Application of tandem mass spectrometry in the screening and diagnosis of mucopolysaccharidoses.

Mucopolysaccharidoses (MPSs) are caused by a deficiency in the enzymes needed to degrade glycosaminoglycans (GAGs) in the lysosome. The storage of GAGs leads to the involvement of several systems and even to the death of the patient. In recent years, an increasing number of therapies have increased the treatment options available to patients. Early treatment is beneficial in improving the prognosis, but children with MPSs are often delayed in their diagnosis. Therefore, there is an urgent need to develop a method for early screening and diagnosis of the disease. Tandem mass spectrometry (MS/MS) is an analytical method that can detect multiple substrates or enzymes simultaneously. GAGs are reliable markers of MPSs. MS/MS can be used to screen children at an early stage of the disease, to improve prognosis by treating them before symptoms appear, to evaluate the effectiveness of treatment, and for metabolomic analysis or to find suitable biomarkers. In the future, MS/MS could be used to further identify suitable biomarkers for MPSs for early diagnosis and to detect efficacy.

A Novel, Controllable, and Efficient Method for Building Highly Hydrophobic Aerogels.

Aerogels prepared using freeze-drying methods have the potential to be insulation materials or absorbents in the fields of industry, architecture, agriculture, etc., for their low heat conductivity, high specific area, low density, degradability, and low cost. However, their native, poor water resistance caused by the hydrophilicity of their polymer matrix limits their practical application. In this work, a novel, controllable, and efficient templating method was utilized to construct a highly hydrophobic surface for freeze-drying aerogels. The influence of templates on the macroscopic morphology and hydrophobic properties of materials was investigated in detail. This method provided the economical and rapid preparation of a water-resistant aerogel made from polyvinyl alcohol (PVA) and montmorillonite (MMT), putting forward a new direction for the research and development of new, environmentally friendly materials.

SIRT3 Regulates Levels of Deacetylated SOD2 to Prevent Oxidative Stress and Mitochondrial Dysfunction During Oocyte Maturation in Pigs.

Mammalian oocyte maturation relies on mitochondrial ATP production, but this can lead to damaging reactive oxygen species (ROS). SIRT3, a mitochondrial sirtuin, plays a critical role in regulating mitochondrial redox balance in mouse oocytes under stress; however, its specific roles in porcine oocytes remain unclear. In this study, we utilized the SIRT3 inhibitor 3-TYP to investigate SIRT3's importance in porcine oocyte maturation. Our findings revealed that SIRT3 is expressed in porcine oocytes and its inhibition leads to maturation failure. This was evident through reduced polar body extrusion, arrested cell cycle, as well as disrupted spindle organization and actin distribution. Furthermore, SIRT3 inhibition resulted in a decrease in mitochondrial DNA copy numbers, disruption of mitochondrial membrane potential, and reduced ATP levels, all indicating impaired mitochondrial function in porcine oocytes. Additionally, the primary source of damaged mitochondria was associated with decreased levels of deacetylated superoxide dismutase 2 (SOD2) after SIRT3 inhibition, which led to ROS accumulation and oxidative stress-induced apoptosis. Taken together, our results suggest that SIRT3 regulates the levels of deacetylated SOD2 to maintain redox balance and preserve mitochondrial function during porcine oocyte maturation, with potential implications for improving pig reproduction.

Multiphoton Ionization Reduction of Atoms in Two-Color Femtosecond Laser Fields.

We report the ionization reduction of atoms in two-color femtosecond laser fields in this joint theoretical-experimental study. For the multiphoton ionization of atoms using a 400 nm laser pulse, the ionization probability is reduced if another relatively weak 800 nm laser pulse is overlapped. Such ionization reduction consistently occurs regardless of the relative phase between the two pulses. The time-dependent Schrödinger equation simulation results indicate that with the assisted 800 nm photons the electron can be launched to Rydberg states with large angular quantum numbers, which stand off the nuclei and thus are hard to be freed in the multiphoton regime. This mechanism works for hydrogen, helium, and probably some other atoms if two-color laser fields are properly tuned.

Oral vaccination with recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase elicited a protective immunity in BALB/c mice.

BACKGROUND: Trichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection. METHODOLOGY/PRINCIPAL FINDINGS: The growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA' plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer's patches were evidently increased at 2-4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group. CONCLUSIONS: TsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice.

Biological properties and roles of a Trichinella spiralis inorganic pyrophosphatase in molting and developmental process of intestinal larval stages.

Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg2+, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.

Functional characterization of a glutathione S-transferase in Trichinella spiralis invasion, development and reproduction.

The purpose of this study was to determine the biological function of a Trichinella spiralis glutathione S-transferase (TsGST) in larval invasion and development by RNA interference (RNAi). The TsGST-specific siRNA 366 was transfected into T. spiralis muscle larvae (ML) via electroporation. At 1 day following transfection, the larval TsGST mRNA and protein expressions were reduced by 40.09 and 65.22 % (P < 0.05), respectively. The enzymatic activity of natural TsGST in siRNA-transfected ML was also suppressed by 45% compared with PBS group (P < 0.05). Silencing of the TsGST significantly inhibited the ability of larvae to invade intestinal epithelium cells (IECs) and isolated intestine. After challenge with siRNA-366-treated ML, the infected mice exhibited a 62.82% reduction of intestinal adult worms, and 65.03 % reduction of muscle larvae compared to the PBS group. Besides, the length of adults, newborn larvae and muscle larvae was significantly shorter than that of control siRNA and PBS group; the female fecundity of siRNA 366 group was lower than those of control siRNA and PBS group (P <  0.05). The results revealed that the specific RNAi significantly reduced the expression and enzymatic activity of TsGST, inhibited the larval invasive and developmental capacity, and impaired the female fecundity. The results further confirmed that TsGST plays a crucial role in the T. spiralis life cycle and it might be a potential molecular target for anti-Trichinella vaccines.

Molecular characterization of a Trichinella spiralis serine proteinase.

The aim of this study was to investigate the biological characteristics and functions of a Trichinella spiralis serine proteinase (TsSerp) during larval invasion and development in the host. The full-length TsSerp cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and western blotting analyses showed that TsSerp was a secretory protein that was highly expressed at the T. spiralis intestinal infective larva and muscle larva stages and primarily located at the cuticle, stichosome and intrauterine embryos of the parasite. rTsSerp promoted the larval invasion of intestinal epithelial cells (IECs) and the enteric mucosa, whereas an anti-rTsSerp antibody impeded larval invasion; the promotion and obstruction roles were dose-dependently related to rTsSerp and the anti-rTsSerp antibodies, respectively. Vaccination of mice with rTsSerp elicited a remarkable humoral immune response (high levels of serum IgG, IgG1/IgG2a, IgE and IgM), and it also triggered both systemic (spleen) and local intestinal mucosal mesenteric lymph node (MLN) cellular immune responses, as demonstrated by a significant elevation in Th1 cytokines (IFN-γ) and Th2 cytokines (IL-4) after the spleen and MLN cells from vaccinated mice were stimulated with rTsSerp. Anti-TsSerp antibodies participated in the killing and destruction of newborn larvae via ADCC. The mice vaccinated with rTsSerp exhibited a 48.7% reduction in intestinal adult worms and a 52.5% reduction in muscle larvae. These results indicated that TsSerp participates in T. spiralis invasion and development in the host and might be considered a potential candidate target antigen to develop oral polyvalent preventive vaccines against Trichinella infection.

Binding of elastase-1 and enterocytes facilitates Trichinella spiralis larval intrusion of the host's intestinal epithelium.

Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection.

Interaction of a Trichinella spiralis cathepsin B with enterocytes promotes the larval intrusion into the cells.

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis.

