Ketamine Does Not Produce Relief of Neuropathic Pain in Mice Lacking the β-Common Receptor (CD131)

Neuropathic pain (NP) is a debilitating condition associated with traumatic, metabolic, autoimmune and neurological etiologies. Although the triggers for NP are diverse, there are common underlying pathways, including activation of immune cells in the spinal cord and up-regulation of the N-methyl-D-aspartate receptor (NMDAR). Ketamine, a well-known NDMAR antagonist, reduces neuropathic pain in a sustained manner. Recent study has shown that the novel 11-amino acid peptide erythropoietin derivative ARA290 produces a similar, long-lasting relief of NP. Here, we show that both drugs also have similar effects on the expression of mRNA of the NMDAR, as well as that of microglia, astrocytes and chemokine (C-C motif) ligand 2, all-important contributors to the development of NP. Although the effects of ketamine and ARA 290 on NP and its molecular mediators suggest a common mechanism of action, ARA 290 has no affinity for the NMDAR and acts specifically via the innate repair receptor (IRR) involved in tissue protection. We speculated therefore, that the IRR might be critically involved in the action of ketamine on neuropathic pain. To evaluate this, we studied the effects of ketamine and ARA 290 on acute pain, side effects, and allodynia following a spared nerve injury model in mice lacking the β-common receptor (βcR), a structural component of the IRR. Ketamine (50 mg/kg) and ARA 290 (30 µg/kg) produced divergent effects on acute pain: ketamine produced profound antinociception accompanied with psychomotor side effects, but ARA290 did not, in both normal and knock out mice. In contrast, while both drugs were antiallodynic in WT mice, they had no effect on NP in mice lacking the βcR. Together, these results show that an intact IRR is required for the effective treatment of NP with either ketamine or ARA 290, but is not involved in ketamine’s analgesic and side effects.     

Further Psychometric Evaluation of the Self-Stigma Scale-Short: Measurement Invariance across Mental Illness and Gender

BackgroundThis study cross-validated the factor structure of the Self-Stigma Scale-Short (SSS-S) in a cohort of patients with mental illness in southern Taiwan. The measurement invariance of the SSS-S factor structure across mental illness and gender was also examined.MethodsThe sample consisted of 161 patients with schizophrenia (51.6% males; mean age ± SD = 40.53 ± 10.38 years) and 189 patients with other mental illnesses (34.9% males; mean age = 46.52 ± 11.29 years).ResultsThe internal reliability (total score: α = 0.948) and concurrent validity (r = 0.335 to 0.457 with Depression and Somatic Symptoms Scale; r = −0.447 to −0.556 with WHOQOL-BREF) of the SSS-S were both satisfactory, and the results verified that the factor structure in our Taiwan sample (RMSEA = 0.0796, CFA = 0.992) was the same as that of the Hong Kong population. In addition, the results supported the measurement invariance of the SSS-S across mental illness (ΔRMSEAs = −0.0082 to −0.0037, ΔCFAs = 0.000) and gender (ΔRMSEAs = −0.0054 to −0.0008, ΔCFAs = −0.001 to 0.000).ConclusionFuture studies can use the SSS-S to compare self-stigma between genders and between patients with different kinds of mental illnesses.     

Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (T_m) and thermodynamic (−ΔG°) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the −ΔG° and T_m values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson–Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290–350 nm range is correlated linearly with −ΔG° and T_m, but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine–DNA adducts are recognized in nucleotide excision repair.     

