Pisum sativum L.) is an important grain legume consumed both as human food and animal feed. However, productivity in low rainfall regions can be significantly reduced by inferior soils containing high levels of boron and/or salinity. Furthermore, powdery mildew (PM) (Erysiphe pisi) disease also causes significant yield loss in warmer regions. Breeding for tolerance to these abiotic and biotic stresses are major aims for pea breeding programs and the application of molecular markers for these traits could greatly assist in developing improved germplasm at a faster rate. The current study reports the evaluation of a near diagnostic marker, PsMlo, associated with PM resistance and boron (B) tolerance as well as linked markers associated with salinity tolerance across a diverse set of pea germplasm. The PsMlo1 marker predicted the PM and B phenotypic responses with high levels of accuracy (>80%) across a wide range of field pea genotypes, hence offers the potential to be widely adapted in pea breeding programs. In contrast, linked markers for salinity tolerance were population specific; therefore, application of these markers would be suitable to relevant crosses within the program. Our results also suggest that there are possible new sources of salt tolerance present in field pea germplasm that could be further exploited.Field pea ( Pisum sativum L.) is an important grain legume consumed as an inexpensive source of proteins, complex carbohydrates, fiber, B group vitamins, folate and minerals such as calcium, iron and potassium. Peas contain up to 87% total digestible nutrients, which along with their low sodium and fat content   that can-1 in the soil will inhibit plant growth  reported . Four genotypes were sown per pot and the seedlings were thinned to three per genotype 1 week post-emergence. Plants were watered on every third day to 100% field capacity after weighing the pots. The screening experiment contained three replications within a randomized complete block design and was conducted in a polyhouse maintained at 24 ± 4°C with a 16/8 h (light/dark) photoperiod.Six seeds of each genotype were sown in 25 cm square non-draining pots filled with sandy loam soil pre-mixed with 10 mg kgSeven weeks after emergence, seedlings were assessed for B toxicity based on the percentage of leaf area affected per plant. A 1–6 scale was used where 1 had no leaf chlorosis or necrosis, 2 had ≤20% tip chlorosis and necrosis, 3 had 20–30% tip chlorosis and necrosis, 4 had 30–40% chlorosis and necrosis, 5 had 40–50% chlorosis and necrosis, and 6 had >50% chlorosis and necrosis of the total leaf area. A mean score was derived for all plants across all replications and genotypes were considered tolerant it they had a score of one or less or sensitive if the score was greater than 1.0.® potting mix in a glasshouse maintained at 20 ± 2°C, with a 16/8 h (light/dark) photoperiod. The potting mix consisted of 1,000 L of composted pine bark, 1 kg Floranid® N32, 1 kg 8–9 months Osmocote®, 1 kg 3–4 months Osmocote®, 225 g MicroMax® Complete, 225 g SP Quality® FeEDDHA Chelate (6% Fe), 30 kg agricultural lime, and 2 kg Saturaid®. Following the method of Three seeds were sown in 14 cm diameter pots filled with Bio GroSeedlings were assessed for resistance 2 weeks after the introduction of PM infected plants and scored using a 0–4 scale, where 0 had no mycelial growth or sporulation, 1 had <5% mycelial growth and sporulation, 2 had 5–25% mycelial growth and sporulation, 3 had 25–75% mycelial growth and sporulation, and 4 had >75% mycelial growth and sporulation on the plant. Plant scores were averaged for each genotype and considered resistant if they had a mean score of less than or equal to one and were susceptible if they scored greater than one.-1 calcium nitrate. After 10 days a salinity treatment mixed with the hydroponic nutrient solution was applied to all genotypes at an initial rate of 3 dS m-1 NaCl. Subsequently, every third day the concentration of NaCl was increased by 3 dS m-1 with each watering, up to 18 dS m-1, and maintained at this concentration until assessment. All solutions were applied at a rate of 300 ml per pot directly to the growing medium surface, avoiding contact with the leaves. The trial consisted of three replicates in a randomized complete block design. The salinity tolerance score and screening method were based on a visual growth response scale (1–10) as described by Salinity screening was conducted as described by Analysis of variance (ANOVA) was conducted on all phenotyping data using GENSTAT 14th edition for windows.® 96 Plant Kit  according to the manufacturer’s instructions. The concentration of DNA was confirmed using a spectrophotometer  at the two wavelength ratios of A260/230 and A260/280 nm.DNA was extracted from pooled leaf tissue samples harvested from up to three plants per genotype using the DNeasy® 5 software package . The PsMlo1 marker produced an allele size of 296 bp in B tolerant and PM resistant genotypes whereas the allele size of 294 bp was observed for sensitive/susceptible genotypes for both traits (A near-diagnostic marker (PsMlo1) identified for PM that was also closely linked to B tolerance  was asseh traits .TM genotyping chemistry  with some modifications. Primers were designed using 100 bp upstream and downstream flanking sequences around the target SNP variant position (Supplementary Table TM 2x Master Mix (LGC Genomics) and 0.14 μl KASPTM Primer Mix (LGC Genomics). The amplifications were performed in the SureCycler 8800 Thermal Cycler  using a touchdown PCR protocol following manufacturer’s instructions. End-point fluorescence values were determined by scanning the PCR plates in the FLUOstar Omega microplate reader  and the cluster plot analysis was completed in KlusterCallerTM software (LGC Genomics).SNP genotyping for the salt tolerance QTLs as described in P = 0.259). However, the severity of B toxicity on genotypes varied significantly (P < 0.001) from 1 (no symptoms) to 6 (>50% chlorotic and necrotic). Of the 171 genotypes tested for B tolerance, 58 (34%) were tolerant and 113 (66%) were sensitive to B. B tolerant genotypes showed no symptoms of toxicity throughout the 7 weeks of the experiment. The sensitive genotypes showed chlorosis and brown speckles on older leaves as typical symptoms of B toxicity .Plant responses within genotypes were generally consistent, although occasionally segregating. Individual genotypic responses under B treatment were uniform across the three replications and variance among the replications was non-significant (P = 0.762). The severity of PM infection varied significantly on genotypes (P < 0.001) from zero infection (no symptoms) to a highest mean score of 4 (>75% infection). Of the 171 genotypes tested for PM resistance, 61 genotypes (36%) were resistant and 110 (64%) were susceptible to PM disease with conidiospores present on both leaves and stems of the plants .Similar to B responses, plant responses to PM infection within genotypes were generally consistent, although occasionally segregating. Genotypes showed consistent response across the three replicates for PM disease . Fifty-six genotypes were tolerant to B and resistant to PM, and 108 genotypes were found to be sensitive/susceptible to both treatments. Of the 171 genotypes tested, only two genotypes (02-164-2 and 09HP300-10HO2-2) were tolerant to B but susceptible to PM, whereas five genotypes  were sensitive to B but resistant to PM. B and PM phenotyping showed a bimodal distribution with the majority at the extremes of the scoring range, although there were a number of individuals with intermediate scores . The intermediate category contained a total of 24 genotypes for PM resistance and 33 for B tolerance of which 10 were common.The phenotypic scores between B tolerance and PM resistance were highly correlated (Table 1).The results for the marker analysis were highly correlated to the phenotypic results. Of the 171 genotypes tested, marker predictions were accurate for both traits for 135 genotypes (79%). A further seven genotypes were predicted accurately for PM, but not for B, generating 83% correct predictions for PM. These seven genotypes also showed an uncoupling of the two traits (Table 2). Of the PM false positives, two genotypes were segregating for the PM trait and 9 for B tolerance (Tables 1 and 2). The two genotypes segregating for PM resistance were also segregating for B tolerance. All segregating genotypes were identified as breeding lines. Of the 29 genotypes where the marker was not predictive, only 11 were publicly released cultivars. Six of the false positive genotypes were breeding lines with Parafield in the pedigree, which was also a false positive (Table 2).A total of 26 genotypes were deemed false positives for both PM and B. These genotypes contained the resistance/tolerant allele but were susceptible/sensitive phenotypically. Furthermore, another three genotypes were false negatives for both traits, and contained the susceptible/sensitive marker allele and were phenotypically resistant/tolerant (P = 0.669). However, there was a significant (P < 0.001) variation among genotypes for salinity tolerance. Salinity phenotypes also showed a distribution with bimodal characteristics where the majority scored 2 or 9, although a number of individuals had intermediate scores . Based on the salinity symptom scores, genotypes such as Parafield, Yarrum, Collegian, and Dun showed tolerant responses with minor chlorosis on margins of lower leaves, whereas Kaspa, Maki and Bonzer were sensitive to the salinity treatment with severe chlorosis and necrosis on more than 50% of the plant.Plant responses within genotypes were generally consistent, although occasionally segregating. Individual genotypic responses in response to the salinity treatment were uniform across the three replications and variance among the replications was non-significant (Table 3). This indicates that the tolerance in Class 1 (and possibly Class 3) must be from different genetic sources.The genotypes were grouped into four classes according to the alleles from the two loci of additive effect with a total Vp of 31%. Class 1 were the null genotypes where markers indicated that they had the sensitive alleles for both QTLs. This class had a total of 64 genotypes, of which 36 were sensitive or moderately sensitive, while 28 were tolerant or moderately tolerant to salinity. Class 2 had the tolerant allele from the Ps III QTL only and consisted of four genotypes that were phenotypically sensitive. Class 3 had the tolerant allele from Ps VII QTL, and consisted of 69 genotypes. Of these 69 genotypes, 30 were tolerant or moderately tolerant, 39 were sensitive or moderately sensitive. Class 4 had both tolerant QTL alleles and contained 56 genotypes. Of these, 28 were categorized as tolerant or moderately tolerant, while 28 were sensitive or moderately sensitive. Each class contained approximately 50% of genotypes as sensitive and moderately sensitive and 50% as tolerant or moderately tolerant, irrespective of the number of tolerance alleles derived from Parafield . There were 35 genotypes of non-Parafield origin, and of these, only four (c. 11%) were tolerant to salinity, five (c. 14%) were moderately tolerant, four (c. 11%) were moderately sensitive, while the remaining 22 genotypes (c. 63%), were sensitive . Outside of Class 4 there were only three genotypes with Parafield in their pedigree and they belonged to Class 3, containing tolerant allele from Ps III QTL only.Further investigation of the genotypes within Class 4 showed that 21 had Parafield in their pedigree  with the remainder cultivars. However, the selected set of genotypes served the objective of comparison between diagnostic and closely linked markers for breeding applications as the full range of genotypic and phenotypic classes were observed.PsMlo gene in two resistant Australian field pea cultivars, Yarrum and ps1771 based on a 2-bp insertion in intron 11  between PM resistance and B tolerance to confirm these findings. The frequency distribution of PM resistance and B tolerance showed a bimodal pattern in the diverse set of pea germplasm, with few genotypes in the intermediate classes which confirms that both traits are under simple genetic control. Only a few genotypes (4%) of the entire pea set showed intermediate responses for resistance/tolerance to PM and B indicating that other sources of partial resistance/tolerance could be present in the germplasm.The PsMlo marker used in the current study is a SCAR marker that was developed from the genome sequencing of the full-length ntron 11 . The res 11  is widespread within the gene pool. Two additional loci conferring PM resistance in field pea have been reported; er2 locus on Ps III , although their presence or other unknown genetic factors would explain false negative phenotypes.In most scenarios, the PsMlo1 marker correctly predicted the PM and B phenotypic responses  across the pea germplasm set. However, a small proportion of genotypes were observed as false positives and false negatives for both traits. False positives are instances where the marker predicts the presence of the trait; however, this is not reflected in the phenotype. The most likely explanations for these inconsistencies would be a recombination event between the marker and the locus, or the presence of an alternative marker haplotype that is indistinguishable from the expected allele size. False negatives (when the marker predicts the absence of the trait but the phenotype is contradictory) are potentially more interesting as they may also represent a recombination event, or they may have a completely different source of resistance/tolerance available for exploitation by the breeding programs. The total number of false positives and negatives observed for B tolerance was higher compared to PM resistance which is expected as the marker was developed from the PM resistance gene, n Ps III  has effen Ps III  and Er3 . fulvum . HoweverThe number of false positives was higher than the false negatives for both traits, which indicates that these genotypes may have common ancestory. Pedigree relationships of the field pea genotypes were investigated, and 16 of the 26 PM false positives had two common ancestors, Gottschalks Viktoria and breeder line 25/1-22-BI-1. These genotypes were used in the early 1980s, but are no longer available to test if one of these may be the source of the false positive haplotype. There were seven other false positives and six were introductions from the South Australian provincial breeding program. Their pedigree information only goes back to parents or grandparents; however, four of them have common ancestors of Alma and Wirrega. In the 1980s, the provincial breeding programs commonly swapped germplasm so it is possible that the false positive donor was exchanged between breeding programs, or there could have been multiple recombination events. Finally, there was one false positive, 96-235*5, that had a unique pedigree that was solely derived from the cultivars Morgan and Bohatyr. Pedigree information indicated that there were a limited number of germplasm donors that has given rise to the 26 PM false positive genotypes within the diverse set of pea germplasm.In comparison to diagnostic markers for qualitative traits such as PM resistance and B tolerance, linked markers for quantitative traits such as salinity tolerance are much more challenging to validate across diverse germplasm. The markers for salinity tolerance used in the current study were developed from a mapping population of Kaspa × Parafield, in which Parafield, a moderately salt tolerant cultivar, contributed two QTLs explaining 12 and 19% of the phenotypic variation . A low pThe predictive power of the salinity tolerance markers was limited when tested across the diverse set of pea germplasm. This observation, however, is not surprising because these markers are associated with two QTLs of moderate effect in a bi-parental background and there could be greater potential for recombination. Furthermore, the effectiveness of these salinity markers was significantly improved for genotypes that have Parafield, the tolerance donor, in their pedigree. Within Class 4, 19 of the 21 Parafield derived genotypes (90%) were salt tolerant and had tolerant alleles from both QTLs. These results suggest that the markers have strong effects and worked reasonably well within the germplasm derived from Parafield. Similar responses of QTL linked markers have previously been shown in tomato where salinity tolerance QTLs worked well in offspring of populations from which they were originally developed . FurtherThe PsMlo1 marker validation across diverse germplasm demonstrates that it can be used for selection for B tolerance and PM resistance in new germplasm sources different from the one in which the marker association was originally found. The PsMlo1 marker offers potential to be widely adapted to identify PM resistant and B tolerant germplasm, making this marker suitable for implementation within the pea breeding program. The fact that a single marker is associated with both traits is an added advantage. Our results demonstrate that markers linked to salinity tolerance QTLs can be implemented in germplasm derived from Parafield. The presence of other sources of tolerance to salinity suggest that there is potential to exploit other uncharacterized QTLs in the germplasm. Breeding for higher salinity tolerance therefore would require multiple QTLs for introgression into existing donor varieties in pea breeding programs. In view of the quantitative nature of salinity, the Parafield loci may not be sufficient for complete tolerance hence several mapping populations are currently being evaluated for new sources of tolerance to salinity. In addition, other genomic assisted approaches such as genome wide association studies (GWAS) and genomic selection (GS) may offer alternative solutions to understand polygenic traits with smaller genetic effects including salinity tolerance.NC, AS, and SK contributed toward conception and interpretation of work. AL and PK organized peas germplasm for this work. SS and ML conducted genotyping work and data analysis. PK and MJ conducted phenotyping work and data analysis. GR, AS, SK and MJ drafted and critically revised the manuscript for important intellectual content and all authors approve final version of manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Compressive stimulation can modulate articular chondrocyte functions. Nevertheless, the relevant studies are not comprehensive. This is primarily due to the lack of cell culture apparatuses capable of conducting the experiments in a high throughput, precise, and cost-effective manner. To address the issue, we demonstrated the use of a perfusion microcell culture system to investigate the stimulating frequency  effect of compressive loading (20% and 40% strain) on the functions of articular chondrocytes. The system mainly integrates the functions of continuous culture medium perfusion and the generation of pneumatically-driven compressive stimulation in a high-throughput micro cell culture system. Results showed that the compressive stimulations explored did not have a significant impact on chondrocyte viability and proliferation. However, the metabolic activity of chondrocytes was significantly affected by the stimulating frequency at the higher compressive strain of 40% . Under the two compressive strains studied, the glycosaminoglycans (GAGs) synthesis was upregulated when the stimulating frequency was set at 1 Hz and 2 Hz. However, the stimulating frequencies explored had no influence on the collagen production. The results of this study provide useful fundamental insights that will be helpful for cartilage tissue engineering and cartilage rehabilitation. The biological function of cartilage to provide resistance to mechanical loading is primarily attributed to its abundant extracellular matrix (ECM), mainly consisting of glycosaminoglycans (GAGs) and collagen . The homc cells,  scaffoldby using  chondrogOne of the key technical hurdles to functional cartilage tissue engineering is the lack of fundamental understanding of the link between extracellular conditions and chondrocyte functions. Though several studies have been carried out to find out the relationship between articular chondrocyte and environment, the majority of them have focused on exploring the effect of biochemical factors on cellular functions . Physica in vivo loading by intra-articular contact [The effects of mechanical loading on articular chondrocytes are complex. Under physiological mechanical stimulation, the articular chondrocytes in cartilage are subjected to complicated physical events including hydrostatic pressure, tensile stress, shear stress, and compressive stress . Among t contact . Accordi contact  and dyna contact , 6–11. D contact . Numerou contact –10. Neve contact , 10, 13, contact . The lat contact .While tissue engineering scientists are making progress , they miμL), which is not only used to accommodate a cell/biomaterial scaffold for 3D cell culture but also to quantitatively define the volume of such sample loading. The overall sample-loading process was described previously [The microcell culture system is composed of 12 individual microbioreactors . At the bottom of each microbioreactor, there is a cylindrical cavity . In addition, the microcell culture platform was placed on the surface of a transparent indium-tin-oxide- (ITO-) based microheater chip to provide a stable thermal condition of 37 ± 0.2°C for cell culture . To pneu−1. The compressive strain (%) was then measured by the equation: x (the deformation length)/y (the height of 3D cell culture construct)∗100%. In this study, the operating stimulating frequencies  were manipulated through the programmable control circuit and EMVs. For the magnitude of compressive loading, it was modulated by tuning the magnitude of the pneumatic pressure exerted in the pneumatic chamber  through 7 cells mL−1. The mixture was subsequently loaded into the cylindrical cavity in the microbioreactors  for up to 5 days using the established experimental setup  supplemented with 10% foetal bovine serum , 2% antibiotic/antimycotic solution, and 50 μg mL−1 ascorbic acid was continuously perfused to the individual microbioreactors at a flow rate of 60 μL hr−1. After a 5-day perfusion cell culture under compressive stimulation, the cell viability was observed microscopically through fluorescent staining. In addition, both culture medium and 3D cell culture constructs were collected to evaluate the proliferative, metabolic, and biosynthetic activities of articular chondrocytes by measuring the cellular DNA, lactic acid, GAGs, and hydroxyproline levels, respectively.Primary articular chondrocytes were isolated from the femorotibial joint as previously described . The celreactors . After sal setup . In thisμL of the dye reagent containing 1 μM calcein and 2 μM ethidium homodimer-1, prepared according to the manufacturer's instruction, was loaded into each microbioreactor chamber. After 20 min incubation, the images of live (green) and dead (red) cells were captured using a confocal microscope . Cell viability was then quantified by counting the live (green) and dead (red) cells using a software program  [The viability of the articular chondrocytes in alginate hydrogel under different compressive stimulation was evaluated using a fluorescent dye kit . After cell culture, briefly, the remaining culture medium inside the microbioreactor chambers was removed. 10 PA, USA) . In addi−1 made from dissolving lactate sodium salt in deionised water (DI) water was used as standard.The lactic acid produced and released into culture medium was measured using a Lactate Reagent Kit  . The assμg and 1.25 μg was used as standard. Moreover, total collagen production was assayed as hydroxyproline content after hydrolysis. The hydroxyproline content of insoluble collagen was determined according to Stegemann and Stalder [A colorimetric reaction based on Farndale et al.  was used Stalder . In this Stalder .n = 9). One-way ANOVA analysis with a statistical significance level of 0.05 was used to examine the effects of different compressive stimulation on the proliferative, metabolic, and biosynthetic activities of chondrocytes after 5 days of culture. The Tukey Honestly Significant Difference (HSD) post hoc test was used to compare the differences between two compressive loading conditions when the null hypothesis of ANOVA analysis was rejected.Data from at least three separate experiments were analyzed and presented as mean ± standard deviation. For a given experiment, each condition was tested in triplicate  with the increase of the applied pneumatic pressure within the experimental conditions investigated  of pneumatic pressure input. This could accordingly lead to reduced PDMS membrane deformation and thus the lower level of compressive strain is generated. The experiments also found that the maximum frequency and strain that can be obtained in the system are 4 Hz and 50% stain, respectively. In the study, furthermore, a dynamic compressive stimulation was applied onto an articular chondrocytes-encapsulating 3D culture construct for 5 days. For a more accurate investigation, the long-term stability of the compressive loading was experimentally examined. This issue has been generally ignored in several studies based on the same pneumatically-driven membrane-based actuations [P > 0.05, ANOVA) during the 5 days operation under a given operating frequency tested. Furthermore, when the compressive strain generated under different operating frequencies were compared, they were measured to have no statistical difference  at each time point investigated during the 5-day operation. Similar results were also found at the higher compressive strain range of about 40%. Taken together, the above findings might indicate that the proposed mechanism for dynamic compressive loading generation was capable of providing a stable compressive loading during the cell culture period.From above equation, it can be observed that the deformation magnitude of a PDMS membrane is proportional to the magnitude of pneumatic pressure applied. These could reasonably explain the proportional relationship between the applied pneumatic pressure and the resultant compressive strain as observed in tuations , 26, 28.P > 0.05, ANOVA) among the experimental conditions tested. These outcomes were consistent with the previous findings showing that the cell viability of articular chondrocytes is not mechanically affected by the compressive strain being lower than 51.5% [ in vivo. In normal physiological conditions, a human articular cartilage is subjected to the compressive strain ranging from 20% to 30% in vivo [In this study, the primary articular chondrocytes cultured in 3D alginate hydrogel constructs were subjected to various compressive stimulation conditions  for up to 5 days. In addition, the dynamic compressive stimulation regimen was based on 3 consecutive cycles per day, (one cycle is defined as 1 hour of loading followed by 1 hour of relaxation), which was reported to be more physiologically relevant , 31. In an 51.5% . Articul in vivo , 33. In P > 0.05, ANOVA) between the control (no compressive stimulation) and the mechanical compression-treated cases, meaning that the stimulating frequency  and the magnitude  of compressive stimulation play no role in the proliferation of articular chondrocytes within the experimental conditions investigated. The articular chondrocytes, accounting for less than 5% of tissue volume, are the only cell species existing in articular cartilage. It has been reported that articular chondrocytes could not significantly proliferate when they are cultured in 3D culture environment, whereas their proliferative activity might increase while cultured in 2D monolayer format [Furthermore, the DNA content of cells was measured as an indicator of articular chondrocytes proliferation after 5 days of cell culture in this study. As shown in r format . The pher format , 9 or nor format .P < 0.05, ANOVA). The total lactic acid production of chondrocytes cultured under the stimulating frequency of 2 Hz (40% strain) was 18.77% statistically higher than that of the control case (P < 0.05). Nevertheless, the lactic acid production was not statistically different among the cells experiencing stimulating frequency lower than 2 Hz. The dynamic compression at the 40% strain and 2 Hz is close to the compressive condition that human articular cartilage experiences during high intense exercise [In addition, the metabolic activities of articular chondrocytes treated with various ranges of compressive loading were evaluated by measuring the lactic acid production. Regarding articular chondrocytes metabolism, anaerobic glycolysis is the major metabolic pathway , from whexercise , 36. The in vitro investigations revealed that the articular chondrocytes could rearrange their synthesis of ECM components [P < 0.05, ANOVA) both at the two strain magnitudes (20% and 40%) explored. In the ECM of articular cartilage, the GAGs are mainly responsible for the compressive stiffness of such tissue [P < 0.05 for 1 Hz (20% strain) and P < 0.01 for 2 Hz (20% strain) and 1 and 2 Hz (40% strain)) higher than that of the control case. Nevertheless, such phenomenon was not found among the cells experiencing the stimulating frequency lower than 0.5 Hz. The stimulating frequencies of 1 Hz and 2 Hz are about the frequency range of compressive loading that the human articular cartilages are subjected to during walking [P > 0.05, ANOVA) influence the total hydroxyproline production within the experimental conditions investigated.Under compressive stimulation, severalmponents , 5–11. Hmponents , 9. In th tissue , 37. It h tissue . For the walking  and runn walking  conditioAs a whole, the above findings demonstrated that the GAGs synthesis was mechanically upregulated while the collagen production was not affected when 3D cultured chondrocytes were subjected to dynamic compressive stimulation. Reports in literature have also revealed similar research outcomes. For example, some studies have disclosed that dynamic compressive or tensile loading might increase the GAGs biosynthesis of articular chondrocytes but these mechanical stimulations were found to have no positive effect on the synthesis of collagen , 21, 38.In this study, the stimulating frequency  effect of compressive loading (strain: 20 and 40%) on the cell viability and proliferation as well as the metabolic and biosynthetic activities of articular chondrocytes were investigated. It was found that the dynamic compressive loadings explored in this study did not have significant impact on the articular chondrocyte viability and proliferation, in which the cells kept the cell viability as high as 95%. As to the metabolic activity of articular chondrocytes, the frequency of compressive stimulation did have significant influence when the compressive strain was increased to 40%, in which the lactic acid production of cells treated with the stimulating frequency of 2 Hz (40% strain) was 18.77% statistically higher than that of the case without mechanical stimulation. This compressive loading is close to the condition that human articular cartilage experiences during high intense exercise. For the biosynthetic activities of articular chondrocytes, results showed that the GAGs synthesis was mechanically upregulated when the stimulating frequency of compressive loading was set at 1 Hz and 2 Hz. These are about the frequency range of compressive loading that the human articular cartilages subjected to during walking and running conditions, respectively. Nevertheless, the formats of compressive loading including stimulating magnitude and frequency did not have significant impact on the collagen production of articular chondrocytes within the experimental conditions explored. As a whole, the research findings above are found fundamentally important both for articular cartilage tissue engineering and articular cartilage rehabilitation.The video clip demonstrated the dynamic process of compressive loading  working onto the 3D cell culture constructs (light-yellow colored) within the microcell culture system .Click here for additional data file.


