[Establish a whole-process comprehensive surveillance management mode for immune checkpoint inhibitor pneumonitis].

The application of immune checkpoint inhibitors (ICIs) has shown impressive anti-tumor efficacy across multiple malignant tumors, leading to the prolonged survival period of tumor patients. However, immune-related adverse events should not be ignored. Checkpoint inhibitor pneumonitis (CIP) is a pulmonary adverse event that can occur in malignant tumor patients after receiving ICIs treatment. The incidence of CIP has been reported to range from 2.7% to 20.0% in clinical trials and real-world research. Furthermore, some patients might suffer from serious or fatal CIP, and the prognosis of such patients will be poor. Early detection, diagnosis and treatment may improve the prognosis of these patients. The establishment of a whole-process CIP comprehensive surveillance management mode covering the health care system and patients during ICIs treatment might be helpful to improve the early diagnosis and treatment capacity of CIP, which is a key measure to improve the prognosis of these patients.

Effects of wet atmospheric nitrogen deposition on epiphytic lichens in the subtropical forests of Central China: Evaluation of the lichen food supply and quality of two endangered primates.

Over the last few decades, the threat posed to biodiversity and ecosystem function by atmospheric nitrogen (N) deposition has been increasingly recognized. The disturbed nutrient balance and species composition of plants induced by higher N deposition can impact the biodiversity of the organisms that consume the plants. In this research, we implemented several experiments to estimate the effects of increased N deposition on the growth, survival, and nutrients of the dominant epiphytic lichens in the subtropical mountains in Central China to assess the lichen food amount and nutritional quality for two endangered primates endemic to China. Our results indicated that the thallus growth and propagule survival of the lichens were significantly decreased when nitrogen addition changed from 6.25 to 50.0 kg N·ha-1·y-1; it was also shown that lichen biomass could be decreased by 11.2%-70.2% when the deposition addition exceeded 6.25 kg N·ha-1·y-1. Further, our study revealed that increased nitrogen deposition also reduced the nutritional quality of the lichens via reducing the soluble protein and soluble sugar levels and increasing the fiber content, which would substantially affect the diet selection of the plants consumers in the region, particularly the populations of the two lichen-eating endangered primate species, Rhinopithecus roxellana and R. bieti. Our experimental study suggested that the nitrogen pollution derived from anthropogenic activities could cause cascading effects for the whole forest ecosystem of Central China; thus, more studies about nitrogen deposition in this region are required.

Fetal liver-conditioned medium induces hepatic specification from mouse bone marrow mesenchymal stromal cells: a novel strategy for hepatic transdifferentiation.

BACKGROUND: Although different strategies have been established for hepatic differentiation of mesenchymal stromal cells (MSC), further studies are required to define an efficient strategy to produce hepatocytes from stem cells and uncover the mechanisms of hepatic differentiation. METHODS: Bone marrow mesenchymal stromal cells (BMMSC), isolated from ICR mice, were induced by fetal liver-conditioned medium from different developmental stages, embryonic days (ED) 9.5, 11.5 and 13.5 and newborn (1 day). Differentiated cells were characterized by morphologic changes, liver-specific gene expression at mRNA and/or protein levels and in vitro functional features. RESULTS: BMMSC morphologically became epithelioid and binucleated after 7 days' exposure to fetal liver-conditioned medium from ED13.5, expressed liver-specific genes (AFP, HNF-3beta, TTR, CK18, ALB and CK19) at mRNA and/or protein levels and acquired in vitro functions characteristic of liver cells, including glycogen storage, urea production and albumin secretion. Conditioned medium derived from fetal liver at ED13.5 was most efficient on hepatic differentiation of BMMSC compared from the other three developmental stages. DISCUSSION: The present study not only provides a high-performance strategy for hepatic differentiation from BMMSC, but also implies liver at different developmental stages might secrete different types of cytokines that have diverse effects on hepatic differentiation, which could support further investigation to provide insight into fundamental processes that govern development and regeneration of the liver.

Evolution and characterisation of the AhRAF4 NB-ARC gene family induced by Aspergillus flavus inoculation and abiotic stresses in peanut.

