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1.  The effects of Cordyceps sinensis phytoestrogen on estrogen deficiency-induced osteoporosis in Ovariectomized rats 
Isoflavones are naturally occurring plant chemicals belonging to the “phytoestrogen” class. The aim of the present study was to examine the effects of isoflavones obtained from Cordyceps sinensis (CSIF) on development of estrogen deficiency-induced osteoporosis in ovariectomized rats.
After the rats were treated orally with CSIF, serum alkaline phosphatase (ALP), tartarate resistant acid phosphatase (TRAP), serum osteocalcin (OC), homocysteine (HCY), C-terminal crosslinked telopeptides of collagen type I (CTX), estradiol and interferonγ (IFN-γ) level were examined. At the same time, the urine calcium, plasma calcium, plasma phosphorus and the mass of uterus, thymus and body were also examined.
The beneficial effects of CSIF on improvement of osteoporosis in rats were attributable mainly to decrease ALP activity, TRAP activity, CTX level and IFN-γ level. At the same time, CSIF also increase the OC and estradiol level in ovariectomized osteopenic rats. The histological examination clearly showed that dietary CSIF can prevent bone loss caused by estrogen deficiency.
The significant estrogenic activity of CSIF demonstrated that CSIF has significant estrogenic effects in OVX rats.
PMCID: PMC4302055  PMID: 25496560
Cordyceps sinensis; Isoflavones; Osteoporosis; Phytoestrogen; Estrogens
2.  Overactivation of Mitogen-Activated Protein Kinase and Suppression of Mitofusin-2 Expression Are Two Independent Events in High Mobility Group Box 1 Protein–Mediated T Cell Immune Dysfunction 
High mobility group box 1 protein (HMGB1), a critical proinflammatory cytokine, has recently been identified to be an immunostimulatory signal involved in sepsis-related immune dysfunction when released extracellularly, but the potential mechanism involved remains elusive. Here, we showed that the treatment with HMGB1 in vitro inhibited T lymphocyte immune response and expression of mitofusin-2 (Mfn-2; a member of the mitofusin family) in a dose- and time-dependent manner. Upregulation of Mfn-2 expression attenuated the suppressive effect of HMGB1 on T cell immune function. The phosphorylation of both extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) was markedly upregulated by treating with high amount of HMGB1, while pretreatment with ERK1/2 and p38 MAPK-specific inhibitors (U0126 and SB203580) could attenuate suppression of T cell immune function and nuclear factor of activated T cell (NFAT) activation induced by HMGB1, respectively. HMGB1-induced activity of ERK1/2 and p38 was not fully inhibited in the presence of U0126 or SB203580. Interestingly, overexpression of Mfn-2 had no marked effect on HMGB1-mediated activation of MAPK, but could attenuate the suppressive effect of HMGB1 on the activity of NFAT. Thus, the mechanisms involved in HMGB1-induced T cell immune dysfunction in vitro at least partly include suppression of Mfn-2 expression, overactivation of ERK1/2, p38 MAPK, and intervention of NFAT activation, while the protective effect of Mfn-2 on T cell immune dysfunction induced by HMGB1 is dependent on other signaling pathway associated with NFAT, but not MAPK. Taken together, we conclude that overactivation of MAPK and suppression of Mfn-2 expression are two independent events in HMGB1-mediated T cell immune dysfunction.
PMCID: PMC3760027  PMID: 23697559
3.  Characterization of the Intergenic Spacer rDNAs of Two Pig Nodule Worms, Oesophagostomum dentatum and O. quadrispinulatum 
The Scientific World Journal  2014;2014:147963.
The characteristics of the intergenic spacer rDNAs (IGS rDNAs) of Oesophagostomum dentatum and O. quadrispinulatum isolated from pigs in different geographical locations in Mainland China were determined, and the phylogenetic relationships of the two species were reconstructed using the IGS rDNA sequences. The organization of the IGS rDNA sequences was similar to their organization in other eukaryotes. The 28S-18S IGS rDNA sequences of both O. dentatum and O. quadrispinulatum were found to have variable lengths, that is, 759–762 bp and 937–1128 bp, respectively. All of the sequences contained direct repeats and inverted repeats. The length polymorphisms were related to the different numbers and organization of repetitive elements. Different types and numbers of repeats were found between the two pig nodule species, and two IGS structures were found within O. quadrispinulatum. Phylogenetic analysis showed that all O. dentatum isolates were clustered into one clade, but O. quadrispinulatum isolates from different origins were grouped into two distinct clusters. These results suggested independent species and the existence of genotypes or subspecies within pig nodule worms. Different types and numbers of repeats and IGS rDNA structures could serve as potential markers for differentiating these two species of pig nodule worms.