Xanthoria mandschurica was sampled from 21 sites and analyzed using inductively coupled plasma mass spectrometry (ICP-MS). The results show that 1) eight elements  are of atmospheric origin and are highly influenced by the atmospheric transportation from the North China Plain, as well as local mining activities, while 2) the remaining 22 elements are primarily of crustal origin, the concentration of which has been enhanced by local mining and quarrying activities. These results clearly validate the applicability of lichens in biomonitoring of atmospheric elemental deposition and demonstrate the spatial pattern for air pollution in the region.Air pollution is a major concern in China. Lichens are a useful biomonitor for atmospheric elemental deposition but have rarely been used in North China. The aim of this study was to investigate the atmospheric depositions of 30 trace elements  in a region of the Taihang Mountains, Hebei Province, China using lichens as biomonitors. Epilithic foliose lichen  The concentration of elements, particularly trace elements, in lichens has been used as an important parameter in biomonitoring of atmospheric deposition due to the direct correlation between trace element concentration in lichens and the environment132567891011X, NOX and dust) while the data are limited in quantity so as to preclude the determination of spatial trends of atmospheric pollutants. This is particularly true in the Taihang Mountains, where air quality has been influenced by the long-range transport of atmospheric pollutants and local anthropogenic emissions, but levels and spatial patterns of atmospheric trace metal deposition have not been understood. Lichens, therefore, particularly the common and widespread species, could be an alternative or complement to instrumental methods for this purpose2With rapid urbanization and industrialization in recent decades, air pollution associated with atmospheric metals has become an increasing concern in China, especially in the North China Plain where many large cities and industrial complexes are located. This concern has received national attention due to the adverse effects on human and ecosystem healthXanthoria mandschurica collected from 21 sites in Taihang Mountains (The objectives of the present study are 1) to identify the origin of trace elements in lichens and 2) to map the spatial patterns of atmospheric elemental deposition measured by lichen elemental composition. Element concentrations were measured in the lichen ountains . This coUCC) is a measure of the relative contribution of crustal input. EFUCC values > 5 may suggest a significant atmospheric contribution, whereas lower EFs could indicate a crustal originSR) is a measure of the relative contribution of SR input. Both of the EFs can take into consideration the effects of secondary pollution on lichen element composition from contaminated soils.Al was selected to calculate EFs according to UCC values, the elements can be classified into two groups G1 (EFUCC < 5) and G2  to identify the correlations among them. Eight clusters were distinguished at a Euclidean distance of 6.0 . Phosphoet al.SRs using HDT in order to present partial order relationsElements in a cluster are often positively inter-correlated , indicating that they had similar spatial patterns. For the sake of simplicity and brevity, we mapped spatial patterns at cluster scale by using a combination of the Hasse diagram technique (HDT) and GIS techniques described by Pirintsos The concentration patterns for the G1 elements are given in SR patterns for the G2 elements are given in SR pattern  show roughly similar patterns, characterized by higher concentrations near regions with intensive mineral mining activities, for instance, at Fuping (sites S9 to S17), Pingshan (S21) and Tangxian S11; . CorrelaThe mean concentrations for G2 elements in lichens are also higher than or at the upper range of most values found in the literature for different lichen species from diverse ecosystems5678910111215161718192021222324SR at sites S9, S10, S17 and S20 , where seven metals  are frequently mineralized in deep rock layers to varying extents and S20 . These p and S20  and, to  and S20 . This fina Plain . At sitenfluence .SR patterns . The climate is continental temperate monsoon, with a mean annual rainfall of approximately 550 mm. The prevailing winds are from the southeast in summer and northwest in winter. In addition to intensive agricultural cultivation and a well-developed road network supporting heavy traffic, the area is also characterized by intense industrial operations including mining, quarrying, road construction and cement production, etc. In China, this has led to severe air pollution in the form of aerosol smog, particulate storms and acid rain.The study area  lies in the Taihang Mountains in Hebei Province. The area covers approximately 8,000 kmXanthoria mandschurica was collected at each sampling site over the course of two weeks in October 2013. At each site, in an area of 1 km2, at least 50 individuals of X. mandschurica with diameters of 3–5 cm were randomly collected on rocks at a height of 0.5 m above ground level and 5 m from trees or shrubs (if present). SRs were randomly extracted using a hammer, while shallow soil (0–5 cm) and deep soil (20–50 cm) were extracted using a soil gauge. The composite samples were mixed to represent the average status of elemental composition in the site, and then preserved in a paper bag.Twenty-one sampling sites were selected at an interval of 15–20 km  accordinThe outermost part of the lichen thalli (1–1.5 cm) was detached and carefully cleaned for analysis. Because the outermost, 2–5 mm part of the lichen corresponds to parts of the thallus that are one year in age213 and H2O2 for lichens, and in a mixture of HNO3, HF, HCl, and HClO4 for both rock and soil samples. Thirty elements  were analyzed at Hebei Geological Laboratory by inductively coupled plasma mass spectrometry . The analytical quality of the results was evaluated against the following reference materials: the national reference materials of GBW07359, GBW07361, GBW07362 and GBW07365 for rocks; GBW07451, GBW07452 and GBW07457 for soils; and GBW10014 (cabbage), GBW10015 (spinach), GBW10052  and IAEA-336  for lichens. The measured element concentrations were within the confidence intervals of the certified/suggested value. Analytical precision was generally <5% as judged from frequent, duplicate analyses.200–300 mg of each sample were mineralized by microwaves in high pressure Teflon vessels in a mixture of HNOEF has been widely used in determining elemental sources in lichenshttp://folk.uio.no/ohammer/past/) using the Euclidean distance of the Z-score standardized variables to measure dissimilarities and Ward’s criterion to construct the hierarchical tree.A one-sample Kolmogorov-Smirnov test was conducted to test the normal distribution for each element. When the data were normally distributed, a Pearson correlation analysis was conducted between the elements of lichens and local SRs, top soils and deep soils; otherwise, a Spearman rank-order correlation analysis was performed. A CA of the lichen elemental concentration was performed using the software Past 3.10  for 3D visualization of digital elevation map (DEM) data that were derived from the ASTER Global Digital Elevation Model (ASTER GDEM) with a horizontal accuracy of 30 m and obtained from Geospatial Data Cloud (http://www.gscloud.cn/). Other maps  according to the manualThe map in her maps  and 4 weHow to cite this article: Liu, H.-J. et al. Use of the lichen Xanthoria mandschurica in monitoring atmospheric elemental deposition in the Taihang Mountains, Hebei, China. Sci. Rep.6, 23456; doi: 10.1038/srep23456 (2016).


Hepatitis E virus (HEV), the causative agent of hepatitis E, has been reported in a wide variety of animals, including birds, but little is known of HEV infection in pet birds in northwest China. The objective of the present investigation was to examine HEV seroprevalence in three species of pet birds, namely, Eurasian siskin, Oriental skylark, and black-tailed grosbeak from Gansu. Serum samples collected from 685 pet birds from August 2011 to September 2012 were examined independently for the presence of antibodies against HEV. A total of 59 (8.31%) pet birds were tested positive for HEV antibodies by the commercially available enzyme immunoassay kits. Of these, the seroprevalence was diverse in different species pet birds; the most frequent level was 10.83% (39/360) in Eurasian siskin, followed by 6.57% (19/289) in Oriental skylark, and 2.29% (1/36) in black-tailed grosbeak. Age and collecting region of pet birds were the main risk factors associated with HEV infection. The present study firstly revealed the seroprevalence of HEV infection in three species of pet birds in northwest China, which provided the baseline data for taking comprehensive countermeasures and measures for effectively preventing and controlling HEV infection in birds. Hepatitis E virus (HEV), a newfound pathogen belonging to a newly recognized family of RNA viruses (Hepeviridae) , is the Avian hepatitis E virus was first isolated from chickens with big liver and spleen disease or hepatitis-splenomegaly syndrome , 6. AviaCarduelis spinus), Oriental skylark (Alauda gulgula), and black-tailed grosbeak (Coccothraustes migratorius) from Gansu, for the first time, which would provide “baseline” information for assessing the effectiveness of strategies for control of avian HEV infection in pet birds in China.Avian HEV infection in domestic birds has been reported all over the world , 9, 10 aThis study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Pet birds of various species , from which serum samples were collected, were handled with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China. The market owners of pet birds had given permission for the collection of serum samples.The present study was conducted in Gansu (35°50′40.9′′N 103°27′7.5′′E), northwestern China, with an area of 454,000 square kilometres, and the overwhelming majority of its land is quite high . Gansu usually has a semiarid to arid, continental climate, with warm to hot summers and cold to very cold winters. A large proportion of the precipitation is delivered during the summer months.Blood samples were collected from 685 pet birds from 3 pet markets in 3 representative administrative divisions  in Gansu from August 2011 to September 2012. The samples were collected aimlessly from pet markets. Blood samples were collected from the wing vein of pet birds by a veterinary practitioner, taken to the laboratory, kept at room temperature for 2 h, and centrifuged at 3000 g for 10 min, and then clear serum was separated and stored at −20°C until being further tested. The information of animal husbandry practices was obtained from the pet bird market owners by questioning.A commercially available indirect enzyme linked immunosorbent assay (ELISA) kit  was used to examine serum circulating antibodies (CAbs) to avian HEV, according to the manufacturer's instructions . Negativy) of pet birds of different gender (x1), age group (x2), species (x3), and geographical location (x4) was analysed by χ2 test using SAS version 9.1 . In the multivariable regression analysis, each of these variables was included in the binary Logit model as an independent variable. The best model was judged by Fisher's scoring algorithm. All tests were two-sided, and values of P < 0.05 were considered statistically significant. Odds ratios (ORs) and their 95% confidence intervals (95% CIs) were estimated to explore the strength of the association between HEV seropositivity and the conditions investigated.The variation in HEV seroprevalence (P = 0.002), but the difference between male  and female  groups was not statistically significant   of the 685 examined bird serum samples by ELISA . Of thes= 0.416) .P < 0.001) (P < 0.05), but the difference between Eurasian siskin and Oriental skylark was not statistically significant (P = 0.059). The seroprevalence of different species from different locations ranged from 2.29%  (black-tailed grosbeak: Tianshui) to 15%  (Eurasian siskin: Lanzhou)  was significantly higher than that from Longnan  and Tianshui  (< 0.001) . MoreoveLanzhou) .Avian HEV infection has been isolated from the United States , 12, Can Chlamydia psittaci, and others [Pet birds and wild birds play an important role in the public health because they may serve as natural reservoirs for many causative agents, such as Newcastle disease, avian influenza,d others –20, and d others , which iIn the present study, we investigated the seroprevalence of avian HEV in three species of pet birds in Gansu. The overall avian HEV seroprevalence in pet birds was 8.61%, which was lower than that in chickens in Shandong 35.9%)  and Guan%  and GuP = 0.002); this result is probably due to the fact that the adult pet birds had more chance to contact with avian HEV compared to juvenile pet birds and increased the risk of infection. The more epidemiological surveys should be conducted to reveal the relationship between age of pet birds and the HEV infection.Previous studies have indicated that age was not a significant factor during HEV spread . HoweverIn China, a great number of pet birds are raised in a semifree range system, which enhances the opportunity of exposure to food or water contaminated with HEV when they gather together. No statistical difference in HEV seroprevalence was found between male pet birds  and the females  , probablThe diagnosis of avian HEV infection in the field should be done minimally by detecting the specific virus RNA from the chicken samples . Avian HIn summary, the present study revealed for the first time the seroprevalence of HEV infection in three species of pet birds in northwest China, which offered the baseline data for taking comprehensive countermeasures and measures for effectively preventing and controlling HEV infection in birds and we believe that this information will be significant for researching the hepatitis E virus (HEV) infection in birds in China.


It is estimated that 287,000 women worldwide die annually from pregnancy and childbirth-related conditions, and 6.9 million under-five children die each year, of which about 3 million are newborns. Most of these deaths occur in sub-Saharan Africa. The maternal health situation in Tanzania mainland and Zanzibar is similar to other sub-Saharan countries. This study assessed the availability, accessibility and quality of emergency obstetric care services and essential resources available for maternal and child health services in Zanzibar.From October and November 2012, a cross-sectional health facility survey was conducted in 79 health facilities in Zanzibar. The health facility tools developed by the Averting Maternal Death and Disability program were adapted for local use.Only 7.6 % of the health facilities qualified as functioning basic EmONC  facilities and 9 % were comprehensive EmONC facilities. Twenty-eight percent were partially performing basic EmONC and the remaining 55.7 % were not providing EmONC. Neonatal resuscitation was performed in 80 % of the hospitals and only 17.4 % of the other health facilities that were surveyed. Based on World Health Organisation (WHO) criteria, the study revealed a gap of 20 % for minimum provision of EmONC facilities per 500,000 population. The met need at national level  was only 33.1 % in the 12 months preceding the survey. The study found that there was limited availability of human resources in all visited health facilities, particularly for the higher cadres, as per Zanzibar minimum staff requirements.There is a need to strengthen human resource capacity at primary health facilities through training of health care providers to improve EmONC services, as well as provision of necessary equipment and supplies to reduce workload at the higher referral health facilities and increase geographic access. Despite the high priority given to maternal and child health, it is now clear that the realization of Millennium Development Goals (MDGs) by 2015 is unrealistic. Annually, it is estimated that 287,000 women worldwide die from pregnancy and childbirth-related conditions, and 6.9 million under-five children die each year, of which about 3 million are newborns. Most of these deaths occur in sub-Saharan Africa [The maternal health situation in Tanzania mainland and Zanzibar is similar to other sub-Saharan countries . A signiIn Tanzania, estimates of maternal mortality vary based on the sources, but all have wide confidence intervals. For example the Tanzania Demographic Health Survey, (TDHS)  estimateIn committing to MDGs 4 and 5, the Government agreed to reduce the under-five mortality rate by two-thirds and reduce the maternal mortality ratio by three-quarters, by 2015 from 1990 levels. Many countries have been embarking on Emergency Obstetric and Newborn Care (EmONC) needs assessments in order to obtain the data necessary to improve maternal health services , 19. As A cross-sectional health facility survey was conducted in Unguja and Pemba Islands of Zanzibar between October and November, 2012.The study involved public, private for profit, and non-governmental organization health facilities providing maternal and child health (MCH) services. There were 224 health facilities in Zanzibar at the time of the survey. Sixty four percent of these facilities were government owned; 34 % were privately owned, while 2 % were parastatal. The health sector in Zanzibar includes three levels of care and corresponding facilities as follows: a) Primary level: Health Care Units and Centres  b) Secondary level: District Hospitals c) Tertiary level: Mnazi Mmoja National Hospital. PHCUs provide Primary health care services, PHCU+ are selected to provide additional services such as delivery, dental, laboratory and pharmacy services. PHCCs provide the same services as PHCU+ with the addition of inpatient and X-ray services. District hospitals provide second line referral services, including basic surgery and the tertiary hospital  provides referral services.A measure of relative variance was applied to determine the sample number of health facilities required . All secAll higher secondary and tertiary health facilities (43 facilities) were included in the sample as they offer maternity health services. The random sampling procedure was applied to get the remaining sample of the first line PHCUs. The sampling frame included 100 PHCUs in all districts. STATA 12.0 software was used to conduct random sampling required sample size (38 of 100 PHCUs). Our initial sample was 80 health facilities; however, one health facility was dropped due to unavailability of staff to participate in the study.www.mailman.columbia.edu/research/averting-maternal-death-and-disability-amdd) at Columbia University, New York, USA, and United Nations (UN) Partners were adapted for local use. Methods included key informant interviews, observations, and data extraction. A workshop with the Zanzibar Ministry of Health (ZMOH) and other stakeholders was held to make the relevant adaptations to the Zanzibar context [The assessment methods and modules developed by the Averting Maternal Death and Disability (AMDD) program , were recruited, ensuring at least one midwife per team. All interviewers had completed at least secondary school education. Research assistants and supervisors were trained for three days to ensure that they understood the objectives of the survey, the study methods and the content of the modules. During the training, roles of supervisors, research assistants and other study team members were also explained. A training manual was prepared to enhance the effectiveness of training and was also used as a reference for data collectors during the survey.Teams were assigned to move around the districts ensuring data collection from all sampled facilities. With exception of hospitals, data collection required approximately one day per health facility. Supervisors contacted the health facility in-charge, assigned work to RAs, maintained continuous progress in data collection and reviewed the completed questionnaires for errors and omissions on a daily basis. Interviews were conducted with health facility in-charges or midwives at the PHCUs and PHCUs + and in-charges of the maternity wards, pharmacy, laboratory and human resource for PHCC and hospital levels.Data cleaning and consistency checks were conducted after the survey. Data were analyzed using STATA . Analyses were conducted in accordance with UN guidelines for monitoring obstetric services , includiThe World Health Organisation (WHO) handbook on monitoring emergency obstetric and neonatal care EmONC defines the signal functions as 1) administration of parenteral antibiotics, 2) administration of parenteral uterotonics, 3) administration of parenteral anticonvulsants; 4) manual removal of the placenta (MRP); 5) removal of retained products; 6) Assisted Vaginal Delivery (AVD); 7) neonatal resuscitation; 8) blood transfusion; and 9) obstetric surgery. The handbook classifies health facilities that have performed the first seven signal functions in the last three months as basic EmONC (BEmONC) facilities and those providing all nine signal functions are classified as comprehensive EmONC (CEmONC) facilities . FurtherThe availability of EmONC services was measured by determining the number of facilities that performed the complete set of required signal functions in the three-month period prior to the assessment. Only 7.6 % (6/79) of the visited health facilities qualified as fully functioning Basic Emergency Obstetric and Neonatal Care (BEmONC) facilities and 9 % (7/79) were comprehensive EmONC facilities. Twenty eight percent (22/79) of the visited health facilities were classified as partially performing BEmONC and 55.7 % (44/79) were considered non-EmONC facilities Table .Table 1FFigure The WHO handbook on monitoring emergency obstetric care recommends the minimum acceptable number of EmONC facilities as 5 EmONC with at least one CEmONC per 500,000 population . AccordiThe distribution of BEmONC and CEmONC varied across Zanzibar. Basic EmONC facilities were mainly available in North Pemba and South Pemba regions, as well as West Urban regions, as shown in Fig. Overall, 47 %  of all births occurred in EmONC health facilities. The proportion of health facility delivery was slightly higher in Unguja zone as compared to Pemba. The highest proportion of births in health facilities were noted in Chake Chake and South Unguja. South District had the highest proportion of births occurring in EmONC facilities followed by Chake Chake. North B District had a higher proportion of births occurring in non-EmONC facilities as compared to other districts. In the urban districts, women from the outside the catchment areas were often delivering at Mnazi Mmoja National Hospital (MMNH). There were few deliveries occurring in health facilities in Central and West districts and neither had functioning EmONC facilities.According to the WHO handbook on monitoring emergency obstetric care, ‘Met need’ estimates the proportion of all women with major direct obstetric complications who are treated in a health facility providing EmONC services . The intThis indicator calculates caesarean deliveries as a proportion of expected live births in the population in a specified period . It is aThis indicator provides an indication of quality of care in health facilities. According to the WHO handbook on monitoring emergency obstetric care the maximum acceptable level of CFR is less than one percent . Althougn = 553) in all health facilities visited in the 12 months preceding the survey. The district variation was noted for this indicator, the highest being in South and Urban districts with 5.8 % (n = 46) and 2.4 % (n = 377) respectively.Quality of intrapartum and newborn care was also assessed through facility-based perinatal mortality. The WHO handbook on monitoring emergency obstetric care defines the intrapartum and very early neonatal death rate as the proportion of births that result in a very early neonatal death or an intrapartum death (fresh stillbirth) in an EmONC facility. The numerator for this indicator is the sum of intrapartum and very early neonatal deaths within the first 24 h of life and the denominator was all deliveries in the facilities during the same period . The intHealth facilities’ in-charges were asked if the facilities provide obstetric and neonatal care services 24 h a day and 7 days a week. Of the visited health facilities, only 18 % (14/79) provide obstetric and neonatal care continuously on a daily basis.Reviews of three randomly selected Cesarean deliveries from birth registers performed in the last 12 months were conducted at seven health facilities offering Cesarean delivery services, with a total of 21 cases analyzed. Eighteen cesarean cases were classified in the register as emergency and two were elective. The main documented reasons for cesarean delivery were obstructed labour (4 cases), followed by fetal distress (3 cases). However, there was no documentation of the indications for most (14 cases) of the caesarean deliveries reviewed. With regard to the timely management, on average, 67 min elapsed from a decision to the actual performance of cesarean section. The minimum time taken was 30 min and the maximum was 86 min in Mwembeladu and district hospitals respectively. About half (10 cases) of cesarean deliveries were performed by obstetrician/gynecologists, mainly based in the district and private hospitals, while 7 cesareans were conducted by trained Assistant Medical Officers (AMOs) and 4 cases by Medical doctors (MDs). AMOs receive training on emergency obstetric and neonatal care during their pre-service training, but also undergo special three-month competency-based training on cesarean sections and provision of quality EmONC services, while MDs are trained during their pre-service study period and internship.The provision of health services depend to a large extent, on the availability of both human resources and properly equipped health facilities. The study found that there was limited supply of human resources in all visited health facilities, particularly for the higher cadres, in relation to Zanzibar minimum staff requirements For example, at MMNH there was a 50 % gap between the planned versus actual number of medical doctors. The met need for nurses (excluding midwives) was only 22.7 %. However, the planned need of midwives surpassed the required number in most of the visited health facilities. At district level, the human resource situation was similar to that in Mnazi Mmoja Hospital. Medical doctors and general surgeons were not available at any of the district hospitals where six are required by policy. Obstretrician-Gynecologists were only available 33.3 % of staffing establishment at the time of the survey. With regard to the availability of trained AMOs, the study found that only 10.0 % of the AMOs and 11.1 % of anesthesiologists were available at PHCCs. There was also insufficient numbers of the most important cadres to perform duties at PHCU+ level. At private hospitals the availability of human resources was quite similar with exception of greater availability of MDs and Anesthesiologists (MD).The survey team inspected the infrastructure of 35 health facilities offering delivery services to see if they were convenient for both staff and patients. Overall, 91 % (32/35) of the facilities had good infrastructure and nearly all had sufficient light source to perform tasks during the day and night. More than 85 % of health facilities’ toilets were in good condition and well-functioning. Water was available in 86 % (30/35) of the visited facilities offering delivery services. Privacy was well guaranteed with presence of curtains in 86 % (30/35) of facilities.With regard to communication, the study found that 30.4 % (24/79) of all the visited health facilities had a functional mobile phone owned by the facility. Every health facility had individual health providers who had personal, functional mobile phones and 78.5 % (62/79) of the staffs used their own mobile phones to refer patients to the next level of care.Oxytocics and prostaglandins drugs were available at 39 % (31/79) of all health facilities visited. Among those facilities reported to have oxytocics and prostaglandins, oxytocin was highly available at 90 % (28/31) followed by misoprostol at 71 % (22/31). Anticonvulsants drugs were available at 43 % (34/79) in visited health facilities.Overall, general equipment and supplies needed for labor and delivery services were available at 60 % (21/35) of facilities offering delivery services. Fetal stethoscope and kidney basins were equally available at 97.1 % (34/35). The supplies which were reasonably found in all health facilities offering delivery services included: IV cannulae 71.4 % (25/35), clinical oral thermometer 68.6 % (24/35), suture needles/suture materials 65.7 % (23/35) and examination table 77.1 % (27/35). Labor/delivery tables with and without stirrups were available at 45.7 % (16/35). Partographs were available at 51.4 % (18/35) while adult ventilator bags and mask were available at 25.7 % (9/35). Ultrasounds were available at 23 % (8/35).For every 500,000 population, the minimum acceptable level is five EmONC facilities with at least one of which provides comprehensive care . This stAlthough most facility-based deliveries are taking place in EmONC health facilities, few deliveries were noted in Central, North B and West districts. Furthermore, the study found that there was a high volume of deliveries in the urban district. These deliveries might have been conducted in lower health facilities like PHCU + s and PHCCs, hence reducing the workload at tertiary level. Human resource and infrastructural capacity would be needed to ensure quality delivery services at lower level health facilities. Unlike Unguja, Pemba has a relatively even distribution of EmONC facilities; however, most of these EmONC facilities are underutilized for delivery services. Specialists perform most of the caesarean sections as compared to the trained AMO’s. Yet, most of these Caesarean sections are performed in the district and private hospitals. Ideally, specialists at MMNH referral hospital should have attended most of the complicated cases but this was not the case. In comparison, to the mainland, 80 % of Caesarean sections are performed by trained AMO’s , 26.Met need for EmONC is another indicator that reflects a health system’s capacity to respond to women’s obstetric needs when complications develop . The metThe majority of the caesarean sections were for emergencies, which show that these occur at a large extent due to (assumed) poor monitoring of pregnancies, given the types of ‘expected’ common complications. In fact, the issue of delays corroborates these findings as specialists are few but also it seems that even AMOs are too few on the islands. Generally, human resource data found in the survey suggests that there is a gap of specialists in CEmONC health facilities in particular among the higher cadres.Although the WHO handbook on monitoring emergency obstetric care recommends 5 %–15 % of pregnancies are expected to require Cesarean delivery , what maThe maximum acceptable level of direct obstetric case fatality rate can sometimes be misinterpreted due to the lack of comparability among health facilities. For example, women with the most serious complications may be referred from the PHCCs and die at MMNH referral hospital, hence increasing the direct obstetric case fatality rate in the hospital. The high overall CFR suggests poor quality of care in the respective health facilities, which may include delayed and inappropriate referrals. Inadequate human resources, limited availability of equipment and supplies might have contributed to the higher CFR. The study did not capture specific reasons for the alarming high CFR in Wete, Mkoani and Chake Chake districts and deeper understanding of quality of care should therefore be sought.There was also district variation with regard to the intrapartum and very early neonatal deaths, with the highest rates in South, Wete and Urban districts. Poor monitoring of progress of labour and delay in management of complications may be at the root of many of these deaths. Manual vacuum aspiration/dilation and curettage, and AVD were the least performed signal functions in PHCU + s. This reflects both the need for training and improving utilization of services. Similar findings have been reported in other low-income countries , 18.This study has limitations and strengths that should be borne in mind. For instance, if the direct obstetric case fatality rate is acceptable, but EmONC coverage or met need is insufficient, the implication is that women who deliver in EmONC facilities are likely to survive but maternal death outside health facilities might still be common as only half of women deliver in facilities . Hence, Despite Zanzibar having met the minimum standards for EmONC availability per population, the met need is still quite low. With only 50 % delivering in health facilities it will be very hard to reach this without increase in utilization. Evidence about quality of care and availability of human resources and supplies, as well as geographic distribution, might explain why these numbers are still low. Yet it will be essential to also make sure that women seek care in order to make real change in this coverage. In order to improve quality of care it is important to consider other important issues like effective training, and monitor the success through supportive supervision and good leadership by senior physicians, which may also encourage women to use services. The use of evidence-based practices should highly be encouraged at all levels. Audit within medical institutions are needed to review patient charts and determine whether the indications for cesarean delivery are appropriate and to determine the causes behind the high case fatality rates found in this survey .AMDD, Averting Maternal Death and Disability; AMOs, Assistant Medical Officers; AVD, Assisted Vaginal Delivery; BEmONC, Basic Emergency Obstetric Neonatal Care; CeMONC, Comprehensive Emergency Obstetric Neonatal Care; CFR, case fatality rate; DANIDA, Danish International Development Agency; MCH, Maternal and child health; MD, Medical doctor; MDGs, Millennium Development Goals; MMNH, Mnazi Mmoja National Hospital; MRP, manual removal of the placenta; PHCC, Primary Health Care Centers; PHCUs, Primary Health Care Units; RA, Research Assistance; TDHS, Tanzania Demographic Health Survey; UN, United Nations; WHO, World Health Organisation; ZMOH, Zanzibar Ministry of Health.


The river Göta Älv is a source of freshwater for 0.7 million swedes. The river is subject to contamination from sewer systems discharge and runoff from agricultural lands. Climate models projects an increase in precipitation and heavy rainfall in this region. This study aimed to determine how daily rainfall causes variation in indicators of pathogen loads, to increase knowledge of variations in river water quality and discuss implications for risk management.Data covering 7 years of daily monitoring of river water turbidity and concentrations of E. coli, Clostridium and coliforms were obtained, and their short-term variations in relation with precipitation were analyzed with time series regression and non-linear distributed lag models. We studied how precipitation effects varied with season and compared different weather stations for predictive ability.Generally, the lowest raw water quality occurs 2 days after rainfall, with poor raw water quality continuing for several more days. A rainfall event of >15 mm/24-h  was associated with a three-fold higher concentration of E. coli and 30% higher turbidity levels (lag 2). Rainfall was associated with exponential increases in concentrations of indicator bacteria while the effect on turbidity attenuated with very heavy rainfall. Clear associations were also observed between consecutive days of wet weather and decreased water quality. The precipitation effect on increased levels of indicator bacteria was significant in all seasons.Rainfall elevates microbial risks year-round in this river and freshwater source and acts as the main driver of varying water quality. Heavy rainfall appears to be a better predictor of fecal pollution than water turbidity. An increase of wet weather and extreme events with climate change will lower river water quality even more, indicating greater challenges for drinking water producers, and suggesting better control of sources of pollution. Drinking water producers are responsible for providing safe drinking water. One challenge faced by producers is that water supplies, especially surface water sources, may experience temporal variations in water quality. Therefore, the raw water quality needs to be repeatedly tested to validate if the current water treatment technique is sufficient for producing safe and clean drinking water. A common indicator of water quality is turbidity, a measure of water cloudiness, which is relatively easy to quantify with optical devices and is regularly used as a first indicator of levels of microbial contamination. However, turbidity reflects the load of organic and inorganic particles, so additional water samples are needed for more precise analysis of organic contaminants. Density analyses of indicator bacteria, such as coliforms or Escherichia coli, may return a better estimate of levels of human pathogens. Because precise analyses of indicator bacteria are performed in laboratories, there is a delay between sampling and results being available, which is why turbidity monitoring and indicator bacteria samples often complement each other.Several studies have shown relationships with prior weather events, especially wet weather, and raw water quality parameters Within Sweden, the southwest is one of the regions with the highest annual precipitation, with an average of around 1 m per year, and climate change is projected to increase annual precipitation and heavy rainfall events. The river Göta Älv runs through this region, serving as a freshwater supply for 0.7 million people. The river is at risk of pathogen contamination from multiple sources, including runoff from agricultural land and point sources such as sewer system discharges With the use of time series analysis we aim to describe how daily rainfall influences raw water quality parameters in this important freshwater supply in Sweden. We focus on water quality parameters commonly used as indicators of pathogen contamination and study their long term variations and determine precipitation effects on short term variations. We analyze the distribution of lagged effects of precipitation and determine the extent to which there are seasonal effect modifications. Such results will increase knowledge of fluctuations in raw water quality, and provide a causal link behind health effects and the evidence base to develop guidance for risk evaluation of drinking water production.The river Göta Älv originates from Sweden's largest lake (Vänern) and runs 93 km to Kattegatt at the North Sea. Close to the sea, the river divides into a second branch (Nordre Älv), with Göta Älv continuing through the coastal city Gothenburg  . The Cit3/s and varies around 200 to 1000 m3/s. Hydropower stations in the upper region  can cause rapid changes in stream flow. The flow time from Vänern to the sea varies from 1.5 to 5 days, with an average of about 3 days. The rivers total descent is about 44 meters, where the majority takes place at the hydropower stations.The main river arm has an average flow rate of 550 m3/s, with about half the volume for Alelyckan and the other half for the reservoir. This river water intake is closed when information suggest the river water is inadequate for drinking water production. To maintain drinking water production at Alelyckan, the water in the tunnel is then taken back and the tunnel transports freshwater in the other direction. Closures of the river water intake are determined by analysis of indicator bacteria and turbidity levels, or high conductivity caused by inflows of seawater. Intake closures can also be based on events of heavy rainfall or information on upstream events indicating elevated contamination risks, such as releases of untreated sewage water. The river is the recipient for eight wastewater treatment plants upstream of the intake at Alelyckan.Five drinking water utilities use the river as a freshwater supply, distributing water to around 700,000 people. Two