With the demographic shift in age in advanced countries inexorably set to progress in the 21st century, dementia will become one of the most important health problems worldwide. Vascular cognitive impairment is the second most common type of dementia after Alzheimer's disease and is frequently responsible for the cognitive decline of the elderly. It is characterized by cerebrovascular white matter changes; thus, in order to investigate the underlying mechanisms involved in white matter changes, a mouse model of chronic cerebral hypoperfusion has been developed, which involves the narrowing of the bilateral common carotid arteries with newly designed microcoils. The purpose of this paper is to provide a comprehensive summary of the achievements made with the model that shows good reproducibility of the white matter changes characterized by blood-brain barrier disruption, glial activation, oxidative stress, and oligodendrocyte loss following chronic cerebral hypoperfusion. Detailed characterization of this model may help to decipher the substrates associated with impaired memory and move toward a more integrated therapy of vascular cognitive impairment. Subcortical ischemic vascular dementia (SIVD) is characterized by white matter (WM) changes and lacunar infarctions, which occur as a result of a reduction in cerebral blood flow (CBF) over an extended period of time, causing small vessel changes –3. CerebThe rat model of chronic cerebral hypoperfusion is accompanied by cognitive impairment and cholinergic deficits , 13, 17 The aims of the current paper are to provide a comprehensive survey of the experimental evidence that has accumulated since establishment of this mouse BCAS model, in order to extrapolate the results into human neurological conditions, and to consider the particular strengths and pitfalls of the method.Ten-week-old male C57BL/6J mice (24–29 g) are conventionally used to induce chronic cerebral hypoperfusion by BCAS , 20, 21.Mice are anesthetized with 2% halothane or 25–50 mg/Kg sodium pentobarbital and, through a midline cervical incision, both CCAs are exposed and freed from their sheaths. Two 4–0 silk sutures are placed around the distal and proximal parts of the right CCA. The artery is then gently lifted by the sutures and placed between the loops of the microcoil just proximal to the carotid bifurcation Figures . The micThe blood pressure of the surviving mice does not change significantly at any postoperative intervals until 30 days, compared with the sham-operated controls . The mortality rates are reported to range from 10% to 20%: 13% in mice with microcoils of 0.22 mm in diameter, 17% in those of 0.20 mm, and 15–19% in those of 0.18 mm . In contBody weight has been shown to decrease after the surgery, but recover to baseline by day 7, in mice with 0.22, 0.20, and 0.18 mm diameter microcoils. Although the mice with the 0.22, 0.20, and 0.18 mm diameter microcoils placement tended to have a lower body weight than those with sham operation, no significant difference is noted at any postoperative interval. In contrast, the mice with the 0.16 mm diameter microcoil placement showed significantly lower body weight at all postoperative intervals, compared with the sham-operated mice .After placement of the 0.22, 0.20, and 0.18 mm diameter microcoils, the animals regained consciousness within a few hours and occasionally showed transient ptosis but no apparent motor weakness. In contrast, some of the mice with 0.16 mm diameter microcoils placed (~35%) did not regain consciousness, showing rolling or circling movements lasting 2 to 6 hours after awakening, and severe akinesia with a squatting posture .Although anesthetics such as sodium pentobarbital and halothane are known to provide varying degrees of neuroprotection against ischemic injury , the selAlthough 2VO rats develop specific WM changes without any apparent gray matter changes , 12, 2VO18F-fluorodeoxyglucose (FDG) uptake in the cerebral cortex, suggesting that the early 18F-FDG uptake scan can serve as an estimate of CBF. The early 18F-FDG uptake scan in the striatum showed a similar temporal profile to that of the cerebral cortex, suggesting that the CBF values in the cerebral cortex and the striatum change in parallel after BCAS. By contrast, the CBF in the hippocampus did not decrease at 2 hours or 2 months after BCAS but finally decreased at 6 months. The late 18F-FDG scans show that the glucose uptake in the hippocampus did not decrease by 2 months after BCAS but decreased by 20% at 6 months after BCAS. The lack of reductions in hippocampal CBF and metabolism in the early phase after BCAS is probably due to the hippocampus being supplied by both anterior and posterior circulations , 8281, 8 Since cerebral endothelial cells contribute to numerous signaling cascades that help regulate brain homeostasis and function , angio-/The BCAS model characterizing features of vascular cognitive impairment with WM changes may serve as a powerful tool for investigation of the molecular pathology of WM lesions and in the design of therapeutic measures for WM changes induced by chronic cerebral hypoperfusion. Although these models do not (and no other current models can) describe all features of SIVD , the BCA


The mechanical properties of cellular microenvironments play important roles in regulating cellular functions. Studies of the molecular response of endothelial cells to alterations in substrate stiffness could shed new light on the development of cardiovascular disease. Quantitative real-time PCR is a current technique that is widely used in gene expression assessment, and its accuracy is highly dependent upon the selection of appropriate reference genes for gene expression normalization. This study aimed to evaluate and identify optimal reference genes for use in studies of the response of endothelial cells to alterations in substrate stiffness.PLUS, NormFinder, BestKeeper, and the Comparative ΔCt method, were employed to evaluate the expression of nine candidate genes. We observed that the stability of potential reference genes varied significantly in human umbilical vein endothelial cells on substrates with different stiffness. B2M, HPRT-1, and YWHAZ are suitable for normalization in this experimental setting. Meanwhile, we normalized the expression of YAP and CTGF using various reference genes and demonstrated that the relative quantification varied according to the reference genes.Four algorithms, GeNormConsequently, our data show for the first time that B2M, HPRT-1, and YWHAZ are a set of stably expressed reference genes for accurate gene expression normalization in studies exploring the effect of subendothelial matrix stiffening on endothelial cell function. We furthermore caution against the use of GAPDH and ACTB for gene expression normalization in this experimental setting because of the low expression stability in this study. Change in the mechanical property of cells and their microenvironments and the relevance to biological functions have recently attracted increased attention. For example, mechanical microenvironments may regulate cellular functions relevant to development, homeostasis, and disease Among the widely used methods, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a rapid and sensitive method for measuring gene expression. Despite being a very powerful technique to accurately quantify gene expression, several determinants, such as input sample, RNA extraction, efficiency of reverse transcription from RNA to complementary DNA and PCR efficiency, should be taken into account. Therefore, normalization of qRT-PCR data with suitable internal reference genes (RGs) is required In our present study, we investigate reliable reference genes for real-time PCR in human umbilical vein endothelial cells (HUVECs) on substrates with different stiffness. It is well established that the subendothelial layers of blood vessels become stiff in cardiovascular diseases Previous studies demonstrated the importance of substrate stiffness on the gene expression of various cell types in vitro model of the HUVECs in order to explore the effect of subendothelial matrix stiffening on endothelial cell function. Polyacrylamide gels were prepared to obtain three different stiffness values that mimic pathophysiological states  values with three biological and three technical replicates. The box plot of the Ct values of all candidate RGs show the differences in transcript levels between RGs . The expPLUS, Normfinder, Bestkeeper and the Comparative ΔCt method, were employed to further evaluate the expression stability of the candidate RGs individually.Four distinct algorithms, GeNormThe GeNorm algorithm applies a statistical algorithm to calculate the average stability measure (M). A lower value of average expression stability M indicated more stable expression PLUS. Of the candidates, the B2M gene had the lowest M value, followed by HPRT-1. Interestingly, ACTB, although frequently used for gene expression, had the lowest expression stability in this study. Moreover, the V parameter calculated by GeNormPLUS recommended the use of two RGs for reliable normalization in this experimental setting  for the RGs. The lowest S value indicates the most stable RG expression.According to NormFinder analysis, the best ranked RG was B2M, followed by HPRT-1, YWHAZ, and RPL13A . TBP andBestKeeper calculates the gene expression variation for candidate genes based on standard deviation (SD), coefficient of correlation (r) and percentage covariance (CV). The lowest SD value indicates the most stable RG expression.PLUS and BestKeeper results were expected because their statistical algorithms were distinct.As shown in PLUS and Normfinder results.The Comparative ΔCt method assesses the most stable RGs by comparing the relative expression of “pairs of genes” within each tissue sample or each treatment PLUS, Normfinder, BestKeeper, and the Comparative ΔCt method) were compared  in vitro. Of nine candidate RGs, we identified B2M, HPRT-1, and YWHAZ as the most suitable RGs, using four widely recognized algorithms . We further found that both ACTB and GAPDH, which are frequently used for gene expression normalization in many experiments, were not suitable normalization controls in this experimental setting. The results from GeNormPLUS, NormFinder, and the Comparative ΔCt method assessment were more consistent with each other than with the BestKeeper method.Analysis of gene expression using qRT-PCR, which is a powerful method that combines high specificity and sensitivity, is a commonly used technology for gene expression analysis in response to different environmental conditions The three most stably expressed genes in our experimental setting, B2M, HPRT-1, and YWHAZ, encode proteins with independent physiological functions. The protein encoded by B2M is a component of the major histocompatibility complex (MHC) class I Numerous morphological changes, which are accompanied by substantial changes in biochemical processes and metabolism and alteration in structural proteins, occur when cells adapt to substrates with different stiffness. Substrate stiffness regulates actin