Bacillus amyloliquefaciens strain H57 (H57). Scanning electron microscopy of ileal mucosa revealed significant villus damage, including focal erosions of epithelial cells and villous atrophy, while transmission electron microscopy demonstrated severe enterocyte damage and loss of cellular integrity in NE-exposed birds. In particular, mitochondria were morphologically altered, appearing irregular in shape or swollen, and containing electron-lucent regions of matrix and damaged cristae. Apical junctional complexes between adjacent enterocytes were significantly shorter, and the adherens junction was saccular, suggesting loss of epithelial integrity in NE birds. Segmented filamentous bacteria attached to villi, which play an important role in intestinal immunity, were more numerous in birds exposed to NE. The results suggest that mitochondrial damage may be an important initiator of NE pathogenesis, while H57 maintains epithelium and improves the integrity of intestinal mucosa. Potential actions of H57 are discussed that further define the mechanisms responsible for probiotic bacteria’s role in maintaining gut health.Epithelial damage and loss of barrier integrity occur following intestinal infections in humans and animals. Gut health was evaluated by electron microscopy in an avian model that exposed birds to subclinical necrotic enteritis (NE) and fed them a diet supplemented with the probiotic  The intestinal mucosa is covered by mucus, a complex hydrated gel that protects epithelial cells from chemical, enzymatic, microbial, and mechanical damage. The epithelium and its mucus layer permit the selective movement of ions, nutrients and water, but restrict the translocation of microbes and toxins from the lumen2. In particular, intercellular junctional complexes, which are specialized regions of contact between the plasma membranes of epithelial adjacent cells, are crucial for normal intestinal epithelium functioning3. Electron microscopy demonstrates that intercellular junctional complexes have four major components: the tight junction (TJ) or zonula occludens, the adherens junction (AJ) or zonula adherens, the desmosome (DS) or macula adherens, and gap junctions (GJ), or macula communicans (nexus). The TJ and AJ constitute the apical junctional complex (AJC)—the primary structure that regulates the intestinal barrier, and which is linked to the actin cytoskeleton. The DS and gap junctions are involved in cell–cell adhesion, and intracellular communication, respectively4. Figure 5.As in mammals, the intestinal epithelium in birds is a single layer of cells, comprising columnar absorptive cells (enterocytes), goblet and enteroendocrine cells, and various immune cells. The intestinal mucosa projects into the gut lumen via villi, which are composed of an epithelial layer, a core of underlying lamina propria (containing the microvasculature) and a thin layer of smooth muscle (muscularis mucosae). Each villus serves as a functional absorptive unit6. Ultrastructural modifications of intestinal mucosa occur in response to dietary changes7, including the presence of antigens in the diet, and changes in the composition of the gut bacterial community8. Ultrastructural examination of the epithelium, in particular enterocyte cells, and their constituents, such as mitochondria, lysosomes, endoplasmic reticulum, tight junctions and microvilli, can reveal the state of intestinal health. Other cells and molecules of the epithelial barrier unit include immune competent cells, i.e. intraepithelial lymphocytes (IEL) and tissue-related immune cells, and their products, cytokines and antimicrobial proteins. Moreover, mucus, and mucus-linked specific commensal bacteria, play important roles in the regulation of intestinal immunity and physiology9.An intact epithelial barrier is essential for maintaining gut homeostasis in both humans and animals10. Birds with subclinical NE do not display clinical signs of disease, but can experience malabsorption and poor growth11. Partial or complete damage of microvilli and enterocyte necrosis has been reported during spontaneous NE caused by Clostridium perfringens (Cp)12. Enteric pathogens can also change the morphology and functionality of tight junctions15. In mice, intestinal infections cause disruptions of mitochondrial morphology and activity16. Similar changes are likely to occur during subclinical avian NE, however there have been limited reports of intestinal ultrastructure changes in NE disease17. Kaldhusdal et al.12 reported vesiculation and blebbing of the luminal cell membrane, and degenerative changes of cytoplasmic organelles, while Olkowski et al.17 observed disruption and fragmentation of intercellular junctional complexes, and increased cytoplasmic vacuolization, both features of ongoing necrosis.Disruption of the epithelium and disintegration of villi occur following enteric infections, and necrotic enteritis (NE) is an example of an infection disease that can cause severe damage to the small intestinal mucosa in chickens20. The most commonly used model for bacterial intestinal infections in broiler chicks is the NE challenge disease model, and, for the current study, a subclinical NE model was employed. Our recent research demonstrated significant beneficial effects on intestinal mucosa maintenance and improvement of feed efficiency in a subclinical NE disease model in chickens supplemented with the probiotic Bacillus amyloliquefaciens strain H57 (H57)21. For the present study, electron microscopy was employed to evaluate intestinal villus ultrastructure and epithelium integrity of 21 day-old chickens exposed to NE and fed the probiotic H57. The aim was to gain greater insight into the pathogenesis of NE, and the mode of action of the H57 on maintaining intestinal mucosa integrity and function.Probiotic supplementation has shown promising results against various enteric pathogens in humans and animals, including prevention and treatment of necrotizing enterocolitis (NEC) in premature infants. In NEC, probiotics enhance the mucosal barrier by increasing the production of mucus, inhibiting bacterial translocation, and strengthening tight junctionsEimeria and Cp  displayed mild focal intestinal lesions typical of NE on post-mortem examination, confirming subclinical NE disease . Figure p < 0.001) in NE birds (graded as 2.6 degrees) when compared with all other treatments  compared with other groups, including controls  than NE-challenged birds (56/100 µM2), similar to control chicks.Enterocytes and other epithelial cells, including goblet cells, IEL, heterophils, and M-cells were identified in all samples Fig. a–g. Entetes Fig. g, electrtes Fig. d, were ates Fig. a,b. In Ntes Fig. g. Enterools Fig. c. Birds p < 0.05) microvilli (approx. 1.6 µM) than all other treatments (approx. 