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In this article, we study a class of new p(x)-Kirchhoff problem without satisfying the Ambrosetti-Rabinowitz type growth condition. Under some suitable superliner conditions, we introduce new methods to show the boundedness of Cerami sequences. By using the mountain pass lemma and the symmetric mountain pass lemma, we prove that the p(x)-Kirchhoff problem has a nontrivial weak solution and infinitely many solutions.

Objective To identify the immune responses induced by subunit vaccine of Ag85B-ESAT-6 (AE) fusion protein by mucosal route and the protection against Mycobacterium tuberculosis (MTB) infection in mice. Methods AE and AE with c-di-AMP as adjuvant were inoculated intranasally in mice. The generation of specific IgG, cytokines secreted by Th1 cells (IFN-γ, IL-2) and Th2 cells (IL-10) were detected by ELISA. The transcriptional levels of IFN-γ, IL-2, IL-10, and TNF-α were determined using real-time quantitative PCR. After MTB infection by vein, the antibodies level in mice sera and cytokines secretion of splenocytes were detected by ELISA. Histopathological changes in mice lung was illustrated by HE staining, and bacteria burdens of spleen and lung were counted by colony-forming units (CFUs) on plate. Results AE and AE combined with c-di-AMP via nasal mucosal immunization could induce high level specific antibodies in sera, promote splenocyte proliferation, and lead to increased Th1/Th2 cytokines and TNF-α transcription in spleen and lung, and secret more Th1/Th2 cytokines in spleen. After MTB infection, compared with the control group, the specific antibody levels of AE and AE combined with c-di-AMP immunized mice still increased, with enhanced the Th1/Th2 cellular immune responses, inflammatory response in the lung tissues, and reduced bacteria loads in spleen and lung, especially in mice immunized with AE combined with c-di-AMP. Conclusion Intranasal mucosal vaccination of AE subunit vaccine can induce humoral and cellular immune responses, and provide protection against MTB infection in mice, c-di-AMP as an adjuvant can improve the immunogenicity of AE to a certain extent.

Orthodontic tooth movement (OTM) depends on periodontal ligament cells (PDLCs) sensing biomechanical stimuli and subsequently releasing signals to initiate alveolar bone remodeling. However, the mechanisms by which PDLCs sense biomechanical stimuli and affect osteoclastic activities are still unclear. This study demonstrates that the core circadian protein aryl hydrocarbon receptor nuclear translocator–like protein 1 (BMAL1) in PDLCs is highly involved in sensing and delivering biomechanical signals. Orthodontic force upregulates BMAL1 expression in periodontal tissues and cultured PDLCs in manners dependent on ERK (extracellular signal–regulated kinase) and AP1 (activator protein 1). Increased BMAL1 expression can enhance secretion of CCL2 (C-C motif chemokine 2) and RANKL (receptor activator of nuclear factor–κB ligand) in PDLCs, which subsequently promotes the recruitment of monocytes that differentiate into osteoclasts. The mechanistic delineation clarifies that AP1 induced by orthodontic force can directly interact with the BMAL1 promoter and activate gene transcription in PDLCs. Localized administration of the ERK phosphorylation inhibitor U0126 or the BMAL1 inhibitor GSK4112 suppressed ERK/AP1/BMAL1 signaling. These treatments dramatically reduced osteoclastic activity in the compression side of a rat orthodontic model, and the OTM rate was almost nonexistent. In summary, our results suggest that force-induced expression of BMAL1 in PDLCs is closely involved in controlling osteoclastic activities during OTM and plays a vital role in alveolar bone remodeling. It could be a useful therapeutic target for accelerating the OTM rate and controlling pathologic bone-remodeling activities.

Severe zinc dendrite growth is a formidable challenge in aqueous zinc-ion batteries during repeated cycles because of the uneven distribution of electric fields on a post-stripping zinc anode. Herein, we demonstrate a totally new nucleation mechanism by introducing cations with high valence (Ce3+ and La3+) into the electrolyte to homogenize electric fields and regulate the zinc deposition behavior. Intriguingly, a detailed evolution investigation of surface morphology discloses that zinc nucleation shifts to progressive nucleation via the preferential adsorption of cation additives from instantaneous nucleation in the addtive-free electrolyte, thereby resulting in the stable zinc stripping/plating behavior. A symmetric cell with 0.01 M Ce2(SO4)3 can be cycled at 5 A g−1 with a lower voltage hysteresis of 83.3 mV for more than 700 h, which is remarkably longer than that of the pristine one (282 h). A higher capacity retention of Zn//NaV3O8·1.5H2O cell (87.0%) after 250 cycles at 5 A g−1 is also achieved in comparison with that in addtive-free electrolyte (68.1%). This work provides new and deep insights into the effects of cation adsorption on zinc nucleation.