organization, cellular metabolism and protein synthesis in cells Due to the original intention of our research, it is impossible in this study to research all human cell lines, and thus, these conclusions should be corroborated prior to application in other human cell lines. Furthermore, optimization of reference genes for real-time PCR associated with mechanical environments allows us to conduct more standardized biomechanopharmacology studies In conclusion, we validated a stably expressed RG set for use in endothelial cells on a variety of substrate stiffness. B2M, HPRT-1, and YWHAZ were identified as the most stable RGs. Indeed, a combination of 2 genes out of these 3 genes is sufficient to provide accurate qRT-PCR normalization. Our results also demonstrated that special attention must be given to the choice of suitable RGs during the studies of the cell behaviors responding to the mechanical changes of their microenvironments.Polyacrylamide gels were prepared as described previously Force curves were acquired with an atomic force microscope . Silicon nitride cantilever  with a spherical tip, of which the diameter was 16.46 µm, was used for measurements of Soft and Medium PA gels. For Stiff PA gels measurement, the silicon nitride cantilever  with a borosilicate sphere of 11.9 µm in diameter was used. Force curves were obtained on at least seven different locations of each sample and twenty force curves were collected at each sample. Each force curve was taken at a rate of 1 Hz.The force curves were analyzed using the Hertz model for a sphere in contact with a flat surface. In this model, the Young’s modulus E are calculated according to the following equation F is the loading force, E is the Young’s modulus, R is the radius of the silica sphere. Loading force was calculated by using Hook’s law, d is the cantilever deflection. The characterization of the substrates is shown in 2. Cells between passages 2 and 6 were used for all experiments. The cells were plated onto the previously prepared PA gels and grown for 48 hours. The medium was replaced once after 24 h.Primary HUVECs were obtained from ScienCell Research Laboratories  and cultured on plastic flasks coated with 1% gelatin in endothelial cell growth medium-2 (EGM-2) supplemented with 2% FCS and growth factors  at 37°C under 5% COTotal RNA was extracted using the RNeasy mini kit , according to the manufacturer’s instructions. The quantity and quality of RNA samples were determined using a Helios beta spectrometer . RNA samples with 260/280 ratio from 1.9 to 2.1 were used for further analysis.First strand cDNA was synthesized by reverse transcribing 500 ng of total RNA with the RevertAid™ first strand cDNA synthesis kit  in a 20 µl reaction using random primers according to manufacturer’s instructions. The RT reaction sequence consisted of incubation at 25°C for 5 minutes, followed by 60 minutes at 42°C. The reaction was terminated by heating to 70°C for 5 minutes. The cDNA was stored at −20°C until the PCR experiments.The qRT-PCRs were performed in 96-well plates with an ABI StepOne Plus Sequence Detection System  using the SYBR® Green Realtime PCR Master Mix . Thermocycling was performed using the following conditions: 94°C for 1 minutes, followed by 40 cycles of 94°C for 20 s, 59°C for 20 s, and 72°C for 25 s. The Ct values were automatically calculated using commercial software . The details of primer sequences are given in 2) and slope values were obtained from the standard curve, and the corresponding PCR amplification efficiencies (E) were calculated using the slope of the calibration curve according to the following equation: After completion of standard qRT-PCR, melting curve analysis demonstrated a single PCR amplicon for each reaction. The PCR efficiency was evaluated by the dilution series method using a mix of sample cDNAs as the template. A standard curve was generated using linear regression based on the threshold cycle (Ct) values for all dilution points in a series. The correlation coefficients  and analyzed using the Statistical Analysis System (SAS) software. Means of different groups were compared using one way analysis of variance followed by Student-Newman-Keuls text. Figure S1Specificity of qRT-PCR amplification. (a) Amplified fragments were separated by 2% agarose gel. BM2000 represented DNA size marker. The fifth marker band corresponds to 250bp, and the last marker band corresponds to 100bp. (b) Dissociation curves of the nine amplicons showing single peaks.(TIF)Click here for additional data file.Figure S2Expression levels of PAI-1 and tPA in endothelial cells on a variety of substrate stiffness. Genes were normalized to individual and/or combined RGs. Results are mean±SD, n = 3; Means were compared by ANOVA followed by Student-Newman-Keuls text. #P<0.05, Medium vs. Soft. *P<0.05, Stiff vs. Soft. &P<0.05, Stiff vs. Medium.(TIF)Click here for additional data file.Table S1Primer Information for YAP, CTGF, tPA, and PAI-1.(DOC)Click here for additional data file.


Chlamydia spp. are obligate intracellular gram-negative bacteria that cause a wide range of significant diseases in humans and animals worldwide, resulting in significant economic losses. Chlamydial infection in cattle has been reported in many countries including China. However, there has been no survey of chlamydial infection of dairy cattle in Guangzhou, southern China. The objective of the present investigation was to examine the chlamydial seroprevalence in dairy cattle in Guangzhou, subtropical southern China by using an indirect hemagglutination assay (IHA). The overall seroprevalence of chlamydial infection in dairy cattle was 7.25% (29/400). Greater than or equal to eight-yr-old dairy cattle had the highest seroprevalence (10.34%), followed by those that were ≥ 6 years old or < 7 years old dairy cattle (10.20%), although there were no statistically significant differences among different groups (P > 0.05). Dairy cattle with 5 pregnancies had the highest seroprevalence (10.81%). These results indicate that chlamydial infection was present in dairy cattle in Guangzhou, subtropical southern China, and integrated strategies and measures should be executed to control and prevent chlamydial infection and disease outbreak in the study region. Chlamydia spp. are obligate intracellular gram-negative bacterial pathogens that cause a wide range of significant diseases with huge economic losses in humans, birds and other animals worldwide [Chlamydia that comprises nine species according to the new taxonomy in 2011 in the new edition of Bergey’s Manual of Systematic Bacteriology [orldwide -5. Chlameriology . ChlamydChlamydial infection in cattle has been reported in many countries such as Australia, Germany, Ireland, Italian, Sweden, Switzerland -12, as wThe survey was conducted in Guangzhou City which is the capital of Guangdong Province, China and has a subtropical monsoon climate. Crossed by the Tropic of Cancer, it is located between longitude 112°57' to 114°3' east and latitude 22°26' to 23°56' north, bordering on the South China Sea. It is the biggest cosmopolitan city in South China, and also China's Southern Gateway to the world. The annual average temperature is 22.8°C, the average relative humidity is about 68%, and the annual rainfall at the urban area is over 1600 mm. The city covers a total area of 7434.4 square kilometers and has a population of approximately twelve millions.Blood samples were collected randomly from 400 dairy cattle (representing different ages and pregnancies) on 5 representative farms between July 2009 and March 2010 in Guangzhou City, Guangdong Province, China. The dairy cattle populations represented a local breed (Chinese Holstein) and introduced breed . The animals of each herd were randomly selected, and 1 blood sample was collected from each animal. Approximately 5 ml of blood were obtained via a jugular vein. Blood samples were centrifuged at 3,000 rpm for 10 min, and serum was obtained, frozen, and stored at −20°C until further analysis. Biometric data for dairy cattle, including ages, breeds and numbers of past pregnancies were obtained through a questionnaire at the time of blood collection.Chlamydia were determined in sera using an indirect hemagglutination assay (IHA) with a commercially available kit  according to the manufacturer's instructions as described previously [Chlamydia antigen was added to each well, the plate was shaken slightly with a vibrator for 2 min followed by incubation at 37°C for 2 h. The test was considered positive when a layer of agglutinated erythrocytes was formed in wells at dilutions of 1:16 or higher, and positive and negative controls were included in each test.Antibodies to eviously ,14. IHA eviously , which heviously ,14,16. IP < 0.05.Differences in seroprevalence of chlamydial infection among dairy cattle of different age groups and numbers of pregnancies were analyzed using a Chi square test using the SPSS for Windows . The differences were considered statistically significant when A total of 400 serum samples from dairy cattle in Guangzhou, Southern China were examined by IHA for chlamydial antibodies. 29 of 400 (7.25%) examined dairy cattle were seropositive for chlamydial infection by IHA (Table P > 0.05).The ages of the examined dairy cattle ranged between 1 year and 8 years, seroprevalence varied in different age groups, ranging from 1.96% to 10.3% Table . The higChlamydia can infect epithelial cells and monocyte or macrophages of a wide host range. Chlamydial infection in cattle has been reported sporadically all over the world and is implicated in respiratory and reproductive tract diseases [diseases ,5. Thougdiseases .In the present survey, chlamydial antibodies were detected in 29 7.25%) of 400 dairy cattle by IHA Table , and som.25% of 4P > 0.05). The varied seroprevalence in different age groups suggests the possibility of horizontal transmission in the investigated herds. The highest chlamydial seroprevalence in dairy cattle of greater than or equal to eight-yr-old may due to chronic infection or sustain infection, also may due to lower resistance or immunity of the dairy cattle of this age. The association between chlamydial seroprevalence and numbers of pregnancies was also analyzed in the present study (Table P > 0.05).As shown in Table dy Table , and varThe results of the present survey indicated that chlamydial infection is prevalent in dairy cattle in Guangzhou, subtropical southern China, which may represents one of the causes of bovine abortion, pneumonia and arthritis. Therefore, integrated strategies and measures should be performed to control and prevent chlamydial infection and disease outbreak in dairy cattle in the study region.PRC: People’s Republic of China; IHA: Indirect hemagglutination assay; CFT: Complement-fixation test.The authors declare that they have no competing interests.DHZ conceived and designed the study, and critically revised the manuscript. FRZ, HYX and MJX performed the experiments, analysed the data and drafted the manuscript. SYH, HQS and XQZ helped in study design, study implementation and manuscript revision. All authors read and approved the final manuscript.


