Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (R_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (R_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.     

Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1

Misfolding and degradation of CFTR is the cause of disease in patients with the most prevalent CFTR mutation, an in-frame deletion of phenylalanine (F508del), located in the first nucleotide-binding domain of human CFTR (hNBD1). Studies of (F508del)CFTR cellular folding suggest that both intra- and inter-domain folding is impaired. (F508del)CFTR is a temperature-sensitive mutant, that is, lowering growth temperature, improves both export, and plasma membrane residence times. Yet, paradoxically, F508del does not alter the fold of isolated hNBD1 nor did it seem to perturb its unfolding transition in previous isothermal chemical denaturation studies. We therefore studied the in vitro thermal unfolding of matched hNBD1 constructs ±F508del to shed light on the defective folding mechanism and the basis for the thermal instability of (F508del)CFTR. Using primarily differential scanning calorimetry (DSC) and circular dichroism, we show for all hNBD1 pairs studied, that F508del lowers the unfolding transition temperature (T_m) by 6–7°C and that unfolding occurs via a kinetically-controlled, irreversible transition in isolated monomers. A thermal unfolding mechanism is derived from nonlinear least squares fitting of comprehensive DSC data sets. All data are consistent with a simple three-state thermal unfolding mechanism for hNBD1 ± F508del: N(±MgATP) ⇄ I_T(±MgATP) → A_T → (A_T)_n. The equilibrium unfolding to intermediate, I_T, is followed by the rate-determining, irreversible formation of a partially folded, aggregation-prone, monomeric state, A_T, for which aggregation to (A_T)_n and further unfolding occur with no detectable heat change. Fitted parameters indicate that F508del thermodynamically destabilizes the native state, N, and accelerates the formation of A_T.     

Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin

Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT _m = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K_a = 16.2±0.6×10⁷ M⁻¹ where enthalpic change ΔH = −13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = −2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K_a = 3.1±0.4×10⁷ M⁻¹) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.     

The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering

The kinetics of heat-induced and cetyltrimethylammonium bromide induced amorphous aggregation of tobacco mosaic virus coat protein in Na(+)/Na(+) phosphate buffer, pH 8.0, have been studied using dynamic light scattering. In the case of thermal aggregation (52 degrees C) the character of the dependence of the hydrodynamic radius (R(h)) on time indicates that at certain instant the population of aggregates is split into two components. The size of the aggregates of one kind remains practically constant in time, whereas the size of aggregates of other kind increases monotonously in time reaching the values characteristic of aggregates prone to precipitation (R(h)=900-1500 nm). The construction of the light scattering intensity versus R(h) plots shows that the large aggregates (the start aggregates) exist in the system at the instant the initial increase in the light scattering intensity is observed. For thermal aggregation the R(h) value for the start aggregates is independent of the protein concentration and equal to 21.6 nm. In the case of the surfactant-induced aggregation (at 25 degrees C) no splitting of the aggregates into two components is observed and the size of the start aggregates turns out to be much larger (107 nm) than on the thermal aggregation. The dependence of R(h) on time for both heat-induced aggregation and surfactant-induced aggregation after a lapse of time follows the power law indicating that the aggregation process proceeds in the kinetic regime of diffusion-limited cluster-cluster aggregation. Fractal dimension is close to 1.8. The molecular chaperone alpha-crystallin does not affect the kinetics of tobacco mosaic virus coat protein thermal aggregation.     

Cooling-sensitive TRPM8 is thermostat of skin temperature against cooling

We have shown that cutaneous cooling-sensitive receptors can work as thermostats of skin temperature against cooling. However, molecule of the thermostat is not known. Here, we studied whether cooling-sensitive TRPM8 channels act as thermostats. TRPM8 in HEK293 cells generated output (y) when temperature (T) was below threshold of 28.4°C. Output (y) is given by two equations: At T >28.4°C, y = 0; At T <28.4°C, y  =  -k(T – 28.4°C). These equations show that TRPM8 is directional comparator to elicits output (y) depending on negative value of thermal difference (ΔT  =  T – 28.4°C). If negative ΔT-dependent output of TRPM8 in the skin induces responses to warm the skin for minimizing ΔT recursively, TRPM8 acts as thermostats against cooling. With TRPM8-deficient mice, we explored whether TRPM8 induces responses to warm the skin against cooling. In behavioral regulation, when room temperature was 10°C, TRPM8 induced behavior to move to heated floor (35°C) for warming the sole skin. In autonomic regulation, TRPM8 induced activities of thermogenic brown adipose tissue (BAT) against cooling. When menthol was applied to the whole trunk skin at neutral room temperature (27°C), TRPM8 induced a rise in core temperature, which warmed the trunk skin slightly. In contrast, when room was cooled from 27 to 10°C, TRPM8 induced a small rise in core temperature, but skin temperature was severely reduced in both TRPM8-deficient and wild-type mice by a large heat leak to the surroundings. This shows that TRPM8-driven endothermic system is less effective for maintenance of skin temperature against cooling. In conclusion, we found that TRPM8 is molecule of thermostat of skin temperature against cooling.     

Evidence for the formation of start aggregates as an initial stage of protein aggregation

The kinetics of thermal aggregation of glycogen phosphorylase b and glyceraldehyde 3-phosphate dehydrogenase from rabbit skeletal muscles were studied using dynamic light scattering. Use of high concentrations of the enzymes (1-3 mg/ml) provided a simultaneous registration of the native enzyme forms and protein aggregates. It was shown that initially registered aggregates (start aggregates) were large-sized particles. The hydrodynamic radius of the start aggregates was about 100 nm. The intermediate states between the native enzyme forms and start aggregates were not detected. The initial increase in the light scattering intensity is connected with accumulation of the start aggregates, the size of the latter remaining unchanged. From a certain moment in time aggregates of higher order, formed as a result of sticking of the start aggregates, make a major contribution to the enhancement of the light scattering intensity.     

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T₉TKE₁₂ sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from K_d = 25±6 nM to K_d = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (K_d = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the ν_as(P-O) and ν_s(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in ν_as(UO₂)²⁺ vibration (from 923 cm⁻¹ to 908 cm⁻¹) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.     

Probing the Binding Sites of Antibiotic Drugs Doxorubicin and N-(trifluoroacetyl) Doxorubicin with Human and Bovine Serum Albumins

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K _DOX-BSA = 7.8 (±0.7)×10³ M⁻¹, K _FDOX-BSA = 4.8 (±0.5)×10³ M⁻¹ and K _DOX-HSA = 1.1 (±0.3)×10⁴ M⁻¹, K _FDOX-HSA = 8.3 (±0.6)×10³ M⁻¹. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.     

Mechanism of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.     

Plasticity of the thermal requirements of exotherms and adaptation to environmental conditions

In exothermal organisms, temperature is an important determinant of the rate of ecophysiological processes, which monotonically increase between the minimum (t_{d min}) and maximum (t_{d max}) temperatures typical for each species. In insects, t_{d min} and t_{d max} are correlated and there is a approximately 20°C interval (thermal window W_T = t_{d max} − t_{d min}) between them over which insects can develop. We assumed that other exotherms have similar thermal windows because the thermal kinetics of their physiological processes are similar. In this study, we determined the thermal requirements for germination in plants. Seeds of 125 species of Central European wild herbaceous and crop plants were germinated at nine constant temperatures between 5 and 37°C, and the time to germination of 50% of the seeds D and rate of germination R (=1/D) were determined for each temperature and the Lactin model used to determine t_{d min}, t_{d max}, and W_T. The average width of the thermal windows for seeds was significantly wider (mean 24°C, 95% CI 22.7–24.2°C), varied more (between 14.5 and 37.5°C) and development occurred at lower temperatures than recorded for insects. The limiting temperatures for germination, t_{d min} and t_{d max}, were not coupled, so the width of the thermal window increased with both a decrease in t_{d min} and/or increase in t_{d max}. Variation in W_T was not associated with taxonomic affiliation, adult longevity, or domestication of the different species, but tends to vary with seed size. Plants are poor at regulating their temperature and cannot move to a more suitable location and as a consequence have to cope with wider ranges in temperatures than insects and possibly do this by having wider thermal windows. Synthesis: The study indicated specificity of W_T in different exotherm taxa and/or their development stages.     

