate the HRE region by luciferase reporter assay. More importantly, we identified two linkage-disequilibrium SNP sites at exon 203 and 752 of the hif2αb gene in blunt snout bream. Haplotype II (A203A752) and its homozygous diplotype II (A203A203A752A752) appeared frequently in a selected strain of blunt snout bream with hypoxia tolerance. Diplotype II has a lower oxygen tension threshold for loss of equilibrium (LOEcrit) over a similar range of temperatures. Moreover, its erythrocyte number increased significantly (p < 0.05) than those in diplotype I and diplotype III strains at 48h of hypoxia. The enzymes related with hypoxia tolerant traits, i.e., reduced glutathione, superoxide dismutase, and catalase, were also significantly (p < 0.05) induced in diplotype II than in diplotype I or III. In addition, the expression of epo in the liver of diplotype II was significantly (p < 0.01) higher than that in the diplotype I or III strains at 48h of hypoxia. Taken together, our results found that the hypoxia-tolerant-related diplotype II of hif2αb has the potential to be used as a molecular marker in future genetic breeding of hypoxia-tolerant strain.

Tension Gauge Tethers as Tension Threshold and Duration Sensors.

Mechanotransduction, the process by which cells respond to tension transmitted through various supramolecular linkages, is important for understanding cellular behavior. Tension gauge tethers (TGTs), short fragments of double-stranded DNA that irreversibly break under shear-stretch conditions, have been used in live cell experiments to study mechanotransduction. However, our current understanding of TGTs' mechanical responses is limited, which limits the information that can be gleaned from experimental observations. In this study, we quantified the tension-dependent lifetime of TGTs to better understand their mechanical stability under various physiologically relevant stretching conditions. This work has broad applications for using TGTs as tension threshold and duration sensors and also suggests the need to revisit previous interpretations of experimental observations.

Numerical study of opposed zero-net-mass-flow jet-induced erythrocyte mechanoporation

With the advantages of biosafety and efficiency, increasing attention has been paid to the devices for gene and macromolecular drug delivery based on mechanoporation. The transient pore formation on the cell membrane allows cargo transportation when the membrane areal strain is beyond the critical pore value and below the lysis tension threshold. Based on this principle, we propose a method to apply the proper fluid stress on cells moving in a microchannel under the action of zero-net-mass-flux (ZNMF) jets. In this study, an immersed finite element method (IFEM) is adopted to simulate the interaction between the cells and the fluid fields so as to investigate the cell movement and deformation in this mechanoporation system. To evaluate the efficiency of the cargo delivery, a pore integral is defined as the mean pore rate when the cell passes through the jet region. By analyzing the effects of the parameters, including the pressure gradient along the microchannel, the jet amplitude, and the jet frequency, on the pore integrals, a group of optimized parameters for cargo delivery efficiency are obtained. Additionally, the stability and safety of this system are analyzed in detail. These results are helpful in designing the mechanoporation devices and improving their efficiency of drug delivery.

β2-Integrin Adhesive Bond Tension under Shear Stress Modulates Cytosolic Calcium Flux and Neutrophil Inflammatory Response

On arrested neutrophils a focal adhesive cluster of ~200 high affinity (HA) β2-integrin bonds under tension is sufficient to trigger Ca2+ flux that signals an increase in activation in direct proportion to increments in shear stress. We reasoned that a threshold tension acting on individual β2-integrin bonds provides a mechanical means of transducing the magnitude of fluid drag force into signals that enhance the efficiency of neutrophil recruitment and effector function. Tension gauge tethers (TGT) are a duplex of DNA nucleotides that rupture at a precise shear force, which increases with the extent of nucleotide overlap, ranging from a tolerance of 54pN to 12pN. TGT annealed to a substrate captures neutrophils via allosteric antibodies that stabilize LFA-1 in a high- or low-affinity conformation. Neutrophils sheared on TGT substrates were recorded in real time to form HA β2-integrin bonds and flux cytosolic Ca2+, which elicited shape change and downstream production of reactive oxygen species. A threshold force of 33pN triggered consolidation of HA β2-integrin bonds and triggered membrane influx of Ca2+, whereas an optimum tension of 54pN efficiently transduced activation at a level equivalent to chemotactic stimulation on ICAM-1. We conclude that neutrophils sense the level of fluid drag transduced through individual β2-integrin bonds, providing an intrinsic means to modulate inflammatory response in the microcirculation.