Aflatoxin contamination in peanut is a serious food safety issue to human health around the world. Finding disease resistance genes is a key strategy for genetic improvement in breeding to deal with this issue. We identified an Aspergillus flavus-induced NBS-LRR gene, AhRAF4, using a microarray-based approach. By comparison of 23 sequences from three species using phytogenetics, protein secondary structure and three-dimensional structural analyses, AhRAF4 was revealed to be derived from Arachis duranensis by recombination, and has newly evolved into a family of several members, characterised by duplications and point mutations. However, the members of the family descended from A. ipaensis were lost following tetraploidisation. AhRAF4 was slightly up-regulated by low temperature, drought, salicylic acid and ethylene, but down-regulated by methyl jasmonate. The distinct responses upon As. flavus inoculation and the differential reactions between resistant and susceptible varieties indicate that AhRAF4 might play a role in defence responses. Temporal and spatial expression and the phenotype of transformed protoplasts suggest that AhRAF4 may also be associated with pericarp development. Because tetraploid cultivated peanuts are vulnerable to many pathogens, an exploration of R-genes may provide an effective method for genetic improvement of peanut cultivars.

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).

Involvement of tyrosine residue in the inhibition of plant vacuolar H(+)-pyrophosphatase by tetranitromethane.

Plant vacuolar vesicles contain a novel H(+)-translocating pyrophosphatase (H(+)-PPase, EC 3.6.1.1). Modification of tonoplast vesicles and purified vacuolar H(+)-PPase from etiolated mung bean seedlings with tetranitromethane (TNM) resulted in a progressive decline in H(+)-translocating pyrophosphatase activity. The half-maximal inhibition was brought about by 0.6, 1.0, and 0.8 mM TNM for purified and membrane-bound H(+)-PPases, and its associated proton translocation, respectively. The maximal inhibition of vacuolar H(+)-PPase by TNM occurred at a pH value above 8. Loss of activity of purified H(+)-pyrophosphatase followed pseudo-first order rate kinetics, yielding a first-order rate constant (k2) of 0.039 s(-1) and a steady-state dissociation constant of inactivation (Ki) of 0.02 mM. Covalent modification of vacuolar H(+)-PPase by TNM increased Km value of the enzyme for its substrate without a significant effect on Vmax. Double logarithmic plots of the pseudo-first order rate constant (kobs) versus TNM concentration exhibited a slope of 0.88, suggesting that at least one tyrosine residue was involved in the inactivation of H(+)-PPase enzymatic activity. Further spectrophotometric measurements of the nitrated H(+)-pyrophosphatase indicated that TNM could modify approximately two tyrosine residues/subunit of the enzyme. However, Tsou's analysis revealed that only one of those modified tyrosine residues directly participated in the inhibition of enzymatic activity of vacuolar H(+)-PPase. The physiological substrate, i.e., dimagnesium pyrophosphate, provided substantial protection against inactivation by TNM. Moreover, NEM pretreatment of the enzyme decreased the number of subsequent nitration of vacuolar H(+)-PPase. Taken together, we suggest that vacuolar H(+)-pyrophosphatase contains a substrate-protectable tyrosine residue conferring to the inhibition of its activity and this tyrosine residue may be located in a domain sensitive to the modification of Cys-634 by NEM.

Inhibition of plant vacuolar H(+)-ATPase by diethylpyrocarbonate.