This paper is devoted to study a class of semilinear elliptic equations with variable exponent. By means of perturbation technique, variational methods and a priori estimation, the existence of infinitely many sign-changing solutions to this class of problem is obtained.

Bacteria identified in the oral cavity are highly complicated. They include approximately 1000 species with a diverse variety of commensal microbes that play crucial roles in the health status of individuals. Epidemiological studies related to molecular pathology have revealed that there is a close relationship between oral microbiota and tumor occurrence. Oral microbiota has attracted considerable attention for its role in in‐situ or distant tumor progression. Anaerobic oral bacteria with potential pathogenic abilities, especially Fusobacterium nucleatum and Porphyromonas gingivalis, are well studied and have close relationships with various types of carcinomas. Some aerobic bacteria such as Parvimonas are also linked to tumorigenesis. Moreover, human papillomavirus, oral fungi, and parasites are closely associated with oropharyngeal carcinoma. Microbial dysbiosis, colonization, and translocation of oral microbiota are necessary for implementation of carcinogenic functions. Various underlying mechanisms of oral microbiota‐induced carcinogenesis have been reported including excessive inflammatory reaction, immunosuppression of host, promotion of malignant transformation, antiapoptotic activity, and secretion of carcinogens. In this review, we have systemically described the impact of oral microbial abnormalities on carcinogenesis and the future directions in this field for bringing in new ideas for effective prevention of tumors., Oral microbiota has been playing an important role in the development of cancer throughout human body. Thus we try to summarize the current researches about this topic.

The soluble form of Escherichia coli alkaline phosphatase (ECAP), commercialized calf intestinal alkaline phosphatase (CIAP) and biotinylated CIAP (Biotin-AP) were tested as probes for nonspecific adsorptions of proteins on micro/nano materials represented by magnetic submicron particles (MSP). The nonspecific adsorptions of three candidate probes were evaluated by directly measuring the activities of the adsorbed probes with 4-nitrophenylphosphate. The activities of those three candidate probes exhibited reasonable resistance to detergents, but the presences of > 0.1% glycerol in the adsorption systems greatly reduced their nonspecific adsorptions on MSPs. The nonspecific adsorptions of ECAP and Biotin-AP on some MSPs were significant and saturable but became negligible after such MSPs were coated with small zwitterions, bovine serum albumin and/or streptavidin. Only MSPs applicable to chemiluminescence immunoassays showed negligible nonspecific adsorptions of Biotin-AP and ECAP, but all of the tested MSPs exhibited negligible nonspecific adsorptions of CIAP. Therefore, Biotin-AP and ECAP were suitable probes for nonspecific adsorptions of proteins on micro/nano materials.

Potassium is one of the most essential inorganic cations for plant growth and development. The high affinity K+ (HAK)/K+ uptake (KUP)/K+ transporter (KT) family plays essential roles in the regulation of cellular K+ levels and the maintenance of osmotic balance. However, the roles of these genes in the responses of bananas to low-potassium stress are unclear. In this study, 24 HAK/KUP/KT (MaHAK) genes were identified from banana genomic data. These genes were further classified into four groups based on phylogenetic analysis, gene structure and conserved domain analysis. Segmental duplication events played an important role in the expansion of the MaHAK gene family. Transcriptome analysis revealed the expression patterns of MaHAKs in various tissues under different K+ conditions. MaHAK14b was upregulated under both short- and long-term K+-deficient conditions, suggesting that it plays crucial roles in K+ uptake at low K+ concentrations. Furthermore, MaHAK14b mediated K+ uptake when it was heterologously expressed in the yeast mutant R5421 on low K+ medium. Collectively, these findings provide a foundation for further functional analysis of MaHAK genes, which may be used to improve stress resistance in bananas.