1–1.2 µM)  Fig. d. IntereµM) Fig. b’,c’.p < 0.05), and H57 and NE & H57 treatments . Dilatations within the AJC were evident in the AJ and DS regions  was found in NE and NE & H57 birds, compared with control and H57 birds.Change in AJC length and morphology were investigated with TEM, and data are presented in Table ons Fig. f’. TJ wap < 0.05) in NE-challenged, NE & H57, and H57 birds in comparison with control birds , than in NE-challenged birds  is known to produce extracellular enzymes27 for metabolic support, which could have initiated the slight increase in villi density.The findings of the current study provide new insights into the pathogenesis of subclinical NE and effects of probiotic H57 on intestinal health. When compared to NE birds that experienced epithelial damage and necrosis Figs. b,b’,c,c’rds Fig. b, whereards Fig. a. Birds 28, and it can affect scattered single villi, or one or more villi clusters, or may affect all the villi in a section. Eimeria meronts are known to pass through the microvilli membrane to re-infect more enterocytes and alter villus dynamics29. However, in NE, enterocyte lysis by coccidia enables Cp access to binding sites for colonization, and also provides cell debris for bacterial nutrition30. Changes in apical enterocytes may be induced directly by the effect of toxins, and or indirectly due to villus ischemia and subsequent coagulative necrosis28. In the present study, damage to villus tips or the entire villus in chicks exposed to subclinical NE was observed. It should be noted that our samples were collected on day 21, therefore changes to epithelium and lamina propria were not detected and are not discussed here.SEM and TEM confirmed gross necropsy observations of mucosal damage in NE birds Fig. b–d. Ther33. Mitochondria are dynamic organelles that undergo structural alterations to meet changing needs of epithelial cells and to maintain gut homeostasis34. Enterocyte mitochondria are involved in the regulation of numerous aspects of cellular activity, including the regulation of gut functions such as intestinal barrier integrity and mucosal immune responses37. Damage to this organelle will result in decreased energy production and eventually cell death36.Our findings suggest that morphological and density changes in mitochondria may potentially lead to cellular necrosis and lesions in subclinical NE disease. Maintenance of the epithelial barrier is an energy-dependent process, thus in the case of swollen, irregular, vacuolated, or cristae damaged mitochondria (as in NE birds), loss of adenosine triphosphate (ATP) generation and release of oxygen radicals in enterocytes may have occurred. Subsequently, apoptotic necrosis of enterocytes and impaired energy metabolism of epithelial cells may induce a variety of insults, including spread of infection39. In contrast, we think that enterocyte mitochondrial damage during NE is a prerequisite for subsequent cell death and necrosis, and suggest that NE instead could be an intestinal disease associated with mitochondrial damage and energy deficiency. Mitochondrial damage has not been reported previously for subclinical NE, but it has been argued for bacterial enterotoxins33. Loss of epithelial barrier integrity, epithelial cell apoptosis, and bacterial invasion have been demonstrated following mitochondrial dysfunction36. Degenerative changes and mitochondrial dysfunction, including oxidative stress and impaired ATP production, are found in the intestines of patients with inflammatory bowel disease (IBD)41. The direct targeting of mitochondria has been reported for toxins belonging to the clostridial toxin family42. After having gained access to the cytosol, Clostridium difficile toxin B interacts with mitochondria influencing the ATP-sensitive mitochondrial K+ channels43, and subsequently causing mitochondrial swelling, vacuolization and damage.Previous investigators had suggested that the primary morphological changes in NE commence at the basement membrane of epithelium, (i.e. proteolytic damage within the lamina propria), and progress towards the center of the villus44. Enterocytes are more than just an absorbing cell, they respond to antigens in the gastrointestinal tract45, and have a pivotal role in the cross-talk between the IEL and luminal agents46. In response to injury, enterocytes undergo apoptosis and are continuously renewed by stem cells, however in the case of deregulated epithelial replacement, a small physical opening in the intestinal barrier allows bacterial translocation, and local inflammation47. In the current study, enterocyte alteration (enterocyte damage and necrosis) and vacuolization of cytoplasm and organelles were found in NE birds. Multivesicular bodies were also present, confirming cellular paranecrosis and subsequent necrosis in these birds  within enterocytes and intraepithelial immune cells found between enterocytes  suggest that both mitochondrial dysfunction and increased gut permeability affect the overall competence of the intestinal epithelial barrier49, but the stimuli that initiate either process is not known. However, in the current study, enterocytes of NE & H57 birds were intact and with normal-appearing cytoplasm and mitochondria , , Fig. d, and, ip), Fig. c’. Lengt59. The paracellular barrier in healthy intestinal tissue is characterized by high expression levels of TJ proteins and a low paracellular permeability (or a tight epithelium), while mucosal inflammation is frequently associated with decreased expression of junctional proteins (or a leaky epithelium)61. Numerous bacteria have been implicated in altering TJ, in particular Cp species. They use a potent enterotoxin that binds to two members of claudins, making them unstable13. Recent human studies have indicated both downregulation and upregulation of TJ proteins, in particular claudins, in patients with IBD63. Nevertheless, the role of these proteins in modulating TJ and epithelial barrier function is not fully understood.Recent studies have suggested a strong association between the junctional complex and intestinal health4. Our study suggests that dietary addition of H57 in NE-challenged birds maintains the normal morphology of AJC (AJC were significantly longer and less saccular in NE & H57 birds compared with NE birds), and subsequently reduces intestinal damage. In NE birds, the length of AJC was decreased, presumably due to a lack of structural components (proteins) or decreased recruitment of AJ or TJ proteins. Altered or reduced expression of such proteins could favor the influx of luminal antigens and consequently cause inflammatory damage60. Signaling molecules and extracellular stimuli such as cytokines and nutrients regulate the AJC64, while pathogens target structural and regulatory components of the AJC66. Actin, as a part of the cytoskeleton, connects AJC into an integrated network. Numerous bacterial toxins recognize the actin cytoskeleton as a target, as microbes utilize the host cell cytoskeleton for many activities, such as attachment, entry, movement within and between cells, vacuole formation, and avoidance of phagocytosis69. There are toxins (including clostridia toxins) that modify the actin cytoskeleton and alter the function of the junctional complex, thus causing cell lysis and absorption of toxins by the vascular system70. As mentioned above, we did not find morphological changes in TJ, and agree with Jou et al.71 who demonstrated increased paracellular permeability without disruption of TJ protein organization. Another study, however, showed that increased paracellular permeability was associated with significant redistribution of the TJ proteins72. In the current study, we identified differences in actin filaments condensation in the AJ zone by TEM. There is normally little actin condensation evident at the TJ and AJ area73 as actin networks are in a dynamic steady state74. Actin condensation within the host cell occurs most probably due to bacterial-induced host cell signaling directly beneath the groups of adherent bacteria76. Our preliminary data demonstrate that exposure to NE triggers actin condensation, while in NE & H57 birds, the accumulation of actin was reduced. Clostridium toxins prevent actin filament polymerization (elongation), resulting in complete depolymerization of cellular actin and potentially cell death77.In this paper, we demonstrate ultrastructural changes of AJC in a subclinical NE avian model. No morphological changes in TJ were found, however the widening (sacculation) of the AJC (in the AJ and DS parts) was significant Table . AJ and 78  for the generation of T helper 17 (Th17) cell responses83. SFB are found on ileal mucosa, and stimulate Th17, which are capable of producing IL-17 cytokines with a proinflammatory role in the mucosal (IgA) immune response generation84.A fascinating aspect of this study was the appearance and evaluation of the abundance of segmented filamentous bacteria (SFB). There were more SFB attached to villi in birds exposed to NE, NE & H57 and H57 compared with control birds Fig. . SFB are78 Fig. d,d’ that86. There is evidence that probiotics have anti-inflammatory effects, as they downregulate IL-17 production and other proinflammatory Th17-secreted cytokines88. The IL-17 is beneficial in controlling dysbiosis in the gut, but may be harmful if dysregulated, therefore in the case of overgrown SFB, accumulation of Th17 cells in the ileum could lead to the damaging inflammatory effects89 as seen in NE birds. Chickens exposed to stressful condition that predisposes them to NE can be offered diets supplemented with probiotics, as an approach for limiting SFB expansion and the Th17-associated proinflammatory response. Probiotics (including H57 from this study) tend to induce a regulatory response in the context of inflammatory and some autoimmune diseases90.In this study, chicks feed H57, with and without NE disease, had an increased abundance of SFB, when compared to control birds, however the increase in abundance was significantly less than in NE birds. It appears that H57 downregulated SFB abundance in birds exposed to NE. Probiotics favoring SFB could therefore have an effect in regulating the immune response in the gutEimeria and Cp). When birds were exposed to NE, there was mucosal damage, and enterocyte cytoplasmic alteration and necrosis. In particular, mitochondrial morphology and density were impaired. In contrast, birds challenged with NE and fed the probiotic displayed intact villi with normal enterocyte morphology and well-maintained mitochondria. Our findings identify new ultrastructural features involved in the pathogenesis of NE, and unravel some mechanisms of probiotic action, including effects on mitochondria morphology and regulation of SFB abundance on the ileal mucosa. In NE-challenged birds exposed to the probiotic, colonization of the intestinal mucosa with SFB appears to improve their response to subsequent bacterial infection. Since the integrity of epithelial cells is energy-dependent, mitochondrial function is undoubtedly crucial to the maintenance of intestinal physiology. Therefore the role of mitochondria in gut health, in particular, enterocyte mitochondrial bioenergetics during intestinal infections needs to be further explored.Our data highlight the potential role of the probiotic H57 in improving epithelial cell maintenance and integrity of the ileal mucosa during subclinical infection with NE disease . All the experiments complied with ARRIVE guidelines93. The room was located in the Poultry Research Facility on Gatton Campus (University of Queensland), and was thoroughly cleaned and disinfected prior to bird placement. One hundred and ninety two chicks were individually weighed, and randomly placed in 24 cages (8 birds/cage), at a stocking density of 13 birds/m2. Chicks were fed an all-phase wheat-soybean-based mash diet  from day-old until the end of the experiment (day 21). The basal diet was not supplemented with any antimicrobial growth promoters or coccidiostatics, and did not contain any other recognized substances with antibacterial properties. Feed and water was supplied ad libitum, except on treatment days, which required birds to have restricted water and feed supply (days 9 and 14). Strict biosecurity management practices were followed to prevent cross contamination between control and treated birds.Day-old male broiler chicks (Ross 308) were obtained from a commercial breeder . Birds were vaccinated against Marek’s disease, infectious bronchitis, and Newcastle disease at the hatchery. From day one, chicks were kept in an isolated, temperature-controlled room, in which the brooding temperature and a lighting regimen were as recommended by the breeding company8 CFU/g feed  and was fed to groups 3 and 4 throughout the experiment. Shini et al.21 has detailed the NE challenge, to which groups 2 and 3 were exposed. Briefly, birds challenged with NE were exposed to a coinfection with a commercially available anti-coccidiosis vaccine containing 4 strains of Eimeria spp.  at a concentration of 1.6 × 104 oocysts/mL and freshly prepared broth culture material containing a Cp suspension (1.76 × 108 CFU/mL). On day 9 post-hatch, chicks were exposed to Eimeria vaccine  via drinking water, and 5 days later to a virulent Cp strain EHE-NE18  mixed in feed at a ratio 1: 1.5 (vol/wt).Table Immediately, after euthanasia, a 2-cm length of the ileal tissue (middle part), was rinsed with cold Dubleccos Phosphate Buffered Saline (DPBS) and then cut into very thin strips (1 mm × 2 mm). The tissue was fixed in 3% glutaraldehyde in 0.1 M phosphate buffer 7.4 at 4ºC. Subsequent processing was conducted using a microwave processor. For scanning electron microscopy (SEM), specimens were post-fixed in osmium tetroxide, dehydrated in ethanol, and critical point dried using an Autosamdri-815 series point dryer , sputter coated with gold (using a SPI module sputter coater) prior to viewing with a Neoscope JCM-5000 SEM . For TEM, tissue was post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Epon resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a JEM1011  microscope operating at 80 kV.94, two observers took all SEM and TEM measurements independently. SEM images were employed to observe and measure changes on density and conditions of the villi. The density was determined utilizing squares (500 × 500 µM) overlaid on sets of photomicrographs. Adjustments for the scale were made, and Fig. 2. If villi were touching the four perimeter sides of a box, either the two outer sides with villi or two inner sides were counted. The same images were used to evaluate apical damage of villi. The degree of villus damage was graded using a scale developed by Gomide Junior et al.95, and modified for our needs ; low density ( +), when less than five SFB attached to a villus were counted; moderate density (+ +), when between six and fifty SFB were found; and high density (+ + +), when > 50 SFB were counted ds Table . To asseds Table a. The ab2 of cell area.TEM images were used to assess the morphology of epithelial cells, in particular enterocytes and their content and features, such as cytoplasmic organelles (mainly mitochondria), microvilli and the AJC. The morphology and abundance of enterocyte mitochondria was assessed from photomicrographs of six enterocytes/sample or 36/treatment. Enterocytes with regular (uninterrupted) microvilli were evaluated in all occasions Fig. a. Six in22 to maintain consistency in measurements, length of AJC was taken from the apical membrane, where the TJ began and included not only TJ and AJ, but also DS  was determined. As suggested by Karcher and Applegate DS Fig. . Only ce DS Fig. b. Theref DS Fig. a was als DS Fig. c,f, M-ce DS Fig. d, hetero DS Fig. h and int DS Fig. i.p < 0.05 were considered significant. Level of significance is presented in relevant tables and figures. For statistical analyses, the SFB abundance was recorded as categorical data, thereafter transformed into numbers using an encoding technique in Minitab.Data were analyzed using MiniTab 17 software. Comparisons between treatments were made with the one-way ANOVA procedure and Tukey’s multiple comparison test. Differences with