Protective immunity in mice vaccinated with a novel elastase-1 significantly decreases Trichinella spiralis fecundity and infection.

Trichinella spiralis is an important foodborne parasitic nematode that represents an enormous threat to the food safety of pork meat. The development of a preventive vaccine is valuable for the prevention and control of Trichinella infection in domestic pigs to ensure pork safety. Elastase is a trypsin-like serine protease that hydrolyzes the host's diverse tissue components and participates in parasite penetration, and it might be a novel vaccine target molecule. The aim of this study was to assess the protective immunity produced by vaccination with a novel Trichinella spiralis elastase-1 (TsE) in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsE elicited a systemic humoral response (high levels of serum IgG and subclass IgG1/IgG2a and IgA) and significant local enteral mucosal sIgA responses. Anti-rTsE IgG recognized the native TsE at the cuticle, stichosome of intestinal infective larvae and adult worm (AW), and intrauterine embryos of female AW. The rTsE vaccination also produced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of Th1 cytokines (IFN-γ, IL-2) and Th2 cytokines (IL-4, IL-10) after spleen, mesenteric lymph node and Peyer's patch cells from immunized mice were stimulated with rTsE. The immunized mice exhibited a 52.19% reduction in enteral AW and a 64.06% reduction in muscle larvae after challenge infection. The immune response triggered by rTsE vaccination protected enteral mucosa from larval intrusion, suppressed larval development and reduced female fecundity. The results indicate that TsE may represent a novel target molecule for anti-T. spiralis vaccines.

Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.

BACKGROUND: Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis. METHODS: The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA. RESULTS: The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients. CONCLUSIONS: rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.

Apatinib Combined with Local Irradiation Leads to Systemic Tumor Control via Reversal of Immunosuppressive Tumor Microenvironment in Lung Cancer.

PURPOSE: This study aimed to investigate the potential systemic antitumor effects of stereotactic ablative radiotherapy (SABR) and apatinib (a novel vascular endothelial growth factor receptor 2 inhibitor) via reversing the immunosuppressive tumor microenvironment for lung carcinoma. MATERIALS AND METHODS: Lewis lung cancer cells were injected into C57BL/6 mice in the left hindlimb (primary tumor; irradiated) and in the right flank (secondary tumor; nonirradiated). When both tumors grew to the touchable size, mice were randomly divided into eight treatment groups. These groups received normal saline or three distinct doses of apatinib (50 mg/kg, 150 mg/kg, and 200 mg/kg) daily for 7 days, in combination with a single dose of 15 Gy radiotherapy or not to the primary tumor. The further tumor growth/regression of mice were followed and observed. RESULTS: For the single 15 Gy modality, tumor growth delay could only be observed at the primary tumor. When combining SABR and apatinib 200 mg/kg, significant retardation of both primary and secondary tumor growth could be observed, indicated an abscopal effect was induced. Mechanism analysis suggested that programmed death-ligand 1 expression increased with SABR was counteract by additional apatinib therapy. Furthermore, when apatinib was combined with SABR, the composition of immune cells could be changed. More importantly, this two-pronged approach evoked tumor antigen-specific immune responses and the mice were resistant to another tumor rechallenge, finally, long-term survival was improved. CONCLUSION: Our results suggested that the tumor microenvironment could be managed with apatinib, which was effective in eliciting an abscopal effect induced by SABR.

Comparison of anti-inflammatory effects of intense pulsed light with tobramycin/dexamethasone plus warm compress on dry eye associated meibomian gland dysfunction.