of these utilities, Alelyckan and Lackarebäck, are distributing drinking water to the population in Gothenburg. Lackarebäck takes water from a lake reservoir (Delsjön) that under normal conditions is continually supplied with river water through a 9 km tunnel designed to maintain a constant water level in the lake. The river water intake at Alelyckan is located close to the utility and takes about 2 mDespite the northern latitude of the study area, the climate is comparably very mild. The latitude provides distinct winter and summer seasons and daylight spans between 7–17 h/day. On average, February is the coldest month with daily mean temperatures a few degrees Celsius below zero. The river is usually not covered with ice during winter seasons. Precipitation is fairly constant throughout the seasons although the second half of the year generally experiences more and heavier rainfall events.3/s) measured near Lilla Edet were also provided. The time series data of water quality parameters are displayed in Concentrations of the indicator bacteria Escherichia coli (E. coli), Clostridium perfringens and coliforms, together with water turbidity, are routinely monitored to indicate the quality of the river water. Seven stations along the river monitor quality parameters with varying frequencies. The municipal water department in Gothenburg, Department of Sustainable Waste and Water, provided laboratory analyses of concentrations of indicator bacteria, sampled outside the river water intake to Alelyckan during the time period 2004–2010. This location had the highest rate of sampling of indicator bacteria for laboratory analysis, three times weekly, although more frequent sampling may be performed, particularly when high concentrations are detected. Laboratory results of coliforms and E. coli were reported in units of Most Probable Number (MPN), water samples analyzed with Coliert-18/Quanti-Tray (ISO 9308-2:2012), enumerated after an incubation time of 18 hours at 35°C. Clostridium concentrations were reported in Colony Forming Unit (CFU), enumerated after incubation at 44°C for 21±3 hours (ISO/CD 6461-2 2002-12-20). These samples were not preheated with intention to kill vegetal cells. The analytic methods of enumeration of indicator bacteria have been the same during the study period. Turbidity has been continuously monitored at the river water intake and we obtained daily mean values in Formazin Nephelometric Unit (FNU) during the same period (2004–2010). Daily data on stream flow  provided daily weather data. Daily precipitation data were available for three stations along the river: Gothenburg, (south station) the village of Alvhem situated about 30 km upstream from the water intake (middle station) , and theThe associations between daily precipitation and raw water quality were analyzed using time series regression. Raw water quality parameters were log-transformed  and generalized additive regression models (GAM) were fitted Daily mean values of river water turbidity ranged between 1.6 and 33.7 FNU with an overall mean of 7.0 FNU. The median value of E. coli was 96 MPN/100 mL and the highest concentration was enumerated to 2800 MPN/100 mL. Same statistics for coliforms and was 750 MPN/100 mL and 24000 MPN//100 mL. Although these maximum concentrations were detected in June, the best average river water quality occurred in spring and summer (April–July). In general, average water quality was lowest in the darker seasons, as for example maximum observation of turbidity in May was below the average value for November. Coliforms showed less elevated concentrations in December through March relatively the other water quality parameters, and also showed a long-term declining trend throughout the study period.Most precipitation was recorded at the middle station with an average daily precipitation of 3.1 mm, or 7.0 mm if only including days with precipitation, resulting in an average annual precipitation of 1138 mm. The average annual precipitation in the other stations was 872 mm/year (north) and 970 mm/year (south). The maximum daily precipitation was recorded at the south station (59.7 mm), which also had the highest total count of days with any observed precipitation. Short-term variations in all raw water quality parameters were highly associated with prior precipitation regardless which weather stations provided data, although the middle station had the best predictive ability and the north station second best. An event of heavy precipitation decreased water quality over several days, with the effect peak in general two days later. Precipitation effects were significant over numerous lags, and the lag structure on turbidity appeared less peaky and to be affected a few days longer compared to indicator bacteria.Precipitation events of 15 mm/24-h or more (n = 142) were associated with a three-fold increase in E. coli concentrations : 146–242%) two days later (lag 2) using a DLNM model. A similar model, with turbidity as outcome, estimated an average increase of 32% (CI: 27–37%) at lag 2. A DLNM model with precipitation as continuous predictor, and allowing for non-linear associations, fitted increased concentrations with increased amount of precipitation analyzing indicator bacteria, while the effect on turbidity attenuated with extreme rainfall. An event of 40 mm/24-h was estimated to increase E. coli concentrations two days later by 580% (CI: 443–754%), and an extreme event of 50 mm/24-h was associated with increased concentrations by 11 times . The precipitation effects at lag 2 are illustrated in Analyzing the effect of consecutive wet or dry weather days and raw water quality also exposed clear relationships. On average, consecutive wet weather for more than a week increased E. coli concentrations four-fold compared to one week of dry weather . As estimates for singular precipitation events, this precipitation predictor also indicated a lagged effect after wet weather days. Analysis of seasonal effects indicated that precipitation influenced concentrations of indicator bacteria all year around and seasonal effect modifications were quite moderate. The effect of precipitation across the seasons on turbidity appeared to be modified more than for indicator bacteria, with the smallest relative effect during summer. 2) for precipitation predictors logically indicated a similar seasonal pattern as the seasonal moving average of precipitation, but the variability differed between water quality parameters. Precipitation explained around 50 % of the variation in coliform concentrations in river water during fall, and decreased to around 20 % in winter and spring. When analyzing turbidity in summer periods the R2 values were low, especially when using precipitation data from the south station. R2 values of precipitation across season, from different weather stations, are displayed in The extent to which short-term variations in raw water parameters were explained with prior precipitation varied over the year. The coefficient of determination . The DLNM models were fit with natural cubic splines in lag space and used 6 df, with knots distributed at a log scale. DLNM models with continuous precipitation predictor were fitted with a natural-cubic spline in exposure space and according to AIC scores, the log transformed turbidity data was best fit with 3 df in space of precipitation, log-Clostridium was associated with precipitation using 2 df, whereas a linear association with precipitation was sufficient when analyzing log-concentrations of E. coli and coliforms. Decreased snow cover (Vänersborg) was related to a short-term decrease in raw water quality, most clear regarding turbidity and just about significant regarding coliforms. Including such predictor in the models did not influence effects or lag structure of precipitation, and was not included in final models. Daily mean temperature showed none, or only small effects, on short-term variation and were not included in final models. Turbidity data contained a week day pattern which could be linked to scheduled maintenance of the measuring device (3 times weekly); an average increase of ∼5% per day after filter cleaning was observed, and this effect was adjusted for in all turbidity models.When comparing estimates between DLNM models and unconstrained distributed lag models  we conclIn this large data study clear relationships between precipitation and decreased river water quality were uncovered. Regression models showed that concentrations of indicator bacteria, but not turbidity, increase exponentially with the amount of observed precipitation , which sWhen comparing the effect of precipitation across seasons, additional differences between turbidity and indicator bacteria were observed, with a weakened effect on turbidity during summer . This efThe water quality parameters also showed varying seasonal averages, which to some extent could be explained by prolonged dry or wet weather, but it's likely a result of a combination of factors which might differ with water quality parameter. The inactivation rate of E. coli increases with higher temperatures, but the increased concentrations during winters could also be a consequence of less sunlight intensity 3/s) at these events. DLNM models on coliforms and Clostridium showed also higher effects after events of extreme rainfall when using a more relaxed association with precipitation (3 df), compared to the models presented and suggested by AIC scores. This indicates that the presented estimated effects of precipitation on levels of Clostridium and coliforms are actually conservative at extreme events. Models on E. coli were stable independently of the flexibility parameter, and E. coli is also argued to be the best indicator of bacteriological quality of water As closures of the river water intake to Alelyckan are determined by concentrations of indicator bacteria (and other causes), the regression models can predict expected closures due to precipitation. Closure of intake due to E. coli counts is protocolled at 400 MPN/100 mL, which the DLNM model predicts to occur two days after precipitation events of 30 mm/24-h. The limit of accepted counts of coliforms are 7000 MPN/100 mL, which DLNM models predict to take place two days after events of 45 mm/24-h, while Clostridium counts of more than 50 CFU/100 mL are predicted to occur two days after events of 39 mm/24-h. These predictions use an average water quality as baseline (i.e. average concentrations are present before the precipitation event) and represent estimates of expected concentrations; peaks in indicator bacteria concentrations can be considerably higher and behave differently than suggested by the estimated lag structure. For example, some model outliers include the maximum observations of E. coli counts (2800 MPN/100 mL and 1400 MPN/100 mL) which were sampled the same day (lag 0) as two of the five heaviest precipitation observations (44.6 and 44.5 mm/24-h). This day had also a turbidity value below the overall mean value, and normal stream flows were registered . Top row shows observations of river water quality parameters measured near Alelyckan (Gothenburg). A: daily mean turbidity, B: E. coli, C: coliforms and F: Clostridium. Bottom row from left: E: maximum precipitation observation from the three weather stations, F: consecutive wet and dry (negative) days where a wet day was defined as any observed precipitation in any station, G: daily mean temperature observed in Gothenburg and H: stream flow measured in Lilla Edet.(TIFF)Click here for additional data file.Figure S2Precipitation effects. Relative effect of daily precipitation (0–54 mm) along 0–15 lags on raw water quality. A: turbidity, B: E. Coli, C: coliforms, D: Clostridium perfringens.(TIFF)Click here for additional data file.Figure S3Seasonal effect modifications. Relative effect of daily precipitation (>15 mm/24-h) along 0–8 lags on raw water quality across seasons. A: turbidity, B: E. Coli, C: coliforms, D: Clostridium perfringens.(TIF)Click here for additional data file.Figure S42-seasonality patterns.R Variation explained  across seasons by non-linear precipitation predictors 0–8 day prior observations of river water parameters A: turbidity, B: E. Coli, C: coliforms, D: Clostridium perfringens. Colors represent the three different precipitation stations and horizontal dotted lines represent the average R2.(TIFF)Click here for additional data file.Figure S5Stream flow effect modifications. Estimated relative effect on turbidity of a rainfall event of >15 mm along 0–15 lags days at different stream flows (quartiles). Vertical bars represent 95% CI. Vertical lines represent estimated effect peaks.(TIFF)Click here for additional data file.Figure S6Turbidity, precipitation and indicator bacteria. Associations  between turbidity and indicator bacteria (lag 0) and precipitation and indicator bacteria (lag 2) , together with model residuals (dots). Blue dotted lines represent mean levels, and red dotted lines represent unaccepted levels for open raw water intake at Alelyckan drinking water utility.(TIFF)Click here for additional data file.Table S1Monthly statistics of observations in river water during 2004–2010.(DOCX)Click here for additional data file.Text S1Statistical details.(DOCX)Click here for additional data file.