Treatment of the tonoplast H(+)-ATPase from mung bean seedlings (Vigna radiata L.) with histidine-specific modifier, diethyl pyrocarbonate (DEP), caused a marked loss of the ATP hydrolysis activity and the proton translocation in a concentration-dependent manner. The reaction order of inhibition was calculated to be 0.98, suggesting that at least one histidine residue of vacuolar H(+)-ATPase was modified by DEP. The absorbance of the vacuolar H(+)-ATPase at 240 nm was progressively increased after incubation with DEP, suggesting that N-carbethoxyhistidine had been formed. Hydroxylamine, which could break N-carbethoxyhistidine, reversed the absorbance change and partially restored the enzymic activity. The pK(a) of modified residues of vacuolar H(+)-ATPase was kinetically determined to be 6.73, a value close to that of histidine. Thus, it is assuredly concluded that histidine residues of the vacuolar H(+)-ATPase were modified by DEP. Kinetic analysis showed that V(max) but not K(m) of vacuolar H(+)-ATPase was decreased by DEP. This result is interpreted as that the residual activity after DEP inhibition was primarily due to the unmodified enzyme molecules. Moreover, simultaneous presence of DEP and DCCD (N,N'-dicyclohexyl-carbodiimide), an inhibitor modified at proteolipid subunit of vacuolar H(+)-ATPase, did not induce synergistic inhibition, indicating their independent effects. The stoichiometry studies further demonstrate that only one out of four histidine residues modified was involved in the inhibition of vacuolar H(+)-ATPase by DEP. Mg(2+)-ATP, the physiological substrate of vacuolar H(+)-ATPase, but not its analogs, exerted preferentially partial protection against DEP, indicating that the histidine residue involved in the inhibition of enzymatic activity may locate at/or near the active site and directly participate in the binding of the substrate.

Functional size of the thylakoid phosphatases determined by radiation inactivation.

Radiation inactivation technique was employed to determine the functional size of phosphatases from thylakoid membrane. The enzymatic activities of phosphatases decayed in a simple function with the increase of radiation dosage. D37 values of 18.8 +/- 2.4-14.1 +/- 1.5 Mrad were obtained, using phosphoserine, phosphothreonine, p-nitrophenol phosphate, and phospho-histone V-S, respectively, as substrates. The molecular masses of 48.2 +/- 6.3-61 +/- 5.7 kDa were yielded by target theory analysis. We thus speculate that the thylakoid alkaline phosphatase is probably a monomer while acid phosphatase is functionally a dimer in situ.

Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation.

A radiation inactivation technique was employed to determine the functional size of pyrophosphatase (PPase) from the chromatophores of Rhodospirillum rubrum. The activities of hydrolysis and synthesis reactions of pyrophosphatase and its coupled proton translocation decayed in a simple exponential function with the increase of radiation dosages. D37 values of 5.2 +/- 0.7 and 5.8 +/- 0.8 Mrads were obtained for pyrophosphate hydrolysis and its associated proton translocation yielding molecular masses of 167.7 +/- 30.7 and 156.3 +/- 26.6 kDa, respectively. Similarly, a D37 value of 4.4 +/- 0.6 Mrads was measured for the acid-base induced pyrophosphate synthesis resulting in a radiation sensitive size of 196.3 +/- 31.9 kDa.

Radiation inactivation analysis of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings.

Radiation inactivation analysis was employed to determine the functional masses of enzymatic activity and proton translocation of H(+)-pyrophosphatase from submitochondrial particles of etiolated mung bean seedlings. The activities of H(+)-pyrophosphatase decayed as a simple exponential function with respect to radiation dosage. D(37) values of 6.9+/-0.3 and 7.5+/-0.5 Mrad were obtained for pyrophosphate hydrolysis and its associated proton translocation, yielding molecular masses of 170+/-7 and 156+/-11 kDa, respectively. In the presence of valinomycin and 50 mM KCl, the functional size of H(+)-pyrophosphatase of tonoplast was decreased, while that of submitochondrial particles remained the same, indicating that they are two distinct types of proton pump using PP(i) as their energy source.

Cimifoetisides VI and VII Two new cyclolanostanol triterpene glycosides from the aerial parts of Cimicifuga foetida.

Two new cyclolanostanol triterpene glycosides, cimifoetiside VI (1) and cimifoetiside VII (2), and one known compound were isolated from the aerial parts of Cimicifuga foetida L. On the basis of spectral and chemical evidences, the structures of 1 and 2 were elucidated to be (23R,24S)-24-O-acetylisodahurinol-3-O-beta-d-galactopyranoside and (23R,24R)-24-O-acetylshengmanol-3-O-beta-d-glucopyranosyl-(1'' --> 2')-beta-d-xylopyranoside. The known compound was identified as (23R,24R)-24-O-acetylshengmanol-3-O-beta-d-galactopyranoside (3).