The cross-linked structure of butadiene rubber (BR) vulcanizate presents a challenge to the environmentally benign recycling of waste tires. The high scission of BR vulcanizate was realized by thermo-oxidation in the presence of soybean oil. The effects of the mass ratio of rubber to oil (2:1, 1:1, 2:3, 1:2 and 2:5), oxidation time (0.5, 1, 2, 3, 5, 7 h) and temperature (130, 140 and 160 °C) on degradation were investigated by measuring the evolution of the sol fraction and cross-link density of degraded BR. The results showed that the BR vulcanizate was degraded effectively under thermo-oxidation (140 °C, 3 h), resulting in the highest sol fraction of 70.7%. Furthermore, the structural evolution of degraded BR was studied by gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FTIR). Oxidation, scission and recombination of the cross-linked network of BR occurred simultaneously during thermo-oxidation. The scission of the BR vulcanizate was enhanced by suppressing the recombination of BR chains in the presence of soybean oil.

The covalently crosslinking structure between rubber matrix and carbon black (CB) fillers present a significant challenge to analyze the structure and properties of bound rubber in vulcanizate. This work provides a layered exfoliation method to destruct cross-linked network and separate bound rubber encapsulated CB particles out of vulcanizate. A layer of loosely bound rubber was observed by using transmission electron microscopy (TEM), and the existence of tightly bound rubber layer was proved by thermogravimetric analyzer (TGA), atomic force microscopic (AFM) and dispersion experiment. The chemical components of studied layers were further analyzed by using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS), where the detection of S, N and Zn elements indicated the vulcanization reaction occurred in it, and the content of polysulfide and disulfide bonds was higher than that of monosulfide. The existence of oxygenic groups is resulted from the occurrence of oxidative scission reaction.

Periodontitis, a bacterium-induced inflammatory disease that is characterized by alveolar bone loss, is highly prevalent worldwide. Elucidating the underlying mechanisms of alveolar bone loss in periodontitis is crucial for understanding its pathogenesis. Classically, bone cells, such as osteoclasts, osteoblasts and bone marrow stromal cells, are thought to dominate the development of bone destruction in periodontitis. Recently, osteocytes, the cells embedded in the mineral matrix, have gained attention. This review demonstrates the key contributing role of osteocytes in periodontitis, especially in alveolar bone loss. Osteocytes not only initiate physiological bone remodeling but also assist in inflammation-related changes in bone remodeling. The latest evidence suggests that osteocytes are involved in regulating bone anabolism and catabolism in the progression of periodontitis. The altered secretion of receptor activator of NF-κB ligand (RANKL), sclerostin and Dickkopf-related protein 1 (DKK1) by osteocytes affects the balance of bone resorption and formation and promotes bone loss. In addition, the accumulation of prematurely senescent and apoptotic osteocytes observed in alveolar bone may exacerbate local destruction. Based on their communication with the bloodstream, it is noteworthy that osteocytes may participate in the interaction between local periodontitis lesions and systemic diseases. Overall, further investigations of osteocytes may provide vital insights that improve our understanding of the pathophysiology of periodontitis.

This paper is devoted to study a class of semilinear elliptic equation with variable exponent. By means of perturbation technique, the method of invariant sets for the descending flow and necessary estimates, the existence of infinitely many sign-changing solutions to this problem is obtained.

Objective Chondrogenesis and endochondral ossification in mandibular condyle play crucial roles in maxillofacial morphogenesis and function. Circadian regulator brain and muscle arnt‐like 1 (BMAL1) is proven to be essential for embryonic and postnatal development. The goal of this study was to define the functions of BMAL1 in the embryonic and postnatal growth of mandibular condylar cartilages (MCC). Materials and Methods Micro‐CT, TUNEL staining and EdU assay were performed using BMAL1‐deficient mice model, and in vitro experiments were performed using rat chondrocytes isolated from MCC. RNA sequencing in mandibular condyle tissues from Bmal1 ‐/‐ mice and the age‐matched wild‐type mice was used for transcriptional profiling at different postnatal stages. Results The expression levels of BMAL1 decrease gradually in MCC. BMAL1 is proved to regulate sequential chondrocyte differentiation, and its deficiency can result in the impairment of endochondral ossification of MCC. RNA sequencing reveals hedgehog signalling pathway is the potential target of BMAL1. BMAL1 regulates hedgehog signalling and affects its downstream cascades through directly binding to the promoters of Ptch1 and Ihh, modulating targets of hedgehog signalling which is indispensable for endochondral ossification. Importantly, the short stature phenotypes caused by BMAL1 deficiency can be rescued by hedgehog signalling activator. Conclusions Collectively, these results indicate that BMAL1 plays critical roles on chondrogenesis and endochondral ossification of MCC, giving a new insight on potential therapeutic strategies for facial dysmorphism.