Powders made using hydrolysed proteins were classified as Type III powders (n = 3). Type II powders exhibited similar flow characteristics to Type I powders despite having significantly (p < 0.05) smaller particle size, lower circularity, and greater elongation. Type III powders exhibited lowest particles size, highest surface free fat, and poorest flow properties . Upon reconstitution of powders (12.5% w/w), no significant difference (p < 0.05) in apparent viscosity was observed between Type I and II powders. Reconstituted Type III powders had relatively poor stability to separation compared to Type I and II powders, caused by large starch granules and/or poor emulsification by hydrolysed proteins. Overall, this study illustrated the range of physical behaviour and structures present in commercial IF powders. In particular, the effect of dry addition of lactose and the hydrolysis of protein were found to have major effects on physical properties. The physical properties of 15 commercially available infant formulas (IF) and follow-on (FO) formulas were analysed. Powders made with intact milk proteins were classified into two groups; Type I—homogenous mixtures of milk powder particles ( Infant formula (IF) powders are dehydrated emulsions consisting of protein, fat carbohydrate, vitamins, and minerals necessary to nourish infants in the absence of breast milk. As an infant grows and solid foods are introduced, complimentary follow-on (FO) formulas can be used as supplementary food. Most IF and FO powders are made with intact bovine proteins, however, specialised products also exist e.g., IF made with hydrolysed caseins and whey proteins for infants showing adverse reactions to standard formulations . The U.SProcess-composition interactions during manufacture have large effects on the physical quality of dairy and IF powders ,4,5,6,7.Large powder particle sizes generally increase flowability due to a reduction in the area of contact between particles during flow ,15. HoweRehydration properties of powders are also influenced by manufacturing conditions. Wettability, the time it takes a given quantity of powder to sink below the surface of water at a certain temperature, improves with increasing particle size and is adversely affected by high surface free fat content ,19,20. TIn recent years, there has been an increase in the publications dealing with IF manufacture ,26,27,28A total of 15 IF powders were studied. Of these, 12 samples were purchased, and 3 samples were donated by H&H Group . Particle size distribution was measured by a Mastersizer 3000 . For this, 10 kPa air pressure was applied to the induction pipe, meaning the vacuum applied to the induction system was mostly responsible for powder induction. This low air pressure was necessary for agglomerated powders so as not to break the agglomerate structure. Powder particle size distributions were distributed normally and did not show any shoulders extending into larger particle size regions, such as is observed when non-agglomerated powders  are not sufficiently dispersed. Refractive indices used for dispersant (air) and particles were 1 and 1.45, respectively . Particl3 of powder was dispersed. It was observed for Type II powders that when using 30 mm3 of dispersed powder, there was over twice the amount of particles on the microscope plate which were not adequately separated; therefore, for Type II powders, the sample volume was reduced to 15 mm3. Surface free fat was determined as per GEA Niro method  gives the diameter of a sphere with the same volume to surface area ratio as the whole powder distribution , yet powder 5 was a more flowing powder. Several factors could account for the difference in flowability. Powder 5 was a more spherical powder, with a tighter size distribution, both of which could impart better flow characteristics. Surface free fat in powder 5 was lower which could also have had a positive effect, although it is hard to gauge the significance of the difference (Δ = 0.15 g free fat 100 ghis case . This ilp < 0.05) difference in compressibility was observed between Type I and II powders. Compressibility measured during flow index testing was always higher than compressibility measured by tapping, indicating higher compressive force in the former measurement. Correlation between the two tests was high for Type I powders (r = 0.99) but for Type II powders no correlation was observed (r = 0.16). The exact reason why there was strong correlation for Type I powders and no correlation for Type II powders is unclear but may be related to the presence of lactose crystals in Type II powders.Compressibility was measured by two means; a—difference in powder volume before and after tapping 100 times; and b—difference in volume before and after flow index analysis. No significant  in 40 °C water in a sealed glass bottle. Formulations were allowed to settle for 20 s, after which the bottles were emptied and inspected for deposits. Three of the six reconstituted Type II powders exhibited a thin deposit which had a gritty texture which was observed under the microscope to be crystalline particles with pyramidal or tomahawk shape. Deposits were not observed upon rehydration of Type I powders. Crystal sizes in the range of 200–300 μm were observed under polarised light in Type II powders, which according to Lowe and Patterson  compared to Type I and II powders. PCA was employed to determine grouping of powders based on a transformation of the measured variables into principle components. Differentiation between powders was not possible based on stage b. This iThe current study showed that, as hypothesised, significant variation is present in the physical properties and behaviour of commercial IF and FO products. Interpretation of the data as a function of powder structure and/or composition yielded the most interesting observations. For example, structural analysis of IF powders using the relatively simple technique of polarised light microscopy gave important information regarding the powder structure, which was instructive for interpretation of data, as well as providing interesting information on the process used during manufacture. The presence of large lactose tomahawk crystals in IF powders indicated a manufacturing process where at least some degree of dry-blending of lactose was utilised. Physical behaviour of these powders was different to powders manufactured without dry-blending of lactose and, furthermore, presence of α-lactose monohydrate crystals resulted in behaviour which was not in keeping with often reported correlations between flowability, compressibility, and particle size. With regards to composition, hydrolysis of the protein fraction was found to significantly affect the reconstituted behaviour of powders. Emulsification in hydrolysed formulations was inferior to formulations with intact proteins, indicating emulsion stability could be problematic during manufacture of hydrolysed IF.Overall, the results presented in this study provide a useful framework for IF researchers and product developers. The ranges of physical qualities present in IF and FO products were presented, and their effect on analysis and interpretation of results was demonstrated. In particular, the importance of contextualising bulk powder and reconstituted properties as a function of powder structure and/or composition was highlighted.