To quantify the levels of tissue inhibitor of metalloproteinase 4 (TIMP4) and its ratios with free metalloproteinases (MMP) in the aqueous humor of patients with primary open angle glaucoma (POAG), pseudoexfoliation syndrome (PXS) and pseudoexfoliative glaucoma (PXG) and to evaluate a possible imbalance between MMPs and TIMPs in these samples.Free MMP2, MMP3, MMP9, TIMP1, TIMP2, TIMP4 concentrations and active levels of MMP2 and MMP3 were determined with immunoassay ELISA and activity assay kits in 168 aqueous samples.TIMP4 was elevated in glaucoma patients(POAG: 0.95 ± 0.49 PXG: 1.28 ± 1.38 pg/ml. p < 0.001). POAG, PXS and PXG samples demonstrated higher MMP2, TIMP1 and TIMP2 concentrations (p < 0.001). Samples from the PXS and PXG groups had a lower total/active MMP2 ratio (p < 0.004 and p < 0.008 respectively). Stoichiometric analysis showed an overbalance of TIMPsover MMPs in both POAG & PXG groups,especially of TIMP4.TIMP4 elevation is a novel finding in glaucomatous eyes. A disregulation of extracellular matrix homeostasis is suggested in POAG, PXS and PXG. The most important risk factor for the development and progression of glaucoma is the elevation of the intraocular pressure (IOP) . IOP regIn glaucoma, the normal architecture and function of the juxtacanalicular TM is altered by the pathological accumulation of connective tissue and the partial loss of endothelial cells ,6. OtherPseudoexfoliation syndrome (PXS) is an age-related systemic disorder characterized by the abnormal accumulation of extracellular matrix (ECM) material in extraocular and ocular tissues. The pseudoexfoliative material might congest the outflow pathways of the TM contributing to the elevation of the IOP and may be involved in the development of pseudoexfoliative glaucoma (PXG) ,10.ECM remodeling is primarily controlled by endogenous enzymes, known as matrix metalloproteinases (MMPs). MMPs are a family of endopeptidases that contain an active Zn2+ site which is responsible for their enzymatic activity . They arIt is well documented that MMPs and TIMPs play an important role in many ocular pathological processes, including glaucoma . In vitrWithin this context, the objectives of this study were: a) to trace and estimate the levels of TIMP4 in the aqueous humor, b) to investigate the ratios of TIMP4 to other free MMPs and TIMPs in the aqueous humor, and c) to investigate the potential imbalance of free forms of MMPs and TIMPs in eyes with POAG, PXS and PXG.This was a clinic-based, cross-sectional study. The protocol adhered to the tenets of the Helsinki Declaration and written informed consent was given by all participants. The institutional review board of the Democritus University of Thrace approved the protocol and the study was conducted at the Department of Ophthalmology at the University Hospital of Alexandroupolis, Greece.Participants were recruited from the outpatient service of the Department of Ophthalmology at the University Hospital of Alexandroupolis, Greece, in a consecutive-if-eligible basis. All participants were divided into 3 study groups: POAG, PXS, and PXG. The diagnosis of glaucoma and pseudoexfoliation syndrome was made by a glaucoma specialist (GL or VPK) after an evaluation that included slit-lamp examination with mydriasis, gonioscopy, tonometry, central corneal thickness measurement, visual field testing and optic disc assessment with a 90 diopters non-contact lens. Visual field defects were assessed with Humphrey perimetry, using the SITA Standard 30–2 program. The diagnosis of glaucoma and pseudoexfoliation syndrome were made according to the European Glaucoma Society guidelines .Exclusion criteria for all participants were: a) history of optic neuropathy other than glaucoma, b) retinal pathology, c) corneal opacity, d) penetrating eye surgery or other ocular operations any time in the past, e) previous ocular inflammation and f) systemic disorders such as diabetes mellitus and autoimmune or inflammatory diseases that could have affected the optic nerve. All study groups were compared with an age-matched sample of control participants (CG) recruited from the list of patients scheduled for routine cataract surgery after an examination by a glaucoma expert (GL). Controls had to have no other ocular pathology except age-related cataract. They were also excluded if they had any of the systemic disorders listed above. Exclusion criteria for the PXS group were: IOP > 21 mmHg at any time point according to the participant’s medical records, optic disc suspicious for glaucoma, visual field defects and the use of antiglaucoma medications.All aqueous humor samples were collected by the same surgeon (VPK) at the initial steps of scheduled cataract or combined cataract-trabeculectomy surgery. Preoperative chemoprophylaxis included only topical use of 5% povidone iodine solution for 30 seconds immediately before the operation. Anterior chamber paracentesis was made through clear cornea with a 27G tuberculin syringe avoiding contact with other ocular tissues. The samples (mean quantity 150 μl) were stored in -73° Celsius. After collecting the aqueous humor sample, the routine steps of the planned operation were performed.In order to assess the free total (proform and active) concentrations of MMP2, MMP3 and MMP9 in aqueous humor, 96 well enzyme immunoassay ELISA kits were used . Similar ELISA kits  were used to determine the concentration of total TIMP1, TIMP2 and TIMP4. All ELISA measurements included free forms of molecules. The quantification of active MMP2 and MMP3 was performed with activity assay kits . The latter quantified endogenously activated enzyme levels and total levels (proform and active) without taking into account complexed forms. This strategy may have helped increase the sensitivity and validity of the study. Sample preparation, dilution and enzyme detection were performed according to the specific assay instructions. More specifically, a 10-fold dilution was used for MMP2, MMP3, TIMP2 and TIMP4, while a 100-fold dilution was used for TIMP1. MMP9 quantification required both 10- and 100-fold dilutions in order to confirm the results. The aqueous quantity added to each well was appointed by each ELISA kit (Table Graphic curves were designed using the Origin 6 software (OriginLab Corporation) according to sample optical densities and concentrations. After averaging the duplicate readings for each standard, control and sample, the average zero standard optical density was subtracted. By plotting the optical density for the standards versus the concentration of the standards, the best curve was drawn. The corresponding concentration for each enzyme was calculated, after multiplication by the dilution factor. The detection sensitivity of each kit is presented in Table The SPSS statistical software  was used for the analysisof results. Sample normality was assessed with the Kolmogorov Smirnoff test. Multiple independent samples tests were performed with the Kruskal-Wallis test and confirmed by two independent Mann–Whitney rank sum tests. All tests were two-tailed and statistical significance was considered for p < 0.01 after Bonferroni correction.One hundred and sixty eight eyes from 168 white participants  were included. There was no statistical difference in the mean age of the groups. Demographic and clinical parameters are shown in Table TIMP4 levels were higher in both glaucoma groups (POAG and PXG) when compared to controls (p = 0.001). In contrast, in the PXS group, TIMP4 concentrations presented no statistical difference from controls p = 0.936)  compared to controls  and active MMP3 (p < 0.001) than controls. PXS and PXG samples though, had lower ratios of active/total MMP2 (p = 0.004 and p = 0.008) , lower TIMP4 ratios [MMP2/TIMP4 (p < 0.001) and MMP9/TIMP4 (p < 0.001)] and higher MMP3 levels (p = 0.008). No differences between POAG and PXG eyes were detected.MMPs play a critical role in TM tissue remodeling. They do not only function as catalytic proteases, but also as extracellular processing enzymes that influence cell- to-cell and cell-to-matrix signaling . Their aGlaucoma and MMP regulation have been highly associated since most of the proposed pathogenic mechanisms suggest enzyme dysfunction ,25. The ECM disorders within the TM and surrounding tissues concur in POAG, PXS and PXG ,18. In PRecent evidence suggests that trabecular cellular function is compromised especially at the juxtacanalicular and Schlemm’s canal region . In trabIn this study, using a recently developed ELISA immunostaining kit, we were able, for the first time, to quantify TIMP4 in aqueous humor samples. We found that TIMP4 was only elevated in glaucoma patients. In PXS patients, TIMP4 levels were statistically similar to controls. Furthermore, both POAG and PXG patients showed a decreased ratio of MMP9/TIMP4, a result not seen in PXS samples. Instead, in PXS patients the MMP2/TIMP4 ratio is increased. POAG and PXG patients had similar MMP2/TIMP4 and MMP3/TIMP4 ratios with controls even though there was an increase in the concentration of MMP2 in the POAG group and MMP3 in the PXG group. The increase in TIMP4 levels seems to compensate (or even over-compensate)for the respective increase in MMP concentrations in eyes with POAG and PXG . Thus, bIn POAG, several studies have shown that the levels of total MMP2 and MMP3 and the levels of TIMP1 and TIMP2 are generally up-regulated. Furthermore, in PXS and PXG, total MMP-2, total MMP-3, TIMP-1 and TIMP2 levels in aqueous humor were also significantly elevated compared to samples from conntrols ,18. Our The importance of stoichiometric balance between MMP2/TIMP2 was shown by a number of reports ,17. In oAmong the objectives of our study was to reveal potential differences within study groups. A recent study with POAG and PXG patients indicated that MMPs/TIMPs ratios from TMs pecimens are decreased in PXG, while in samples of POAG eyes, the MMP-1/TIMP-1 ratio and the total MMP1 + 2 + 3 + 9/total TIMP1 + 2 + 3ratio were increased when compared to PXG eyes . MoreoveRegarding the comparison between the PXG and PXS groups, the most important differences were found in the TIMP4 concentrations and ratios. No differences were discovered in active MMPs concentrations, or in MMP9/TIMP1-2 ratios. Another notable difference was that MMP3 levels were significantly elevated in PXG, but not in PXS samples. These results suggest that TIMP4 and MMP3 may have a different regulatory role in pseudoexfoliative eyes with and without glaucoma.Our study has certain limitations. Firstly, there was a wide range of MMP levels within the study groups. This wide variation may reflect differences in phenotypes, disease stage or disease duration. Secondly, the majority of patients were under treatment with topical medication  that could potentially affect aqueous composition and MMP concentration .Our study suggests that there is an imbalance between MMP-related proteolytic activity and its regulation by members of the TIMP family of enzymes in eyes with POAG and PXG. Our novel finding is the elevation of TIMP4 in the aqueous samples of glaucomatous eyes. TIMP4, as a potential inhibitor of MMP activity, could be implicated in ECM homeostasis disorders in glaucoma. Its further role in glaucoma pathology merits further investigation.The authors declare that they have no competing interests.NF drafted the manuscript. He was also involved in study design, data collection, quantitative analysis of the samples and result interpretation. GL participated in the design of the study and patient examination. He also reviewed the manuscript for important intellectual content. AA participated in data acquisition and drafted part of the manuscript. AK participated in the analysis and interpretation of results and reviewed the draft for important intellectual content. IT carried out the immunoassays and participated in the analysis of data. Al.K coordinated the laboratory procedures and designed the biochemical approach of the study. VPK conceived of the study and was involved in study design, sample collection and patient examination. He also critically reviewed the manuscript for important intellectual content and gave the final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/13/69/prepub