Efficiently whole-genomic mutagenesis approach by ARTP in blunt snout bream (Megalobrama amblycephala)

The atmospheric and room temperature plasma (ARTP) mutagenesis technique was first used in the present study to produce whole-genomic mutants in blunt snout bream (Megalobrama amblycephala). The optimal ARTP mutation parameters for semen were established according to sperm motility and abnormal embryonic rate. A total of 2026 mutated offsprings were produced with normal and abnormal individuals with a ratio of approximately 1:1. Among them, 384 normal mutant fish (~20%), after three months of cultivation, demonstrated improved performance with either fast growth or hypoxia tolerance, and 89 mutant individuals with both hypoxia tolerance and fast growth (H&F). The average bodyweight of the H&F fish increased by 26.48% compared with the control group. The average oxygen tension threshold for loss of equilibrium (LOEcrit) of the H&F fish was 0.45 mg/L, significantly (p < 0.05) lower than 0.86 mg/L in the control group. Meanwhile, the red blood cell count, hemoglobin concentration, and Na+/K+-ATPase enzyme activity of the H&F group increased significantly (p < 0.05), whereas, the gill tissue cell apoptosis rate decreased considerably (p < 0.05), suggesting that they are hypoxia tolerant. According to the genome resequencing analysis, 3,195,434 SNPs and 927,930 InDels were found in the H&F group, which were significantly higher than the 9127 SNPs and 2908 InDels in the control group. The average mutation rate in the H&F fishes reached 2.98 × 10−3 at the genome level, approximately 350 times that of the control group. Importantly, 3651 special non-synonymous SNP mutations were found in the H&F fish, which are located in 1223 genes. Among them, eight hypoxia- and growth-related genes with non-synonymous mutations at one or more sites were differentially expressed (DE) in H&F fish. Taken together, our results establish a novel whole-genomic mutagenesis approach using ARTP in blunt snout bream, which should be useful for future genetic breeding.

Peat depth as a control onSphagnummoisture stress during seasonal drought

AbstractPeatlands are globally important long‐term sinks of carbon, however there is concern that enhanced peat decomposition and moss moisture stress due to climate change mediated drought will reduce moss productivity making these ecosystems vulnerable to carbon loss and associated long‐term degradation. Peatlands are resilient to summer drought moss stress because of negative ecohydrological feedbacks that generally maintain a wet peat surface, but where feedbacks may be contingent on peat depth. We tested this ‘survival of the deepest’ hypothesis by examining water table (WT) position, near‐surface moisture content, and soil water tension in peatlands that differ in size, peat depth, and catchment area during a summer drought. All shallow sites (&lt;40 cm depth) lost their WT (i.e., the groundwater well was dry) for considerable time during the drought period. Near‐surface soil water tension increased dramatically at shallow sites following WT loss, increasing ~5–7.5× greater at shallow sites compared to deep sites (≥40 cm depth). During a mid‐summer drought intensive field survey, we found that 60–67% of plots at shallow sites exceeded a 100 mb tension threshold used to infer moss water stress. Unlike the shallow sites, tension typically did not exceed this 100 mb threshold at the deep sites. Using species dependent water content – chlorophyll fluorescence thresholds and relations between volumetric water content and WT depth, Monte Carlo simulations suggest that moss had nearly twice the likelihood of being stressed at shallow sites (0.38 ± 0.24) compared to deep sites (0.22 ± 0.18). This study provides evidence that mosses in shallow peatland may be particularly vulnerable to warmer and drier climates in the future, but where species composition may play an important role. We argue that a critical ‘threshold’ peat depth specific for different hydrogeological and hydroclimatic regions can be used to assess what peatlands are especially vulnerable to climate change mediated drought.

Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, many protective polymorphisms such as sickle cell trait having been selected to high frequencies in malaria endemic regions1,2. Recently, it was shown that the blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3–5. This is a similar degree of protection to sickle cell trait and considerably greater than the best malaria vaccine, but until now the protective mechanism has been unknown. Here, we demonstrate a significant impact of Dantu on Plasmodium falciparum-merozoite RBC invasion. Dantu was associated with extensive changes to the RBC surface protein repertoire, but unexpectedly, inhibition did not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we found a strong link between RBC tension and merozoite invasion and identified a tension threshold above which invasion rarely occurred, even in non-Dantu RBCs. Dantu RBCs had higher average tension, meaning that a greater proportion resisted invasion. These findings provide both an explanation for the malaria-protective effect of Dantu, and fresh insights into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs both within and between individuals.

EGFR activation attenuates the mechanical threshold for integrin tension and focal adhesion formation.

Mechanical forces, growth factors and the extracellular matrix all play crucial roles in cell adhesion. To understand how epidermal growth factor receptor (EGFR) impacts the mechanics of adhesion, we employed tension gauge tether (TGT) probes displaying the integrin ligand cRGDfK and quantified integrin tension. EGF exposure significantly increased spread area, cell circularity, integrated integrin tension, mechanical rupture density, radial organization and size of focal adhesions in Cos-7 cells on TGT surfaces. These findings suggest that EGFR regulates integrin tension and the spatial organization of focal adhesions. Additionally, we found that the mechanical tension threshold for outside-in integrin activation is tunable by EGFR. Parallel genetic and pharmacologic strategies demonstrated that these phenotypes are driven by ligand-dependent EGFR signaling. Our results establish a novel mechanism whereby EGFR regulates integrin activation and cell adhesion, providing control over cellular responses to the environment.This article has an associated First Person interview with the first author of the paper.

Membrane stiffness is one of the key determinants of E. coli MscS channel mechanosensitivity

Mechanosensitive (MS) channels have an intimate relationship with membrane lipids that underlie their mechanosensitivity. Membrane lipids may influence channel activity by directly interacting with MS channels or by influencing the global properties of the membrane such as elastic area expansion modulus or bending rigidity. Previous work has implicated membrane stiffness as a potential determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing elastic area expansion modulus. Increasing dioleoylphosphatidylethanolamine (DOPE) content of azolectin liposomes made it more difficult to activate EcMscS by membrane tension (i.e. increased gating threshold). This effect was exacerbated by stiffer forms of phosphatidylethanolamine such as the branched chain lipid diphytanoylphosphoethanolamine (DPhPE) or the fully saturated lipid distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Furthermore, a comparison of the branched chain lipid diphytanoylphosphocholine (DPhPC) to the stiffer DPhPE indicated again that it was harder to activate EcMscS in the presence of the stiffer DPhPE. We show that these effects are not due to changes in membrane bending rigidity as the membrane tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 remained the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes caused a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data suggests that membrane stiffness (elastic area expansion modulus) is one of the key determinants of the mechanosensitivity of EcMscS.

Vegetation source water identification using isotopic and hydrometric observations from a subhumid mountain catchment

AbstractThis study coupled long‐term hydrometric and stable water isotope data to identify links between subsurface water storage and vegetation in a subhumid mountain catchment in Arizona, USA. Specific observations included catchment‐scale hydrologic fluxes and soil water storage and stable water isotopes from stream water, soil water, groundwater, and sap water from Arizona pine (Pinus arizonica) and Douglas fir (Pseudotsuga menziesii) individuals. Here, we find that tightly bound soil water was sufficient to meet dry period vegetation water demand when the former was defined in terms of field capacity as opposed to a matric tension threshold. This water was a mixture of summer and winter precipitation that predominates in both shallow and deep soil waters, and contributed significantly to streamflow. We also identified a less common mobile water type that did not contribute significantly to streamflow and was related to infiltration during isotopically depleted precipitation events. Although each water type was used by both Arizona pine and Douglas fir vegetation, the second water type was dominant in Douglas fir sap water. Therefore, we conclude that Arizona pine and Douglas fir can occupy different ecohydrological niches at this subhumid mountain location. Further, a lack of isotopic distinction between tightly bound and inferred mobile soil water signals that the ecohydrological water source separation hypothesis is not entirely applicable at this site. The results of this study broadly highlight how alternative definitions of tightly bound water can influence interpretation of data, and contribute to a more thorough understanding of interactions between subsurface storage and plant water dynamics.

Quantifying tension in tension-free hiatal hernia repair: a new intra-operative technique.

A similar technique to measure crural closure tension has not been described before and with this method there is now a possibility to optimise this operation with objective measures, a hundred years after it was first described. The aims of this study were to develop a reliable method for measuring the tension of crural closure during hiatal hernia repair and to describe the tension characteristics of crural closure. 50 patients underwent crural tension measurement. Hiatal surface area (HSA) was measured intraoperatively and a Sauter FH 50 Universal Digital Force Gauge was used to measure the tension of crural closure during cruroplasty. Outcome measures included the mean tension of the crural closure and the presence of any muscle splitting during the cruroplasty. A combined total of 148 interrupted cruroplasty sutures were performed in all fifty patients. Each interrupted suture had three tension measurements recorded. The mean standard deviation amongst 148 sets of tension measurements was 0.27. Age, hiatal width and HSA were positively correlated with crural tension with r values of 0.44 (p = 0.0015), 0.81 (p < 0.0001) and 0.78 (p < 0.0001), respectively. Strength of association was low for age (r2 = 0.19) but moderate for hiatal width and HSA (r2 = 0.65 and 0.61, respectively). The presence of muscle splitting occurred at higher crural closure tension (5.3N vs. 1.62N, p < 0.0001). The lowest observed mean crural closure tension causing muscle splitting was 3.52N (IQR 3.93-6.77N). We have developed a technique for measuring the tension of crural closure during laparoscopic repair of hiatal hernia which is reproducible, quick, of low cost and requires only minimal additional equipment. Initial findings suggest that crural closure tension up to ~ 4N could be the permissible tension threshold for suture cruroplasty and higher tension often results in muscle splitting during cruroplasty.

Imaging Integrin Tension and Cellular Force at Submicron Resolution with an Integrative Tension Sensor.

Molecular tension transmitted by integrin-ligand bonds is the fundamental mechanical signal in the integrin pathway that plays significant roles in many cell functions and behaviors. To calibrate and image integrin tension with high force sensitivity and spatial resolution, we developed an integrative tension sensor (ITS), a DNA-based fluorescent tension sensor. The ITS is activated to fluoresce if sustaining a molecular tension, thus converting force to fluorescent signal at the molecular level. The tension threshold for ITS activation is tunable in the range of 10-60 pN that well covers the dynamic range of integrin tension in cells. On a substrate grafted with an ITS, the integrin tension of adherent cells is visualized by fluorescence and imaged at submicron resolution. The ITS is also compatible with cell structural imaging in both live cells and fixed cells. The ITS has been successfully applied to the study of platelet contraction and cell migration. This paper details the procedure for the synthesis and application of the ITS in the study of integrin-transmitted cellular force.

Determining Integrin Molecular Tension for the Recruitment and the Activation of Focal Adhesion Kinase

Focal adhesion kinase (FAK), a member of protein tyrosine kinase family, is an important mediator of signals transduced by integrins (major mechanosensitive transmembrane receptors transmitting cellular forces at cell matrix interface). FAK regulates various cellular functions such as cell adhesion, proliferation, differentiation and spreading. Although the biochemical role of FAK in integrin signaling is relatively more understood, the biomechanical processes for FAK's recruitment and activation by integrins remains less clear. To study the mechanobiology of FAK, Our group adopted two molecular tension tools, tension gauge tether (TGT) and integrative tension sensor (ITS), both having a designable tension threshold, selectable in the range of 10-60 pN. TGT globally knocks down integrin tensions in live cells to a designed force level, while ITS visualizes integrin tension by fluorescence imaging with high resolution. With TGT, ITS and a FRET-based biosensor reporting FAK activation, we manipulated and mapped integrin tension in live cells, and studied the consequent FAK aggregation and activation. ITS and FAK live cell imaging revealed that integrin tension higher than 12 pN was produced after FAK aggregation, indicating that FAK recruitment to focal adhesions does not require integrin tension higher than 12 pN. TGT and FAK biosensor revealed that FAK was not activated on TGT surfaces with tension tolerance (Ttol) of 12, 23 and 33 pN, but on the surfaces with Ttol of 43 pN and 54 pN. These results suggest that FAK activation by integrin signaling requires integrin tension higher than 33 pN. Overall, our study revealed that FAK recruitment and FAK activation have distinct requirement for integrin tensions at separate force levels.