Strategies to promote bone regeneration have been always the focus of investigations since the skeleton plays important roles like mechanical support, organ protection, and mineral homeostasis maintenance in the normal physiological activities of the human body. Magnetic fields have shown to be highly influential in the regeneration process, arousing tremendous interest in utilizing magnetic materials to enhance osteogenesis. In this work, we attempt a more comprehensive and detailed review of magnetic materials in promoting bone regeneration by including not only the mechanisms of bone regeneration, the history and basic concepts of magnetism, but also the types of magnetic materials as well as their influence parameters, designs and fabrication techniques with a focus on their usage in the field of bone regeneration like 3D printed scaffolds and implants. In addition, we provide some possible ideas on the synergistic action between magnetic and other materials on bone tissue. Finally, we propose the development trend of magnetic materials in the field of bone regeneration in the future. There is a huge demand for a more effective and less traumatic way to accelerate bone regeneration of the patients with diseases like fractures, tumors and osteoporosis that cause severe pains, bone loss, limb deformations, and restrictions of mobility. Magnetic materials have substantial potential to be manufactured into novel clinical applications with the ability to increase the bone regeneration efficiency.

Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes Fusarium wilt of banana, the most devastating disease on a banana plant. The genome of Foc TR4 encodes many candidate effector proteins. However, little is known about the functions of these effector proteins on their contributions to disease development and Foc TR4 virulence. Here, we discovered a secreted metalloprotease, FocM35_1, which is an essential virulence effector of Foc TR4. FocM35_1 was highly upregulated during the early stages of Foc TR4 infection progress in bananas. The FocM35_1 knockout mutant compromised the virulence of Foc TR4. FocM35_1 could interact with the banana chitinase MaChiA, and it decreased banana chitinase activity. FocM35_1 induced cell death in Nicotiana benthamiana while suppressing the INF1-induced hypersensitive response (HR), and its predicted enzymatic site was required for lesion formation and the suppression to INF1-induced HR on N. benthamiana leaves. Importantly, treatment of banana leaves with recombinant FocM35_1 accelerates Foc TR4 infection. Collectively, our study provides evidence that metalloprotease effector FocM35 seems to contribute to pathogen virulence by inhibiting the host immunity.

The aetiology of paroxysmal nocturnal haemoglobinuria (PNH) is a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIGA), resulting in global deficiency of glycosyl phosphatidylinositol–anchored proteins (GPI-APs). This study applied RNA-sequencing to examine functional effects of the PIGA mutation in human granulocytes. CXCR2 expression was increased in GPI-AP− compared to GPI-AP+ granulocytes. Macrophage migration inhibitory factor, a CXCR2 agonist, was significantly higher in plasma of PNH patients. Nuclear factor-κB phosphorylation was upregulated in GPI-AP− compared with GPI-AP+ granulocytes. Our data suggest novel mechanisms in PNH, not obviously predicted by decreased production of the GPI moiety.

Developing chemotherapeutic resistance affects clinical outcomes of oxaliplatin treatment on various types of cancer. Thus, it is imperative to explore alternative therapeutic strategies to improve the efficacy of oxaliplatin. Here, it is shown that circadian regulator period 2 (PER2) can potentiate the cytotoxicity of oxaliplatin and boost cell apoptosis by inhibiting DNA adducts repair in human oral squamous cell carcinoma (OSCC) cells. The circadian timing system is closely involved in controling the activity of DNA adducts repair and gives it a 24 h rhythm. The mechanistic dissection clarifies that PER2 can periodically suppress proliferating cell nuclear antigen (PCNA) transcription by pulling down circadian locomotor output cycles kaput–brain and muscle arnt‐like 1 heterodimer from PCNA promoter in a CRY1/2‐dependent manner, which subsequently impedes oxaliplatin‐induced DNA adducts repair. Similarly, PER2 is capable of improving the efficacy of classical DNA‐damaging chemotherapeutic agents. The tumor‐bearing mouse model displays PER2 can be deployed as an oxaliplatin administration timing biomarker. In summary, it is believed that the chronochemotherapeutic strategy matching PER2 expression rhythm can efficiently improve the oxaliplatin efficacy of OSCC., Circadian regulator period 2 (PER2) potentiates the cytotoxicity of oxaliplatin depending on suppressing proliferating cell nuclear antigen (PCNA) transcription periodically by pulling down circadian locomotor output cycles kaput–brain and muscle arnt‐like 1 heterodimer from PCNA promoter in a CRY1/2‐dependent manner, which subsequently impedes oxaliplatin‐induced DNA adducts repair. Oxaliplatin administration in synergy with PER2 can efficiently improve chemotherapeutic efficacy of oral squamous cell carcinoma (OSCC).