This study answered the question of whether biophysical mechanisms for microparticle shedding discovered in platelets and erythrocytes also apply to nucleated cells: cytoskeletal disruption, potassium efflux, transbilayer phospholipid migration, and membrane disordering. The calcium ionophore, ionomycin, disrupted the actin cytoskeleton of S49 lymphoma cells and produced rapid release of microparticles. This release was significantly inhibited by interventions that impaired calcium-activated potassium current. Microparticle release was also greatly reduced in a lymphocyte cell line deficient in the expression of scramblase, the enzyme responsible for calcium-stimulated dismantling of the normal phospholipid transbilayer asymmetry. Rescue of the scrambling function at high ionophore concentration also resulted in enhanced particle shedding. The effect of membrane physical properties was addressed by varying the experimental temperature (32–42°C). A significant positive trend in the rate of microparticle release as a function of temperature was observed. Fluorescence experiments with trimethylammonium diphenylhexatriene and Patman revealed significant decrease in the level of apparent membrane order along that temperature range. These results demonstrated that biophysical mechanisms involved in microparticle release from platelets and erythrocytes apply also to lymphocytes. In thisThe extent to which these various mechanisms apply to nucleated cells has not yet been adequately addressed . Some evIonomycin, 1-(trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), Alexa Fluor 488 Phalloidin Conjugate, and 6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalene chloride (patman) were obtained from Life Technologies . Ionomycin and MC540 were dissolved in dimethyl sulfoxide (DMSO) as stock solutions, while TMA-DPH was suspended in dimethylformamide. Quinine was purchased from Sigma .2). Raji human Burkitt's lymphoma cells were grown at 5% CO2 in RPMI . Prior to experiments, unless otherwise stated, cells were isolated through centrifugation then washed and suspended in MBSS  at a density of 0.4–3.0 × 106 cells/mL. Unless stated otherwise, experiments were conducted at 37°C.S49 mouse lymphoma cells were cultured in DMEM (10% horse serum) at 37°C in humidified air (10% COWashed cell samples (2 mL) were equilibrated 5 min in quartz fluorometer sample cells in either a Fluoromax 3  or PC-1  spectrofluorometer prior to data acquisition. Sample homogeneity was maintained by magnetic stirring, and temperature was regulated with circulating water baths.Microparticle release was assayed by light scatter at 500 nm . After dI435 and I500 are the emission intensities at 435 and 500 nm. The intensity data were smoothed by nonlinear regression to an arbitrary function  prior to calculation of GP.The fluorescence emission of patman was observed at 435 and 500 nm  by rapid (3 s resolution) sluing of the emission monochromator mirror. The probe was added to cell samples after measuring background intensity for 100 s, and the fluorescence intensity was then monitored for several hundred seconds until steady state was reached. The polarity of patman's environment was assessed by calculating the generalized polarization (GP) as follows :(1)GP=IThe steady-state anisotropy of TMA-DPH  was assessed using Glan-Thompson polarizers. Probe was equilibrated with cell samples for 10 min prior to acquisition of data with excitation and emission polarizers alternatively oriented parallel and then perpendicular to each other. Anisotropy was calculated as described previously, and at least 20 points were averaged in determining values for the figures and statistical analyses .Cells were washed and treated with ionomycin as in other experiments. The treated cells were then simultaneously fixed, permeabilized, and stained with fluorescent phalloidin (Alexa Fluor© 488) according to the manufacturer's protocol (Life Technologies). After being mounted onto microscopy slides, cells were stained with a solution containing 165 nM phalloidin, 3.7% formaldehyde, 1% bovine serum albumin, and 0.1 mg/mL lyso-PC  for 20 min at 4°C. Slides were then washed with buffer before coverslips were mounted. Images were collected on an Olympus FluoView FV 300 confocal laser scanning microscope using a 60x oil immersion objective lens. A 3x digital zoom was also applied. The excitation light source was a 488 nm argon laser, and a 505–525 nm bandpass filter was used on the emission detector.P < 0.0001, one-sample t-test with H0 = 0). Moreover, repetition of the experiment in the presence of a calcium chelator (EGTA) inhibited the response to ionomycin and therefore demonstrated that microparticle release was dependent on calcium and therefore not an artifact of the ionophore itself . These data argued that PS exposure is also important for microparticle release in nucleated cells.Previous genetic data and experiments with a pharmacological inhibitor of scramblase  demonstRecent studies suggested a role for membrane lipid order and fluidity in determining the ability of erythrocytes to release microvesicles in response to calcium . To explP < 0.025, r2 > 0.44, n = 11 temperatures, by linear regression, values apply to both patman GP and TMA-DPH anisotropy). Overall, The relative level of membrane order was also assessed at these temperatures by fluorescence spectroscopy using patman GP and TMA-DPH anisotropy. The data with both probes Figures  showed aIt appears that the basic mechanisms governing microparticle shedding in platelets and erythrocytes may apply broadly to all cell types. This finding implies that microparticle release may simply be the consequence of a reduction in the stability of the cell membrane. Hence, factors that regulate the release process are biophysical in nature and represent withdrawal of elements that normally maintain stability. Based on this study and those focused on platelets and erythrocytes, these critical elements include the cellular osmotic balance, cytoskeletal attachments, the asymmetric distribution of phospholipids across the two faces of the membrane, and the elastic properties of the bilayer , 41, 42.The importance of osmotic balance is obvious because of associated effects on cell volume. A cell volume reduction would be required to compensate for the loss of plasma membrane surface area as microparticles are released. Moreover, the volume change would presumably promote membrane budding because of the stress imposed by surface area mismatch. In the case of calcium-induced particle release, the volume reduction appears to involve potassium efflux.As shown in The role of transbilayer asymmetry of phospholipid species in maintaining membrane stability seems less obvious. Loss of that asymmetry during internal calcium accumulation results in exposure of PS on the outer surface. Much attention has been paid to that exposure because of the roles of PS as a signal mechanism in hemostasis and recognition of apoptotic cells by macrophages . Why extP = 0.05, r2 = 0.047 by linear regression, n = 5–15 per temperature, 83 total points).The role of membrane order differed from that of potassium current, cytoskeleton breakdown, and scramblase activation. Each of the latter three appeared required, though not individually sufficient, for microparticle release. In contrast, membrane order, at least at the level accessible to experimental manipulation in living cells, functioned only as a modulator of the rate of release. Only a minimal trend in the apparent amount of membrane particles shed was observed across the temperature range, suggesting that there is a limit on how much is or can be released , loss of membrane phospholipid asymmetry, and cytoskeletal disruption. Moreover, the level of membrane lipid order appears to modulate the release process with greater rates of release occurring from a more fluid membrane. This apparent effect of membrane order suggests that conditions in which these physical properties are altered may promote enhanced or inappropriate microparticle shedding. The obvious example is during apoptosis when the cell membrane becomes disordered and more fluid prior to membrane blebbing and microparticle shedding . As a se


All components, produced using either method, met CBS quality control criteria. There were no significant differences in platelet yield between components produced with and without mixing. A significant difference was seen for RBC hemolysis at expiry (P = 0.03), but for both groups, levels met quality control requirements. Noninferiority of components produced without mixing was confirmed for all parameters. Manual mixing is laborious and has a risk of repetitive strain for production staff and its significance is unclear. Elimination of this step will improve process efficiencies without compromising quality.The elimination of a thorough manual mixing of whole blood (WB) which takes place following the overnight hold, but before the first centrifugation step, during buffy coat component production at Canadian Blood Services (CBS) was investigated. WB was pooled after donation and split. Pairs of platelet, red blood cell (RBC), and plasma components were produced, with half using the standard method and half using a method in which the mixing step was eliminated. Quality assessments included yield, pH, CD62P expression and morphology for platelets, hemoglobin, hematocrit, hemolysis, and supernatant K Whole blood (WB) collection and storage and processing conditions can have critical consequences not only for the immediate quality of the resulting components but also for their long-term quality during storage. At Canadian Blood Services (CBS), production of pooled platelet components (PCs) is a semiautomated process in which WB is held overnight and then separated by a hard centrifugation step into RBCs, plasma, and buffy coats (BCs), from which PCs are subsequently produced , 2. CBS In contrast with other mixing steps, such as the proper mixing of WB with anticoagulant at donation , 4, therEthical approval for this study was granted by CBS Research Ethics Board. The study was designed as a noninferiority study which used a pool and split design to generate homogenous starting samples. WB donations were collected from consenting healthy blood donors into 70 mL citrate-phosphate-dextrose anticoagulant. Following a minimum 3-hour rest period on cooling trays at room temperature (18–24°C), eight or nine ABO-matched units were docked together using sterile tubing with multiple connectors (MacoPharma) and an automated Compodock  and drained into a 5 L pooling bag . Following gentle but thorough mixing, the pool was split back into the original blood bags in random order, with volumes based on the net weight of the WB pool (minus 3% for losses incurred during pooling and splitting) divided by the number of units in the pool. WB units were prelabeled either mixed (M) or nonmixed (NM) and were stored for approximately 8 h at room temperature prior to component production the following day.WB units from each pool were processed into leukoreduced pooled PCs in plasma, leukoreduced RBCs in saline-adenine-glucose-mannitol preservative, and frozen plasma following the