Scaling of limb proportions and limb bone diameters in three species of Chinese snub-nosed langurs (genus Rhinopithecus).

In this study, aspects of the structural mechanics of the upper and lower limbs of the three Chinese species of Rhinopithecus were examined. Linear regression and reduced major axis (RMA) analyses of natural log-transformed data were used to examine the dimensions of limb bones and other relationships to body size and locomotion. The results of this study suggest that: (1) the allometry exponents of the lengths of long limbs deviate from isometry, being moderately negative, while the shaft diameters (both sagittal and transverse) show significantly positive allometry; (2) the sagittal diameters of the tibia and ulna show extremely significantly positive allometry--the relative enlargement of the sagittal, as opposed to transverse, diameters of these bones suggests that the distal segments of the fore- and hindlimbs of Rhinopithecus experience high bending stresses during locomotion; (3) observations of Rhinopithecus species in the field indicate that all species engage in energetic leaping during arboreal locomotion. The limbs experience rapid and dramatic decelerations upon completion of a leap. We suggest that these occasional decelerations produce high bending stresses in the distal limb segments and so account for the hypertrophy of the sagittal diameters of the ulna and tibia.

An essential arginyl residue in the tonoplast pyrophosphatase from etiolated mung bean seedlings.

Tonoplast membrane of etiolated mung bean (Vinga radiata. L.) seedlings contained H(+)-translocating pyrophosphatase (PPase). Modification of tonoplast vesicles and partially purified PPase from etiolated mung bean seedlings with arginine-specific reagents, phenylglyoxal (PGO) and 2,3-butanedione (BD), resulted in a marked decline in H(+)-translocating PPase activity. The half-maximal inhibition was brought about by 20 millimolar PGO and 50 millimolar BD for membrane bound and 1.5 millimolar PGO and 5.0 millimolar BD for soluble PPase, respectively. The substrate, Mg(2+)-pyrophosphate, provided partial protection against inactivation by these reagents. Loss of activity of partially purified PPase followed pseudo-first order kinetics. The double logarithm plots of pseudo-first order rate constant versus reagent concentrations gave slopes of 0.88 (PGO) and 0.90 (BD), respectively, suggesting that the inactivation may possibly result from reaction of at least one arginyl residue at the active site of H(+)-translocating PPase.

The two binding sites for DCMU in Photosystem II.

Measurements on the fluorescence induction of Triton X-100 extracted Photosystem II (PSII) particles confirmed the existence of the two sites of inhibition in PSII for the herbicide DCMU. The two sites were located on the reducing and oxidizing sides of PSII, respectively. The inhibition on the oxidizing side, unlike that on the reducing side which was of the "none or all" type, was found only to slow down the electron donation at low concentrations of DCMU. The results also suggested that the inhibitions of DCMU at these two sites were mutually exclusive, i.e., the binding on one site prevented the binding on the other site.

Classification and evolution of Asian colobines.

In order to study the differentiation of Asian colobines, 14 variables measured on 123 skulls, including Rhinopithecus, Presbytis, Presbytiscus (Rhinopithecus avunculus), Pygathrix and Nasalis were analyzed by one-way, cluster and discriminant function analyses. Information on paleoenvironmental changes in China and southeast Asia since the late Tertiary was used to examine the influences of migratory routes and range of distribution in Asian colobines. A cladogram for 6 genera of Asian colobines was constructed from the results of various analyses. Some new points or revisions were suggested: (1) Following one of two migratory routes, ancient species of Asian colobines perhaps passed through Xizang (Tibet) along the northern bank of the Tethys sea and through the Heng Duan Shan regions of Yunnan into Vietnam. An ancient landmass linking Yunnan and Xizang was already present on the east bank of the Tethys sea. Accordingly, Asian colobines would have two centers of evolutionary origin: Sundaland and the Heng Duan Shan regions of China. (2) Pygathrix shares more cranial features with Presbytiscus than with Rhinopithecus. This differs somewhat from the conclusion reached by Groves. (3) Nasalis (karyotype: 2n = 48) may be the most primitive genus among Asian colobines. Certain features shared with Rhinopithecus, e.g. large body size, terrestrial activity and limb proportions, can be interpreted as symplesiomorphic characters. (4) Rhinopithecus, with respect to craniofacial features, is a special case among Asian colobines. It combines a high degree of evolutionary specialization with retention of some primitive features thought to have been present in the ancestral Asian colobine.

Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.

Radiation inactivation analysis of chloroplast CF0-CF1 ATPase.

Radiation inactivation technique was employed to measure the functional size of adenosine triphosphatase of spinach chloroplasts. The functional size for acid-base-induced ATP synthesis was 450 +/- 24 kilodaltons; for phenazine methosulfate-mediated ATP synthesis, 613 +/- 33 kilodaltons; and for methanol-activated ATP hydrolysis, 280 +/- 14 kilodaltons. The difference (170 +/- 57 kilodaltons) between 450 +/- 24 and 280 +/- 14 kilodaltons is explained to be the molecular mass of proton channel (coupling factor 0) across the thylakoid membrane. Our data suggest that the stoichiometry of subunits I, II, and III of coupling factor 0 is 1:2:15. Ca2+- and Mg2+-ATPase activated by methanol, heat, and trypsin digestion have a similar functional size. However, anions such as SO3(2-) and CO3(2-) increased the molecular mass for both ATPase's (except trypsin-activated Mg2+-ATPase) by 12-30%. Soluble coupling factor 1 has a larger target size than that of membrane-bound. This is interpreted as the cold effect during irradiation.

Alanine-scanning mutagenesis along membrane segment 4 of the yeast plasma membrane H+-ATPase. Effects on structure and function.

Membrane segment 4 of P-type cation pumps has been suggested to play a critical role in the coupling of ATP hydrolysis to ion translocation. In this study, structure-function relationships in M4 of the yeast (Saccharomyces cerevisiae) plasma membrane H+-ATPase have been explored by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles, as described previously (Nakamoto, R. K., Rao, R. , and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). One substitution (I329A) led to arrest of the enzyme at an early stage of biogenesis, and three others (G333A, L338A, G349A) reduced ATP hydrolysis to near-background levels. The remaining 26 mutants were expressed well enough in secretory vesicles (44-121% of wild type) and had sufficient ATPase activity (16-123% of wild type) to be characterized in detail. When acridine orange fluorescence quenching was used to measure rates of ATP-dependent proton pumping over a range of ATP concentrations, only minor changes were seen. In kinetic studies, however, seven of the mutant enzymes (I331A, I332A, V334A, V336A, V341A, V342A, and M346A) were resistant to vanadate inhibition, and three of them (I332A, V336A, and V341A) also had a decreased Km and increased pH optimum for ATP hydrolysis. Limited trypsinolysis was used to probe the structure of two different Val-336 substitutions, V336A, described above, and V336R, which displayed little or no ATPase activity. Both were cleaved at a relatively normal rate to give a pattern of fragments essentially identical to that seen with the wild-type enzyme. However, while vanadate, ADP, and ATP were able to protect the wild-type and V336A enzymes against trypsinolysis, the V336R ATPase was protected only by ADP and ATP. Taken together, the data suggest that key residues in the M4 segment may help to communicate the E1-E2 conformational change to ion-binding sites in the membrane.

Mechanical properties of bone-implant interface: an in vitro model for the comparison of stability parameters affecting various stages during osseointegration for dental implant.

Radiographic examination and palpation have been two of most common the methods often used in clinical assessment for implant stability for years. However, radiographs are two-dimensional and difficult to standardize in dental clinical diagnosis. In current study, an in vitro model for dental implant during osseointegration was designed and tested. To attain optimal healing range prior to frequency measurement, removal torque measurements of the initial as well as plateau mechanical bounding force will be exam for various stiffness of the base materials. Resonance frequency measurements will then be taken at predetermined healing intervals on implants placed in experimental animal. Significance between mechanical stability and increase in resonance frequency will be observed in search of its correlation with the stability of the implant-tissue interface.

The high affinity binding site for calcium on the oxidizing side of photosystem II.

Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169-173). Inclusion of 50 mM Ca++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca++ if, Ca++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides.