Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern.

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

Examining the spatial networks of an urban agglomeration through the lens of multi-dimensional element flow: Evidence from western China.

The Chengdu-Chongqing urban agglomeration (CCUA), as the only national-level city cluster in southwestern China, serves as a strategic support for the Yangtze River Economic Belt and an important demonstration area for promoting new urbanization in the country. The study of the networked characteristics of the CCUA contributes to a systematic understanding of its spatial connectivity patterns, optimization of spatial structure and layout, and promotion of high-quality regional development. In this study, we constructed models for traffic flow, information flow, migration flow, and composite flow to calculate the strength of connections between cities and the total flow of various elements in the CCUA. ArcGIS spatial visualization tools were used to depict the spatial connectivity patterns of the element flows within the CCUA. Additionally, social network analysis methods, including network density, centrality, and cohesive subgroups analysis, were employed to reveal the spatial network structure characteristics of the CCUA. The findings are as follows: (1) The overall structure of the cities within the CCUA is relatively loose, with significant differences in connectivity strength. It exhibits a west-strong and east-weak pattern, with Chengdu-Chongqing, Chengdu-Deyang, Chengdu-Mianyang, and Chengdu-Meishan occupying the top tier, while Zigong and Ya'an have relatively weak connections with other cities. Chengdu and Chongqing have prominent positions in the CCUA, with Chengdu having a more prominent core position compared to Chongqing, resulting in an overall hierarchical distribution of "1 + 1+7 + 7". (2) The network density of the element flows in the CCUA is relatively low, indicating a generally weak element connectivity. The centrality of cities other than Chengdu and Chongqing is at a moderate to lower level, suggesting a weak overall resource connectivity capacity in the CCUA. (3) Most cities tend to form cohesive subgroups based on geographic proximity, while the cohesive subgroup in Chongqing is still in its early stages of development due to administrative boundaries. The research results quantitatively depict the spatial network structure characteristics of the CCUA, providing theoretical references for its development.

Performance and Fouling Study of Asymmetric PVDF Membrane Applied in the Concentration of Organic Fertilizer by Direct Contact Membrane Distillation (DCMD).

This study proposes using membrane distillation (MD) as an alternative to the conventional multi-stage flushing (MSF) process to concentrate a semi-product of organic fertilizer. By applying a unique asymmetric polyvinylidene fluoride (PVDF) membrane, which was specifically designed for MD applications using a nonsolvent thermally induced phase separation (NTIPS) method, the direct contact membrane distillation (DCMD) performance was investigated in terms of its sustainability in permeation flux, fouling resistance, and anti-wetting properties. It was found that the permeation flux increased with increasing flow rate, while the top-surface facing feed mode was the preferred orientation to achieve 25% higher flux than the bottom-surface facing feed mode. Compared to the commercial polytetrafluoroethylene (PTFE) membrane, the asymmetric PVDF membrane exhibited excellent anti-fouling and sustainable flux, with less than 8% flux decline in a 15 h continuous operation, i.e., flux decreased slightly and was maintained as high as 74 kg·m-2·h-1 at 70 °C. Meanwhile, the lost flux was easily recovered by clean water rinsing. Overall 2.6 times concentration factor was achieved in 15 h MD operation, with 63.4% water being removed from the fertilizer sample. Further concentration could be achieved to reach the desired industrial standard of 5x concentration factor.

Effect of grafted graphene nanosheets on morphology evolution and conductive behavior of poly(methyl methacrylate)/poly(styrene-co-acrylonitrile) blends during isothermal annealing.

A facile method was developed for directly grafting poly(methyl methacrylate) (PMMA) to graphene oxide (GO) without surface modification, with the resultant insulating PMMA-g-GO nanosheets further reduced in situ to give conductive grafted reduced graphene oxide (RGO) nanosheets. The effect of PMMA-g-RGO nanosheets on the morphological evolution and conductive behavior of partially miscible blends of poly(methyl methacrylate)/poly (styrene-co-acrylonitrile) (PMMA/SAN) upon annealing above their phase-separation temperature was investigated using phase-contrast microscopy (PCM) with a real-time online digital picoammeter. With phase separation of the blend matrix, the well-dispersed PMMA-g-RGO nanosheets in the homogeneous matrix preferentially migrated to the SAN-rich phase and showed remarkably little aggregation. Surface grafting of PMMA-g-RGO might inhibit the aggregation of nanosheets in the blend matrix and weaken the retardation effect of nanosheets on the morphology evolution of the blend matrix. Furthermore, the percolation behavior of dynamic resistivity for ternary nanocomposites was attributed to the formation of a PMMA-g-RGO conductive network in the SAN-rich phase. The activation energy of conductive pathway formation was closer to the activation energy of flow for PMMA than that of SAN.

Assessing the ecological vulnerability of the upper reaches of the Minjiang River.

The upper reaches of the Minjiang River (URMR), located on the eastern edge of the Tibetan Plateau in southwestern China, are an important component of the ecological barrier of the Upper Yangtze River Basin. Climate change and human activities have increased the ecological sensitivity and vulnerability of the region, which may pose a threat to the ecological security of the Yangtze River Basin and have negative impacts on local social and economic development. In this study, we analyzed land use and cover change (LUCC) of the URMR between 2000 and 2010, and found that the total rate of LUCC was less than 0.50% during this period. In addition, net primary production (NPP) was employed to describe the changes in ecosystem sensitivity and vulnerability, and the results demonstrated that slightly and moderately sensitive and vulnerable zones occupied the largest area, distributed mainly in forest, shrub, and grassland ecosystems. However, compared with the period from 2000 to 2005, the ecological sensitivity and vulnerability showed a worsening trend in the period 2005-2010. Exploring the relationship between vulnerability/sensitivity and environmental factors, we found that sensitivity and vulnerability were positively correlated with precipitation (>700 mm) and aridity index (>36 mm/°C). The results highlight that the future ecological sensitivity and vulnerability of URMR should be further investigated, and that the LUCC induced by human activities and climate change have caused alteration of in ecosystem vulnerability.

Aldehyde dehydrogenase 1: a specific cancer stem cell marker for human colorectal carcinoma.

Since the identification of cancer stem cells (CSCs) a new understanding of tumor occurrence and development has evolved. According to the stem cell (SC) theory, colorectal carcinoma (CRC) SCs may be derived from mutations in normal intestinal cells. CSCs can be defined by their cell of origin (SCs or early progenitor cells). Thus, through a shared stem cell marker between CSCs and SCs, it is possible to investigate the association between its expression and the various clinicopathological features in patients with CRC. Aldehyde dehydrogenase 1 (ALDH1) is an appropriate marker. The present study was performed to examine the role of ALDH1 in CRC. Through indirect fluorescence antibody staining, the association between ALDH1 protein expression and various clinicopathological parameters was investigated. Furthermore, enzyme­linked immunosorbent assay (ELISA) was used to investigate the differing content of ALDH1 between CRC tissues and normal colorectal tissues. The results revealed that ALDH1 expression was markedly associated with tumor stage, Dukes' stage and the level of tumor cell differentiation. Using ELISA, it was demonstrated that there was a greater level of ALDH1 in CRC tissue than in normal colorectal tissue. Therefore, ALDH1 levels can be used as a useful parameter for pathological evaluation of tissue histology and to predict disease prognosis.

Expression and prognostic value of tumor stem cell markers ALDH1 and CD133 in colorectal carcinoma.

The identification of cancer stem cells (CSCs) has improved the understanding of tumor occurrence and development. According to CSC theory, colorectal carcinoma (CRC) may be derived from these few cells. Thus, markers for CSCs may lead to the identification of CSCs and investigation of the correlation with various clinicopathological features and survival time in human CRC patients. Aldehyde dehydrogenase 1 (ALDH1) and CD133 (also known as Prominin-1 or AC133) were involved in the current study. The aim of the present study was to identify CSCs through markers of CSCs and to explore the value of the CSC markers, ALDH1 and CD133, in human CRC. The correlation between ALDH1 and CD133 protein expression and the various clinicopathological parameters were investigated through immunohistochemistry (IHC). In addition, the Kaplan-Meier method was used to estimate patients' overall survival. Correlation of the survival differences between ALDH1- or CD133-positive expression and negative controls was analyzed by the log-rank test. Furthermore, the correlation between the expression of ALDH1 and CD133 was assessed by Spearman's rank correlation. A marked correlation between the differentiation degree and expression of ALDH1 in tumor cells was demonstrated, but not with CD133 expression. In addition, it was demonstrated that low-stage tumors exhibit a higher expression of ALDH1 or CD133 staining compared with high-stage tumors. Meanwhile, CD133 expression was associated with lymph node metastasis-positive cases, but ALDH1 expression was not. Furthermore, compared with negative cases, ALDH1-positive patients exhibited a poor prognosis. However, no significant difference was identified between CD133-positive and -negative cases in terms of survival time. Overall, the results of the present study indicated that ALDH1 and CD133 may serve as useful markers of CSC to predict disease prognosis and clinicopathological characteristics of human CRC.

Genomic dissection and prioritizing of candidate genes of QTL for regulating spontaneous arthritis on chromosome 1 in mice deficient for interleukin-1 receptor antagonist.

Rheumatoid arthritis is a heterogeneous disease with clinical and biological polymorphisms. IL-1RN is a protein that binds to interleukin-1 (IL-1) receptors and inhibits the binding of IL-1-alpha and IL-1-beta. IL-1RN levels are elevated in the blood of patients with a variety of infectious, immune, and traumatic conditions. Balb/c mice deficient in IL-1ra (mouse gene of IL-1RN) develop spontaneous autoimmune arthritis while DBA/1 mice deficient in IL-1ra do not. Previously, we identified a major QTL that regulates the susceptibility to arthritis in Balb/c mice with IL-1ra deficiency. In this study, we found that the QTL may contain two peaks that are regulated by two sets of candidate genes. By haplotype analysis, the total genomic regions of candidate genes were reduced from about 19 Mbp to approximately 9 Mbp. The total number of candidate genes was reduced from 208 to 21.

PDGFRß triggered by bFGF promotes the proliferation and migration of endothelial progenitor cells via p-ERK signalling.

We have examined the effects of bFGF (basic fibroblast growth factor) on p-ERK (phosphorylated extracellular signal-regulated kinase) through PDGFRß (platelet-derived growth factor receptor ß) in the proliferation and migration of EPCs (endothelial progenitor cells). EPC migration was detected using the Transwell system. The expression of PDGFRß mRNA and protein, total ERK and p-ERK proteins was respectively assessed by real-time PCR and Western blottings. bFGF promote the proliferation and migration of EPCs, the effects of bFGF being implemented by activating ERK signalling through the expression of PDGFRß, whereas an anti-bFGF antibody and inhibitor of PDGF (platelet-derived growth factor) receptor kinase (AG1296) could respectively decrease the expression of PDGFRß mRNA and protein and p-ERK protein. Total ERK protein did not change under the same experimental conditions, and an inhibitor of p-ERK (PD98059) inhibited the proliferation and migration of EPCs. The findings strongly suggest that a PDGFRß/p-ERK signalling pathway triggered by bFGF plays an important role in the proliferation and migration of EPCs.

Differential gene expression between wild-type and Gulo-deficient mice supplied with vitamin C.

The aim of this study was to test the hypothesis that hepatic vitamin C (VC) levels in VC deficient mice rescued with high doses of VC supplements still do not reach the optimal levels present in wild-type mice. For this, we used a mouse scurvy model (sfx) in which the L-gulonolactone oxidase gene (Gulo) is deleted. Six age- (6 weeks old) and gender- (female) matched wild-type (WT) and sfx mice (rescued by administering 500 mg of VC/L) were used as the control (WT) and treatment (MT) groups (n = 3 for each group), respectively. Total hepatic RNA was used in triplicate microarray assays for each group. EDGE software was used to identify differentially expressed genes and transcriptomic analysis was used to assess the potential genetic regulation of Gulo gene expression. Hepatic VC concentrations in MT mice were significantly lower than in WT mice, even though there were no morphological differences between the two groups. In MT mice, 269 differentially expressed transcripts were detected (≥ twice the difference between MT and WT mice), including 107 up-regulated and 162 down-regulated genes. These differentially expressed genes included stress-related and exclusively/predominantly hepatocyte genes. Transcriptomic analysis identified a major locus on chromosome 18 that regulates Gulo expression. Since three relevant oxidative genes are located within the critical region of this locus we suspect that they are involved in the down-regulation of oxidative activity in sfx mice.

[Effect of Schwann cells on differentiation of rat bone marrow mesenchymal stem cells at different ages].

OBJECTIVE: Bone marrow mesenchymal stem cells (BMSCs) are multipotent and thus are able to differentiate into a number of different cell types under certain culture condition. However, the effect of age on the differentiation remains unknown. To explore the effect of the microenvironment formed by Schwann cells (SCs) on BMSCs differentiation into neurons and oligodendrocytes in rats at different ages in vitro. METHODS: SCs were extracted and purified from the distal sciatic nerves of neonatal Wistar rats. BMSCs were isolated from bone marrow of Wistar rats (aged 1 month, 6 months, and 12 months, respectively) and cultured in vitro. The cells were identified by immunofluorescent staining. The BMSCs at passage 2 were labeled by PKH26 and cocultured with SCs at passage 3 in equal proportions in two layer Petri dish. According to the BMSCs from the rats at different ages, experiment was divided into 3 groups: SCs were cocultured with 1-month-old rat BMSCs (group A), 6-month-old rat BMSCs (group B), and 12-month-old rat BMSCs (group C), respectively. The morphological changes of cocultured BMSCs were observed by inverted phase contrast microscope, the expressions of neuron-specific enolase (NSE) and myelin basic protein (MBP) in the cocultured BMSCs were tested by immunofluorescent staining, and the expression of neuregulin 1 (NRG1) was detected by ELISA method. RESULTS: SCs and BMSCs were isolated and cultured successfully. The identification of SCs showed positive expression of S-100 and BMSCs showed positive expressions of CD29, CD44, and CD90. At 7 days after coculture, the BMSCs in group A began retraction, and became round or tapered with the processes and had a nerve cells or oligodendrocytes-like morphology, but most BMSCs in groups B and C showed no obvious morphological changes under inverted phase contrast microscope. Immunofluorescent staining showed that the positive expression rates of NSE in groups A, B, and C were 22.39% +/- 2.86%, 12.89% +/- 1.78%, and 2.69% +/- 0.80%, respectively, and the positive expression rates of MBP in groups A, B, and C were 16.13% +/- 2.39%, 6.33% +/- 1.40%, and 0.92% +/- 0.17%, respectively. There were significant differences in terms of NSE and MBP positive expression rates among 3 groups (P < 0.05). ELISA analysis showed that NRG1 in the supernatant of group A was increased after coculture in a time-dependent manner. At 6, 9, and 12 days of coculture, NRG1 content was higher in group A than in groups B and C, and in group B than in group C, showing significant differences (P < 0.05). CONCLUSION: The microenvironment formed by SCs can promote BMSCs differentiation into neurons and oligodendrocytes, but the differentiation capability of BMSCs decreases with aging, and the variety of growth factors secreted by SCs is likely important factors that induce the differentiation of BMSCs into neurons and oligodendrocytes.

Differential gene expression between wild-type and Gulo-deficient mice supplied with vitamin C

The aim of this study was to test the hypothesis that hepatic vitamin C (VC) levels in VC deficient mice rescued with high doses of VC supplements still do not reach the optimal levels present in wild-type mice. For this, we used a mouse scurvy model (sfx) in which the L-gulonolactone oxidase gene (Gulo) is deleted. Six age- (6 weeks old) and gender- (female) matched wild-type (WT) and sfx mice (rescued by administering 500 mg of VC/L) were used as the control (WT) and treatment (MT) groups (n = 3 for each group), respectively. Total hepatic RNA was used in triplicate microarray assays for each group. EDGE software was used to identify differentially expressed genes and transcriptomic analysis was used to assess the potential genetic regulation of Gulo gene expression. Hepatic VC concentrations in MT mice were significantly lower than in WT mice, even though there were no morphological differences between the two groups. In MT mice, 269 differentially expressed transcripts were detected (> twice the difference between MT and WT mice), including 107 up-regulated and 162 down-regulated genes. These differentially expressed genes included stress-related and exclusively/predominantly hepatocyte genes. Transcriptomic analysis identified a major locus on chromosome 18 that regulates Gulo expression. Since three relevant oxidative genes are located within the critical region of this locus we suspect that they are involved in the down-regulation of oxidative activity in sfx mice.

Functional and histological improvement of the injured spinal cord following transplantation of Schwann cells transfected with NRG1 gene.