standard semiautomated BC production method currently in place at CBS, as described previously , 2. The PCs were sampled on Day 1 and Day 5 and were tested on Day 1 , Day 5  and morphology), and after expiry (Days 7-8 of storage for pH and platelet yield). Unit weight was used to calculate volume based on a specific gravity of 1.03 g/mL. Calculations for platelet yield on Day 1 and after expiry were made using this calculated volume. A total of 24 PCs was tested .Platelet yield (platelet count/unit) was calculated using the component volume and the platelet count, measured on an ADVIA 120 Hematology Analyzer , as described previously , and steAssessment of platelet degranulation, a marker of platelet activation, was conducted using CD62P expression by flow cytometry on a FACS Canto II , as described previously , 11. PlaRBCs were tested at Day 3 and Day 43 after collection. Unit volume was determined based on the net RBC unit weight and a specific gravity of 1.06 g/mL. Components were sampled and prepared for residual leukocyte count within 24 hours of production. In total, forty (20 M/20NM) RBCs were tested.T and Hct were determined using the ADVIA 120 automated hematology analyzer. HbS levels were measured using a HemoCue Plasma/Low automated hemoglobin analyzer . For both methods, percentage hemolysis was calculated using the following equation:Total hemoglobin (g/L) was measured on an ADVIA 120 automated hematology analyzer and g/unit determined using the calculated volume of the RBC unit. RBCs were tested for rWBC levels using a flow cytometric approach as described previously  and were+ concentrations were determined by indirect potentiometry using ion selective electrodes on a Beckman Coulter DXC 800 .Extracellular KThe volume of 36 (18 M/18 NM) plasma components was measured. Plasma was placed into storage at ≤−20°C and 14 randomly selected components (7 M/7 NM) were shipped frozen for FVIII activity testing within 8 weeks of production, as described previously , 16. + for RBCs), noninferiority margins were determined and applied  were produced. A paired There was no significant difference in volume between pooled PCs produced with or without mixing , indicating that pooling and splitting produced uniform components. Both the M and NM components produced from one pool did not meet QC criteria for platelet yield after expiry, possibly due to low donor platelet counts in that specific WB pool . OtherwiP < 0.01) with NM pooled platelets displaying lower ESC compared to M pooled platelet products. No difference between M and NM groups was seen at expiry . Bacterial growth was not detected in any unit, and all QC criteria were met with 100% pass rates for all parameters for both M and NM samples . P = 0.03), with the NM units showing greater hemolysis. Despite this, noninferiority was confirmed, as the upper bound of 95% confidence interval of 0.029% was less than the margin (0.06%).No significant difference was observed for rWBCs/unit between M and NM groups . Total h+ between the 2 arms at Day 3 (P = 0.02) with higher supernatant K+ observed in the NM units, but no difference at Day 43. Early in storage, levels of supernatant K+ are low (~5 mM), and they rise steadily during hypothermic storage [Measurement of hemolysis using the Drabkin method showed no significant differences between the M and NM arms and noninferiority was again confirmed . There w storage . TherefoP = 0.08), and all units passed QC requirements for sterility. FVIII activity was examined as this is one of the most labile of the coagulation factors, is often used as an indicator of plasma quality [P = 0.41). Noninferiority of the NM units was confirmed.Volumes were not significantly different between plasma produced from M or NM WB . Among the centres we contacted, none have transitioned from a process that mixed WB prior to centrifugation to one that did not, and so we are not aware of any validation or investigation of the effect of this process change. The results of the current study demonstrate that elimination of the mixing step is not detrimental to product quality, and components produced without mixing of the WB will meet all necessary QC criteria. In terms of process efficiency, and also for the health and safety of the production staff processing the blood, removal of this step may indeed be beneficial, and at CBS we are currently planning to implement this change.


PCR-DGGE, sequencing and microscopic analysis revealed remarkable bacterial and fungal diversity differences between the snow samples and the outdoor air samples. In addition, DGGE banding profiles for the snow samples collected were also shown distinctly different from one another. Absent from the outdoor air, certain human, plant, and insect fungal pathogens were found in the snow samples. By calculation, culturable bacteria accounted for an average of 3.38% (±1.96%) of TOC for the snow samples, and 0.01% for that of outdoor air samples. The results here suggest that snow precipitations are important sources of fungal pathogens and ice nucleators, thus could affect local climate, human health and agriculture security.In this study, the bacterial and fungal abundances, diversities, conductance levels as well as total organic carbon (TOC) were investigated in the snow samples collected from five different snow occurrences in Beijing between January and March, 2010. The collected snow samples were melted and cultured at three different temperatures . The culturable bacterial concentrations were manually counted and the resulting colony forming units (CFUs) at 26°C were further studied using V3 region of 16 S rRNA gene-targeted polymerase chain reaction -denaturing gradient gel electrophoresis (PCR-DGGE). The clone library was constructed after the liquid culturing of snow samples at 26°C. And microscopic method was employed to investigate the fungal diversity in the samples. In addition, outdoor air samples were also collected using mixed cellulose ester (MCE) filters and compared with snow samples with respect to described characteristics. The results revealed that snow samples had bacterial concentrations as much as 16000 CFU/ml for those cultured at 26°C, and the conductance levels ranged from 5.6×10 Microorganisms attached to Saharan dust were found to be transported across the Atlantic Ocean There have been increasing evidences that the biological agents could be transported globally 4 to 5.4×106 cells per liter in their snow samples collected For the microbial pollutant transport, the snow precipitation as aforementioned might play an important role. According to Christner and co-workers (2008), DNA-containing cells between 0.3 and 15 µm in diameter had a concentration of 1.5× 10In 2010, consecutive snow precipitations occurred in Beijing between January and March. Taking this opportunity, we carried out this work to investigate if snow precipitations could represent an important source of microbial species and infections. In addition, we also investigated if the snow samples differed from one another with respect to biological contents. Besides, we also measured the conductance and TOC (bacteria are important contributor to both) for snow samples and outdoor air samples. Bacterial diversity was studied by analyzing the colony forming units using 16 S rRNA gene-targeted PCR-DGGE and constructing clone library after sequencing, and fungal genera were identified through optical microscopic observation. All samples collected were cultured simultaneously at three different temperatures .There were five consecutive snow precipitations in Beijing on Jan 1, Feb 7, Mar 1, Mar 8, Mar 14, 2010. In addition, snow samples were also collected on Feb 27, 2011. For each snow occurrence on a different date, fresh snow samples (on the top of the snow layer) in the middle of or right after the snow precipitation were collected from the top layer into 50 ml tubes from five different locations (to mimimize the variations from the snow samples from each occurrence) within the campus of Peking University, located at the 4th Ring and 5th Ring of Beijing city. For each snow occurrence, the snow samples collected from five different places within the campus were pooled together for analysis. Considering upper atmospheric temperature, the snow samples were stored at −70°C in a freezer  before analysis. In addition, for microbial diversity analysis the outdoor air samples within Peking University campus were also collected using an Aerosol Button Sampler (SKC) with mixed cellulose ester (MCE)  filters at a flow rate of 13.5 L/min for 40 min on a date  between these five snow occurrencies. High flow rate rather than its standard 4 L/min was used to collect enough air samples for limiting dessication effects of longer sampling time. For culturable concentration study, the filter sample was placed in a corning tube  filled with 4 ml DI water , and then extracted using a sonicator  for about 20 min. The resulting suspension was stored at −4°C for subsequent experiments. For denaturing gradient gel electrophoresis (DGGE study), three filter samples collected at 10 L/min for 20 min were also used. Blank agar plates or DI water were used as negative controls for culturing and PCR-DGGE analysis.Immediately before analysis, the snow samples were melted at room temperature. Then, 2 ml of the melted snow was taken out from the snow tube and diluted 10 times using DI water. For the snow samples (pooled together) collected on each occurrence on a particular date, 80 µl of the original suspesion (diluted 10 times) was pipetted onto on a Trypticase Soy Agar (TSA)  plate for bacteria culturing. Likewise, 80 µl of the original suspesion without dilution was pipetted onto a Malt Extract Agar  plate for fungal culturing at 26°C. For bacteria, the snow samples were grown at three different temperatures: 4°C, 26°C and 37°C. Bacterial and fungal aerosols are often cultured at 26°C and 37°C, while use of 4°C could readily activate those bioaerosols acting as ice nucleators. For each sample, three replicates were perfromed. The colony forming units (CFUs) were manually counted and the bacterial concentration was calculated as CFU/ml of melted snow suspenison. For culturable concentrations, 100 µl of filter extraction liquid was plated onto an agar plate with three replicates for the outdoor air sample. For DGGE study, the fiter samples were directly placed on agar plates for culturing at 26°C.5'-ATTACCGCGGCTGCTGG-3') and P3 (5'-GCclamp-CCTACGGGAGGCAGCAG-3') as used by Muyzer et al. (1993) 2O. The cycle conditions were set as : 94°C for 3 min, 30 cycles of , and 72°C for 10 min. DI water was used as the negative control in the PCR experiments. The DGGE analysis of PCR amplicons was performed using a Bio-Rad DcodeTM mutation detection system  according to the manufacturer’s instructions. In details, approximately 20 µl PCR product was transferred to each well of 8% polyacrylamide (acrylamide/bisacrylamide: 35.7∶0.8) gels which contain a linear 30–65% gradient of urea and formamide increasing in the direction of electrophoresis. The electrophoresis was conducted in 1× TAE buffer for 540 min at a constant voltage of 100 V at 60°C. DNA bands in gels were stained by GelRed solution (10000 x diluted with DI water)  and photographed  under ultraviolet lamp at the wavelength of 302 nm. Data were recorded and analyzed with Quantity One software (Bio-Rad). Here, the bands were marked by an arrow, and all lanes were compared with each other. The DGGE bands for the snow samples and the outdoor air sample were further compared using dendrograms obtained using the BioRad built-in software functions “band detection” and “unweighted pair group method with arithmetic mean (UPGMA)” clustering, and the