Night-interrupting light inhibits diapause induction in the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae)

The inhibitory effects of the timing, intensity (I_I) and period (I_T) of night-interrupting light on diapause induction of the Kanzawa spider mite (Tetranychus kanzawai) were investigated in a series of laboratory experiments. During a light and dark period of 8 and 16 h d⁻¹, respectively, a single 1-h night-interrupting light was applied at early (E), middle (M), and late (L) parts of the dark period: i.e., at 3, 7.5, and 12 h after the start of the dark period, respectively. No interrupting light was applied in the control treatment. The incidence of diapause was significantly lower in the M treatment (63%) compared to the control treatment (100%). In the E and L treatments, more than 90% of females entered diapause, which was comparable to the control treatment. Since the longest consecutive dark period during the E and L treatments was longer than the critical dark period (CDP) of 10.5-11 h d⁻¹, during which 50% of females entered diapause, the night-interrupting light probably failed to prevent diapause induction. However, in the M treatment, the longest consecutive dark period was shorter than the CDP; therefore, the night-interrupting light inhibited diapause induction. Moreover, the inhibitory effects of night-interrupting light in the M treatment increased as I_I and I_T increased. The dose of night-interrupting light (I_I × I_T) was significantly negatively related to the incidence of diapause. The median effective dose for 50% disturbance of diapause induction was 2.5 kJ m⁻² at wavelengths between 350 and 1050 nm. Our results suggest that the longest consecutive dark period and the dose of night-interrupting light should both be considered when a lighting-based physical control is applied to inhibit diapause induction and consequent overwintering of T. kanzawai in commercial agricultural fields.     

Biochemical comparison of Anopheles gambiae and human NADPH P450 reductases reveals different 2'-5'-ADP and FMN binding traits

NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2′, 5′-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k _cat) of 105 s⁻¹ and 88 s⁻¹, respectively, for mosquito and human CPR. However, the inhibitory effects of 2′,5′-ADP on activity were different; the IC₅₀ value of AgCPR for 2′, 5′ –ADP was significantly higher (6–10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2′, 5′- ADP. This was confirmed by isothermal titration calorimetry where binding of 2′,5′-ADP to AgCPR (K _d = 410±18 nM) was ∼10 fold weaker than human CPR (K _d = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K_d = 83±2.0 nM) than the equivalent human domain (K_d = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC₅₀ = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of ‘smart’ insecticides and synergists that selectively target mosquito CPR.     

Burkholderia dabaoshanensis sp. nov., a Heavy-Metal-Tolerant Bacteria Isolated from Dabaoshan Mining Area Soil in China

Heavy-metal-tolerant bacteria, GIMN1.004^T, was isolated from mine soils of Dabaoshan in South China, which were acidic (pH 2–4) and polluted with heavy metals. The isolation was Gram-negative, aerobic, non-spore-forming, and rod-shaped bacteria having a cellular width of 0.5−0.6 µm and a length of 1.3−1.8 µm. They showed a normal growth pattern at pH 4.0–9.0 in a temperature ranging from 5°C to 40°C.The organism contained ubiquinone Q-8 as the predominant isoprenoid quinine, and C_16∶0, summed feature 8 (C_18∶1 ω7c and C_18∶1 ω6c), C_18∶0, summed feature 3 (C_16∶1 ω7c or iso-C_15∶0 2-OH), C_17∶0 cyclo, C_18∶1 ω9c, C_19∶0 cyclo ω8c, C_14∶0 as major fatty acid. These profiles were similar to those reported for Burkholderia species. The DNA G+C % of this strain was 61.6%. Based on the similarity to 16S rRNA gene sequence, GIMN1.004^T was considered to be in the genus Burkholderia. The similarities of 16S rRNA gene sequence between strain GIMN1.004^T and members of the genus Burkholderia were 96−99.4%, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia soli DSM 18235^T (99.4%); Levels of DNA-DNA hybridization with DSM 18235^T was 25%. Physiological and biochemical tests including cell wall composition analysis, differentiated phenotype of this strain from that closely related Burkholderia species. The isolation had great tolerance to cadmium with MIC of 22 mmol/L, and adsorbability of 144.94 mg/g cadmium,and it was found to exhibit antibiotic resistance characteristics. The adsorptive mechanism of GIMN1.004^T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd²⁺.Therefore, the strain GIMN1.004^T represented a new cadmium resistance species, which was tentatively named as Burkholderia dabaoshanensis sp. nov. The strain type is GIMN1.004^T ( = CCTCC M 209109^T =  NRRL B-59553^T ).     

Calcineurin-inhibitor minimization in liver transplant patients with calcineurin-inhibitor-related renal dysfunction: a meta-analysis

Background

Introduction of calcineurin-inhibitor (CNI) has made transplantation a miracle in the past century. However, the side effects of long-term use of CNI turn out to be one of the major challenges in the current century. Among these, renal dysfunction attracts more and more attention. Herein, we undertook a meta-analysis to evaluate the efficacy and safety of calcineurin-inhibitor (CNI) minimization protocols in liver transplant recipients with CNI-related renal dysfunction.