Circadian rhythms (CR) are a series of endogenous autonomous oscillators generated by the molecular circadian clock which acting on coordinating internal time with the external environment in a 24-h daily cycle. The circadian clock system is a major regulatory factor for nearly all physiological activities and its disorder has severe consequences on human health. CR disruption is a common issue in modern society, and researches about people with jet lag or shift works have revealed that CR disruption can cause cognitive impairment, psychiatric illness, metabolic syndrome, dysplasia, and cancer. In this review, we summarized the synchronizers and the synchronization methods used in experimental research, and introduced CR monitoring and detection methods. Moreover, we evaluated conventional CR databases, and analyzed experiments that characterized the underlying causes of CR disorder. Finally, we further discussed the latest developments in understanding of CR disruption, and how it may be relevant to health and disease. Briefly, this review aimed to synthesize previous studies to aid in future studies of CR and CR-related diseases.

For a linear response in a competitive bioaffinity assay of a ligand, an optimized system requires CRT greater than 3-fold of CPT, CPT greater than 50-fold of KdR, and KdR greater than 260-fold of KdX, based on chemometrics for bioaffinity interactions (CRT and CPT are the concentrations of the probe and the biomacromolecule and KdX and KdR are the dissociation constants of complexes with the ligand and the probe, respectively). These criteria were tested for a competitive bioaffinity assay of biotin. The probe was a conjugate of monomethyl-poly-(ethylene glycol)-5000, 1-naphthyl-ethylenediamine and biotin. The complex of the probe with streptavidin was quantified by the fluorescence at 430 nm based on Forster resonance energy transfer with tryptophan residues as the intrinsic donors. By fluorometric titration, KdR of the probe was 5.4 ± 1.4 nM (n = 4). At 1.5 μM probe plus 0.50 μM streptavidin, there was a linear decrease of fluorescence at 430 nm for biotin concentrations ranging from ∼36 to ∼500 nM; the linear response slope was consistent with that for the fluorescence at 430 nm to the concentration of the complex of streptavidin and the probe. Biotin at 81 and 414 nM was estimated with variation coefficients below 7%. These proposed criteria may be universally applicable for linear responses in competitive assays of ligands.

BMAL1 is a core component of the circadian clock loop, which directs the sophisticated circadian expression of clock-controlled genes. Skeletal Bone development is a complex biological process involving intramembranous ossification, endochondral ossification and bone remodeling, as well as specific cells, such as mesenchymal cells, osteoblasts, osteoclasts, chondrocytes, etc. Growing evidences suggest that BMAL1 is indispensable for hard tissue development, including bone, cartilage and teeth. Loss of BMAL1 in animals can inhibit bone and cartilage development, and result in abnormal bone mass. In mesenchymal cells, BMAL1 defect inhibits osteoblastic and chondrocytic differentiation. Inactivation of BMAL1 also can promote the differentiation and formation of osteoclasts and increase bone resorption. Specifically, preclinical data demonstrate that the abnormity of BMAL1 expression is associated with skeletal disorders such as skeletal mandibular hypoplasia, osteoarthritis, osteoporosis, etc. In this review, we systemically describe the impact of BMAL1 in skeletal development and homeostasis, and devote to searching new therapy strategies for bone disorders.

Three dimensional chemically cross-linked polymer networks present a great challenge for recycling and reutilization of waste tire rubber. In this work, the covalently cross-linked networks of ground tire rubber (GTR) were degraded heterogeneously under 150 °C due to the synergistic effects of the soybean oil and controlled oxidation. The degradation mechanism was discussed using Horikx theory and Fourier transformation infrared spectroscopy (FTIR). The results showed that the structural evolution of sol and gel parts, which indicated that the sols consisted of degraded GTR chains with low molecular weight, while the gels were mainly composed of bound rubber coated carbon black, which are separated from the cross-linked network of GTR in a high degradation degree. The degraded GTR compound demonstrated an excellent reinforcing effect on solution styrene-butadiene rubber (SSBR), due to the presence of core-shell structured carbon black. This work provide an efficient and economic approach to degrade GTR and transform it into useful products.