Childcare workers in Denmark have high prevalence of musculoskeletal pain (MSP) and sickness absence, but the existing knowledge of their physical work demands is limited, hampering preventive initiatives. This study aimed to assess the physical work demands with accelerometers and workplace observations of childcare workers handling children age 0–3.Data collection consisted of an electronic survey, anthropometric measurements, accelerometer measurements providing information of physical activity types and postures with Acti4 software from five consecutive workdays, as well as 4-h visual workplace observation per childcare worker from 16 Danish nurseries.In total, 199 childcare workers were enrolled in the study. A total of 4181 working hours of accelerometer measurements and 722 h of workplace observations were carried out. Accelerometer measurements showed that they spent about half of the working day (44.8%) in sedentary postures, and the rest standing (22.8%), moving (13.0%), walking (14.6%), running (0.1%), and climbing stairs (0.7%), with 4.1% in knee straining postures (kneeling and squatting) and 4.3% forward trunk inclination >60°. Workplace observations showed that they carried children 1.8% of the working hours.Physical work demands of Danish childcare workers are characterized by about half of the workday being sedentary, and the remaining of the workday being quite evenly distributed between standing and dynamic activities, with low exposures to carrying children. Their exposure to forward bending of the trunk and knee straining postures could impose a risk for MSP and sickness absence, and preventive initiatives should be considered. The main aim of childcare work in Danish day nurseries is to provide safe, healthy, and developing environment for children age 0–3 years. On a daily basis, childcare workers perform a variety of tasks meeting the children’s basic needs, e.g. diaper changes, feeding hungry mouths, getting down to eye level, providing comfort, and changing clothes. These work tasks unavoidable involve physical activities such as lifting, carrying, and supporting the children’s weight, as well as requiring the childcare workers to do postures and movements such as bending forward, squatting, kneeling, and sitting on the floor.High physical work demands increase the risk of musculoskeletal pain (MSP) and sickness absence  which aims to build a greater research-based knowledge regarding physical work demands among childcare workers, and to test whether it is possible to reduce their perceived physical exertion and MSP through a participatory ergonomic intervention. Details regarding the recruitment procedures and inclusion criteria are presented in the protocol paper  (kg m−2)], and blood pressure (mmHg) (seated with flexed elbow at heart level) were performed and accelerometers were attached. Five AX3 accelerometers  were attached on the childcare worker’s skin with adhesive tape  and secured with transparent adhesive film  for five workdays. The accelerometers were mounted at the following positions: (i) the trunk, on the spine just below the processus spinosus at the level of T1–T2, (ii) the dominant arm, laterally and 3 cm distal to the deltoid insertion, (iii) the right thigh at the most muscular part of the quadriceps femoris, midway on the line between the anterior inferior iliac spine and the top of the patella, and (iv) the right calf and the left calf on the flat part of the soleus and gastrocnemius aponeurosis just distal to the lateral and medial heads of the gastrocnemius. In a paper diary, childcare workers recorded what time they ‘woke up’, ‘arrived at work’, ‘left from work’, ‘went to sleep’ and/or if any of the devices were detached in a diary for all days of accelerometer measurements.On Monday or Tuesday, baseline objective physical measures of body height, body weight (kg), body mass index [body weight/ and portable ergonomic observation approach (POE)  were useQuestionnaire data were exported to an Excel spreadsheet  upon answering. If the childcare workers had missing data, they were contacted after 2 weeks and 1 month via text messages and phone calls to fill in missing data fields.Anthropometric data were carefully entered in an Excel spreadsheet, and subsequently double-checked for correctness.The manufacturer’s software  was used for initialization of accelerometers at a sampling rate of 25 Hz and data download. For further analyses, a custom-made MatLab-based software  was used to determine physical work demands  (−1 of work (28–31 h week−1) compared with previously studied groups. This was rejected as this would primarily include the days when the accelerometers were attached or detached from the subject, which were seen as high-risk days for biased behaviour. The ≥4 h criteria primarily included full working days. Due to different amounts of valid workdays between subjects, the dataset was normalized to an average working day for each subject. Thus, for each subject each unique accelerometer derived variable was aggregated into a new dataset as a single mean of the valid workday(s) for each subject.The diaries were digitized and used to differentiate the accelerometer measurements into ‘working hours’, ‘leisure time’, and ‘sleep’ in the Acti4 software. Only accelerometer measurements from ‘working hours’ ’) was used for the analysis of the occupational physical work demands. Then valid workdays for each subject were determined. The criteria for a valid workday were ≥4 h of accelerometer measurements, which were in line with previous studies  to derive duration and frequency of observed physical work demands.All processed data were imported into SPSS  and derived variables  were first calculated for each childcare worker and subsequently the percentile descriptive data were generated from eligible childcare workers for each variable.Demographics and health characteristics of the 199 childcare workers enrolled in the study are presented in On average, the childcare workers worked 34.9 h weekly and the majority (57.8%) were childcare workers, 36.2% assistant childcare worker, and 6% had a temporary hire or were an intern. The largest proportion of the childcare workers (28.6%) had a seniority of 12–60 months in their current job. The majority (68.8%) stated having either a good (42.9%) or a very good health (25.9%). The mean self-rated physical exertion at work was 5.9 on a 0–10 scale .The accelerometer-based physical work demands are presented in On average, the primary part of the working hours (51.3%) were spent by the childcare workers in physical activities such as standing (22.8%), moving (13.0%), walking (14.6%), running (0.1%), and climbing stairs (0.7%). About half of the working hours (44.8%) were spent sedentary, including sitting (43.0%) or lying (1.8%). Of this time spent sedentary, the workplace observations showed that the childcare workers were sitting on the floor in 12.4% of the working hours.Moreover, 4.1% of the time measured from the 175 eligible childcare workers were used in the knee straining postures, kneeling (2.5%) and squatting (1.6%). Furthermore, the accelerometer measurement showed that the childcare workers had their arms elevated >60° and >90° in 5 and 1.1% of the working hours, respectively. Moreover, the 179 eligible childcare workers had a forward trunk inclination >30° and >60° in 10.6 and 4.8% of the working hours, respectively.On average, the childcare workers carried children or objects in 2.6% of the working time, on average 2.5 times per hour. The childcare workers carried children 1.7% of the working hours, on average 1.7 times per hour. Moreover, the childcare workers were handling children or objects (while the child/object has contact to the surface) in 8.2% of the workday, corresponding to 12.6 times per hour.Percentage childcare workers performing particularly demanding physical work demands in relatively long time of the workday (≥10%) are presented in To our knowledge, this is the largest study of childcare workers with accelerometer measurements over several consecutive workdays and workplace observations of physical work demands. Of the 199 childcare workers included in the study, a total of 4181 h of accelerometer measurements over an average of 3.6 workdays on 181 eligible childcare workers and 722 h of workplace observations with an average of 3.7 h for 195 eligible childcare workers were carried out. The presented findings contain information of the physical works demands among childcare workers going far beyond the existing research literature. Thus, we consider the findings from this study valuable for improving our knowledge about the need for, and where to target, preventive workplace interventions among childcare workers.Based on the accelerometer measurements over several workdays, we found that the childcare workers spent about half of the workday (44.8%) sedentary (i.e. sitting and lying). Compared with similar accelerometer measurements and analyses of sedentary behaviour in other job groups, the childcare workers spend more of their working hours sedentary than cleaners and manufacturing workers . The exposure to these work demands are of shorter duration than seen in other occupational groups with comparable accelerometer measurements, such as cleaners  and manufacturers . Considering that the childcare workers are spending about half of the workday sedentary, and the quite even distribution between standing and dynamic activities, we do not consider them to impose a risk for health impairments, such as low back pain among the childcare workers.The percent working time spent on physical activity demands with higher intensity like stair climbing and running (<1%) is of too low duration to be likely to influence the health of the childcare workers. Our finding of a low duration of physical activity of higher intensity at work among childcare workers is in line with the previous accelerometer study among childcare workers , while being considerably lower than among occupational groups like different types of construction work  having between 10 and 12% of the workday with the arm elevated >60° (The exposure to forward trunk inclination >60° while on feet was 4.3% of the workday among the childcare workers. This exposure to forward trunk inclination is comparable to what have previously been found with the same measurement methodology among cleaners (4.6% of workday), while being a little higher than for manufacturing workers (3.1%) . About hThe accelerometer measurements of the knee straining postures, kneeling and squatting, revealed that the childcare workers spent 2.5% of the workday kneeling and 1.6% squatting. Moreover, 7.4% performed the knee straining postures (squatting and kneeling) more than 10% of the workday. Occupational exposure to kneeling and squatting is well known to increase risk for knee pain and disorders . However, based on the accelerometer measurements and workplace observations, the physical work demands of the childcare workers were characterized by almost half of the workday being sedentary, and the remaining of the workday being quite evenly distributed between moving, standing, and walking, with low exposure to carrying and more intensive activities like running and stair climbing. Overall, these exposures are lower than we have found with similar accelerometer measurements (except for sedentary time being higher) among cleaners and manufacturing workers . We would also like to acknowledge Klaus Hansen, Dorte Ekner, Jørgen Skotte, Pernille Kold Munch, and the TOY-projects student helper team for their effort during the data collection.AHO, PFH, and CNR participated in the discussion and drafting of the conceptual design of the study and wrote the initial protocol as well as the application for funding. PFH, KGS, MAS, CNR, and AHO participated in finalizing the measurement and observational protocols. MAS was responsible for planning and organizing the team carrying out data collection. KGS was part of the data collection team. PFH started drafting the paper and AHO wrote the manuscript. All authors have read and commented on the draft version as well as approved the final version of the manuscript.The authors declare that they have no competing interests.


The aim of this study was to evaluate the effect of intestinal microbiota metabolites in colorectal cancer patients on HT29 cell line using MTT assay.Colorectal cancer is one of the most common malignant tumors. Human guts harbor abundant microbes that adjust many aspects of the host physiology. Increasing studies suggest that gut microbiota play a significant role in the incidence and expansion of CRC, as a result of virulence factors, bacterial metabolites, or inflammatory pathways.In this cross-sectional study, 60 biopsy samples including 30 cancerous and 30 adjacent healthy tissues were collected from patients with CRC during 2017. Biopsy samples were first cultured on Thioglycollate broth medium for 24hr after which the microbiota metabolites were filtered and stored at -20 C° for further evaluation. HT29 cells were treated by microbiota metabolites at different times  and its viability was assessed by MTT assay. The cells treated with microbiota metabolites showed increased viability and proliferation in time-dependent analysis by MTT assay, but there was not significant differences between the two groups. It seems that microbial metabolites are able to induce proliferation and increase cell viability and thus induce colorectal cancer. Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death in the world (1). CRC is the fourth most common cancer in men following gastric, bladder, and prostate cancers and is the second most common cancer in women after breast cancer in Iran . The incClostridium and bacterioides, as the predominant population of the intestinal microbiota, produce bacterial metabolites such as bile acids, fatty acids, β-galactosidase, β-glucosidase, reductase, decarboxylase, and protease which can also increase the risk of colorectal cancer , 2% non-essential amino acid , and 1% antibiotics. The cells were passaged for several generations in special flasks using trypsin-EDTA (0.25%). Trypsinized cells were incubated at 37 ° C and 5% CO2 atmosphere.MTT test To investigate the effect of cytotoxicity or proliferation of metabolites obtained from microbiota of CRC patients, cell viability was assessed using MTT Assay kit  after complete growth of HT29 cells for 3 days. A total of 100μ of filtered bacterial metabolites was shed on the cells and incubated at 3, 6, 12, and 18 hrs at 37° C as well as atmosphere containing 5% Co2. Thereafter, 100µL of MTT solution was added to the plates and incubated for 4 hours at 37°C and 5% Co2 atmosphere. Then, 1 ml of the solubilization solution was added to the plates and incubated 15-20 min at 37° C with a 5% Co2 atmosphere. Next, reads at 570 nm and 650 nm, as a reference wavelength, using ELISA Reader, . Cell viability was