Methods

We included randomized trials with no year and language restriction. All data were analyzed using random effect model by Review Manager 5.0. The primary endpoints were glomerular filtration rate (GFR), serum creatinine level (sCr) and creatinine clearance rate (CrCl), and the secondary endpoints were acute rejection episodes, incidence of infection and patient survival at the end of follow-up.

Results

GFR was significantly improved in CNI minimization group than in routine CNI regimen group (Z = 5.45, P<0.00001; I² = 0%). Likely, sCr level was significantly lower in the CNI minimization group (Z = 2.84, P = 0.005; I² = 39%). However, CrCl was not significantly higher in the CNI minimization group (Z = 1.59, P = 0.11; I² = 0%). Both acute rejection episodes and patient survival were comparable between two groups (rejection: Z = 0.01, P = 0.99; I² = 0%; survival: Z = 0.28, P = 0.78; I² = 0%, respectively). However, current CNI minimization protocols may be related to a higher incidence of infections (Z = 3.06, P = 0.002; I² = 0%).

Conclusion

CNI minimization can preserve or even improve renal function in liver transplant patients with renal impairment, while sharing similar short term acute rejection rate and patient survival with routine CNI regimen.     

Maximum diastolic potential of human induced pluripotent stem cell-derived cardiomyocytes depends critically on I(Kr)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl₂ (n = 22). Patch-clamp techniques were used to record I_Kr and I_K1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl₂, at concentrations that selectively block I_K1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible I_K1 in 53% (20/38) of the cells studied, but presence of I_Kr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of I_K1 in the majority of cells, but robust expression of I_Kr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on I_Kr due to the absence of I_K1. Thus, efforts should be directed at producing more specialized and mature hiPSC-CM for future therapeutic applications.     

Methanocella conradii sp. nov., a thermophilic, obligate hydrogenotrophic methanogen, isolated from Chinese rice field soil

Background

Methanocellales contributes significantly to anthropogenic methane emissions that cause global warming, but few pure cultures for Methanocellales are available to permit subsequent laboratory studies (physiology, biochemistry, etc.).

Methodology/Principal Findings

By combining anaerobic culture and molecular techniques, a novel thermophilic methanogen, strain HZ254^T was isolated from a Chinese rice field soil located in Hangzhou, China. The phylogenetic analyses of both the 16S rRNA gene and mcrA gene (encoding the α subunit of methyl-coenzyme M reductase) confirmed its affiliation with Methanocellales, and Methanocella paludicola SANAE^T was the most closely related species. Cells were non-motile rods, albeit with a flagellum, 1.4–2.8 µm long and by 0.2–0.3 µm in width. They grew at 37–60°C (optimally at 55°C) and salinity of 0–5 g NaCl l⁻¹ (optimally at 0–1 g NaCl l⁻¹). The pH range for growth was 6.4–7.2 (optimum 6.8). Under the optimum growth condition, the doubling time was 6.5–7.8 h, which is the shortest ever observed in Methanocellales. Strain HZ254^T utilized H₂/CO₂ but not formate for growth and methane production. The DNA G+C content of this organism was 52.7 mol%. The sequence identities of 16S rRNA gene and mcrA gene between strain HZ254^T and SANAE^T were 95.0 and 87.5% respectively, and the genome based Average Nucleotide Identity value between them was 74.8%. These two strains differed in phenotypic features with regard to substrate utilization, possession of a flagellum, doubling time (under optimal conditions), NaCl and temperature ranges. Taking account of the phenotypic and phylogenetic characteristics, we propose strain HZ254^T as a representative of a novel species, Methanocella conradii sp. nov. The type strain is HZ254^T ( = CGMCC 1.5162^T = JCM 17849^T = DSM 24694^T).

Conclusions/Significance

Strain HZ254^T could potentially serve as an excellent laboratory model for studying Methanocellales due to its fast growth and consistent cultivability.     

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome‐blocking complexes

Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington''s, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer''s disease models, nematodes with muscle or pan-neuronal Aβ_1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10⁻⁶). In worms expressing Aβ_1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10⁻⁶). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.     