Bacillus fastidious uricase (BF uricase) containing 322 amino acid residues exhibited high stability under physiological conditions. Its crystal structure was solved to 1.4-Å resolution, showing homotetramer containing two homodimers. After the intersubunit antiparallel β-sheet in its homodimer, each subunit had a total of 18 C-terminus residues forming an α-helix (Q305-A313) and random coil (S314-L322) on surface to bury other two α-helices (I227-T238 and I244-R258). In comparison, reported crystal structures of Arthrobacter globiformis and Aspergillus flavus uricases had atomic coordinates of only some C-terminus residues, while the crystal structures of all the other uricases accessible before September 2014 missed atomic coordinates of all their C-terminus residues, after the intersubunit antiparallel β-sheets. In each homodimer of BF uricase, H-bonds were found between E311 and Y249 and between Y319 and D257; electrostatic interaction networks were found to surround D307 plus R310 and intersubunit R3, K312 plus D257, E318 plus K242, and L322 plus R258. Amino acid mutations that disrupted those interactions when R3 and D307 were reserved caused moderate decreases of activity at pH 9.2 while negligible decreases of activity at pH 7.4, but destroyed stability at pH 7.4 while slightly decreased stability at pH 9.2. Such structural information guided the fusion of 6His-tag to the C-terminus of the mutant L322D with SNSNSN as a linker to reserve the activity and stability. Hence, the folding of the C-terminus residues is crucial for thermal stability of BF uricase under physiological conditions; these new structural insights are valuable for molecular engineering of uricases.

By approximating maximum activities of six-histidine (6His)-tagged enzyme/mutants adsorbed on Ni2+-NTA-magnetic-submicron-particle (Ni2+-NTA-MSP), a facile approach was tested for comparing enzyme specific activities in cell lysates. On a fixed quantity of Ni2+-NTA-MSP, the activity of an adsorbed 6His-tagged enzyme/mutant was measured via spectrophotometry; the activity after saturation adsorption (Vs) was predicted from response curve with quantities of total proteins from the same lysate as the predictor; Vs was equivalent of specific activity for comparison. This approach required abundance of a 6His-tagged enzyme/mutant over 3% among total proteins in lysate, an accurate series of quantities of total proteins from the same lysate, the largest activity generated by enzyme occupying over 85% binding sites on Ni2+-NTA-MSP and the minimum activity as absorbance change rates of 0.003 min(-1) for analysis. The prediction of Vs tolerated errors in concentrations of total proteins in lysates and was effective to 6His-tagged alkaline phosphatase and its 6His-tagged mutant in lysates. Notably, of those two 6His-tagged enzymes, Vs was effectively approximated with just one optimized quantity of lysates. Hence, this approach with Ni2+-NTA-MSP worked for comparison of specific activities of 6His-tagged enzyme/mutants in lysates when they had sufficient abundance among proteins and activities of adsorbed enzymes were measurable.

Novel bio-based composite nanoparticles were prepared through the electrostatic interaction between lysozyme (Lys) and amphiphilic dopamine-modified poly (γ-glutamic acid) (PGA-DA). The composite nanoparticles were used as particulate emulsifiers to stabilize white oil, forming gel-like emulsions. When the concentration of PGA-DA and Lys was 0.75 mg mL−1, the mean diameter of the composite nanoparticles was smallest (423 nm) with excellent stability. The influences of pH values on the properties of the composite nanoparticles, emulsifying performances and the activity of Lys in the emulsions were carefully investigated. Scanning electron microscopy (SEM) and laser scanning confocal microscopy (CLSM) characterization demonstrated that the composite nanoparticles adsorbed at the oil–water interface. Rheological measurements provided a possible stabilizing mechanism, which depended on the network structure of the adsorption layer at the interface-phase. Emulsifying properties were significantly improved with increasing the pH value of the solution. In addition, the bioactivity of emulsions at pH 6.2 was highest and well retained during a storage period of three months.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder originating from hematopoietic stem cells and is a life-threating disease characterized by intravascular hemolysis, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene (PIG-A) on the X chromosome, which blocks synthesis of the glycolipid moiety and causes deficiency in GPI-anchored proteins. PNH is closely related to aplastic anemia, in which T cells mediate destruction of BM. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, we applied RNA-seq to examine the transcriptome of T cell subsets (CD4+ naive, CD4+ memory, CD8+ naive, and CD8+ memory) from PNH patients and healthy control subjects. Differentially expressed gene analysis in four different T cell subsets from PNH and healthy control subjects showed distinct transcriptional profiles, depending on the T cell subsets. By pathway analysis, we identified novel signaling pathways in T cell subsets from PNH, including increased gene expression involved in TNFR, IGF1, NOTCH, AP-1, and ATF2 pathways. Dysregulation of several candidate genes (JUN, TNFAIP3, TOB1, GIMAP4, GIMAP6, TRMT112, NR4A2, CD69, and TNFSF8) was validated by quantitative real-time RT-PCR and flow cytometry. We have demonstrated molecular signatures associated with positive and negative regulators in T cells, suggesting novel pathophysiologic mechanisms in PNH. These pathways may be targets for new strategies to modulate T cell immune responses in BM failure.

