Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.     

Radiation damage to φX174 DNA and biological effects

Summary Dilute aqueous solutions of biologically active DNA can serve as a simplified model system of the cell. As a biological endpoint the survival of the DNA (after transfection to E. coli spheroplasts) is used. Damage in the DNA, irradiated in water with gamma rays, can be ascribed to reactions with primary waterradicals. By introducing additives in such solutions, which will scavenge the primary waterradicals, competition between a scavenger and DNA for such radicals can be studied. Comparison of different additives makes it possible to decide whether a compound behaves like a simple scavenger, radiosensitizer or like a radioprotector. In this context work has been done with the electron-affinic radiosensitizers metronidazole, misonidazole and nifuroxime. We have found that these wellknown cellular sensitizers do not enhance the inactivation of biologically active DNA. They act as simple competitive scavenger for waterradicals. However, if besides a sensitizer a trace of a metalloporphyrin containing compound (e.g. cyt. c) is present during irradiation an enhanced DNA inactivation, which can be interpreted as sensitization, is observed. Without sensitizer metalloporphyrins induce an enhanced protection of DNA.Apart from these effects the consequences of both chemical-(sulphy-dryl) and enzymatic-(excision; recombination) repair has been studied. It has been found that sulphydryl compounds are able to react with DNA radicals, modifying the radiation damage in such a way that e.g. breaks are prevented. Further in double-stranded DNA a considerable amount of OH and also H radical damage appeared to be reparable by the excision-repair mechanism. However, post-replication repair had only very small or no effect on the amount of damage.     

Inhibition of MAP4K4 signaling initiates metabolic reprogramming to protect hepatocytes from lipotoxic damage

The primary hepatic consequence of obesity is non-alcoholic fatty liver disease (NAFLD), affecting about 25% of the global adult population. Non-alcoholic steatohepatitis (NASH) is a severe form of NAFLD characterized by liver lipid accumulation, inflammation, and hepatocyte ballooning, with a different degree of hepatic fibrosis. In the light of rapidly increasing prevalence of NAFLD and NASH, there is an urgent need for improved understanding of the molecular pathogenesis of these diseases. The aim of this study was to decipher the possible role of STE20-type kinase MAP4K4 in the regulation of hepatocellular lipotoxicity and susceptibility to NAFLD. We found that MAP4K4 mRNA expression in human liver biopsies was positively correlated with key hallmarks of NAFLD (i.e., liver steatosis, lobular inflammation, hepatocellular ballooning, and fibrosis). We also found that the silencing of MAP4K4 suppressed lipid deposition in human hepatocytes by stimulating β-oxidation and triacylglycerol secretion, while attenuating fatty acid influx and lipid synthesis. Furthermore, downregulation of MAP4K4 markedly reduced the glycolysis rate and lowered incidences of oxidative/endoplasmic reticulum stress. In parallel, we observed suppressed JNK and ERK and increased AKT phosphorylation in MAP4K4-deficient hepatocytes. Together, these results provide the first experimental evidence supporting the potential involvement of STE20-type kinase MAP4K4 as a component of the hepatocellular lipotoxic milieu promoting NAFLD susceptibility.     

Novel cell subtypes of SPP1 + S100P+, MS4A1-SPP1 + S100P+ were key subpopulations in intrahepatic cholangiocarcinoma

BackgroundIn this study, we integrated single-cell RNA sequencing (scRNA-seq) data to investigate cell heterogeneity and utilized MSigDB and CIBERSORTx to explore the pathways of major cell types and the relationships between different cell subtypes. Subsequently, we explored the correlation of cell subtypes with survival and used Gene Set Enrichment Analysis (GSEA) analyses to assess the pathways associated with the infiltration of specific cell subtypes. Finally, multiplex immunohistochemistry in tissue microarray cohort were performed to validate differences in protein level and their correlation with survival.ResultsiCCA presented a unique immune ecosystem, with increased proportions of Epi (epithelial)-SPP1–2, Epi-S100P-1, Epi-DN (double negative for SPP1 and S100P expression)-1, Epi-DN-2, Epi-DP (double positive for SPP1 and S100P expression)-1, Plasma B-3, Plasma B-2, B-HSPA1A-1, B-HSPA1A-2 cells, and decreased proportions of B-MS4A1. High level of Epi-DN-2, Epi-SPP1–1, Epi-SPP1–2, B-MS4A1, and low level of Epi-DB-1, Epi-S100P-1, and Epi-S100P-2 was significantly associated with longer overall survival (OS), and high level of B-MS4A1_Low_Epi-DN-2_Low was associated with the shortest OS. Moreover, the results of MsigDB and GSEA suggest that bile acid metabolism is a crucial process in iCCA. Finally, we found that S100P+, SPP1+, SPP1 + S100P+, and MS4A1-SPP1 + S100P+ were highly expressed, whereas MS4A1 was lowly expressed in iCCA, and patients with high level of S100P+, SPP1 + S100P+, and MS4A1-SPP1 + S100P+ exhibited shorter survival.ConclusionsWe identified the cell heterogeneity of iCCA, found that iCCA is a unique immune ecosystem with many cell subtypes, and showed that the novel cell subtypes of SPP1 + S100P+ and MS4A1-SPP1 + S100P+ were key subpopulations in iCCA.     

