Exploring Novel Sensor Design Ideas through Concentration-Induced Conformational Changes in PEG Single Chains.

Polyethylene glycol (PEG) is an artificial polymer with good biocompatibility and a low cost, which has a wide range of applications. In this study, the dynamic response of PEG single chains to different ion concentrations was investigated from a microscopic point of view based on single-molecule force spectroscopy, revealing unique interactions that go beyond the traditional sensor-design paradigm. Under low concentrations of potassium chloride, PEG single chains exhibit a gradual reduction in rigidity, while, conversely, high concentrations induce a progressive increase in rigidity. This dichotomy serves as the cornerstone for a profound understanding of PEG conformational dynamics under diverse ion environments. Capitalizing on the remarkable sensitivity of PEG single chains to ion concentration shifts, we introduce innovative sensor-design ideas. Rooted in the adaptive nature of PEG single chains, these sensor designs extend beyond the traditional applications, promising advancements in environmental monitoring, healthcare, and materials science.

Phenoxy Group-Containing Asymmetric Non-Fullerene Acceptors Achieved Higher VOC over 1.0 V through Alkoxy Side-Chain Engineering for Organic Solar Cells.

Alkoxy side chain engineering on the ß-position of the thienothiophene units of Y6 derivatives plays a vital role in improving photovoltaic performances with simultaneously increasing open-circuit voltage (Voc) and fill factor (FF). In this work, we prepared a series of asymmetric non-fullerene acceptors (NFAs) by introducing alkoxy side chains and phenoxy groups on the state-of-the-art Y6-derivative BTP-BO-4F. For the comparison, 2O-BO-4F with a symmetric alkoxy side chain on the outer thiophene units and BTP-PBO-4F with an asymmetric N-attached phenoxy alkyl chain on the pyrrole ring are synthesized from BTP-BO-4F. Thereafter, we construct four asymmetric NFAs by introducing different lengths of linear/branched alkoxy chains on the ß-position of the thienothiophene units of BTP-PBO-4F. The resulting NFAs, named L10-PBO, L12-PBO, B12-PBO, and B16-PBO (L = linear and B = branched alkoxy side chains), are collectively called OR-PBO-series. Unexpectedly, all OR-PBO NFAs exhibit strong edge-on molecular packing and weaker π-π interactions in the film state, which diminish the charge transfer in organic solar cell (OSC) devices. As a consequence, the optimal devices of OR-PBO-based binary blends show poor photovoltaic performances [power conversion efficiency (PCE) = 6.52-9.62%] in comparison with 2O-BO-4F (PCE = 12.42%) and BTP-PBO-4F (PCE = 15.30%) reference blends. Nevertheless, the OR-PBO-based binary devices show a higher Voc and smaller Vloss. Especially, B12-PBO- and B16-PBO-based devices achieve Voc over 1.00 V, which is the highest value of Y-series OSC devices to the best of our knowledge. Therefore, by utilizing higher Voc of OR-PBO binary blends, B12-PBO and B16-PBO are incorporated into the PM6:BTP-PBO-4F-based binary blend and fabricated ternary devices. As a result, the PM6:BTP-PBO-4F:B12-PBO ternary device delivers the best PCE of 15.60% with an increasing Voc and FF concurrently.

Quantitative PCR of string-test collected gastric material: A feasible approach to detect Helicobacter pylori and its resistance against clarithromycin and levofloxacin for susceptibility-guided therapy.