calculated using the following formula: Viability (%) = (OD test/OD control) x100Statistical analysis Statistical calculations were performed to compare the mean and standard deviation using SPSS Ver.20 and t-Test with P <0.05 considered statistically significant (P <0.05). The cultures of HT29 cells were treated with metabolites after cell growth and differentiation . MTT assThe cytotoxic effect of bacterial metabolites on the HT29 cell line was assessed by ELISA reader on differentiated cell supernatants. These results confirm the lack of toxic effects of the investigated metabolites on differentiated cell lines at 6, 3, 12, 18 h . As depiCancer is one of the leading causes of death worldwide. According to WHO projections by 2030, more than 21 million new cancer cases and more than 13 million cancer deaths will occur globally. Meanwhile, colon cancer is also considered to be the third most common cancer in the world (Microbial pathogens are known to initiate 20% of tumorigenesis and a considerable number of malignancies are associated with dysbiosis . IntestiIn the present study, the role of intestinal microbiota in CRCs was investigated on the lethality or cellular proliferation on HT29 cells. Based on the results, the microbiota metabolites of these patients induced cell proliferation in cell culture compared to blank cell culture. The mean percentage of cellular proliferation in metabolites obtained from tumor specimens was slightly higher than in normal tissue samples but no significant difference was observed between the two groups. Both normal and tumor specimens were taken from one patient; although some studies have reported differences between microbiota in cancer and normal tissue, most studies have indicated that the structure of the gut microbiota often forms before three years of age and has a stable structure throughout their lives .Certain factors such as antibiotic use, specific diet, chemotherapy, etc. can disrupt the gut microbiota structure . In a stEnterococcus faecalis, Enterotoxin bacteroides Fragillis or fadA in Fusobacterium nucleatum have the ability to break down DNA and thus induce intestinal cell pre-proliferation and increase in intestinal microbiota in microbiota studies (In a study examining effect of fecal filtered fluid on caco2 cell culture using comet test, Venturi and colleagues observed that in normal samples they had no detrimental effect on DNA breakage, but in samples with intestinal problems such as intestinal polyps, DNA breakage was observed. . DNA bre studies .Given the effect of bacterial metabolites isolated from CRC patients on increasing proliferation of the HT29 cell line, it seems that bacterial metabolites may play a central role in the relationship between the microbiota and associated colon cancer. Bacteria can alter the expression of genes involved in carcinogenesis during infection or their inflammation or directly by the production of intestinal microbiota metabolites. More studies are required to study the role of gut microbiota in CRC patients.The authors declare that they have no conflict of interest.


Cancers may be treated by selective targeting of the genes vital for their survival. A number of attempts have led to discovery of several genes essential for surviving of tumor cells of different types. In this work, we tried to analyze genes that were previously predicted to be essential for melanoma surviving. Here we present the results of transient siRNA-mediated knockdown of the four of such genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, in melanoma cell line A375, then assayed the cells for their viability, proliferation and ability to migrate in vitro. In our study, the knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating. Possible explanation for such counterintuitive results may include insufficiency of the predicting computational models or the necessity of a multiplex knockdown of the genes.To examine the hypothesis of essentiality of hypomutated genes for melanoma surviving we have performed knockdown of several genes in melanoma cell line and analyzed cell viability and their ability to migrate.Knockdown was performed by siRNAs transfected by Metafectene PRO. The levels of mRNAs before and after knockdown were evaluated by RT-qPCR analysis. Cell viability and proliferation were assessed by MTT assay. Cell migration was assessed by wound healing assay.The knockdown of the genes predicted as essential for melanoma survival does not lead to statistically significant changes in cell viability. On the other hand, for each of the studied genes, mobility assays showed that the knockdown of each of the target genes accelerates the speed of cells migrating.Our results do not confirm initial hypothesis that the genes predicted essential for melanoma survival as a matter of fact support the survival of melanoma cells. Cancer is a group of abnormal cell growth diseases with the potential to invade or spread to other body locations. In USA, cancer is the second leading cause of death in both men and women of all ages . Many trOne of the most important premises of the personalized medicine is thorough understanding of underlining genetics. Genetics of cancer cells defines their resistance to drugs, aggressiveness and ability to metastasize. In particular, it is critical to understand the difference between cancer and normal cells genomes, distinguish them successfully and selectively destroy tumor cells.A majority of the studies of cancer genetics concentrate on the genes either mutated  or diffeHere, we assessed the role of some of these genes on survivability of melanoma cell line by performing their siRNA-mediated knockdown followed by investigation of the cell viability, proliferation and migration rate in vitro. While we hypothesized that melanoma cells lacking the transcripts of these essential genes would show decrease in both the proliferation and viability, experiments proved otherwise.Human malignant melanoma A375 and Human Embrionic Kidney HEK293T cells were cultured under standard conditions. In brief, Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) and l-glutamine were placed in humidified atmosphere with 5% CO2 at 37 °C. Before transfection, cells were trypsinized, resuspensed, diluted to 1 ml, and counted by Partec Flow Cytometer.siRNAs were designed according to recommendations  with theTotal RNA was extracted from cells using phenol–chloroform method according to Chomczynski and Sacchi . RNA was3 cells were transfected in 96-well plates in five replicated wells. After 24 h, cells were trypsinized, counted and seeded at 2×102 cells/well. Every 24 h, five wells with cells were treated with MTT solution and incubated 3 h at 37 °C. After removal of media, formazan pellets were dissolved in 200 μl of DMSO. For each pellet, absorbance of formazan solution was analyzed at 570 nm in Multimode Plate Reader (PerkinElmer).Cell viability was determined in 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. In brief, 103 cells/well in 96-well plates. 