Evening salivary cortisol and alpha-amylase at 14 months and neurodevelopment at 4 years: Sex differences

Stress system activity in early life can have long-term effects on neurodevelopment. The main aim of this study was to assess the association of child evening salivary cortisol and alpha-amylase basal levels at 14 months of age with longer-term neuropsychological development at 4 years in a low-risk population-based birth cohort derived from the INMA (Environment and Childhood) project in Spain. We included 186 parent-children pairs with information on both stress system activity and neurodevelopment. Both stress markers at 14 months of age showed an association with neuropsychological development at 4 years. Salivary cortisol showed a sex-specific pattern of association. In girls, cortisol levels at 14 months were negatively associated with cognitive development [long-term declarative memory (β = − 17.8, p = 0.028; 95% CI = − 33.2 to − 2.5); executive function (β = − 9.8, p = 0.08; 95% CI = − 21 to 1)] and gross motor development (β = − 13; p = 0.022; 95% CI = − 24 to − 2), whereas in boys cortisol levels were negatively associated with socioemotional development [autistic-like behaviours: Incidence Rate Ratio (IRR) = 1.6, p = 0.039; 95% CI = 1.01 to 2.41]. Salivary alpha-amylase was positively associated with socioemotional development in boys only [social competence (β = 2.11, p = 0.013; 95% CI = 0.47 to 3.72), autistic-like behaviours (IRR = 0.93, p = 0.042; 95% CI = 0.87 to 0.99) and hyperactivity symptoms (IRR = 0.81, p = 0.021; 95% CI = 0.69 to 0.97)]. These results suggest that stress system activity in early life is associated with longer-term neurodevelopment and that sex is an important factor in this relationship.     

Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4 °C temperature

Temporal storage of ovaries can provide opportunity to rescue oocytes from ovaries of endangered felids. The objective of the study was to examine the effect of different storage periods (2, 24 and 48 h) of ovaries at 4 °C for maturation of cat oocytes in vitro. Ovaries were collected from 25 domestic cats at various stages of the estrous cycle by routine ovariohysterectomy following anesthesia at different local veterinary clinics, and maintained in physiological saline at 4 °C for 2, 24 or 48 h until oocytes recovery. Selected COCs were maturated at 38 °C for 48 h in four-well petri dishes, which included 500 μL modified synthetic oviduct fluid (mSOF) medium under mineral oil in a humidified 5% CO₂, 5% O₂, and 90% N₂ atmosphere incubator. After the in vitro maturation period, there were no differences between the rate of oocytes matured at MII stages in 2 and 24 h storage groups (50.7% and 48.2% respectively, p > 0.05). However, the same result for the 48 h group was significantly lower than the 2 and 24 h groups (28.0%, p < 0.001).Our results suggest that while 2 or 24 h storage of ovaries at 4 °C does not affect the meiotic competence of oocytes in vitro, 48 h storage of ovaries decrease the results dramatically.     

Charge separation in photosystem II core complexes induced by 690-730 nm excitation at 1.7 K

The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on Pheo_D1. These shifts arise from the formation of metastable plastoquinone anion (Q_A⁻) configurations. Spectra of concentrated samples identify absorption in the 700-730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690-730 nm region can be described by the ‘trap’ CP47 absorption at 689 nm, with dipole strength of ∼1 chlorophyll a (chl a), partially overlapping a broader feature near 705 nm with a dipole strength of ∼0.15 chl a. This absorption strength in the 700-730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690-700 nm region are similar to those obtained with green illumination but fall significantly in the 700-730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally ‘dark’ charge-transfer excitation of the ‘special pair’ P_D1-P_D2. The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.     

Radiation chemists look at damage in redox proteins induced by X‐rays

The three‐dimensional structure of proteins, especially as determined by X‐ray crystallography, is critical to the understanding of their function. However, the X‐ray exposure may lead to damage that must be recognized and understood to interpret the crystallographic results. This is especially relevant for proteins with transition metal ions that can be oxidized or reduced. The detailed study of proteins in aqueous solution by the technique of pulse radiolysis has provided a wealth of information on the production and fate of radicals that are the same as those produced by X‐ray exposure. The results reviewed here illustrate how the products of the interaction of radiation with water or with solutes added to the crystallization medium, and with proteins themselves, are formed, and about their fate. Of particular focus is how electrons are produced and transferred through the polypeptide matrix to redox centers such as metal ions or to specific amino acid residues, for example, disulfides, and how the hydroxyl radicals formed may be converted to reducing equivalents or scavenged.     