Coral uptake of inorganic phosphorus and nitrogen negatively affected by simultaneous changes in temperature and pH

The effects of ocean acidification and elevated seawater temperature on coral calcification and photosynthesis have been extensively investigated over the last two decades, whereas they are still unknown on nutrient uptake, despite their importance for coral energetics. We therefore studied the separate and combined impacts of increases in temperature and pCO₂ on phosphate, ammonium, and nitrate uptake rates by the scleractinian coral S. pistillata. Three experiments were performed, during 10 days i) at three pH_T conditions (8.1, 7.8, and 7.5) and normal temperature (26°C), ii) at three temperature conditions (26°, 29°C, and 33°C) and normal pH_T (8.1), and iii) at three pH_T conditions (8.1, 7.8, and 7.5) and elevated temperature (33°C). After 10 days of incubation, corals had not bleached, as protein, chlorophyll, and zooxanthellae contents were the same in all treatments. However, photosynthetic rates significantly decreased at 33°C, and were further reduced for the pH_T 7.5. The photosynthetic efficiency of PSII was only decreased by elevated temperature. Nutrient uptake rates were not affected by a change in pH alone. Conversely, elevated temperature (33°C) alone induced an increase in phosphate uptake but a severe decrease in nitrate and ammonium uptake rates, even leading to a release of nitrogen into seawater. Combination of high temperature (33°C) and low pH_T (7.5) resulted in a significant decrease in phosphate and nitrate uptake rates compared to control corals (26°C, pH_T = 8.1). These results indicate that both inorganic nitrogen and phosphorus metabolism may be negatively affected by the cumulative effects of ocean warming and acidification.     

Prognostic significance of vitamin D receptor polymorphisms in head and neck squamous cell carcinoma

Background

In patients with advanced non-small-cell lung cancer, vitamin D receptor (VDR) polymorphisms and haplotypes are reported to be associated with survival. We hypothesized that a similar association would be observed in patients with head and neck squamous-cell carcinoma (HNSCC).

Methods

In a post-hoc analysis of our previous prospective cohort study, VDR polymorphisms including Cdx2 G/A (rs11568820), FokI C/T (rs10735810), BsmI A/G (rs1544410), ApaI G/T (rs7976091), and TaqI T/C (rs731236) were genotyped by sequencing in 204 consecutive patients with HNSCC who underwent tumor resection. Progression-free survival was compared between VDR polymorphisms using Kaplan-Meier survival curves with log-rank tests and Cox proportional hazard models adjusting for age, gender, smoking status, primary tumor sites, postoperative stages, existence of residual tumor, and postoperative treatment with chemotherapy or radiotherapy.

Results

During a median follow-up of 1,047 days, tumor progression and death occurred in 76 (37.3%) and 27 (13.2%) patients, respectively. The FokI T/T genotype was associated with poor progression-free survival: median survival for T/T was 265 days compared with 1,127 days for C/C or C/T (log-rank test: P = 0.0004; adjusted hazard ratio, 3.03; 95% confidence interval, 1.62 to 5.67; P = 0.001). In contrast, the other polymorphisms (Cdx2, BsmI, ApaI, TaqI) showed no significant association with progression-free survival. The A-T-G (Cdx2-FokI-ApaI) haplotype demonstrated a significant association with a higher progression rate (P = 0.02).

Conclusion

These results suggest that VDR polymorphisms and haplotypes may be associated with prognosis in patients with HNSCC, although the sample size is not large enough to draw definitive conclusions.     

Pop up satellite tags impair swimming performance and energetics of the European eel (Anguilla anguilla)

Pop-up satellite archival tags (PSATs) have recently been applied in attempts to follow the oceanic spawning migration of the European eel. PSATs are quite large, and in all likelihood their hydraulic drag constitutes an additional cost during swimming, which remains to be quantified, as does the potential implication for successful migration. Silver eels (L_T = 598.6±29 mm SD, N = 9) were subjected to swimming trials in a Steffensen-type swim tunnel at increasing speeds of 0.3–0.9 body lengths s⁻¹, first without and subsequently with, a scaled down PSAT dummy attached. The tag significantly increased oxygen consumption (MO₂) during swimming and elevated minimum cost of transport (COT_min) by 26%. Standard (SMR) and active metabolic rate (AMR) as well as metabolic scope remained unaffected, suggesting that the observed effects were caused by increased drag. Optimal swimming speed (U _opt) was unchanged, whereas critical swimming speed (U _crit) decreased significantly. Swimming with a PSAT altered swimming kinematics as verified by significant changes to tail beat frequency (f), body wave speed (v) and Strouhal number (St). The results demonstrate that energy expenditure, swimming performance and efficiency all are significantly affected in migrating eels with external tags.