similarity of the culturable microbial aerosol diversities was analyzed using the scale bar produced by the methods.For bacterial diversity analysis, the CFUs from the agar plates both for the outdoor air and snow samples were washed off using DI water to a total volume of 5 ml. Then, 1 ml of the bacterial suspension was used for DNA extraction by a bacterial DNA extraction kit  following the manufacturer’s instruction. The extracted DNA samples were further resuspended into 50 µl DI water. The V3 region of the 16 S rRNA gene was amplified using primers P2 . After the culturing, the same procedure described for agar plate culturing was adopted in applying the PCR-DGGE analysis to the CFUs. In addition, the diversity was studied directly by PCR-DGGE procedure without the culturing, in which 1 ml of the melted snow sample was used for direct DNA extraction.Besides, we also constructed clone libraries of V3 region of 16S rRNA gene followed by sequence analyses. The enrichment of melted snow samples was performed using liquid culturing method. After three days’ culturing, 1 ml of each cultured snow sample were extracted for DNA. The V3 region of 16 S rRNA gene of all the extracted DNA were amplified as mentioned above. The PCR products were purified using an agarose gel DNA purification kit , ligated into a pEASY-T1 simple cloning vector, and then transformed into Trans1-T1 phage resistant chemically competent cell following the manufacturer’s instructions . The presence and size of inserts were checked by blue-white selection method followed by PCR and gel electrophoresis. These amplicons were further sequenced using the M13 primers . All sequences were aligned using the MEGA 5.0 software For culturable fungal aerosols, microscopy method was used for fungal genus identification in both snow and outdoor air samples. Such a method is widely used in other studies In this study, the conductance levels in snow samples were also measured using a lock-in amplifier . Before the sample analysis, different frequencies of 1 kHz to 100 kHz were automatically tested using a LabView program developed in our lab. For each snow sample, 20 µl of melted snow sample was used for the measuremnt, and total 500 measurements were made (1 measurement per 300 millisecond). The optimal frequency from those tested at which the conductance level remains relatively stable was selected for the final sample conductance analysis. Using the optimal frequency, the average conductance levels of the mixture from five different snow samples collected on each occurrence on a particular date were measured. In addition, the total organic carbon concentations for all samples (an outdoor air sample after filter extraction and the mixtures of five snow samples for each date) were also measured using a TOC analyzer . Here, we also calculated the bacterial contribution to the total oragnic carbon using a conversion factor of 17 pg carbon per bacterium that was established in a previous study The concentration levels of culturable bacteria of snow samples and outdoor air samples under different culturing temperatures, and the conductance levels of all snow samples were analyzed using paired t-test and Analysis of Variance (ANOVA). These data are normally distributed. A p-value of less than 0.05 indicated a statistically significant difference at a confidence level of 95%.3. These data suggested that bacteria were abundant in the snow samples collected, and bacteria in the snow samples can be more active at lower temperatures than those (likely in the state of viable but not culturable) in outdoor air samples, implying they might play possible roles in ice nucleation and cloud formation. ANOVA tests indicated there were statistically significant differences in the bacterial concentrations obtained for five snow samples for different occurrences at different incubation temperatures .This study investigated the biological contents, conductance levels as well as total organic carbons in snow samples collected from five snow occurrences that happened during January-March 2010 in Beijing, and the relevant results were compared among them as well as with those in outdoor air samples. 3 to 2.3×104 cells/ml 4 and 5.4×106 cells/L In this work, the bacterial concentration levels for the snow samples collected in Beijing were found in similar magnitudes with those in snow samples reported in previous studies. In a previous study, it was found that the bacterial concentration in snow samples collected from Mt. Everest ranged from 5.7×10PCR-DGGE analysis also showed distinct differences in bacterial diversity among the snow samples from different occurrences as well as those in outdoor air samples. In our work, different culturing methods for the bacterial diversity analysis were also investigated in addition to the agar plate culturing followed by PCR-DGGE. Bacillus. On one hand, this finding might due to the liquid culturing method used to retrieve the bacteria of snow samples. Spore-forming organisms, such as Bacillus species and other Gram-positives, tend to dominate culture-dependent surveys of airborne microbial diversity Bacillus species show high resistance to environmental stresses, including UV light exposure, desiccation, and the presence of oxidizers Bacillus species had been collected from spacecraft and facility surfaces, and Bacillus pumilus was found to be the second most dominant species among the aerobic spore-forming bacteria Bacillus flexus was also isolated from surface snow samples collected from Antarctica Bacillus species could serve as an efficient ice nucleator.Here, a V3 region of 16 S rRNA gene clone library was constructed from four snow samples collected in 2010 and one snow sample collected in 2011 to characterize the diversity of the snow bacterial communities and to identify the dominant taxonomic groups. As shown in In a recent study, it was also shown that the bacterial communities found in the air appear to be distinctly different from the bacterial communities found in freshly fallen snow samples Cladosporium, Penicillium, Aspergillus, Alternaria were found abundant in most samples studied. For the outdoor air samples, Alternaria, Aspergillus, Penicillium, Cladosporium, Stemphylium and Phyllosticta species found to dominate the community. Likewise, we also detected Cladosporium, Penicillium, Aspergillus, Alternaria species in the snow samples collected. However, it should be noted that there were some species belonging to Entomophthorales (insects pathogen), Saccharomyces cerevisiae (human opportunistic pathogen Pseudocercospora (plant pathogen), Blastocladiales, and Leptomitales found to be abundant in the snow samples, but not detected in the outdoor air sample. As indicated in In this study, the dominant fungal species in the snow samples and the outdoor air sample were also analyzed based on the morphology using the microscopy methods. Dothideomycetes and Eurotiomycetes of fungi not by common species such as Alternaria, Cladosporium, and Penicillium spp. They attributed this to the culturing bias. For the outdoor air sample, they found 1∶1 ratio of bacterial and fungal sequences. In another study, it was demonstrated that species diversity of fungi in the snow was much broader than in aerosols Alternaria and Cladosporium genera in addition to others such as Botrytis spp. and Ulocladium spp. in their snow samples. Laboratory studies suggest that fungi can also act as the ice nuleator, initiating the snow precipitation In a recent study, they also found fungal species in the snow samples collected 2<0.1). In our another study, we have shown that the bacterial suspension condutance is an exponential function of bacterial concentration for pure bacterial cultures 2+ was found the key ion in determining the conductivity of the surface Bacteria and fungi are important contributor to both conductance and total organic carbon It was shown that bacterial and fungal species contribute to the total carbon in the atmosphere Pseudomonas syringae, are plant pathogens Increasing evidences suggest that the atmosphere is a habitat for bioaerosols where these biological particles may affect the precipitation cycle and/or their own precipitation during the atmospheric transport For the snow samples collected in this study, they might have captured biological particles during their transport to the ground. However, this process would not affect the conclusion from this study. In this work, all snow samples went through the same process, and distinct bacterial diversities were also found in the snow samples. In this study, 16 S rRNA gene-targeted PCR-DGGE was performed on both snow samples and the outdoor air sample. The results obtained here could be negatively impacted by certain DGGE limitations such as the intraspecies operon heterogeneities. In addition, use of microscopy methods, despite of wide use, for fungal identification would have some limitations on the morphology identification and corresponding comparison. Therefore, the results obtained for fungal diversity in this study might have its method limitations. In this study, MCE filter was used for the outdoor air sample analysis. In our recent work, we have found out that highest bacterial diversity was obtained when MCE filter with air samples was directly placed on agar plates for culturing compared to other available bioaerosol sampling methods Bacillus species could possibly serve as an efficient ice nucleator. The precipiation of these foreign biological agents via snowfall or rainfall could have non-negligible impacts on local climate, human health and agriculure security. In addition, snow precipitation could be one of major contributors to the TOC in local environments. In this study, small sample size, i.e., only five snow occurrences, despite of unusual high frequency for Beijing within a year, could have possible limitations on the conclusion generated.Bioaerosols are increasingly being recongonized as a player in the cloud formation, atmospheric chemical process, and climate change. In this study, the bacterial and fungal abundance and diversities in the snow samples collected in Beijing were investigated. The results have shown that there were not only high levels of bacterial concentrations, but also distinct differences in both bacterial and fungal diversities including human, insect and plant pathogens from different snow samples. These results suggested that the microbial structure in the cloud could change dynamically over the time, likely due to a microbial flux transported from other places. In addition, similar to a previous sudy


Lysophosphatidic acid (LPA) through activating its G protein-coupled receptors (LPAR 1–6) exerts diverse cellular effects that in turn influence several physiological processes including reproductive function of the female. Studies in various species of animals and also in humans have identified important roles for the receptor-mediated LPA signaling in multiple aspects of human and animal reproductive tract function. These aspects range from ovarian and uterine function, estrous cycle regulation, early embryo development, embryo implantation, decidualization to pregnancy maintenance and parturition. LPA signaling can also have pathological consequences, influencing aspects of endometriosis and reproductive tissue associated tumors. The review describes recent progress in LPA signaling research relevant to human and ruminant reproduction, pointing at the cow as a relevant model to study LPA influence on the human reproductive performance. The present paper focuses particularly on one of the simplest and most potent lysophospholipids, lysophosphatidic acid (LPA), and summarizes recent knowledge on its biological impact on human and ruminant reproduction.Lysophosphatidic acid is a simple phospholipid that exerts many physiological and pathological actions on various cell types, such as cell proliferation and differentiation , cytoskeSo far LPA has been detected in many various biological fluids such as serum and plasma –7, tears2), PS-PLA1, and lecithin-cholesterol acyltransferase (LCAT), and then the LPL is converted to LPA by ATX [While the precise mechanism of LPA metabolism within most types of cells is still unclear, two general pathways of LPA production have been demonstrated. In the first pathway phosphatidic acid (PA) is produced from phospholipids (PLs) by phospholipase D (PLD), also called autotaxin (ATX) or from diacylglycerol by diacylglycerol kinase . In bothA by ATX . The firA by ATX , pregnanA by ATX , hyperteA by ATX , prostatA by ATX . MoreoveA by ATX , 27.γ [In mammals, LPA exerts its action via at least six high affinity, transmembrane G-protein-coupled receptor (GPCR) types, LPAR1–LPAR6, and possibly through a nuclear receptor PPARγ , 28–31. γ . For exaγ , LPAR2 iγ , and LPAγ , 34, 35.γ , LPAR5 eγ . Howeverγ , 38.The influence of LPA on the reproductive system function of the female has been examined and described for about 30 years. Since the first reports published by Jarvis et al.  in women in vitro [Physiologically, LPA and its active LPARs have been documented to be present in female reproductive organs, such as uterus , 41, 42,in vitro . Interesin vitro , suggest in vitro fertilization the amount of LPA increases with the time of incubation with ATX in 37°C [ in vitro fertilization. LPAR4 was also highly expressed in the cortex of the human ovary [LPA signaling has been extensively studied in the physiology of human ovary. Follicular fluid of the human preovulatory follicle contains lysophospholipase D—ATX which is responsible for local LPA production from lysophosphatidylcholine (LPCs) . However in 37°C . Serum A in 37°C . Moreove in 37°C  demonstran ovary . in vitro fertilization [LPA signaling plays many crucial roles in ovarian function such as ovulation, for example. Before ovulation LPA elevated the level of IL-8, expressed its chemotactic activity for neutrophils, started the inflammatory reaction, and in consequence led to tissue degradation and rupture of the follicle . During lization . Thus, tlization . in vitro. Moreover, Jarvis et al. [ in vitro which resulted in sensitization of myometrial cells to OT. In uterus in the end of gestation the sensitivity of myometrium to OT increases robustly, which in turn induces uterine contractions leading to labor. Other important process in induction and maintenance of smooth muscle contraction is stress fiber formation. LPA enhanced stress fiber formation in human myometrial cells in vitro and thereby increased the efficiency of uterine contractions in the beginning of labor [More than a decade ago Tokumura et al.  suggestes et al.  documents et al. . Moreoves et al.  demonstrof labor .α, respectively, and thereby contributed to regulation of the innate immunity of the fetus [ in vitro model of embryo implantation LPA increased the outgrowth of embryos on decidual cells [The process of embryo implantation includes interaction between the endometrium and the embryo (adhesion and invasion), inflow of innate immune cells, and vascularization. LPA has been presented to be involved in implantation on many various levels. In human decidual cells, LPA increased embryo outgrowth as the result of RhoA signaling . It alsohe fetus . Iwasawahe fetus  documental cells . Throughal cells .The pleiotropic roles of LPA in the function of the reproductive tract are demonstrated not only by the increased amount of LPA in body fluids and in the area of reproductive organs but also by the tissue-specific, regulated expression of its receptors. Wei et al.  demonstrLPA signaling can also have adverse effects during pregnancy. Li et al.  detected in vivo may have been involved in tumor cell proliferation [LPA signaling may also play a role in pathogenesis of both benign and malignant endometrial tumors. Billon-Denis et al.  presenteferation . There aferation , 65. Rapferation  used theferation .LPA signaling may also play a role in breast cancer progression. Kitayama et al.  suggesteOvarian cancer is the most thoroughly studied cancer with respect to LPA signaling in carcinogenesis. There are two types of data in the literature, demonstrating the direct and indirect roles of LPA in the pathogenesis of the tumors. Sutphen et al.  and Xu eMoreover, the regulation of LPARs may also play a significant role in LPA signaling in ovarian cancer. Wang et al.  proved tOverlooking the available data in the literature on the role of LPA in human reproduction we can summarize that the past decade shaded new light on the importance of LPA signaling not only under physiological but also pathological conditions. LPA is produced locally in human reproductive tissues and controls ovarian cycle and pregnancy as well as various abnormalities in the female reproductive tract. Engagement of LPA in the maintenance of pregnancy manifests by its action on the uterus, ovary, fetal membranes, and placenta. LPA can also affect the fetus itself by controlling the process of implantation and vascularization of the embryo. However, there are also reports on adverse effects of LPA in the female reproduction, especially during tumorigenesis. Since in the available literature there are often registered opposite effects of LPA on the reproductive performance in human as well as the research on human tissues is ethically restricted and new LPA-targeted therapeutic strategies are demanded, it is important to find a good animal model to study LPA influence on the function of the female reproductive organs.Properly designed studies to examine the effects of LPA on the reproductive performance in humans should be done in human subjects. However, this is very hard to accomplish, since human studies are difficult to carry out because of their typical complexity and dependence on mostly retrospective data rather than the treatment-based outcomes measured in animal models including the bovine one. Moreover, all the complications in the design and interpretation of human studies, combined with the ethical issues regarding experimentation in humans, continuously increase interest in studies that utilize animal models. On the other hand, the relevance of studies performed in animal models to human health has been questioned many times in the literature, since in almost all animals used as a model many weak points can be found. Taking above arguments into consideration, it has been well documented in the literature that the cow can be one of the quite relevant animal models for studying human reproduction. In the bovine reproduction we can find many similar aspects in the ovarian physiology, early embryo development, pregnancy as well as assisted reproductive techniques , 80. TheIn the bovine ovary, Boruszewska et al.  demonstrIn ruminant uterus, the influence of LPA on the endometrial function was studied for the first time by Liszewska et al.  in sheepIn the cow, we were the first to demonstrate that LPA is locally produced and released from bovine endometrium during estrous cycle and early pregnancy . We foun2 were immunoexpressed both in epithelial and stromal cells [2 expression was higher in epithelial than instromal cells in bovine endometrium. This study demonstrated that epithelial cells are the main source of LPA in the bovine endometrium [Studying intracellular localization of the enzymes responsible for endometrial LPA synthesis, we demonstrated that ATX and PLAal cells  also fouometrium . Similarometrium  found apometrium  also docometrium . Howeverometrium . The datτ action during early pregnancy [The potential role of LPA depends on both its local concentration and the distribution of LPARs in the area of reproductive tissues. In the bovine follicle, Boruszewska et al.  detectedregnancy , or moduregnancy . The datregnancy  in the bregnancy , who sho in vitro, whereas LPAR3 was found only in the cell membrane in both systems. In the ovine uterus, LPAR1 was predominantly present in the stromal tissue, whereas LPAR3 was mostly detected in the epithelial structures [2 synthesis only in the in vitro cultured stromal cells [ via LPAR1.In sheep, Liszewska et al.  demonstrructures . In the ructures . LPAR1 eructures . On the ructures . Boruszeructures  found hial cells , 40. The in vitro and a specific antagonist of LPAR1 and LPAR3 receptors (VPC32183) blocked this effect. This study directly evidenced that LPARs operate and are functionally coupled to signal transduction mechanisms in trophectoderm cells [ in vitro. Therefore the authors suggested that LPA may be involved in the mechanisms regulating morphological changes both in the embryo and the uterus during conceptus adhesion to the uterus in ewes during the implantation process Liszewska et al. [Studying receptor and intracellular mechanism of LPA action in the ovine trophectoderm cells, Liszewska et al.  found thrm cells . In otherm cells . Thereform cells  claim thrm cells , 85 alsoa et al. , 85.2 production, cell viability, and intracellular calcium ion mobilization in the cultured stromal endometrial cells via LPAR1 receptor activation [The studies concerning receptor and intracellular mechanisms of LPA action in the bovine endometrial cells revealed that LPA stimulated PGEtivation .In ruminants, the dynamic LPA synthesis and LPARs expression and action in the follicle, CL and uterus suggest that LPA plays autocrine and/or paracrine roles in the reproductive tract acting via various active LPARs.2 synthesis and secretion in the granulosa cells  and 17β-hydroxysteroid dehydrogenase- (17β-HSD-) catalyzed reactions [β-HSD mRNA expression in granulosa cells  expression in steroidogenic CL cells [In ruminants the cow , 40. In he blood . Woclawehe blood  also shool group . The posa et al. . The autCL cells .α and IFNγ actions at the late luteal stage—when the luteolysing cytokines act the most. It has been documented before that TNFα together with IFNγ can serve as mediators of luteolytic actions of PGFα2 via inhibiting P4 production and stimulating apoptosis of the cultured bovine luteal cells [α and IFNγ but also fibroblasts and endothelial cells [α and IFNγ rise significantly just after initiation of luteolysis, as the reason of a great amount of lymphocytes infiltrating the CL at this time. Moreover, Skarzynski et al. [α in low concentrations caused luteolysis (decreased P4 level), which could be augmented by various factors, including IFNγ. Concerning the possibility of LPA-dependent modulation of TNFα and IFNγ actions at the late luteal stage, Woclawek-Potocka et al. [α and IFNγ on P4 synthesis in the cultured bovine steroidogenic cells. These data are consistent with previous data obtained in vivo that LPA administered into aorta abdominalis or intravaginally increased P4 secretion in the cows during the luteal phase of the estrous cycle [ in vivo were also reported before [α and IFNγ.We also found that LPA did not express only direct luteotropic action  but alsoal cells –96. Physal cells , 98. Penal cells  and Sakual cells  demonstri et al.  demonstra et al.  demonstrus cycle , 40. In d before . Woclawed before  hypothesα- and IFNγ-induced luteal cell apoptosis  also belongs to the TNF super family receptors which together with Fas ligand (FasL) transmit basic signals controlling intercellular apoptosis pathway [α and IFNγ did not stimulate Fas and FasL expression in the cultured steroidogenic luteal cells on days 8–12 of the estrous cycle. Moreover, it has been documented before that the inhibition of intraluteal P4 action by various specific antagonists amplified Fas L-mediated apoptosis viathe increase of Fas and initiation of caspase (Casp)8 and Casp3 expressions as well as Casp3 activity in cultured bovine luteal cells [α and IFNγ [α and IFNγ could not induce apoptosis  and 2,5′-oligoadenylate synthase (OAS1) expression in the steroidogenic cells of the bovine CL (The interactions between LPA and IFNa et al.  in bovinin vitro . Kowalczin vitro  found thse cells . These ra et al.  proved tovine CL . These tThe data obtained in cows prove that LPA can be an additional auxiliary luteotropic factor acting in the CL and in the endometrium during both the estrous cycle and early pregnancy establishment.