ARA 290, a Nonerythropoietic Peptide Engineered from Erythropoietin,Improves Metabolic Control and Neuropathic Symptoms in Patients with Type 2 Diabetes

Although erythropoietin ameliorates experimental type 2 diabetes with neuropathy, serious side effects limit its potential clinical use. ARA 290, a nonhematopoietic peptide designed from the structure of erythropoietin, interacts selectively with the innate repair receptor that mediates tissue protection. ARA 290 has shown efficacy in preclinical and clinical studies of metabolic control and neuropathy. To evaluate the potential activity of ARA 290 in type 2 diabetes and painful neuropathy, subjects were enrolled in this phase 2 study. ARA 290 (4 mg) or placebo were self-administered subcutaneously daily for 28 d and the subjects followed for an additional month without further treatment. No potential safety issues were identified. Subjects receiving ARA 290 exhibited an improvement in hemoglobin A_1c (Hb A_1c) and lipid profiles throughout the 56 d observation period. Neuropathic symptoms as assessed by the PainDetect questionnaire improved significantly in the ARA 290 group. Mean corneal nerve fiber density (CNFD) was reduced significantly compared with normal controls and subjects with a mean CNFD >1 standard deviation from normal showed a significant increase in CNFD compared with no change in the placebo group. These observations suggest that ARA 290 may benefit both metabolic control and neuropathy in subjects with type 2 diabetes and deserves continued clinical evaluation.     

Muscle Oxygen Changes following Sprint Interval Cycling Training in Elite Field Hockey Players

This study examined the effects of Sprint Interval Cycling (SIT) on muscle oxygenation kinetics and performance during the 30-15 intermittent fitness test (_IFT). Twenty-five women hockey players of Olympic standard were randomly selected into an experimental group (EXP) and a control group (CON). The EXP group performed six additional SIT sessions over six weeks in addition to their normal training program. To explore the potential training-induced change, EXP subjects additionally completed 5 x 30s maximal intensity cycle testing before and after training. During these tests near-infrared spectroscopy (NIRS) measured parameters; oxyhaemoglobin + oxymyoglobin (HbO2+ MbO2), tissue deoxyhaemoglobin + deoxymyoglobin (HHb+HMb), total tissue haemoglobin (tHb) and tissue oxygenation (TSI %) were taken. In the EXP group (5.34±0.14 to 5.50±0.14m.s^-1) but not the CON group (pre = 5.37±0.27 to 5.39±0.30m.s^-1) significant changes were seen in the 30-15_IFT performance. EXP group also displayed significant post-training increases during the sprint cycling: ΔTSI (−7.59±0.91 to −12.16±2.70%); ΔHHb+HMb (35.68±6.67 to 69.44±26.48μM.cm); and ΔHbO2+ MbO2 (−74.29±13.82 to −109.36±22.61μM.cm). No significant differences were seen in ΔtHb (−45.81±15.23 to −42.93±16.24). NIRS is able to detect positive peripheral muscle oxygenation changes when used during a SIT protocol which has been shown to be an effective training modality within elite athletes.     

Effects of temperature on orthophosphate absorption by excised corn roots

The uptake of orthophosphate (³²P) by excised corn roots, Zea mays L. was studied using roots grown on 0.2 mm CaSO₄. Nine concentrations of KH₂PO₄ from 1 to 256 μm were used at temperatures of 20°, 30°, and 40°. Enzyme kinetic analysis was applied to the data obtained. Two apparent mechanisms (sites) of phosphate uptake were observed, 1 dominating at high P concentrations and 1 at low P concentrations. A Km of 1.36 × 10⁻⁴ and a Vmax of 177 × 10⁻⁹ moles per gram of roots per hour at 30° was calculated for the mechanism dominating at high P concentrations. Similar calculations gave a Km of 6.09 × 10⁻⁶ and a Vmax of 162 × 10⁻⁹ moles per gram of roots per hour at 30° for the mechanism dominating at low P concentrations. The Q₁₀ for both mechanisms was approximately 2. Calculation of thermodynamic values from the data gave ΔF of − 5200 cal, ΔH of − 950 to − 1400 cal, and a enthalpy of activation (A) of 10,300 to 13,800 cal per mole for the mechanism dominating at high P concentrations. Similar calculations from the data for the mechanism dominating at low P concentrations gave a ΔF of − 7300 cal, ΔH of − 10,700 to − 8200 cal, and a A of 9300 to 18,900 cal per mole. If the dual mechanism interpretation of this kind of data adequately describes this system, then both mechanisms of P absorption by corn roots involve chemical reactions.     