This compound is obtained by partial or total deacetylation of chitin in acidic solution. The chitosan-based hemostatic agents have been gaining much attention in the management of bleeding. The aim of this study was to evaluate in vitro hemagglutination activity of chitosan nanoparticles using human erythrocytes. The preparation of nanoparticles was achieved by ionotropic gelification technique followed by neutralization with NaOH 1 mol/L−1. The hemagglutination activity was performed on a solution of 2% erythrocytes (pH 7.4 on PBS) collected from five healthy volunteers. The hemolysis determination was made by spectrophotometric analysis. Chitosan nanoparticle solutions without NaOH addition changed the reddish colour of the wells into brown, suggesting an oxidative reaction of hemoglobin and possible cell lysis. All neutralized solutions of chitosan nanoparticles presented positive haemagglutination, without any change in reaction color. Chitosan nanoparticles presented hemolytic activity ranging from 186.20 to 223.12%, while neutralized solutions ranged from 2.56 to 72.54%, comparing to distilled water. Results highlight the need for development of new routes of synthesis of chitosan nanoparticles within human physiologic pH.Chitosan is a polysaccharide composed of randomly distributed chains of  The formation process occurs by partial or total deacetylation of chitin on acid solution. The grade of acetylation must be lower than 20% and molecular weight around 200 kDa. The acetylated remaining portions on chains are responsible for compound solubility [Polymers, including chitosan, eventually cause haemagglutination –3. Chitoβ-1- D-glucoslubility .Chitosan could be found on different forms, chain length, and grades of deacetylation. This diversity increases exponentially by chemical modifications which been evaluated . One of Inert chitosan seems to be nontoxic biopolymer. The binding properties of this polymer to diverse surfaces increase its usefulness and potential application in medical sciences . Recent Hemagglutinating capacity of chitosan turned a promised hemostatic agent on reduced pre and postoperatory bleeding. The indication should be to elective and urged situations when the managing of bleeding is a challenge. Since pharmaceutical products might react with red blood cells and induce some undesirable reactions such as hemolysis, this must be addressed when evaluating the biocompatibility of any material intended for in vivo use. Acute effects of the hemolytic reactions are the impairment of the oxidative stress, induction of hypertensive peaks, and nitric oxide depletion, whereas chronic effects are dysregulated iron metabolism in the liver, atherosclerosis, and renal failure . VariousThe production of chitosan nanoparticles was made using inotropic gelification of chitosan solution with TPP suggested by Calvo et al. . For thiμL/min) into chitosan solution over magnetic agitation, staying for more 15 minutes after dropping process. The dispersions were diluted on 0.4; 1; 1.6; and 3 mg/mL, and part of this volume was neutralized by addition of 1 mol/L−1 NaOH. Dispersed nanoparticles on solution 1 were named solution A and dispersed on solution 2 and were named solution B. Solubilization of chitosan on solutions 1 and 2 was satisfactory and had an increased opalescence of the solution after TPP dropping over magnetic agitation, suggesting nanoparticle formation.Adding, 0.2% of TPP was dropped , dried overnight in an oven at 27°C, and observed on transmission electron microscopy TecNai G2 Spirit TEM (FEI). The particles presented a medium size of 10 nm with a round shape .Chitosan nanoparticles were sterilized by direct exposition to UV radiation during 20 minutes. The effectiveness of this method was proved by direct microbiologic assays using BHI and Sabouraud Agar mediums to bacteria and fungi growth, respectively. The Petri dishes with chitosan nanoparticles were incubated on 37°C, for 24 h and 48 h, without any evidence of CFU formation for all tested samples.Five healthy subjects, with 18 to 40 years of age, without hemoglobinopathies and without relatives on the first grade with these diseases were recruited. Peripheral blood samples were obtained by venopuncture on heparinized tubes (BD Vaccutainer).After total blood collection, following a methodology described by Rao and Sharma , the samμL of this dilution was transferred to “U” bottom 96-well microplates. The inoculation was made at room temperature. The proportion of erythrocytes and reagents was 1 : 1 (v : v) and performed in duplicate.Hemagglutination assay was made according to Banerjee et al.  protocol−1 NaOH) and nonneutralized solutions 1 and 2, 1 mol/L−1 NaOH alone, saline solution (0.9% NaCl), phosphate buffer saline , and distilled water.Solutions A and B were evaluated as test groups following the previous concentrations, with and without pH neutralization. Comparative groups also tested were 0.2% TPP, neutralized  on 560 nm wavelength filter. Positive and negative controls were made by respective addiction of distilled water and physiologic solution to blood. The % of hemolysis was obtained using the following formula:Hemolytic activity was performed following Singhal and Alok  method. as SA, PA, and NA corresponded to sample absorbance, positive absorbance, and negative absorbance, respectively. Hartmann et al.  index waAs presented in −1 alone—without chitosan nanoparticles—presented a green-yellowish color. Distilled water induced the appearance of an opaque supernatant on incubated erythrocytes. The conditions of color change and medium opacity interfered the determination of the hemagglutination index of these samples.Interestingly, the samples incubated with A, B, 1 and 2 solutions prepared under acid pH, and samples incubated with NaOH 1 mol/LAfter determing the hemagglutination index, the supernants were transfered to other 96-well plates and subjected to spectrophotometric analysis (560 nm wavelenght). As observed in − and P3O5−10, the last being responsible for the binding with chitosan amino groups [In this study, chitosan nanoparticle solutions were produced by ionotropic gelification protocol. The ionic interaction between 1% acetic acid solubilized chitosan and TPP dissociated in distilled water generates some polyanions such as OHo groups .Synthesis of chitosan nanoparticles showed progressive opalescence after TPP dripping which suggests that small particles would be in the suspension . Also, tThe potential use of chitosan as an hemostatic agent has been demonstrated in literature. Previously, rabbit and human erythrocytes formed aggregates with disrupted shape when incubated with acetic acid solubilized chitosan particles, without the addition of TPP . Other s−1 presented a brownish colour, distinct from the standard redish colour typically observed when samples were incubated with chitosan nanoparticles in neutral pH. This variation in the superanatant's colour impaired the visual examination of the samples and suggested the occurrence of an hemoglobin oxidative phenomenon, consequently an hemolytic event [All the erythrocytes incubated with A and B solutions, solutions 1 and 2 diluted in acid pH, and NaOH 1 mol/Lic event . Hemolysic event . In ordeic event . The bro2Odistilled. This percentage of hemolysis would be considered insignificant to cause any side effect in humans [All tested solutions presented elevated hemolytic activity, except in neutralized B chitosan solution 0.4 mg/mL, at which hemolytic activity was calculated to be 2.56% of the values of samples incubated with Hn humans . When coThe other important issue to be discussed is that pH itself interferes with hemagglutination process by decreasing erythrocyte binding forces. Generally, better agglutination results were found when erythrocytes were incubated with solutions which pH ranged from 6.4 to 8.4—close to the physiological pH . DespiteMoreover, the deacetylation degree of chitosan particles must be considered when evaluating the physicochemical and biological properties of this polymer. The majority of the commercial chitosan polymers have average degree of deacetylation of 70–90% . Higher The strategy of simply acid neutralization by addition of a strong base would not be the ideal choice for chitosan nanoparticle production for human use. A more controlled strategy might be applied to maintain chitosan nanoparticle stability and human biocompatibility. Authors have adopted the ultracentrifugation technique to diminish the residual amount of acetic acid and other solvents in shrimp-derived chitosan nanoparticle synthesis . AlthougThe ionotropic gelification method was efficient and affordable enough for the synthesis of large volumes of chitosan nanoparticle solutions. The pH neutralization of these solutions induced higher hemagglutination index and lower hemolytic activity in human erythrocytes than chitosan nanoparticles conventionally synthetized under acid pH. Noteworthy, the development of new routes for the production of large volumes of chitosan nanoparticles is needed to achieve clinical applications of these solutions in a neutral pH compatible with human blood cells and tissues.