Radiation damage to bull sperm motility. I. X-ray effects and target theory

Diluted bull semen samples were irradiated with 180 kv X-rays. Dose response curves were measured for the survival fraction of the spermatozoa, and for the average velocity of the surviving cells. The dose response curves did not show a sensitivity threshold. The half-value dose was determined as 11 kr for the survival fraction and 10 kr for the average velocity. Target theory was adapted specially to explain the form of the measured dose response curves. From this target theory it was found that a small sensitive element is present in the sperm cell with a volume of approximately 0.7⁵ × 10^-15 cm³.     

Excitation energy flow at 77 K in the photosynthetic apparatus of overwintering evergreens

The flow of excitation energy from the antennae to photosynthetic reaction centre complexes at 77 K was studied in leaves of two evergreen species, namely, snow gum (Eucalyptus pauciflora Sieb. ex Spreng.) and a hemiparasitic mistletoe (Amyema miquelii, Lehm. ex Miq.). The leaves that were naturally acclimated to winter conditions of freezing temperatures and high irradiance displayed the recently discovered cold‐hard‐band or CHB feature of the chlorophyll a fluorescence spectra (Gilmore & Ball, Proc. Nat. Acad. Sci. USA 97:11098–11101, 2000). A streak‐camera‐spectrograph was used and the double convolution integral method for global analysis was applied to simultaneously acquire and simulate, respectively, the time‐ and wavelength‐dependence of all major chlorophyll a components (Gilmore et al. Phil. Trans. Roy. Soc. B‐London 355:1371–1384, 2000). The CHB coincided with changed amplitudes and decreased excited state lifetimes for the main F685 nm and F695 nm emission bands from the photosystem II (PSII) core‐inner‐antenna. The CHB dissipates energy as heat separate from PSII while also reducing the PSII quantum yield by competing for both photon absorption and antenna excitation. The CHB did not correlate with changes in the decay kinetics of the PSI antenna F740 nm band. The spectral‐kinetic features of the altered energy flow were similar in the unrelated evergreen species. These results are consistent with a functional association between the CHB, PSII energy dissipation and protective storage of chlorophyll in overwintering evergreens.     

L-asparaginase release from Escherichia coli cells with K2HPO4 and Triton X100

A method to release L-asparaginase (EC 3.5.1.1) from ATCC Escherichia coli 11303 cells by chemical permeabilization was studied. It was found that a combination of K2HPO4 and Triton X100 was effective. The influences of K2HPO4 concentration, Triton concentration, E. coli cell concentration and pH on the release of enzyme and proteins were investigated in detail. Experimental results showed that 12.5% (w/v) K2HPO4, 2% (w/v) Triton X100 and 3 x 10(8) cells/mL made the amount of enzyme released over 70%. L-Asparaginase in K2HPO4 and Triton solution could remain stable at least for 24 h. The release effect of K2HPO4 and Triton X100 used simultaneously was better than that of K2HPO4 and Triton X100 used separately in succession. Electron microscopy indicated that the chemical treatment altered the surface structure of E. coli cells but did not break them. As the method does not produce a large amount of cell fragments and the amount of enzyme released is relatively high, it can be thought to be an valuable and economic method to release intracellular enzyme.     

Delayed fluorescence observed in the nanosecond time region at 77 K originates directly from the photosystem II reaction center

The excited-state dynamics of delayed fluorescence in photosystem (PS) II at 77 K were studied by time-resolved fluorescence spectroscopy and decay analysis on three samples with different antenna sizes: PS II particles and the PS II reaction center from spinach, and the PS II core complexes from Synechocystis sp. PCC 6803. Delayed fluorescence in the nanosecond time region originated from the 683-nm component in all three samples, even though a slight variation in lifetimes was detected from 15 to 25 ns. The relative amplitude of the delayed fluorescence was higher when the antenna size was smaller. Energy transfer from the 683-nm pigment responsible for delayed fluorescence to antenna pigment(s) at a lower energy level was not observed in any of the samples examined. This indicated that the excited state generated by charge recombination was not shared with antenna pigments under the low-temperature condition, and that delayed fluorescence originates directly from the PS II reaction center, either from chlorophyll a_D1 or P680. Supplemental data on delayed fluorescence from spinach PS I complexes are included.     

AgsA response to cadmium and copper effects at different temperatures in Escherichia coli

Small heat shock proteins (sHsps), present from prokaryotes to eukaryotes, are a highly conserved molecular chaperone family. They play a crucial role in protecting organisms against cellular insults from single or multiple environmental stressors including heavy metal exposure, heat or cold shock, oxidative stress, desiccation, etc. Here, the toxicity of cadmium and copper, and their ability to modify the cellular growth rate at different temperatures in Escherichia coli cells were tested. Also, the response mechanism of the sHSP aggregation‐suppressing protein (AgsA) in such multiple stress conditions was investigated. The results showed that the half effect concentration (EC₅₀) of cadmium in AgsA‐transformed E. coli cells at 37°C, 42°C, and 50°C were 11.106, 29.50, and 4.35 mg/L, respectively, and that of the control cells lacking AgsA were 5.05, 0.93, and 0.18 mg/L, respectively, while the half effect concentration (EC₅₀) of copper in AgsA‐transformed E. coli cells at 37°C, 42°C, and 50°C were 27.3, 3.40, and 1.28 mg/L, respectively, and that of the control cells lacking AgsA were 27.7, 5.93, and 0.134 mg/L, respectively. The toxicities of cadmium and copper at different temperatures as observed by their modification of the cellular growth rate and inhibitory effects were in a dose‐dependent manner. Additionally, biochemical characterization of AgsA protein in cells subjected to cadmium and copper stresses at different temperatures implicated suppressed aggregation of cellular proteins in AgsA‐transformed E. coli cells. Altogether, our data implicate the AgsA protein as a sensitive protein‐based biomarker for metal‐induced toxicity monitoring.     

Exciton Annihilation in the Two Photosystems in Chloroplasts at 100°K

The fluorescence yield (F) of spinach chloroplasts at 100°K measured at 735 nm (photosystem I fluorescence—F 735) and at 685 nm (photosystem II fluorescence—F 685) has been determined with different modes of laser excitation. The modes of excitation included a single picosecond pulse, sequences of picosecond pulses (4, 22, and 300 pulses spaced 5 ns apart) and a single nonmode-locked 2-μs pulse (MP mode). The F 735/F 685 intensity ratios decrease from 1.62 to 0.61 when a single picosecond pulse (or low-power continuous helium-neon laser) is replaced by excitation with the 300-ps pulse train (PPT mode) or MP mode. In the PPT mode of excitation, the 735-nm fluorescence band is quenched by a factor of 45 as the intensity is increased from 10¹⁵ to 10¹⁸ photons/cm² per pulse train and the 685-nm fluorescence is quenched by a factor of 10. In the MP mode, the quenching factors are 25 and 7, respectively, in the same intensity range. Fluorescence quantum yield measurements with different picosecond pulse sequences indicate that relatively long-lived quenching species are operative, which survive from one picosecond pulse to another within the pulse train. The excitonic processes possible in the photosynthetic units are discussed in detail. The differences in the quenching factors between the MP and PPT modes of excitation are attributed to singlet-singlet annihilation, possible when picosecond pulses are utilized, but minimized in the MP mode of excitation. The long-lived quenchers are identified as triplets and/or bulk chlorophyll ions formed by singlet-singlet annihilation. The preferential quenching in photosystem I is attributed to triplet excitons. The influence of heating effects, photochemistry, bleaching, and two-photon processes is also considered and is shown to be negligible.     

The inhibitory effects of UV-B radiation (280–315 nm) on Gunnera magellanica growth correlate with increased DNA damage but not with oxidative damage to lipids

Damage to DNA and disruption of membrane integrity by lipid peroxidation processes are two of the proposed causes of UV‐B‐induced growth inhibition in plants. However, the relative significance of these different types of molecular damage has not been established in experiments carried out under realistic physiological conditions. Plants of Gunnera magellanica (a native herb from southern Patagonia) were exposed to a gradient of biologically effective UV‐B doses (from 0 to 6.5 kJ m^?2 d^?1 of UV‐B_be) in a greenhouse study. Leaf expansion was measured and sensitive techniques were used to detect damage to DNA (in the form of cyclobutane pyrimidine dimers; CPDs) and lipid peroxidation (via electronic‐paramagnetic resonance; EPR). Leaf expansion decreased and the CPD density increased with increasing UV‐B doses, but the degree of lipid peroxidation remained unaffected. The highest UV‐B dose induced a transient oxidative stress situation (as evaluated using the ratio of ascorbyl radical to ascorbate, A^·/AH^–), which was rapidly controlled by an increase in the ascorbate pool. The present results suggest that under a range of UV‐B_be doses that overlaps the range of doses that G. magellanica plants experience in their natural environment, growth inhibition is better explained by DNA damage than by increased lipid peroxidation.