PMCID: PMC4147281  PMID: 25197691
4.  Characterization of the complete mitochondrial genomes of Nematodirus oiratianus and Nematodirus spathiger of small ruminants 
Parasites & Vectors  2014;7:319.
Nematodirus spp. are among the most common nematodes of ruminants worldwide. N. oiratianus and N. spathiger are distributed worldwide as highly prevalent gastrointestinal nematodes, which cause emerging health problems and economic losses. Accurate identification of Nematodirus species is essential to develop effective control strategies for Nematodirus infection in ruminants. Mitochondrial DNA (mtDNA) could provide powerful genetic markers for identifying these closely related species and resolving phylogenetic relationships at different taxonomic levels.
In the present study, the complete mitochondrial (mt) genomes of N. oiratianus and N. spathiger from small ruminants in China were obtained using Long-range PCR and sequencing.
The complete mt genomes of N. oiratianus and N. spathiger were 13,765 bp and 13,519 bp in length, respectively. Both mt genomes were circular and consisted of 36 genes, including 12 genes encoding proteins, 2 genes encoding rRNA, and 22 genes encoding tRNA. Phylogenetic analyses based on the concatenated amino acid sequence data of all 12 protein-coding genes by Bayesian inference (BI), Maximum likelihood (ML) and Maximum parsimony (MP) showed that the two Nematodirus species (Molineidae) were closely related to Dictyocaulidae.
The availability of the complete mtDNA sequences of N. oiratianus and N. spathiger not only provides new mtDNA sources for a better understanding of nematode mt genomics and phylogeny, but also provides novel and useful genetic markers for studying diagnosis, population genetics and molecular epidemiology of Nematodirus spp. in small ruminants.
PMCID: PMC4105107  PMID: 25015379
Nematodirus oiratianus; Nematodirus spathiger; Mitochondrial genome; Phylogenetic analyses
5.  Chrysin Suppressed Inflammatory Responses and the Inducible Nitric Oxide Synthase Pathway after Spinal Cord Injury in Rats 
Chrysin (CH), a natural plant flavonoid, has shown a variety of beneficial effects. Our present study was conducted to evaluate the therapeutic potential of CH three days after spinal cord injury (SCI) in rats and to probe the underlying neuroprotective mechanisms. SCI was induced using the modified weight-drop method in Wistar rats. Then, they were treated with saline or CH by doses of 30 and 100 mg/kg for 26 days. Neuronal function was assessed with the Basso Beattle Bresnahan locomotor rating scale (BBB). The water content of spinal cord was determined after traumatic SCI. The NF-κB p65 unit, TNF-α, IL-1β and IL-6 in serums, as well as the apoptotic marker, caspase-3, of spinal cord tissues were measured using commercial kits. The protein level and activity of inducible nitric oxide synthase (iNOS) were detected by western blot and a commercial kit, respectively. NO (nitric oxide) production was evaluated by the determination of nitrite concentration. The rats with SCI showed marked reductions in BBB scores, coupled with increases in the water content of spinal cord, the NF-κB p65 unit, TNF-α, IL-1β, IL-6, iNOS, NO production and caspase-3. However, a CH supplement dramatically promoted the recovery of neuronal function and suppressed the inflammatory factors, as well as the iNOS pathway in rats with SCI. Our findings disclose that CH improved neural function after SCI in rats, which might be linked with suppressing inflammation and the iNOS pathway.
PMCID: PMC4139843  PMID: 25014398
chrysin; spinal cord injury; neuroprotection; inflammation; inducible nitric oxide synthase
6.  Investigation of the Profile Control Mechanisms of Dispersed Particle Gel 
PLoS ONE  2014;9(6):e100471.
Dispersed particle gel (DPG) particles of nano- to micron- to mm-size have been prepared successfully and will be used for profile control treatment in mature oilfields. The profile control and enhanced oil recovery mechanisms of DPG particles have been investigated using core flow tests and visual simulation experiments. Core flow test results show that DPG particles can easily be injected into deep formations and can effectively plug the high permeability zones. The high profile improvement rate improves reservoir heterogeneity and diverts fluid into the low permeability zone. Both water and oil permeability were reduced when DPG particles were injected, but the disproportionate permeability reduction effect was significant. Water permeability decreases more than the oil permeability to ensure that oil flows in its own pathways and can easily be driven out. Visual simulation experiments demonstrate that DPG particles can pass directly or by deformation through porous media and enter deep formations. By retention, adsorption, trapping and bridging, DPG particles can effectively reduce the permeability of porous media in high permeability zones and divert fluid into a low permeability zone, thus improving formation profiles and enhancing oil recovery.
PMCID: PMC4065096  PMID: 24950174
7.  First report of Cryptosporidium spp. in white yaks in China 
Parasites & Vectors  2014;7:230.
Cryptosporidium is an enteric apicomplexan parasite, which can infect yaks, leading to reduction of milk production and poor weight gain. White yak (Bos grunniens) is a unique yak breed inhabiting only in Tianzhu Tibetan Autonomous County, Gansu province, northwestern China. The objective of the present study was to molecularly determine Cryptosporidium infection and species in white yaks.
Seventy-six fecal samples from white yaks in Tianzhu Tibetan Autonomous County, Gansu province were collected. The small subunit ribosomal RNA (SSU rRNA) gene of each sample was amplified using nested PCR and sequenced. The Cryptosporidium species was determined by comparison of the obtained sequences with that of corresponding Cryptosporidium sequences available in GenBank by BLAST ( and phylogenetic analysis with maximum likelihood (ML) using PAUP*. The overall prevalence of Cryptosporidium infection in white yak was 5.26% (4/76). Species identification showed C. andersoni in one sample (collected in September), and C. bovis in three samples (one collected in November and two collected in September).
The present investigation revealed the existence of Cryptosporidium infection in white yaks in China, for the first time, and two Cryptosporidium species, namely C. andersoni and C. bovis, were identified. These findings extend the host range for Cryptosporidium spp., and also provide base-line information for further studies of molecular epidemiology and control of Cryptosporidium infection in the unique white yaks.
PMCID: PMC4033679  PMID: 24885747
Cryptosporidium spp; Genetic characterization; Prevalence; White Yak; China
8.  Development of Genetically Stable Escherichia coli Strains for Poly(3-Hydroxypropionate) Production 
PLoS ONE  2014;9(5):e97845.
Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In our previous study, a pathway for P3HP production was constructed in recombinant Esecherichia coli. Seven exogenous genes in P3HP synthesis pathway were carried by two plasmid vectors. However, the P3HP production was severely suppressed by strain instability due to plasmid loss. In this paper, two strategies, chromosomal gene integration and plasmid addiction system (PAS) based on amino acid anabolism, were applied to construct a genetically stable strain. Finally, a combination of those two methods resulted in the best results. The resultant strain carried a portion of P3HP synthesis genes on chromosome and the others on plasmid, and also brought a tyrosine-auxotrophy based PAS. In aerobic fed-batch fermentation, this strain produced 25.7 g/L P3HP from glycerol, about 2.5-time higher than the previous strain with two plasmids. To the best of our knowledge, this is the highest P3HP production from inexpensive carbon sources.
PMCID: PMC4023983  PMID: 24837211
9.  The expression dynamics of IL-17 and Th17 response relative cytokines in the trachea and spleen of chickens after infection with Cryptosporidium baileyi 
Parasites & Vectors  2014;7:212.
Cryptosporidium baileyi is the dominant Cryptosporidium species in birds causing emerging health problems in the poultry industry, and is also a model to study the biology of Cryptosporidium spp.. IL-17 (also called IL-17A) is a hallmark pro-inflammatory cytokine of Th17 cells that plays an important role in several human autoimmune diseases and microbial infection disease of many animals, and it may play a role in Cryptosporidium infection.
The present study examined the mRNA level of IL-17 and Th17 response relative cytokines in the trachea and spleen of C. baileyi-infected chickens at different time points using real-time quantitative PCR (qPCR).
All examined cytokines in the trachea were up-regulated in the infected chickens compared with the uninfected control during C. baileyi infection. Significant increased IL-17 mRNA level in the trachea was observed as early as 12 h post infection (pi), peaking at 24 h pi and 10 d pi, and declining thereafter. The transcription levels of IL-17 and Th17 response relative cytokines in spleen were also significantly increased at different time points during the infection.