BACKGROUND: As the reduced eradication rate of Helicobacter pylori (H. pylori), we introduced string-test and quantitative PCR (qPCR) for susceptibility-guided therapy innovatively. The practicality of the string test was evaluated. METHODS: It was an open-label, non-randomized, parallel, single-center study, in which subjects tested by 13 C- urea breath test (UBT) and string-qPCR were enrolled. Based on the results of string-qPCR, we calculated clarithromycin and levofloxacin resistance rates and gave 13 C-UBT positive patients 14 days susceptibility-guided bismuth quadruple therapy. In the empirical therapy group, we retrospectively analyzed the treatment results of 13 C-UBT positive patients also treated with bismuth quadruple at Shenzhen Luohu People's Hospital from January 2021 to May 2022. The eradication rate was compared between susceptibility-guided therapy and empirical therapy groups. RESULTS: The diagnosis of H. pylori infection using the string-qPCR had an overall concordance rate of 95.9% with the 13 C-UBT results. Based on the results of string-qPCR, the clarithromycin and levofloxacin resistance rates were 26.1% and 31.8%, respectively. The patients who were given 14 days susceptibility-guided bismuth-based quadruple therapy achieved a high H. pylori eradication rate of 91.8%. Retrospective analysis of patient treatment data from January 2021 to May 2022 available in the hospital database revealed an overall success rate of 82.3% for those who received empirical bismuth-based quadruple therapies, which is marginally significantly lower than that of the string-qPCR susceptibility-guided group (p = 0.084). CONCLUSION: The high treatment success rate of 91.8% indicates that the string-qPCR test is a valuable and feasible approach for clinical practice to help improve H. pylori treatment success rate.

Age-Related Changes in Trabecular Bone Score and Bone Mineral Density in Chinese Men: A Cross-Sectional and Longitudinal Study.

Purpose: This study was designed to explore age-related changes in trabecular bone score (TBS) and bone mineral density (BMD) in Chinese men through cross-sectional and longitudinal studies. Patients and Methods: We included adult men who had at least twice TBS and BMD examinations in our hospital between January 2013 and December 2020. All men were divided into an age subgroup per 10 years, comparing differences in baseline lumbar spine (LS) TBS and BMD at various parts between each age group and analyzing age-related changes in TBS and BMD during follow-up. Results: Baseline data showed that in men aged 36 to 85 years, BMD in the hip region showed a decreasing trend with age (P for trend < 0.01). However, TBS reached a high value around the age of 50, after which it decreased with age (P for trend = 0.03). During a mean follow-up of 3 years, the average annual change rate at TBS was -0.17% in men aged 36 to 85 years, with the fastest decrease rate -1.08% at 66 to 75 years (P < 0.05). The mean annual rate of change in LS BMD in different age subgroups increased with age (P for trend = 0.001). There was no significant decrease in mean annual change in BMD in hip regions. Conclusion: In men aged 36~85 years, the trend of TBS was inconsistent with BMD. Men experience a high value of LS TBS around age 50, later than the commonly believed age of peak BMD, which may reflect developmental differences between bone microstructure and bone minerals. The TBS may be used as a better indicator of changes in bone strength than BMD in adult men at short-term follow-up. The rapid loss of TBS at age 66 to 75 may have implications for the prevention and medication of osteoporosis in men.

Effects of Leisure Obstacles, Job Satisfaction, and Physical and Mental Health on Job Intentions of Medical Workers Exposed to COVID-19 Infection Risk and Workplace Stress.

The purpose of this study was to investigate the effects of leisure obstacles, job satisfaction, physical and mental health, and work intentions of medical workers in Taiwan. SPSS 26.0 and AMOS 24.0 statistical software were used to analyze 208 questionnaires by basic statistical tests, t-tests, and structural model analysis. Results: Under the epidemic, medical workers were unable to develop job identity due to low promotion opportunities and low job achievement. The lack of recreational exercise skills, time, and information created leisure obstacles. In addition, they were unable to express their true selves freely at work, which led to health problems such as reduced enthusiasm, mental weakness, and emotional irritability. In particular, female medical workers felt more strongly about the issues of leisure obstacles and the intention to stay in their jobs. The study found that the higher their job satisfaction, the higher their intention to stay in the job, while the more pronounced the leisure obstacles and physical and mental health problems, the more pronounced their intention to leave.

Numerical Study of T-Shaped Micromixers with Vortex-Inducing Obstacles in the Inlet Channels.

To enhance fluid mixing, a new approach for inlet flow modification by adding vortex-inducing obstacles (VIOs) in the inlet channels of a T-shaped micromixer is proposed and investigated in this work. We use a commercial computational fluid dynamics code to calculate the pressure and the velocity vectors and, to reduce the numerical diffusion in high-Peclet-number flows, we employ the particle-tracking simulation with an approximation diffusion model to calculate the concentration distribution in the micromixers. The effects of geometric parameters, including the distance between the obstacles and the angle of attack of the obstacles, on the mixing performance of micromixers are studied. From the results, we can observe the following trends: (i) the stretched contact surface between different fluids caused by antisymmetric VIOs happens for the cases with the Reynolds number (Re) greater than or equal to 27 and the enhancement of mixing increases with the increase of Reynolds number gradually, and (ii) the onset of the engulfment flow happens at Re≈125 in the T-shaped mixer with symmetric VIOs or at Re≈140 in the standard planar T-shaped mixer and results in a sudden increase of the degree of mixing. The results indicate that the early initiation of transversal convection by either symmetric or antisymmetric VIOs can enhance fluid mixing at a relatively lower Re.

[Effect of indwelling drainage tube and extubation time on recessive hemorrhage and functional recovery after total hip arthroplasty].

OBJECTIVE: To study and compare the effect of indwelling drainage tube and extubation time on occult hemorrhage and functional recovery after total hip arthroplasty(THA). METHODS: From July 2017 to June 2018, 123 patients who underwent THA in our hospital for the first time were selected as the subjects of study. According to whether the drainage tube was retained or not and the time of extubation, they were divided into three groups:in group A, 41 patients (24 males, 17 females, age 53 to 77 years) did not put drainage tube after THA;in group B, 41 patients were removed 24 hours after THA, 26 males and 15 females, aged 55 to 74 years;in group C, 41 patients were removed 48 hours after THA, 25 males and 16 females, aged 52 to 75 years. The VAS score of pain 72 hours after THA, the total and recessive blood loss, the time of starting functional exercise, and the incidence of postoperative limb swelling were recorded. All the patients were followed up for one year after discharge. Harris hip score was used to evaluate the degree of hip function recovery one year after operation. RESULTS: The occult blood loss of group A, B and C were(513.6±25.3), (521.7±33.4), (519.3±29.8) ml, respectively, with no significant difference(P>0.05). There was no significant difference in blood loss in operation among the three groups(P>0.05). In group B and C, the postoperative apparent blood loss was more than that in group A (P<0.05). There was no significant difference in VAS scores of the three groups before and 72 hours after operation (P>0.05). The time of getting out of bed in group A was shorter than that in group B and C (P<0.05), and that in group B was shorter than that in group C(P<0.05). The Harris hip score at 1 year after operationof the three groups was significantly higher than that of before operation (P<0.05). There was no significant difference in Harris hip score before and after operation among three groups (P>0.05). There was no significant difference in the incidence of complications among three groups (P>0.05). CONCLUSION: Whether the drainage tube is left or not and the time of extubation have no significant effect on the latent blood loss and functional recovery after THA, but without drainage tube after THA can reduce the apparent blood loss, patients can get out of bed at 6 hours after THA, which is more conducive to the recovery and nursing of patients.

Molecular Mechanisms of the Ultra-Strong Inhibition Effect of Oxidized Carbon Dots on Human Insulin Fibrillation.

Amyloid fibrillation of protein is associated with a great variety of pathologic conditions. The aggregation of protein is a complicated process with multisteps, whereas most of the inhibitors with elaborately designed structures can show an inhibition effect only on the nucleation stages of protein fibrillation. Herein, oxidized carbon dots (CDs) were achieved to study the relationship between the surface properties of CDs and their inhibition effect on human insulin (HI) fibrillation. More oxygen-containing function groups can be obtained after oxidation reaction of CDs, such as -OH and -COOH. The results show that 10-1 CDs (the mass ratios of CD/KMnO4 is 10:1), with the highest carboxyl group content, possess the best inhibition ability. All the nucleation, growth, and final phases can be retarded by 10-1 CDs, which have been studied in detail by fluorescence spectra. However, CDs without oxidation can show only a weak inhibition effect on the nucleation stage. The 10-1 CDs is demonstrated to binding with HI monomers much stronger than that of CDs by isothermal titration calorimetry (ITC). Moreover, molecular dynamics simulations (MD) studies indicate that CDs with more carboxyl groups can show stronger affinities with native or unfolded HI monomers, which may be mainly derived from the active binding sites of histidine residues (His5 and His10) on B-chain through electrostatic interaction. Because the unfolding of B-chain in HI is prior to that of A-chain in our MD simulations, the later aggregation of HI can be inhibited effectively by the stronger binding forces between 10 and 1 CDs and the B-chain of HI.