ARA 290 Improves Symptoms in Patients with Sarcoidosis-Associated Small Nerve Fiber Loss and Increases Corneal Nerve Fiber Density

Small nerve fiber loss and damage (SNFLD) is a frequent complication of sarcoidosis that is associated with autonomic dysfunction and sensory abnormalities, including pain syndromes that severely degrade the quality of life. SNFLD is hypothesized to arise from the effects of immune dysregulation, an essential feature of sarcoidosis, on the peripheral and central nervous systems. Current therapy of sarcoidosis-associated SNFLD consists primarily of immune suppression and symptomatic treatment; however, this treatment is typically unsatisfactory. ARA 290 is a small peptide engineered to activate the innate repair receptor that antagonizes inflammatory processes and stimulates tissue repair. Here we show in a blinded, placebo-controlled trial that 28 d of daily subcutaneous administration of ARA 290 in a group of patients with documented SNFLD significantly improves neuropathic symptoms. In addition to improved patient-reported symptom-based outcomes, ARA 290 administration was also associated with a significant increase in corneal small nerve fiber density, changes in cutaneous temperature sensitivity, and an increased exercise capacity as assessed by the 6-minute walk test. On the basis of these results and of prior studies, ARA 290 is a potential disease-modifying agent for treatment of sarcoidosis-associated SNFLD.     

Mitochondrial Networks in Cardiac Myocytes Reveal Dynamic Coupling Behavior

Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ_m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ_m depolarization as well as synchronized limit cycle oscillations of ΔΨ_m in the network. The functional impact of ΔΨ_m instability on cardiac electrophysiology, Ca²⁺ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ_m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10⁻² ± 0.98 × 10⁻² s⁻¹) and pyruvate (7.49 × 10⁻² ± 1.65 × 10⁻² s⁻¹) than lactate (4.83 × 10⁻² ± 1.25 × 10⁻² s⁻¹) or β-hydroxybutyrate (4.11 × 10⁻² ± 0.62 × 10⁻² s⁻¹). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ_m instability, ultimately scaling to produce organ level dysfunction.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

In this work, we integrated terminal truncation and N-terminal oligopeptide fusion as a novel protein engineering strategy to improve specific activity and catalytic efficiency of alkaline α-amylase (AmyK) from Alkalimonas amylolytica. First, the C terminus or N terminus of AmyK was partially truncated, yielding 12 truncated mutants, and then an oligopeptide (AEAEAKAKAEAEAKAK) was fused at the N terminus of the truncated AmyK, yielding another 12 truncation-fusion mutants. The specific activities of the truncation-fusion mutants AmyKΔC500-587::OP and AmyKΔC492-587::OP were 25.5- and 18.5-fold that of AmyK, respectively. The k_cat/K_m was increased from 1.0 × 10⁵ liters · mol⁻¹ · s⁻¹ for AmyK to 30.6 × and 23.2 × 10⁵ liters · mol⁻¹ · s⁻¹ for AmyKΔC500-587::OP and AmyKΔC492-587::OP, respectively. Comparative analysis of structure models indicated that the higher flexibility around the active site may be the main reason for the improved catalytic efficiency. The proposed terminal truncation and oligopeptide fusion strategy may be effective to engineer other enzymes to improve specific activity and catalytic efficiency.     

Pure-Culture Growth of Fermentative Bacteria, Facilitated by H2 Removal: Bioenergetics and H2 Production

We used an H₂-purging culture vessel to replace an H₂-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (ΔG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H₂ production changed such that ΔG remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate⁻¹ for S. lipocalidus and −58.2 ± 1.0 kJ mol alanine⁻¹ for A. colombiense). The cellular maintenance energy, determined from the ΔG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for S. lipocalidus at 55°C and 6.2 × 10⁻¹³ kJ cell⁻¹ day⁻¹ for A. colombiense at 37°C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Increased Circulating Endothelial Apoptotic Microparticle to Endothelial Progenitor Cell Ratio Is Associated with Subsequent Decline in Glomerular Filtration Rate in Hypertensive Patients

Background

Recent research indicates hypertensive patients with microalbuminuria have decreased endothelial progenitor cells (EPCs) and increased levels of endothelial apoptotic microparticles (EMP). However, whether these changes are related to a subsequent decline in glomerular filtration rate (GFR) remains unclear.

Methods and Results

We enrolled totally 100 hypertensive out-patients with eGFR ≥30 mL/min/1.73 m². The mean annual rate of GFR decline (△GFR/y) was −1.49±3.26 mL/min/1.73 m² per year during the follow-up period (34±6 months). Flow cytometry was used to assess circulating EPC (CD34⁺/KDR⁺) and EMP levels (CD31⁺/annexin V⁺) in peripheral blood. The △GFR/y was correlated with the EMP to EPC ratio (r = −0.465, p<0.001), microalbuminuria (r = −0.329, p = 0.001), and the Framingham risk score (r = −0.245, p = 0.013). When we divided the patients into 4 groups according to the EMP to EPC ratio, there was an association between the EMP to EPC ratio and the ΔGFR/y (mean ΔGFR/y: 0.08±3.04 vs. −0.50±2.84 vs. −1.25±2.49 vs. −4.42±2.82, p<0.001). Multivariate analysis indicated that increased EMP to EPC ratio is an independent predictor of ΔeGFR/y.

Conclusions

An increased circulating EMP to EPC ratio is associated with subsequent decline in GFR in hypertensive patients, which suggests endothelial damage with reduced vascular repair capacity may contribute to further deterioration of renal function in patients with hypertension.     

Kinetics and thermodynamics of activation by pretreatment with guanosine 5′-[β-imido]triphosphate of smooth-muscle adenylate cyclase

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5′-[βγ-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2°C. Activation (C→C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37°C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37°C. Over the range 6–24°C, activation by pretreatment with p[NH]ppG followed simple Michaelis–Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased K_m for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37°C) was moderately endergonic (ΔH⁰ ~8kJ·mol⁻¹) and accompanied by an increase in entropy (ΔS⁰ ~146J·mol⁻¹·K⁻¹). The β-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the K_m at every temperature. This result suggested that formation of the receptor–hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the β-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.     

Interaction between Genetic Predisposition to Adiposity and Dietary Protein in Relation to Subsequent Change in Body Weight and Waist Circumference

Background

Genetic predisposition to adiposity may interact with dietary protein in relation to changes of anthropometry.

Objective

To investigate the interaction between genetic predisposition to higher body mass index (BMI), waist circumference (WC) or waist-hip ratio adjusted for BMI (WHR_BMI) and dietary protein in relation to subsequent change in body weight (ΔBW) or change in WC (ΔWC).

Design

Three different Danish cohorts were used. In total 7,054 individuals constituted the study population with information on diet, 50 single-nucleotide polymorphisms (SNPs) associated with BMI, WC or WHR_BMI, as well as potential confounders. Mean follow-up time was ∼5 years. Four genetic predisposition-scores were based on the SNPs; a complete-score including all selected adiposity- associated SNPs, and three scores including BMI, WC or WHR_BMI associated polymorphisms, respectively. The association between protein intake and ΔBW or ΔWC were examined and interactions between SNP-score and protein were investigated. Analyses were based on linear regressions using macronutrient substitution models and meta-analyses.

Results

When protein replaced carbohydrate, meta-analyses showed no associations with ΔBW (41.0 gram/y/5 energy% protein, [95% CI: −32.3; 114.3]) or ΔWC (<−0.1 mm/y/5 energy % protein, [−1.1; 1.1]). Similarly, there were no interactions for any SNP-scores and protein for either ΔBW (complete SNP-score: 1.8 gram/y/5 energy% protein/risk allele, [−7.0; 10.6]) or ΔWC (complete SNP-score: <0.1 mm/y/5 energy% protein/risk allele, [−0.1; 0.1]). Similar results were seen when protein replaced fat.