Acupuncture in the treatment of temporomandibular muscle dysfunction

ABSTRACT BACKGROUND AND OBJECTIVES: Temporomandibular dysfunction consists of frequent non-dental pain in the orofacial region of multifactorial origin and interdisciplinary treatment, among them, acupuncture. The treatment of temporomandibular dysfunction acts both in muscle relaxation and pain control, trying to achieve the physical, mental, and emotional balance of the patient, thus reducing anxiety and improving the quality of life. The objective of this study was to evaluate acupuncture as a treatment for temporomandibular dysfunction. METHODS: A total of 34 volunteers screened and selected at the Federal University of Mato Grosso do Sul, diagnosed with muscle dysfunction according to the Research Diagnostic Criteria. They were randomly divided into two equal groups: group 1 treated with occlusal plaque, massage, thermotherapy and self-care guidelines; and group 2 treated with six acupuncture sessions lasting 30 minutes each. The pain was evaluated by the visual analog scale, and an algometer to assess the muscular tension of the temporal and masseter muscles. The limitation of mouth opening was measured with the use of calipers. The Mann-Whitney test was used for the non-normal distribution (visual analog scale and tension threshold) between the two groups (G1 and G2), and the Friedman test to compare the assessment periods (beginning of the treatment, after six weeks and four months) with a significance level of 5%. RESULTS: There was no difference in mouth opening, visual analog scale scores, or muscle tension threshold in relation to the type of treatment used. Both groups improved after six weeks of treatment. There was no statistical difference in the values obtained after six weeks and after four months. CONCLUSION: The statistical results showed that acupuncture increased the muscle tension threshold, improved the mouth opening and reduced pain, being as effective as the most commonly used conventional therapies.

Mapping Single Platelet Forces Directly by Fluorescence Imaging

Platelets are important blood cells mediating hemostasis and thrombosis. Integrin tension plays a critical role in most platelet functions, such as adhesion, activation, aggregation and contraction. Visualizing and measuring single platelet forces are desired in both research and diagnosis of platelet functions. Here we developed integrative tension sensor (ITS) which converts integrin molecular tension to fluorescent signal, therefore enabling cellular force mapping directly by fluorescence imaging. With the ITS, we mapped integrin-transmitted platelet force at 0.4 µm resolution during platelet adhesion and contraction. We found that platelet force distribution has strong polarization which is sensitive to treatment with anti-platelet drugs, suggesting that the ITS force map can report anti-platelet drug efficacy. Furthermore, the tension threshold required for ITS activation is tunable, allowing the selective imaging of integrin tension at different force levels in cells. This feature helped to reveal two distinct integrin tension levels in platelets: 12~54 piconewton tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful tension sensor for measuring and mapping integrin tension in platelets and many other adherent cells, therefore holding great potential in the study of cell mechanobiology.

The voltage-dependence of MscL has dipolar and dielectric contributions and is governed by local intramembrane electric field

Channels without canonical voltage sensors can be modulated by voltage acting on other domains. Here we show that besides protein dipoles, pore hydration can be affected by electric fields. In patches, both WT MscL and its V23T mutant show a decrease in the tension midpoint with hyperpolarization. The mutant exhibits a stronger parabolic dependence of transition energy on voltage, highly consistent with the favourable dielectric contribution from water filling the expanding pore. Purified V23T MscL in DPhPC droplet interface bilayers shows a similar voltage dependence. When reconstituted in an asymmetric DOPhPC/DPhPC bilayer carrying a permanent bias of ~130 mV due to a dipole potential difference between the interfaces, the channel behaved as if the local intramembrane electric field sets the tension threshold for gating rather than just the externally applied voltage. The data emphasize the roles of polarized water in the pore and interfacial lipid dipoles in channel gating thermodynamics.