α2 is the major luteolytic agent, whereas PGE2 has luteoprotective and antiluteolytic properties [α2 to PGE2 ratio is essential for endometrial receptivity, maintenance of CL action, and P4 secretion as well as accurate pregnancy establishment [2 and LPA act similarly and that PGFα2 and LPA act differently during early pregnancy in cow [In ruminants, uterine PGs are crucial components in the regulation of estrous cycle and early pregnancy. Prostaglandin Foperties , 117. Thlishment . The dynlishment  and posslishment , 120 werlishment  and humalishment  but also2 synthesis via PGES mRNA stimulation only in stromal cells on days 8–10 and 16–18 of the estrous cycle and pregnancy [α2 synthesis via PGFS mRNA stimulation only in epithelial cells on days 8–10 and 16–18 of pregnancy [2 stimulates CL function [α2 synthesis during early pregnancy. The above data seem to be important, because the examined time frames are crucial phases during early pregnancy. First, days 8–10 represent the time of immunological pregnancy establishment as shown by Kelemen et al. [τ production by the conceptus, just before implantation. Therefore, the interactions between LPA and IFNτ at these phases cannot be excluded.There are also data in the literature on the intracellular and enzymatic mechanisms of LPA- dependent stimulation of PG synthesis in the bovine endometrium , 40. In regnancy , 40. Morregnancy , 40. Thuregnancy  suggestefunction , 111 andfunction , 124, LPfunction  also sugn et al. , Barnea n et al. , and Majn et al. . Moreoven et al. . On the α2 and PGE2 release [2 that resulted in a burst of PG synthesis independent of any modifications in gene expression. However, there are also reports in human and rats that LPA increased PGE2 synthesis in human monocytic and ovarian cancer cells [2 and PGI2 [The data obtained in cows is consistent to a certain extent with data obtained in ovine trophectoderm cultured cells, in which LPA induced PGF release . However release  claimed  release  in Jurka release  hypotheser cells , 131 as er cells , 133 viaand PGI2 .Despite different intracellular mechanisms of LPA-induced PG synthesis in the cow and ewe, a new biological function for LPA interaction with PGs in the contribution of pregnancy establishment in cows and in the regulation of the implantation process and embryonic development in ewes was designated in ruminants.There is overwhelming evidence in many studies using a ruminant model that LPA signaling can have significant consequences for reproductive health. The effects of LPA depend on many various conditions such as the target tissue and physiological status of the animal as well as the phase of the estrous cycle or pregnancy. However, the most important issue connected with LPA signaling is the fact that there is the possibility of LPA synthesis directly in the area of the reproductive tissues. Therefore, it is crucial to examine carefully the effects of this biologically active compound on reproductive outcomes using animal models that can the most closely mimic reproductive processes in human.In spite of many limitations in conducting well-designed human studies, information gathered from already published ones combined with the large number of the studies already available in ruminants, clearly demonstrate that LPA has the ability to influence the reproductive performance of an adult female.


The citrate reduction method for the synthesis of gold nanoparticles (GNPs) has known advantages but usually provides the products with low nanoparticle concentration and limits its application. Herein, we report a facile method to synthesize GNPs from concentrated chloroauric acid (2.5 mM) via adding sodium hydroxide and controlling the temperature. It was found that adding a proper amount of sodium hydroxide can produce uniform concentrated GNPs with low size distribution; otherwise, the largely distributed nanoparticles or instable colloids were obtained. The low reaction temperature is helpful to control the nanoparticle formation rate, and uniform GNPs can be obtained in presence of optimized NaOH concentrations. The pH values of the obtained uniform GNPs were found to be very near to neutral, and the pH influence on the particle size distribution may reveal the different formation mechanism of GNPs at high or low pH condition. Moreover, this modified synthesis method can save more than 90% energy in the heating step. Such environmental-friendly synthesis method for gold nanoparticles may have a great potential in large-scale manufacturing for commercial and industrial demand. Gold nanoparticles (GNPs), also named as gold colloids, have attracted increasing attention due to their unique properties in multidisciplinary research fields ,2. AlthoIn the past decades, though many synthetic strategies have been developed to prepare GNPs in organic or aqueous solvents -24, the 4) via adding sodium hydroxide and controlling the temperature. We demonstrated that adding a proper amount of sodium hydroxide to the reaction mixture could produce uniform GNPs with a narrow size distribution after the reduction by sodium citrate at boiling sate. The low reaction temperature was helpful to control the nanoparticle formation rate, and uniform GNPs could be obtained at different temperature by adding a proper amount of alkali. The pH change resulting from the addition of alkali showed a critical role in the influence on the particle size distribution, which might be related to the different formation mechanism of GNPs under different pH conditions.Herein, to meet the need of high concentrations, we modified the classical citrate reduction method and synthesized uniform GNPs from tenfold concentrated precursor  was purchased from Sigma-Aldrich Shanghai Trading Co Ltd, Shanghai, China, while sodium citrate  and sodium hydroxide  were obtained from Shanghai Chemical Co., Shanghai, China. Deionized water (resistance > 18.2 MΩ) was prepared by an ultrapure water system in our laboratory. All chemicals were used as received without any purification.Hydrochloroauric acid trihydrate  were rapidly introduced into the flask with drastic stirring, respectively. The mixtures were continuously heated for a certain period till a ruby-red color appeared.GNPs were first synthesized from HAuClThe concentrations of chloroauric acid and sodium citrate in the final mixture were respectively fixed to 2.5 and 5.0 mM, while that of NaOH was changed. The reaction temperature was selected to be boiling state, 85°C and 70°C. For example, 2.0 mL chloroauric acid (25 mM) was mixed with 5.3 to 10.2 mL of 20 mM NaOH solution, followed by adding the calculated volume of water to a total volume of 20 mL. The flask was put into an oil bath at 110°C for 30 min to balance the reaction mixture to 85°C. Then, 0.6 mL sodium citrate solution (50 mg/ml) was rapidly introduced into the flask under vigorous stirring. After different reaction time, samples were taken out for characterization. The reaction at the boiling state and 70°C was similarly performed, respectively.In the synthesis process of GNPs, a portion of the reaction mixture (0.5 to 1 mL) was taken out from the flask at different reaction time and immediately poured into 9 mL ice-cooled water at 0°C. Such an operation can basically cease the formation process of GNPs due to the low temperature surrounding and the dilution effect, so it was called here as a "sample-frozen" operation. Then, the transmission electron microscopy (TEM) samples were prepared at the earliest time and the ultraviolet-visible (UV-vis) spectra were recorded.UV-vis spectra were recorded on a U-3010 UV-visible spectrophotometer  to collect the surface plasmon resonance (SPR) information of GNPs, in which the highly concentrated samples were diluted pro rata by deionized water to adapt the measurement limitation. TEM samples were prepared by dropping the diluted gold colloids on carbon-coated copper grids, followed by natural drying; then, the samples were observed on a JEM-2010 microscope .4 from 0.25 mM to decreased values was studied [4 are not stable and become black precipitates after hours; this is partially ascribed to the colloidal instability at high ionic strength.In Turkevich's work, the influence of reactant concentration of HAuClIn recent published work, pH control was reported to produce monodisperse GNPs with low polydispersity ,43,44. IBasically, the chemical reaction rate drastically depends on temperature, so the high rate of nanoparticle formation can be decreased at a low temperature. In this work, the nanoparticle synthesis was therefore performed at 85°C and 70°C with a defined range of NaOH amount. It was found that the formation rate of GNPs slowed as expected at lower temperatures. The color of the colloids obtained at 85°C , 7.8 mM , and 9.0 mM (a high pH) were used to prepare reaction-time-dependent samples under different pH conditions. UV-vis spectra and photos  by the citrate reduction method via combined temperature and pH controls. The addition of a proper amount of sodium hydroxide can produce uniform GNPs with a narrow size distribution. The low reaction temperature is helpful to control the nanoparticle formation rate, and uniform GNPs can be obtained at different temperatures in presence of an optimized NaOH dosage. The pH analysis demonstrates that uniform GNPs can be obtained at around neutral conditions. The modified citrate reduction method can produce concentrated gold colloid dispersions and save more than 90% energy in the heating step. Such environmental-friendly synthesis method for gold nanoparticles may have a great potential in large-scale manufacturing to match the increasing commercial and industrial demands.The authors declare that they have no competing interests.CL and GW took the tasks of experimental, basic data collection, and the draft writing; DL gave his contributions on the experimental guidance and TEM observation, as well as the main paper organization; JX took some spectrometric works; and WH took the contributions on the research guidance, discussion, and paper modification.DL is a Ph.D. major in Physical Chemistry, Shandong University, China. He has focused his research interest on the gold nanomaterials especially on the polymer modified gold nanoparticles for more than 6 years from his postdoc careers in Institute of Chemistry, Chinese Academy of Sciences, China and in the Max-Planck Institute of Colloids and Interfaces, Germany. His published papers involved the core/shell nanostructures of the thermosensitive/pH-responsive polymer and amphiphilic polymer grafted gold nanoparticles toward multifunctional nanocarriers and nanosupports.Sample photos, supplementary TEM images, SPR peak changes and UV-vis spectra. Sample photos of concentrated GNPs prepared at different conditions, supplementary TEM images of a selected sample of aggregated Au colloids, SPR peak changes of gold colloids prepared after different reaction time, and the temporal changes of UV-vis spectra and photos in the formation process of GNPs.Click here for file