A fluorometric titration approach was proposed for the calibration of the quantity of monoclonal antibody (mcAb) via the quench of fluorescence of tryptophan residues. It applied to purified mcAbs recognizing tryptophan-deficient epitopes, haptens nonfluorescent at 340 nm under the excitation at 280 nm, or fluorescent haptens bearing excitation valleys nearby 280 nm and excitation peaks nearby 340 nm to serve as Förster-resonance-energy-transfer (FRET) acceptors of tryptophan. Titration probes were epitopes/haptens themselves or conjugates of nonfluorescent haptens or tryptophan-deficient epitopes with FRET acceptors of tryptophan. Under the excitation at 280 nm, titration curves were recorded as fluorescence specific for the FRET acceptors or for mcAbs at 340 nm. To quantify the binding site of a mcAb, a universal model considering both static and dynamic quench by either type of probes was proposed for fitting to the titration curve. This was easy for fitting to fluorescence specific for the FRET acceptors but encountered nonconvergence for fitting to fluorescence of mcAbs at 340 nm. As a solution, (a) the maximum of the absolute values of first-order derivatives of a titration curve as fluorescence at 340 nm was estimated from the best-fit model for a probe level of zero, and (b) molar quantity of the binding site of the mcAb was estimated via consecutive fitting to the same titration curve by utilizing such a maximum as an approximate of the slope for linear response of fluorescence at 340 nm to quantities of the mcAb. This fluorometric titration approach was proved effective with one mcAb for six-histidine and another for penicillin G.

A resonant-light-scattering (RLS) method was proposed to quantify phosphate for screening inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE). In acidified mixtures of phosphate, papaverine and molybdate, there were aggregates exhibiting micrometre sizes, no absorbance peaks over 360 nm but strong RLS peaks at 392 nm; Mie scattering thus accounted for the RLS signals. When papaverine was added before molybdate to acidified samples of phosphate, RLS signals at 392 nm were stable from 5 to 25 min since the addition of molybdate; after optimization, phosphate from 0.40 to 3.60 μM was quantifiable. This RLS method tolerated 60 mg L −1 proteins besides common PDE inhibitors and dimethyl sulfoxide in acidified samples of phosphate; the integration of this RLS method with the coupled action of a phosphomonoesterase on PDE product was thus rational to measure PDE activities without the removal of proteins in samples. By quantifying activities of a truncated mutant of human PDE4B2 via this RLS method, Michaelis–Menten constant, inhibition constants of rolipram, papaverine and theophylline varied over three magnitudes and were consistent with those estimated by an improved malachite green assay of phosphate, respectively. Hence, this novel RLS method was promising for screening inhibitors of PDE isozymes.

At pH from 5.5 to 7.6, absorptivity of 4-nitro-1-naphthol at 450 nm is over 2.1-fold of that of para-nitrophenol at 405 nm and over 9.6-fold of that of ortho-nitrophenol at 415 nm. On 4-nitro-1-naphthyl-β-D-galactopyranoside at pH 7.4, catalytic efficiency of Escherichia coli β-D-galactosidase is 3-fold of that on para-nitrophenyl-β-D-galactopyranoside and about 40% of that on ortho-nitrophenyl-β-D-galactopyranoside, and produces a lower quantification limit of penicillin G by enzyme-linked-immunoabsorbent-assay. Hence, 4-nitro-1-naphthol is favorable to prepare chromogenic substrates of hydrolytic enzymes of neutral or slightly acidic optimum pH.