IL-17 was indicated to participate in the induction of inflammation during infection of some intracellular protozoan parasites. The results in the present study suggest that IL-17 may play a role in immunity against Cryptosporidium infection, and provide basic information for determining the role of Th17 cell in Cryptosporidium infection.
PMCID: PMC4036416  PMID: 24886047
Cryptosporidium baileyi; IL-17; Chicken; Immunity
10.  L-Serine Treatment May Improve Neurorestoration of Rats after Permanent Focal Cerebral Ischemia Potentially Through Improvement of Neurorepair 
PLoS ONE  2014;9(3):e93405.
The present study was conducted to clarify whether treatment with L-serine can improve the brain repair and neurorestoration of rats after permanent middle cerebral artery occlusion (pMCAO). After pMCAO, the neurological functions, brain lesion volume, and cortical injury were determined. GDNF, NGF, NCAM L1, tenascin-C, and Nogo-A levels were measured. Proliferation and differentiation of the neural stem cells (NSCs) and proliferation of the microvessels in the ischemic boundary zone of the cortex were evaluated. Treatment with L-serine (168 mg/kg body weight, i.p.) began 3 h after pMCAO and was repeated every 12 h for 7 days or until the end of the experiment. L-Serine treatment: 1) reduced the lesion volume and neuronal loss; 2) improved the recovery of neurological functions; 3) elevated the expression of nerve growth-related factors; and 4) facilitated the proliferation of endogenous NSCs and microvessels activated after pMCAO and increased the number of new-born neurons. 5) D-cycloserine, an inhibitor of serine hydroxymethyltransferase, blunted the effects of L-serine on NSC proliferation, differentiation, microvascular proliferation. In conclusions, L-serine treatment in pMCAO rats can reduce brain injury and facilitate neurorestoration which is partly associated with the improvement of proliferation of NSCs and microvessels, reconstruction of neurovascular units and resultant neurorepair. The effects of L-serine on endogenous NSC proliferation and microvascular proliferation are partly mediated by the action of L-serine as a substrate for the production of one-carbon groups used for purine and pyrimidine synthesis and modulation of the expression of some nerve growth-related factors.
PMCID: PMC3966884  PMID: 24671106
11.  Septic shock due to community-acquired Pseudomonas aeruginosa necrotizing fasciitis: A case report and literature review 
Necrotizing fasciitis is a rare but fatal infection, characterized by the rapid progression of necrosis of the fascia, skin, soft tissue and muscle. The most common bacteria associated with necrotizing fasciitis is group A streptococcus, although other pathogens have also been implicated. In the present study, a case of community-acquired necrotizing fasciitis, complicated with septic shock and multiple organ dysfunction syndromes due to Pseudomonas aeruginosa, is presented. Despite intensive medical treatment, the condition of the patient deteriorated rapidly and the patient subsequently succumbed to multiple organ failure. In view of the rapid progression and high mortality rate of this disease, early surgery, as well as novel therapeutic approaches for septic shock are required to improve the outcome for patients.
PMCID: PMC4043593  PMID: 24926341
necrotizing fasciitis; pseudomonas aeruginosa; septic shock
12.  Whole-Genome Sequencing of Tibetan Macaque (Macaca thibetana) Provides New Insight into the Macaque Evolutionary History 
Molecular Biology and Evolution  2014;31(6):1475-1489.
Macaques are the most widely distributed nonhuman primates and used as animal models in biomedical research. The availability of full-genome sequences from them would be essential to both biomedical and primate evolutionary studies. Previous studies have reported whole-genome sequences from rhesus macaque (Macaca mulatta) and cynomolgus macaque (M. fascicularis, CE), both of which belong to the fascicularis group. Here, we present a 37-fold coverage genome sequence of the Tibetan macaque (M. thibetana; TM). TM is an endemic species to China belonging to the sinica group. On the basis of mapping to the rhesus macaque genome, we identified approximately 11.9 million single-nucleotide variants), of which 3.9 million were TM specific, as assessed by comparison two Chinese rhesus macaques (CR) and two CE genomes. Some genes carried TM-specific homozygous nonsynonymous variants (TSHNVs), which were scored as deleterious in human by both PolyPhen-2 and SIFT (Sorting Tolerant From Intolerant) and were enriched in the eye disease genes. In total, 273 immune response and disease-related genes carried at least one TSHNV. The heterozygosity rates of two CRs (0.002617 and 0.002612) and two CEs (0.003004 and 0.003179) were approximately three times higher than that of TM (0.000898). Polymerase chain reaction resequencing of 18 TM individuals showed that 29 TSHNVs exhibited high allele frequencies, thus confirming their low heterozygosity. Genome-wide genetic divergence analysis demonstrated that TM was more closely related to CR than to CE. We further detected unusual low divergence regions between TM and CR. In addition, after applying statistical criteria to detect putative introgression regions (PIRs) in the TM genome, up to 239,620 kb PIRs (8.84% of the genome) were identified. Given that TM and CR have overlapping geographical distributions, had the same refuge during the Middle Pleistocene, and show similar mating behaviors, it is highly likely that there was an ancient introgression event between them. Moreover, demographic inferences revealed that TM exhibited a similar demographic history as other macaques until 0.5 Ma, but then it maintained a lower effective population size until present time. Our study has provided new insight into the macaque evolutionary history, confirming hybridization events between macaque species groups based on genome-wide data.
PMCID: PMC4032132  PMID: 24648498
Tibetan macaque; whole-genome sequencing; SNVs; genetic divergence; introgression; demographic trajectories
13.  Chabertia erschowi (Nematoda) is a distinct species based on nuclear ribosomal DNA sequences and mitochondrial DNA sequences 
Parasites & Vectors  2014;7:44.
Gastrointestinal nematodes of livestock have major socio-economic importance worldwide. In small ruminants, Chabertia spp. are responsible for economic losses to the livestock industries globally. Although much attention has given us insights into epidemiology, diagnosis, treatment and control of this parasite, over the years, only one species (C. ovina) has been accepted to infect small ruminants, and it is not clear whether C. erschowi is valid as a separate species.
The first and second internal transcribed spacers (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) and the complete mitochondrial (mt) genomes of C. ovina and C. erschowi were amplified and then sequenced. Phylogenetic re-construction of 15 Strongylida species (including C. erschowi) was carried out using Bayesian inference (BI) based on concatenated amino acid sequence datasets.
The ITS rDNA sequences of C. ovina China isolates and C. erschowi samples were 852–854 bp and 862 -866 bp in length, respectively. The mt genome sequence of C. erschowi was 13,705 bp in length, which is 12 bp shorter than that of C. ovina China isolate. The sequence difference between the entire mt genome of C. ovina China isolate and that of C. erschowi was 15.33%. In addition, sequence comparison of the most conserved mt small subunit ribosomal (rrnS) and the least conserved nad2 genes among multiple individual nematodes revealed substantial nucleotide differences between these two species but limited sequence variation within each species.
The mtDNA and rDNA datasets provide robust genetic evidence that C. erschowi is a valid strongylid nematode species. The mtDNA and rDNA datasets presented in the present study provide useful novel markers for further studies of the taxonomy and systematics of the Chabertia species from different hosts and geographical regions.
PMCID: PMC3937141  PMID: 24450932
Chabertia spp; Nuclear ribosomal DNA; Internal transcribed spacer (ITS); Mitochondrial DNA; Phylogenetic analysis
14.  Investigation of Preparation and Mechanisms of a Dispersed Particle Gel Formed from a Polymer Gel at Room Temperature 
PLoS ONE  2013;8(12):e82651.
A dispersed particle gel (DPG) was successfully prepared from a polymer gel at room temperature. The polymer gel system, morphology, viscosity changes, size distribution, and zeta potential of DPG particles were investigated. The results showed that zirconium gel systems with different strengths can be cross-linked within 2.5 h at low temperature. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) results showed that the particles were polygonal particles with nano-size distribution. According to the viscosity changes, the whole preparation process can be divided into two major stages: the bulk gel cross-linking reaction period and the DPG particle preparation period. A polymer gel with a 3-dimensional network was formed in the bulk gel cross-linking reaction period whereas shearing force and frictional force were the main driving forces for the preparation of DPG particles, and thus affected the morphology of DPG particles. High shearing force and frictional force reduced the particle size distribution, and then decreased the zeta potential (absolute value). The whole preparation process could be completed within 3 h at room temperature. It could be an efficient and energy-saving technology for preparation of DPG particles.
PMCID: PMC3855795  PMID: 24324817
15.  Modified Uterine Allotransplantation and Immunosuppression Procedure in the Sheep Model 
PLoS ONE  2013;8(11):e81300.