In this study, we implanted Schwann cells (SCs) transfected with Neuregulin 1 (NRG1) gene into rats with hemisection spinal cord injury, determined its effects on the repair of spinal cord injury and investigated the underlying mechanisms. Primary SCs were cultured, purified, and transfected with NRG1 gene. SCs and SCs transfected with NRG1 gene were implanted, respectively, into rats with hemisection spinal cord injury. Behavior, imaging, electrophysiology, and immuno-histological analyses were performed to evaluate the effect of NRG1 gene-transfected SCs on the repair of spinal cord injury. In vitro studies showed that NRG1 protein was highly expressed in SCs transfected with NRG1 gene. In addition, the receptors for NRG1, ErbB2, and ErbB4, were upregulated in a time-dependent manner. NRG1-transfected SCs secreted large amount of NRG1 proteins in vivo, which efficiently promoted the expression of ErbB2 and ErbB4 in the neurons and neuroglia cells. Moreover, the number of NSE- and GFAP-positive cells was increased. After cell transplantation, many transplanted cells survived and migrated to the areas with spinal cord injuries. The injuries were recovered in all the experimental groups, but the most significant recovery was observed in the group of rats implanted with SCs transfected with NRG1 gene. We conclude that NRG1-transfected SCs can significantly increase the effect on the repair of spinal cord injury. This repair effect is achieved via the upregulation of ErbB receptor in the target cells, increased proliferation of glial cells, and protection of neurons from apoptosis.

Simulated microgravity promotes cellular senescence via oxidant stress in rat PC12 cells.

Microgravity has a unique effect on biological organisms. Organs exposed to microgravity display cellular senescence, a change that resembles the aging process. To directly investigate the influence of simulated microgravity on neuronal original rat PC12 cells, we used a rotary cell culture system that simulates the microgravity environment on the earth. We found that simulated microgravity induced partial G1 phase arrest, upregulated senescence-associated beta-galactosidase (SA-beta-gal) activity, and activated both p53 and p16 protein pathways linked to cell senescence. The amount of reactive oxygen species (ROS) was also increased. The activity of intracellular antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was all significantly increased at 12h after the microgravity onset, yet decreased at 96h. Furthermore, concomitant block of ROS by the antioxidant N-acetylcysteine significantly inhibited the microgravity-induced upregulation of SA-beta-gal activity. These results suggest that exposure to simulated microgravity induces cellular senescence in PC12 cells via an increased oxidant stress.

[Effects of expression of fatty acid synthetase and phosphoenolpyruvate carboxykinase in offspring rats by high protein intake in pregnancy].

OBJECTIVE: To investigate the effect of pregnant nutritional manipulation on offspring mRNA expression of fatty acid synthetase (FAS) and phosphoenolpyruvate carboxykinase (PEPCK). METHODS: Wistar pregnant rats fed on standard diet and high protein diet (HPD), respectively. The male offspring were divided into control and high protein group (HP) in accordance with the diets of their dams. Offspring were cross-fostered and fed on standard diet after weaning. At adult, some offspring randomly selected from control group, namely as high fat control (HFC), HFC and HP group rats fed on high-fat diet (HFD) to induce obesity. At different stages, tissue was collected for analyzing expression of FAS and PEPCK mRNA by fluorescent quantitation PCR. RESULTS: In HP, body weight and obesity rates were decreased after weaning and HFD (P < 0.05). The abundance of PEPCK mRNA was persistently decreased in HP, and the abundance of FAS mRNA in HP was down-regulated before and after HFD vs. control or HFC. CONCLUSION: The increase of protein intake during pregnancy could decrease the expression of FAS and PEPCK mRNA, and prevent offspring from HFD-induced obesity in adult.

Inhibition of telomerase activity in renal cell carcinoma cells by doxorubicin and anti-Fas monoclonal antibody.

Telomerase, the ribonucleoprotein enzyme that maintains the telomeres of eukaryotic chromosomes, is an attractive target for a mechanism-based therapeutic approach as its activation has been associated with unlimited proliferation in most types of cancer cells. Here, we investigated the effect of chemotherapeutic or immunotherapeutic agents and anti-Fas monoclonal antibody (mAb) on telomerase activity in renal cell carcinoma (RCC) cells. Primary RCC cells were surgically obtained from 24 patients with RCC. Telomerase activity and cytotoxicity were determined by the telomeric repeat amplification protocol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Telomerase activity was positive in 20 (83.3%) of the 24 primary RCC cell cultures. Treatment of ACHN human RCC cells with anti-Fas mAb in combination with vinblastine, interferon-α or interferon-γ did not affect telomerase activity. However, a combination of anti-Fas mAb and doxorubicin resulted in marked down-regulation of telomerase activity in conjunction with a synergistic cytotoxicity. This inhibitory effect on telomerase activity was also observed in 16 telomerase-positive primary RCC cell cultures. These findings suggest that telomerase may be a promising molecular target for combination therapy using biological response modifiers and doxorubicin for RCC.