To determine whether time-resolved immunofluorescence assay (TRIFA) shares the similar accuracy and specificity with automatic chemiluminescence immunoassay (CMIA) in analyzing HBeAg levels in hepatitis B. A total of 157 serum samples were collected from outpatients with infection of HBV in Xiangya Hospital, Central South University. CMIA and TRIFA were used to analyze HBeAg quantitation and HBeAg/HBeAb qualitative detection, respectively. The linear regression equation for the two methods was Y=0.72779X-4.0551 (r=0.712, P0.001). Compared with the CMIA, the sensitivity and specificity in detection of HBeAg by TRIFA were 89.89% and 100%, respectively, and the coincidence rate of HBeAg was 94.27% by two assays. Similarly, the sensitivity and specificity in detection of HBeAb by TRIFA were 100% and 95.45%, respectively. The coincidence rate was 97.45% by two assays. TRIFA has similar accuracy, sensitivity, and specificity with CMIA in quantitative detection of HBeAg, and their coincidence rate in detection of HBeAg/HBeAb is high.目的：探讨国产时间分辨免疫荧光法(time-resolved immunofluorescence assay，TRIFA)是否与全自动化学发光免疫分析法(automatic chemiluminescence immunoassay，CMIA)在定量检测HBeAg/HBeAb中具有同样的准确性和特异性。方法：157份来自中南大学湘雅医院感染病科门诊的HBV感染患者的血清标本，分别用CMIA和TRIFA两种方法进行HBeAg定量及HBeAg/HBeAb定性检测，并对结果进行对比分析。结果：两种方法定量检测HBeAg的直线回归方程为：Y=0.72779X–4.0551(r=0.712，P0.001)。与CMIA比较，TRIFA检测HBeAg定性结果的灵敏度和特异性分别为89.89%和100%，两种方法诊断HBeAg符合率为94.27%。TRIFA检测 HBeAb定性结果的灵敏度和特异性分别为100%和95.45%，两种方法诊断HBeAb的符合率为97.45%。结论：TRIFA在HBeAg/HBeAb定量检测上的准确性、灵敏度和特异性与CMIA基本相当，二者HBeAg/HBeAb诊断符合率高。.

An integration strategy was investigated to measure initial rates of horse butyrylcholinesterase (BChE) at 50.0 µmol L−1 butyrylthiocholine (BTCh) for detecting irreversible inhibitors as pollutants in environment and foods with dimethoate as a model. In this integration strategy: (a) if BTCh consumption within 5.0 min was >60%, BChE initial rates were derived from maximal reaction rates, estimated by an improved integrated method, according to Michaelies-Menten kinetics at 47.0 µmol L−1 BTCh and Michaelis-Menten constant at 94.0 µmol L−1; (b) or else initial rates were determined by the classical initial rate method. Thus, the differences in BChE initial rates without and after dimethoate treatment indexed final dimethoate contents in reaction mixtures to treat BChE. Results supported that this integration strategy determined BChE activities with a linear range about two magnitudes and an upper limit about twice that by the classical initial rate method alone at 2.0 mmol L−1 BTCh. The coefficient of vari...

At 0.12 mmol/L γ-glutamyl p-nitroaniline (GGPNA), an improved integrated method was developed for kinetic analysis of γ-glutamyltransferase (GGT) reaction process and the integration with the classical initial rate method to measure serum GGT. For the improved integrated method, an integrated rate equation, which used the predictor variable of reaction time and considered inhibitions by both GGPNA and products, was nonlinearly fit to GGT reaction processes. For the integration strategy, classical initial rates were estimated when GGPNA consumption percentages were below 50%; otherwise, maximal reaction rates of GGT were estimated by the improved integrated method and converted into initial rates according to the differential rate equation at 0.11 mmol/L GGPNA. The integration strategy was validated using optimized GGT kinetic parameters and 10-s intervals to record reaction curves within 8.0 min. By the integration strategy, there was a linear response from 0.9 to 32.0 U/L GGT, coefficients of variation were below 3.5% for GGT from 8.0 to 32.0 U/L (n=5), and GGT activities in clinical sera responded linearly to their classical initial rates at 2.00 mmol/L GGPNA with an expected slope. Therefore, the integration strategy was successful in measuring GGT at 0.12 mmol/L GGPNA.