To develop an orthotopic, allogeneic, uterine transplantation technique and an effective immunosuppressive protocol in the sheep model.
In this pilot study, 10 sexually mature ewes were subjected to laparotomy and total abdominal hysterectomy with oophorectomy to procure uterus allografts. The cold ischemic time was 60 min. End-to-end vascular anastomosis was performed using continuous, non-interlocking sutures. Complete tissue reperfusion was achieved in all animals within 30 s after the vascular re-anastomosis, without any evidence of arterial or venous thrombosis. The immunosuppressive protocol consisted of tacrolimus, mycophenolate mofetil and methylprednisolone tablets. Graft viability was assessed by transrectal ultrasonography and second-look laparotomy at 2 and 4 weeks, respectively.
Viable uterine tissue and vascular patency were observed on transrectal ultrasonography and second-look laparotomy. Histological analysis of the graft tissue (performed in one ewe) revealed normal tissue architecture with a very subtle inflammatory reaction but no edema or stasis.
We have developed a modified procedure that allowed us to successfully perform orthotopic, allogeneic, uterine transplantation in sheep, whose uterine and vascular anatomy (apart from the bicornuate uterus) is similar to the human anatomy, making the ovine model excellent for human uterine transplant research.
PMCID: PMC3838404  PMID: 24278415
16.  Effect of admission time on mortality in an intensive care unit in Mainland China: a propensity score matching analysis 
Critical Care  2013;17(5):R230.
The relationship between admission time and intensive care unit (ICU) mortality is inconclusive and influenced by various factors. This study aims to estimate the effect of admission time on ICU outcomes in a tertiary teaching hospital in China by propensity score matching (PSM) and stratified analysis.
A total of 2,891 consecutive patients were enrolled in this study from 1 January 2009 to 29 December 2011. Multivariate logistic regression and survival analysis were performed in this retrospective study. PSM and stratified analysis were applied for confounding factors, such as Acute Physiology and Chronic Health Evaluation II (APACHE II) score and admission types.
Compared with office hour subgroup (n = 2,716), nighttime (NT, n = 175) subgroup had higher APACHE II scores (14 vs. 8, P < 0.001), prolonged length of stay in the ICU (42 vs. 24 h, P = 0.011), and higher percentages of medical (8.6% vs. 3.3%, P < 0.001) and emergency (59.4% vs. 12.2%, P < 0.001) patients. Moreover, NT admissions were related to higher ICU mortality [odds ratio (OR), 1.725 (95% CI 1.118–2.744), P = 0.01] and elevated mortality risk at 28 days [14.3% vs. 3.2%; OR, 1.920 (95% CI 1.171–3.150), P = 0.01]. PSM showed that admission time remained related to ICU outcome (P = 0.045) and mortality risk at 28 days [OR, 2.187 (95% CI 1.119–4.271), P = 0.022]. However, no mortality difference was found between weekend and workday admissions (P = 0.849), even if weekend admissions were more related to higher APACHE II scores compared with workday admissions.
NT admission was associated with poor ICU outcomes. This finding may be related to shortage of onsite intensivists and qualified residents during NT. The current staffing model and training system should be improved in the future.
PMCID: PMC4055975  PMID: 24112558
17.  Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement 
PLoS ONE  2013;8(9):e75554.
The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher Kcat/Km value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.
PMCID: PMC3779250  PMID: 24073271
18.  Identification and characterization of microRNAs in Baylisascaris schroederi of the giant panda 
Parasites & Vectors  2013;6:216.
Baylisascaris schroederi is one of the most significant threats to the giant panda’s survival, responsible for half of the deaths reported from 2001 to 2005. MicroRNA (miRNA) has been identified as one of the key factors for gene regulations at the post-transcriptional level, and also considered as a potential control and treatment target against infectious diseases.
The present study investigated the miRNA profile of B. schroederi via high throughput sequencing and real-time quantitative PCR.
A total of 18.07 million raw reads were obtained and 18.01 million were identified with high quality. By analysis of standard stem-loop structures, 108 miRNA candidates were predicted, including 60 known miRNAs and 48 novel ones. Target prediction revealed that the “chitinase” was the most abundant target with 483 sequences, and 263 targets were related to ovarian and egg development. The ribosomal protein related sequences occupied 449 sequences.
Previous studies have shown that some parasites secrete chitinases for exsheathment and/or for penetrating the peritrophic matrix of the host. It therefore seems that B. schroederi may be effectively regulated by miRNAs for development, invasion, and reproduction. Given that chitinases have been identified as important biological control agents for pests, identification of microRNAs in B. schroederi of the giant panda would provide useful information for the development of biological control strategies and/or vaccines against B. schroederi infection in the giant panda.
PMCID: PMC3726319  PMID: 23883822
Baylisascaris schroederi; Giant panda; microRNA (miRNA); miRNA target
19.  Characterization of Haemaphysalis flava (Acari: Ixodidae) from Qingling Subspecies of Giant Panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by Morphology and Molecular Markers 
PLoS ONE  2013;8(7):e69793.
Tick is one of important ectoparasites capable of causing direct damage to their hosts and also acts as vectors of relevant infectious agents. In the present study, the taxa of 10 ticks, collected from Qinling giant pandas (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains of China in April 2010, were determined using morphology and molecular markers (nucleotide ITS2 rDNA and mitochondrial 16S). Microscopic observation demonstrated that the morphological features of these ticks were similar to Haemaphysalis flava. Compared with other Haemaphysalis species, genetic variations between Haemaphysalis collected from A. m. qinlingensis and H. flava were the lowest in ITS2 rDNA and mitochondrial 16S, with sequence differences of 2.06%–2.40% and 1.30%–4.70%, respectively. Phylogenetic relationships showed that all the Haemaphysalis collected from A. m. qinlingensis were grouped with H. flava, further confirmed that the Haemaphysalis sp. is H. flava. This is the first report of ticks in giant panda by combining with morphology and molecular markers. This study also provided evidence that combining morphology and molecular tools provide a valuable and efficient tool for tick identification.
PMCID: PMC3716770  PMID: 23894541
20.  Improvement in Regional CBF by L-Serine Contributes to Its Neuroprotective Effect in Rats after Focal Cerebral Ischemia 
PLoS ONE  2013;8(6):e67044.
To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF). Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1) reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2) improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3) increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca2+-activated K+ channels on the cerebral blood vessel endothelium.
PMCID: PMC3692549  PMID: 23825613
21.  Direct Electrochemistry of Hemoglobin at a Graphene Gold Nanoparticle Composite Film for Nitric Oxide Biosensing 
Sensors (Basel, Switzerland)  2013;13(6):7492-7504.
A simple two-step method was employed for preparing nano-sized gold nanoparticles-graphene composite to construct a GNPs-GR-SDS modified electrode. Hemoglobin (Hb) was successfully immobilized on the surface of a basal plane graphite (BPG) electrode through a simple dropping technique. Direct electrochemistry and electrocatalysis of the hemoglobin-modified electrode was investigated. The as-prepared composites showed an obvious promotion of the direct electro-transfer between hemoglobin and the electrode. A couple of well-defined and quasi-reversible Hb CV peaks can be observed in a phosphate buffer solution (pH 7.0). The separation of anodic and cathodic peak potentials is 81 mV, indicating a fast electron transfer reaction. The experimental results also clarified that the immobilized Hb retained its biological activity for the catalysis toward NO. The biosensor showed high sensitivity and fast response upon the addition of NO, under the conditions of pH 7.0, potential -0.82 V. The time to reach the stable-state current was less than 3 s, and the linear response range of NO was 0.72-7.92 μM, with a correlation coefficient of 0.9991.
PMCID: PMC3715233  PMID: 23748173
hemoglobin; graphene; gold nanoparticles; nitric oxide; biosensor
22.  Expressional time phase of leukocyte molecules induced by allogenic cardiac antigen and cyclosporin A in rats’ in vitro model 
The immunosuppressive agent cyclosporin A has been proven to reduce the rejection rate and prolong the survival time of transplanted hearts. But some reports showed that cyclosporine A did not completely suppress the rejection. We performed in vitro studies to model a time course to observe the effect of cyclosporin A. Methods: The experiment was divided into a control group (group I), an antigen group (group II), a cyclosporin A group (group III) and an antigen + cyclosporin A group (group IV). After transplantation, at 2 h, 6 h, 12 h, 24 h, 48 h and 72 h, leukocyte molecules were monitored. Results: The expression of IL-2R peaked at 12 h in group II and at 6 h in group III. There was a gradual decline in the expression of the P59 gene in group I, positive expression at 2 h and between 12 h and 24 h in group II, in group IV, there was a decrease at 48 h. The expression of the CD4 gene was lowest at 2 h in group I and at 6 h in group II. CD4 expression then quickly increased to a maximum at 48 h in group III, at 2 h in group IV. There was a minimal expression was reached at 12 h in group I and IV and at 6 h in group III in the expression of the CD8 gene. Conclusions: Alloantigen induced lymphocytes to release IL-2R and P59 and stimulated the induction of the CD4 gene’ transcription for 6 h. Cyclosporin A stimulated the release of IL-2R for 2 h. These results provide an in vitro basis for describing the time phases of rejection inhibited by cyclosporin A.
PMCID: PMC3664009  PMID: 23724161
Transplantation rejection model in vitro; early expression’ time phase; cyclosporin A
23.  Genotyping Cryptosporidium andersoni in Cattle in Shaanxi Province, Northwestern China 
PLoS ONE  2013;8(4):e60112.
The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.
PMCID: PMC3613348  PMID: 23560072
24.  Molecular Characterization of Cyclospora-like Organisms from Golden Snub-nosed Monkeys in Qinling Mountain in Shaanxi Province, Northwestern China 
PLoS ONE  2013;8(2):e58216.
Cyclospora spp. have been identified as one of the most important intestinal pathogens causing protracted diarrhea in animals and human beings. To determine the Cyclospora species in the non-human primate Rhinopithecus roxellanae, a total of 71 fecal samples from 19 endangered snub-nosed monkeys in Shaanxi province were collected and examined using Sheater’s sugar flotation technique and by sequencing the fragments of 18S rDNA. Only two Cyclospora isolates from 2 golden snub-nosed monkeys (R. roxellanae) were obtained and identified between July 2011 and August of 2012. The sequences of the 18S rDNA for the two Cyclospora isolates were 477 bp, with no nucleotide variation between them. Phylogenetic analysis based on the 18S rDNA sequences revealed that the two Cyclospora isolates were posited into the clade Cyclospora spp. and sistered to C. colobi. These results first showed that Cyclospora infection occurred in R. roxellanae in hot and rainy weather, which would provide useful information for further understanding the molecular epidemiology of Cyclospora spp. and the control of Cyclospora infection in non-human primates as well as in human beings.
PMCID: PMC3585237  PMID: 23469155
25.  Discovery and verification of gelsolin as a potential biomarker of colorectal adenocarcinoma in a Chinese population: Examining differential protein expression using an iTRAQ labelling-based proteomics approach 
Despite the wide range of available colorectal cancer (CRC) screening tests, less than 50% of cases are detected at early stages. However, the identification of differentially expressed proteins or novel protein biomarkers in CRC may have some utility and, ultimately, improve patient care and survival. Proteomics combined with mass spectroscopy and liquid chromatography are emerging as powerful tools that have led to the discovery of potential markers in cancer biomarker discovery in several types of cancers. This article describes a novel technology that uses isotopic reagents to tag selected proteins that show a consistent pattern of differential expression in CRC.
To identify and validate potential biomarkers of colorectal adenocarcinoma using a proteomic approach.
Multidimensional liquid chromatography/mass spectrometry was used to analyze biological samples labelled with isobaric mass tags for relative and absolute quantitation to identify differentially expressed proteins in human colorectal adenocarcinoma and paired normal mucosa for the discovery of cancerous biomarkers. Cancerous and noncancerous samples were compared using online and offline separation. Protein identification was performed using mass spectrometry. The downregulation of gelsolin protein in colorectal adenocarcinoma samples was confirmed by Western blot analysis and validated using immunohistochemistry.
A total of 802 nonredundant proteins were identified in colorectal adenocarcinoma samples, 82 of which fell outside the expression range of 0.8 to 1.2, and were considered to be potential cancer-specific proteins. Immunohistochemistry revealed a complete absence of gelsolin expression in 86.89% of samples and a reduction of expression in 13.11% of samples, yielding a sensitivity of 86.89% and a specificity of 100% for distinguishing colorectal adenocarcinoma from normal tissue.
These findings suggest that decreased expression of gelsolin is a potential biomarker of colorectal adenocarcinoma.
PMCID: PMC3275404  PMID: 22288069
Biomarker; Colorectal cancer; Gelsolin; Proteomic

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