Bone mineral density and trabecular bone score in Chinese subjects with sarcopenia.

BACKGROUND: As the general population is aging worldwide, the incidence of sarcopenia and osteoporosis is also rapidly increasing. Studies have found the link between sarcopenia and osteoporosis, but the relationship between sarcopenia and osteoporosis, especially bone microarchitecture, remains unclear. AIMS: To investigate the relationship between components of sarcopenia (muscle mass, handgrip strength, and gait speed) and components of osteoporosis [bone mass measured by bone mineral density (BMD) and bone microarchitecture measured by trabecular bone score (TBS)] in Chinese subjects. METHODS: 318 Chinese men and 203 Chinese women were included in our study. Muscle mass and BMD were measured by dual-energy X-ray absorptiometry (DXA). TBS iNsight® software was used for TBS. Jamar hydraulic hand dynamometer was used to assess muscle strength, and gait speed was used to assess physical performance. RESULTS: We found that the relative appendicular skeletal muscle mass (RASM) in both genders and handgrip strength in women correlated positively with TBS, RASM in men and handgrip strength in women correlated positively with BMDs. In the multiple linear regression model, RASM was positively associated with TBS in both genders, but no significant association was observed between RASM and BMDs. Interestingly, handgrip strength showed positive association with all evaluated BMDs and TBS in women, but not in men. Women with sarcopenia had lower TBS and BMDs at all evaluated sites. Men with sarcopenia had lower BMDs only at femur neck and total hip. CONCLUSIONS: The reduction of muscle mass and strength was significantly associated with decreased bone mass and deteriorated bone microarchitecture. More importantly, low muscle mass is an independent risk factor for bone microarchitecture in Chinese subjects.

The correlation of muscle thickness and pennation angle assessed by ultrasound with sarcopenia in elderly Chinese community dwellers.

Background: Sarcopenia is typically defined as the loss of muscle mass, strength and low physical performance with aging. Ultrasound is a safe and easy method for evaluating muscle mass and quality by muscle thickness (MT) and pennation angle (PA), respectively. Although the positive correlations between MT and muscle mass and handgrip strength were observed, the relationship between MT, PA and physical performance remains unclear. Purpose: This study aimed to investigate the correlation of aforementioned ultrasound parameters with muscle mass, muscle strength and physical performance and explore the utility of ultrasound in predicting sarcopenia. Patients and methods: A total of 265 elderly Chinese community dwellers were included. MT of both forearm and lower leg as well as PA of gastrocnemius was assessed by ultrasound. Muscle mass was assessed by dual-energy X-ray absorptiometry. Muscle strength was measured by a Jamar hand dynamometer. Physical performance was assessed by the Short Physical Performance Battery (SPPB). Results: Anterior radial MT in men and regional MTs except posterior fibula in women were negatively correlated with the age. No significant correlation was observed between PA and the age in both genders. Posterior tibial MT and posterior fibula MT were positively correlated with the relative appendicular skeletal muscle mass in men and women, respectively. Anterior ulnar MT was positively correlated with grip strength in both genders. Moreover, gastrocnemius medialis PA showed a positive association with gait speed and SPPB in women but not in men. Conclusion: A combination of posterior fibula MT, anterior ulnar MT and gastrocnemius medialis PA measured by muscle ultrasound is helpful for the assessment of sarcopenia in Chinese elderly women. In addition, a combination of posterior tibial MT and anterior ulnar MT measured by muscle ultrasound is helpful for the assessment of sarcopenia in Chinese elderly men.

Gene delivery of hypoxia-inducible VEGF targeting collagen effectively improves cardiac function after myocardial infarction.

Vascular endothelial growth factor (VEGF) plays important roles in improvement of cardiac function following myocardial infarction (MI). However, the lack of a steerable delivery system of VEGF targeting the infarcted myocardium reduces the therapeutic efficacy and safety. Here, we constructed a series of lentiviral vector systems which could express a fusion protein consisted of a collagen-binding domain (CBD) and hVEGF (CBDhVEGF), under the control of 5HRE-hCMVmp (5HRE), the hypoxia-inducible promoter consists of five copies of the hypoxia-responsive element (HRE) and a human cytomegalovirus minimal promoter (hCMVmp). We demonstrated that 5HRE has the comparable ability to strongly drive CBDhVEGF under hypoxic condition as the ubiquitous CMV promoter, but it can hardly drive target gene under normoxic condition. 5HRE-drived CBDhVEGF specifically bound to type I collagen and significantly promoted the viability of HUVEC cells. Moreover, after injection of lentivirus into heart of mouse with MI, CBDhVEGF was mainly retained in infarcted myocardium where containing rich collagen and significantly improved angiogenesis and cardiac function when compared with hVEGF. Moreover, CBDhVEGF mediated by lentivirus has little leakage from infarcted zone into blood than hVEGF. Taken together, our results indicate that 5HRE-CBDhVEGF lentiviral vector system could improve cardiac function in the collagen-targeting and hypoxia-inducible manners.

[Treatment of Pipkin type I and II femoral head fractures through modified Smith-Peterson approach and modified Hardinge approach-a case-control studies].

OBJECTIVE: To compare clinical results of treatment of Pipkin type I and II femoral head fractures through modified Smith-Peterson(S-P) approach and modified Hardinge approach. METHODS: From July 2005 to July 2014, 42 patients with Pipkin type I and II femoral head fractures were treated with operation. A total of 23 patients in anterior group was treated with modified S-P approach including 17 males and 6 females with an average age of (29.3±9.4) years old, 5 cases of type I by excision of the fragement, 3 cases of type I and 15 cases of type II cases by fixation of the fragement. While a total of 19 patients in the lateral group was treated with modified Hardinge approach including 15 males and 4 females with an average age of (31.4±10.0) years old, 3 cases of type I by excision of the fragement, 4 cases of type I and 12 cases of type II by fixation of the fragement. Operative time, blood loss during operation and fracture healing time were observed and compared. The clinical and radiographic outcomes of the patients were measured using Thompson-Epstein scoring scale. The effect of hip reduction time of less than 6 h, 6 to12 h, and more than 12 h, the effect of surgery time within 24 h and more than 24 h after injury were compared. RESULTS: All patients were followed up from 24 to 60 months with an average of(30.29±6.95) months. The operation time (61.96±12.22) min, blood loss (46.09±18.03) ml, and (74.74±10.06) min, blood loss (72.11±19.88) ml in lateral group in the anterior group were better than those of lateral group(P<0.05). In anterior group, fracture healing time was(12.22±1.70) weeks, the results were excellent in 8 cases, good in 10 cases, fair in 4 cases and poor in 1 case, the excellent and good rate was 78.3%, the incidence of avascular necrosis of femoral head was 8.69%(2/23), and the incidence of heterotopic ossification was 13.04%(3/23). While in lateral group, the fracture healing time was(12.42±1.95) weeks, the results were excellent in 6 cases, good in 7 cases, fair in 3 cases and poor in 3 cases, the excellent and good rate was 68.4%, the incidence of avascular necrosis of femoral head was 10.53%(2/19), and the incidence of heterotopic ossification was 5.26%(1/19). There was no significant difference in fracture healing time, postoperative effect and postoperative complications between the anterior group and lateral group(P<0.05). The effect of patients with reduction time of hip dislocation less than 12 h was significantly better than that of more than 12 h, there was no significant difference in the effect between reduction time within 6 h and 6 to 12 h. There was no significant difference in the outcome between surgical patients within 24 h and more than 24 h after injury. CONCLUSIONS: Dislocated hip of Pipkin type I and II femoral head fractures should be closed reduction within 6 h. If conditions are limited, the reduction time can be accepted within 12 h. Both of modified S-P approach and modified Hardinge approach are effective in treating Pipkin type I and II femoral head fractures, and can obtain excellent outcomes. Moreover, modified S-P approach has advantage of less trauma, less blood loss, shorter operative time.

Functional characterization of TRPM7 in nasopharyngeal carcinoma and its knockdown effects on tumorigenesis.

The aim of this study was to evaluate the association of functional expression of TRPM7 with nasopharyngeal carcinoma (NPC) growth. We examined the correlation of TRPM7 expression with cell growth and proliferation, cell cycle, and apoptosis in vitro in NPC cell lines and NPC tumorigenesis in mice by conducting experiments in mice and by further analyzing the tumor volume and growth. We further explored to see whether there is any positive correlation with the TRPM7 knockdown in NPC cells with their sensitivity to radiation. We found that the functional expression of TRPM7 in nasopharyngeal carcinoma is a critical requirement for physiological processes such as cell cycle, resistance to apoptosis, and cell proliferation. TRPM7 knockdown also enhanced sensitivity to radiotherapy of nasopharyngeal carcinoma. Moreover, we identified TRPM7 as a novel potential regulator of cell proliferation in NPC, through signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway and other anti-apoptotic factors. TRPM7 and STAT3 activation might be critical for the growth of NPC cells and could be an effective target for treatment of nasopharyngeal carcinoma.

Low pH-induced pore formation by the T domain of botulinum toxin type A is dependent upon NaCl concentration.

Botulinum neurotoxins (BoNTs) undergo low pH-triggered membrane insertion, resulting in the translocation of their light (catalytic) chains into the cytoplasm. The T (translocation) domain of the BoNT heavy chain is believed to carry out translocation. Here, the behavior of isolated T domain from BoNT type A has been characterized, both in solution and when associated with model membranes. When BoNT T domain prepared in the detergent dodecylmaltoside was diluted into aqueous solution, it exhibited a low pH-dependent conformational change below pH 6. At low pH the T domain associated with, and formed pores within, model membrane vesicles composed of 30 mol% dioleoylphosphatidylglycerol/70 mol% dioleoylphosphatidylcholine. Although T domain interacted with vesicles at low (50 mM) and high (400 mM) NaCl concentrations, the interaction required much less lipid at low salt. However, even at high lipid concentrations pore formation was much more pronounced at low NaCl concentrations than at high NaCl concentration. Increasing salt concentration after insertion in the presence of 50 mM NaCl did not decrease pore formation. A similar effect of NaCl concentration upon pore formation was observed in vesicles composed solely of dioleoylphosphatidylcholine, showing that the effect of NaCl did not solely involve modulation of electrostatic interactions between protein and anionic lipids. These results indicate that some feature of membrane-bound T domain tertiary structure critical for pore formation is highly dependent upon salt concentration.

Secondary vortex street in the wake of two tandem circular cylinders at low Reynolds number.

The experiments on two tandem circular cylinders were conducted in a horizontal soap film tunnel for the Reynolds number Re=60 , 80, and 100 and the nondimensional center-to-center spacing Gamma ranging in 1 approximately 12. The flow patterns were recorded by a high-speed camera and the vortex shedding frequency was obtained by a spatiotemporal evolution method. The secondary vortex formation (SVF) mode characterized by the formation of a secondary vortex street in the wake of the downstream cylinder was found at large gamma. Moreover, some typical modes predicted by previous investigations, including the single bluff-body, shear layer reattachment, and synchronization of vortex shedding modes, were also revisited in our experiments. Further, numerical simulations were carried out using a space-time finite-element method and the results confirmed the existence of the SVF mode. The mechanism of SVF mode was analyzed in terms of the numerical results. The dependence of the Strouhal number Sr on Gamma was given and the flow characteristics relevant to the critical spacing values and the hysteretic mode transitions were investigated.

Passive oscillations of two tandem flexible filaments in a flowing soap film.

The passive oscillations of flexible filaments in a flowing soap film were investigated to learn the serial interaction between them. When arranged in tandem, the downstream filament flaps at the same frequency as that of the upstream one, but with a larger amplitude, whereas the upstream one is almost unaffected compared to the single filament case. The data analysis shows the downstream filament indeed extracts energy from the vortex street and receives greater force than the upstream one or a single filament in a uniform flow.

Behavior of the deeply inserted helices in diphtheria toxin T domain: helices 5, 8, and 9 interact strongly and promote pore formation, while helices 6/7 limit pore formation.

Diphtheria toxin T domain aids the membrane translocation of diphtheria toxin A chain. When the isolated T domain is deeply membrane-inserted, helices TH 8-9 form a transmembrane hairpin, while helices TH 5-7 form a deeply inserted nontransmembrane structure. Blocking deep insertion of TH 8-9 blocks deep insertion of TH 5-7 ( Zhao, G., and London, E. ( 2005) Biochemistry 44, 4488- 4498 ). We now examine the effects of blocking the deep insertion of TH 5 and TH 6/7. An A282R/V283R dual substitution in TH 5 prevented its deep insertion, significantly decreased the deep insertion of TH 9, and to a lesser degree that of TH 6/7. Blocking deep insertion of TH 6/7 with a L307R mutation had no effect on the deep insertion of TH 5, similar to its previously characterized lack of effect on the deep insertion of TH 8-9. An I364K mutation in TH 9 blocked TH 8-9 deep insertion and greatly reduced pore formation by the T domain, consistent with the role of TH 8-9 in pore formation. The A282R/V283R mutations also reduced the extent of pore formation, but to a lesser degree, suggesting either that TH 5 is part of the pore or that interactions with TH 5 affect the ability of TH 8-9 to form pores. The L307R mutation enhanced the extent of pore formation, suggesting that deeply inserted TH 6/7 may act as a "cork" that partly blocks the pore. Combined, these results indicate that TH 5, 8, and 9 combine to form a deeply inserted scaffold of more strongly associated helices.

Sodium-dependent reorganization of the sugar-binding site of SGLT1.

The sodium-dependent glucose cotransporter SGLT1 undergoes a series of voltage- and ligand-induced conformational changes that underlie the cotransport mechanism. In this study we describe how the binding of external Na changes the conformation of the sugar-binding domain, exposing residues that are involved in sugar recognition to the external environment. We constructed 15 individual Cys mutants in the four transmembrane helices (TMHs) that form the sugar binding and translocation domain. Each mutant was functionally characterized for transport kinetics and substrate specificity. Identification of interactions between mutated residues and hydroxyls on the pyranose ring was assessed by comparing the affinities of deoxy sugars to those of glucose. We determined conformation-dependent accessibility to the mutated residues by both a traditional substituted cysteine accessibility method (SCAM) and a new fluorescence binding assay. These data were integrated to orient the helices and construct a framework of residues that comprise the external sugar binding site. We present evidence that R499, Q457, and T460 play a direct role in sugar recognition and that five other residues are indirectly involved in transport. Arranging the four TMHs to account for Na-dependent accessibility and potential for sugar interaction allows us to propose a testable model for the SGLT1 sugar binding site.

Protein translocation by bacterial toxin channels: a comparison of diphtheria toxin and colicin Ia.

Regions of both colicin Ia and diphtheria toxin N-terminal to the channel-forming domains can be translocated across planar phospholipid bilayer membranes. In this article we show that the translocation pathway of diphtheria toxin allows much larger molecules to be translocated than does the translocation pathway of colicin Ia. In particular, the folded A chain of diphtheria toxin is readily translocated by that toxin but is not translocated by colicin Ia. This difference cannot be attributed to specific recognition of the A chain by diphtheria toxin's translocation pathway because the translocation pathway also accommodates folded myoglobin.