AIM: To compare the anti-inflammatory effects of intense pulsed light (IPL) with tobramycin/dexamethasone plus warm compress through clinical signs and cytokines in tears. METHODS: Eighty-two patients with dry eye disease (DED) associated meibomian gland dysfunction (MGD) were divided into two groups. Group A was treated with IPL, and Group B was treated with tobramycin/dexamethasone plus warm compress. Ocular Surface Disease Index (OSDI), tear film breakup time (TBUT), corneal fluorescein staining (CFS), meibomian gland expressibility (MGE), meibum quality, gland dropout and tear cytokine levels were evaluated before treatment, 1wk and 1mo after treatment. RESULTS: TBUT in Group A was higher (P=0.035), and MGE score was lower than Group B at 1mo (P=0.001). The changes of interleukin (IL)-17A and IL-1ß levels in tears were lower in Group A compared with that in Group B at 1wk after treatment (P=0.05, P=0.005). CONCLUSION: Treatment with IPL can improve TBUT and MGE and downregulate levels of IL-17A and IL-1ß in tears of patients with DED associated MGD better than treatment with tobramycin/dexamethasone plus warm compress in one-month treatment period.

Molecular characterization of a putative serine protease from Trichinella spiralis and its elicited immune protection.

In our previous work, a Trichinella spiralis putative serine protease (TsSP) was identified from ES products of T. spiralis intestinal infective larvae (IIL) and adult worms (AW) by immunoproteomics: it was highly expressed in IIL compared with muscle larvae (ML). In this study, the TsSP biological characteristics in larval invasion and growth were identified and its potential as a vaccine target against Trichinella infection were investigated. Expression of TsSP at various developmental phases (newborn larvae, ML, IIL, and AW) was detected by qPCR, immunofluorescent test and Western blotting. The rTsSP could specifically bind to the intestinal epithelial cell (IEC) membrane and enter into the cytoplasm. Anti-rTsSP serum suppressed the larval invasion of enterocytes in a dose-dependent mode, and killed newborn and ML of T. spiralis, decreased larval infectivity and development in the host by an ADCC-mediated mechanism. Immunization of mice with rTsSP produced a Th2 predominant immune response, and resulted in a 52.70% reduction of adult worms at 5 days post-infection (dpi) and a 52.10% reduction of muscle larvae at 42 dpi. The results revealed there was an interaction between TsSP and the host's IEC; TsSP might be a pivotal protein for the invading, growing and parasiting of this nematode in the host. Vaccination of mice with rTsSP elicited immune protection, and TsSP is a potential target molecule for vaccines against enteral Trichinella infection.

Early detection of Trichinella spiralis DNA in the feces of experimentally infected mice by using PCR.

The aim of this study was to detect Trichinella spiralis DNA in mouse feces during the early stages of infection using PCR. The target gene fragment, a 1.6kb repetitive sequence of T. spiralis genome, was amplified by PCR from feces of mice infected with 100 or 300 larvae at 3-24h post infection (hpi) and 2-28dpi. The sensitivity of PCR was 0.016 larvae in feces. The primers used were highly specific for T. spiralis. No cross-reactivity was observed with the DNA of other intestinal helminths. T. spiralis DNA was detected in 100% (12/12) of feces of mice infected with 100 or 300 larvae as early as 3hpi, with the peak detection lasting to 12-24hpi, and then fluctuating before declining gradually. By 28dpi, the detection rate of T. spiralis DNA in feces of the two groups of infected mice decreased to 8.33% and 25%, respectively. PCR detection of T. spiralis DNA in feces is simple and specific; it might be useful for the early diagnosis of Trichinella infection.

A new screening method based on yeast-expressed human dipeptidyl peptidase IV and discovery of novel inhibitors.

Dipeptidyl peptidase (DPP) IV inhibitors provide a new strategy for the treatment of type 2 diabetes. Human DPP-IV gene was cloned from differentiated Caco-2 cells and expressed in Pichia pastoris. The recombinant enzyme was used in a new system for screening of DPP-IV inhibitors. By high throughput screening, a novel compound (W5188) was identified from 75,000 compounds with an IC(50) of 6.5 microM. This method is highly reproducible and reliable for discovery of DPP-IV inhibitors as shown by Z' value of 0.73 and S/N ratio of 6.89.