Conclusion

This study indicates that the genetic predisposition to general and abdominal adiposity, assessed by gene-scores, does not seem to modulate the influence of dietary protein on ΔBW or ΔWC.     

The ω Subunit of RNA Polymerase Is Essential for Thermal Acclimation of the Cyanobacterium Synechocystis Sp. PCC 6803

The rpoZ gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions (constant illumination at 40 µmol photons m⁻² s⁻¹; 32°C; ambient CO₂) but was heat sensitive and died at 40°C. In the control strain, 71 genes were at least two-fold up-regulated and 91 genes down-regulated after a 24-h treatment at 40°C, while in ΔrpoZ 394 genes responded to heat. Only 62 of these heat-responsive genes were similarly regulated in both strains, and 80% of heat-responsive genes were unique for ΔrpoZ. The RNA polymerase core and the primary σ factor SigA were down-regulated in the control strain at 40°C but not in ΔrpoZ. In accordance with reduced RNA polymerase content, the total RNA content of mild-heat-stress-treated cells was lower in the control strain than in ΔrpoZ. Light-saturated photosynthetic activity decreased more in ΔrpoZ than in the control strain upon mild heat stress. The amounts of photosystem II and rubisco decreased at 40°C in both strains while PSI and the phycobilisome antenna protein allophycocyanin remained at the same level as in standard conditions. The phycobilisome rod proteins, phycocyanins, diminished during the heat treatment in ΔrpoZ but not in the control strain, and the nblA1 and nblA2 genes (encode NblA proteins required for phycobilisome degradation) were up-regulated only in ΔrpoZ. Our results show that the ω subunit of RNAP is essential in heat stress because it is required for heat acclimation of diverse cellular processes.     

Impact of Untreated Obstructive Sleep Apnea on Left and Right Ventricular Myocardial Function and Effects of CPAP Therapy

Background

Obstructive sleep apnea (OSA) has deteriorating effect on LV function, whereas its impact on RV function is controversial. We aimed to determine the effect of OSA and continuous positive airway pressure (CPAP) treatment on left and right ventricular (LV, RV) function using transthoracic echocardiography (TTE) and 2 dimensional speckle tracking (2D ST) analysis of RV deformation capability.

Methods and Results

82 patients with OSA and need for CPAP therapy were prospectively enrolled and underwent TTE at study inclusion and after 6 months of follow up (FU). Multivariate regression analysis revealed an independent association between baseline apical right ventricular longitudinal strain (RV-Sl), BMI and the severity of OSA (apical RV-Sl: P = 0.0002, BMI: P = 0.02). After CPAP therapy, LV functional parameters (LVEF: P<0.0001, LV performance index: P = 0.03, stroke volume: P = 0.042), and apical RV-Sl (P = 0.001) improved significantly. The effect of CPAP therapy was related to severity of OSA (LVEF: AHI 5–14, 66.4±8.8%, 68.5±10.6% [P = ns]; AHI 15–30∶59.8±7.7%, 68.6±9.3% [P = 0.002]; AHI>30∶54.1±12.4%, 68.2±13.6%[P<0.0001]; apical RV-Sl: AHI 5–14: −17.3±8.7%, −16.0±10.8% [P = ns], AHI 15–30: −9.8±6.0%, −15.4±10.9% [P = 0.028], AHI>30: −6.3±5.7%, −17.9±11.2% [P<0.0001]).

Conclusions

OSA seems to have deteriorating effect on LV and RV function. We found a beneficial effect of CPAP on LV and RV functional parameters predominately in patients with severe OSA. 2D speckle tracking might be of value to determine early changes in global and regional right ventricular function.     

Immobilised Histidine Tagged β 2-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β ₂-adrenoceptor (β ₂-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β ₂-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β ₂-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×10³/M for ephedrine and (3.80±0.02) ×10³/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10⁻⁴ M. Thermodynamic studies showed that the binding of the two compounds to β ₂-AR was a spontaneous reaction with exothermal processes. The ΔG^θ, ΔH^θ and ΔS^θ for the interaction between ephedrine and β ₂-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β ₂-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.     

Cold denaturation of monoclonal antibodies

The susceptibility of monoclonal antibodies (mAbs) to undergo cold denaturation remains unexplored. In this study, the phenomenon of cold denaturation was investigated for a mAb, mAb1, through thermodynamic and spectroscopic analyses. tryptophan fluorescence and circular dichroism (CD) spectra were recorded for the guanidine hydrochloride (GuHCl)-induced unfolding of mAb1 at pH 6.3 at temperatures ranging from −5 to 50°C. A three-state unfolding model incorporating the linear extrapolation method was fit to the fluorescence data to obtain an apparent free energy of unfolding, ΔG_u, at each temperature. CD studies revealed that mAb1 exhibited polyproline II helical structure at low temperatures and at high GuHCl concentrations. the Gibbs-Helmholtz expression fit to the ΔG_u versus temperature data from fluorescence gave a ΔC_p of 8.0 kcal mol⁻¹ K⁻¹, a maximum apparent stability of 23.7 kcal mol⁻¹ at 18°C, and an apparent cold denaturation temperature (T_CD) of −23°C. ΔG_u values for another mAb (mAb2) with a similar framework exhibited less stability at low temperatures, suggesting a depressed protein stability curve and a higher relative T_CD. Direct experimental evidence of the susceptibility of mAb1 and mAb2 to undergo cold denaturation in the absence of denaturant was confirmed at pH 2.5. thus, mAbs have a potential to undergo cold denaturation at storage temperatures near −20°C (pH 6.3), and this potential needs to be evaluated independently for individual mAbs.Key words: monoclonal antibodies, thermodynamic stability, cold denaturation, free energy, fluorescence     

More Severe Manifestations and Poorer Short-Term Prognosis of Ganglioside-Associated Guillain-Barré Syndrome in Northeast China

Ganglioside as a neurotrophic drug has been hitherto widely used in China, although Guillain-Barré syndrome (GBS) following intravenous ganglioside treatment was reported in Europe several decades ago. We identified 7 patients who developed GBS after intravenous use of gangliosides (ganglioside+ group) and compared their clinical data with those of 77 non-ganglioside-associated GBS patients (ganglioside− group) in 2013, aiming at gaining the distinct features of ganglioside-associated GBS. Although the mean age, protein levels in cerebrospinal fluid (CSF) and frequency of cranial nerve involvement were similar between the two groups, the Hughes Functional Grading Scale (HFGS) score and the Medical Research Council (MRC) sum score at nadir significantly differed (4.9±0.4 vs 3.6±1.0; 7.7±5.5 vs 36.9±14.5, both p<0.001), indicating a higher disease severity of ganglioside-associated GBS. A higher ratio of patients with ganglioside-associated GBS required mechanical ventilation (85.7% vs 15.6%, p<0.01). The short-term prognosis of ganglioside-associated GBS, as measured by the HFGS score and the MRC sum score at discharge, was poorer (4.3±0.5 vs 2.8±1.1; 17.3±12.9 vs 46.0±13.9, both p<0.001). All the patients in the ganglioside+ group presented an axonal form of GBS, namely acute motor axonal neuropathy (AMAN). When compared with the AMAN patients in the ganglioside− group, more severe functional deficits at nadir and poorer recovery after standard treatment were still prominent in ganglioside-associated GBS. Anti-GM1 and anti-GT1a antibodies were detectable in patients with AMAN while not in patients with the demyelinating subtype of GBS. The concentrations of these antibodies in patients with AMAN were insignificantly different between the ganglioside+ and ganglioside− groups. In sum, ganglioside-associated GBS may be a devastating side effect of intravenous use of gangliosides, which usually manifests a more severe clinical course and poorer outcome.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.