24 h post transfection cell mat was wounded by 200ul pipette tip and photographed every 2–5 h for up to 24 h in total. Photos were processed with software ImageJ [Cell migration was assessed by wound-healing assay as described in . In briee ImageJ . The slop < 0.05 was considered as statistically significant.All statistical analyses were performed using STATISTICA 8.0 software. Measurements were obtained at least in triplicates. Results are reported as the mean ± standard deviation. The Mann–Whitney U and the Kolmogorov–Smirnov tests were used to compare groups of samples. We performed analysis of the 91 genes previously identified in silico as essential for survival of cutaneous melanoma . FANTOM5These 7 genes were analyzed for their expression levels in A375, G361, Sk-mel-1 and Sk-mel-5 melanoma cell lines by qPCR. Four genes that were expressed at detectable level at least in one of these cell lines were selected. Some of them were previously demonstrated to play a role in other cancers , and some were not yet characterized (RHPN2 and ZNFX1). These genes entered further experimental validation.For each of the genes, namely ZNFx1, RHPN2, STK11IP and UNC45A, from 1 to 3 siRNAs were designed and tested in melanoma cell line A375 . Viability assay also revealed no statistically significant changes in cell survival   Fig. .Fig. 3ReRecent advantages in DNA sequencing facilitate analyzing genomes of human tumors. Instead of considering each tumor as a result of a series of point mutations in oncogenes or tumor suppressor genes researches regard the malignancies as completely different tissue type . Cancer According to Fantom5 dataset, just 44 of these genes) were, in fact, expressed in melanoma cell lines. This discrepancy may be explained by the differences could be explained by a difference between expression profiles of real tumors collected from patient and the immortalized cell lines explored in Fantom5. Further whittling down the gene list was performed by exclusion the genes previously associated with any human disease. No consensus trends were observed in subsequent analysis of 18 remaining genes, with some of them behaving in accordance with initial hypothesis, and some in exactly opposite fashion. For instance, previously described knockdowns of UNC45A in ovarian cancer and in myoprogenitor cells led to a decrease in their ability to proliferate . SimilarWe have taken onto account that for some genes effects on cell survival may be tissue-specific, and some other genes may play a role in survival of the cells only in the contexts of multicellular 3D structures, or when cancer cell would interact with immune cells. To minimize effects of the interaction taking place at the cell-to-cell interfaces, we have reduced the list to seven genes, by excluding all genes encoding for the proteins expressed on a surface of human cells.After analyzing expression levels for these 7 genes in available melanoma cell lines sk-mel-1, sk-mel-5, G361 and A375, four genes with the highest expression levels all over, and in the A375 cell line in particular, were selected for knockdowns. Notably, for all four selected genes, namely, UNC45A, STK11IP, RHPN2 and ZNFX1, detected expression levels were extremely low. Nevertheless, we assumed that these genes may be important, for example, they may serve as transcription factors, or other regulatory molecules, and proceeded with wet-lab experiments.The results on knockdown experiments show that the selected genes are not essential for melanoma cells viability or proliferation, but evidently exert their influence at the speed of the cell migration, and, possibly, other processes, not yet explored experimentally. None of these genes were extensively studied by other research groups, hence, current study does add to the current body of evidence for their roles in the functioning of the human body.Gene UNC45A encodes co-chaperone of heat shock protein 90 (Hsp90). This gene is essential for certain types of cancers cells, including breast carcinoma, but is dispensable for normal cells , and forGene STK11IP encodes a leucine-rich repeat containing cytoplasmatic protein functionally linked to the Peutz-Jeghers syndrome kinase LKB1, and capable of interacting with the TGFbeta-regulated transcription factor SMAD4, and a formation of a ternary complex of LKB1- STK11IP-SMAD4 . LKB1 isRhophilin Rho GTPase binding protein 2 (RHPN2) drives mesenchymal transformation of malignant gliomas  and is lZinc finger NFX1-type containing 1 (ZNFX1) encodes an interferon (IFN)-stimulated, mitochondrial-localized dsRNA sensor capable of restricting the replication of RNA viruses . While iIn sum, our finding fail to confirm the hypothesis of Pyatnitskiy et al. Although some genes in certain distinct tumor types are hypomutated, they remain dispensable for the survival of these tumor cells. In accordance with our findings, Pyatnitskiy et al. have corrected their predictive model and published a novel in silico gene set .


The UTOPIA value exhibited an area under the curve value of 0.837 when validated with the independent test dataset. The UTOPIA value displayed good diagnostic performance for predicting urine culture results, which would help to reduce unnecessary culture. Different cutoffs can be used according to the clinical indication.To investigate the association between the results of urinalysis and those of concurrent urine cultures, and to construct a prediction model for the results of urine culture. A total of 42,713 patients were included in this study. Patients were divided into two independent groups including training and test datasets. A novel prediction algorithm, designated the UTOPIA value, was constructed with the training dataset, based on an association between the results of urinalysis and those of concurrent urine culture. The diagnostic performance of the UTOPIA value was validated with the test dataset. Six variables were selected for the equation of the UTOPIA value: age of higher UTI risk , female , nitrite , leukocyte esterase , the number of WBCs (per 1 × 10 Clinical manifestations of UTI are mostly mild; however, the disease could develop serious complications, especially in certain high-risk populations including infants, pregnant women, and aged population3. Therefore, early diagnosis and empirical antimicrobial treatment is essential to improve clinical outcomes of patients with UTI4.Urinary tract infection (UTI) is the most common bacterial infection acquired in the community and in healthcare facilities. The prevalence of UTI is estimated to be 11% of the overall population, and almost half of adult women suffer from UTI at least once in their lifetime5, and an antimicrobial susceptibility profile can be obtained by testing clinical isolates. However, urine culture is a time-consuming procedure, and the microbial spectrum of causative organisms in UTIs is narrow. Therefore, routine cultures are often not necessary to manage patients with uncomplicated UTIs, and only urinalysis either by test strip analysis and/or sediment analysis are recommended for the decision of patient management6. Among the components of test strip analysis, leukocyte esterase (LE) and nitrite are commonly used to diagnose UTI in routine clinical practices. Urine LE positive indicates pyuria, and urine nitrite positive indicates the presence of nitrate-reducing bacteria. However, diagnostic performance of these tests is not sufficiently high to be used alone due to limitations of the test principle7.The gold standard for definitive diagnosis of UTI is detection of the pathogen by bacterial culture of a urine specimen8.Test strip analysis is traditionally done by the dipstick based on physicochemical reactions, and the results are interpreted using a reflectometer. Automated urinalysis systems including sample preparation, aliquot, and reading have been introduced to improve test throughput and efficiency and to reduce labor and time. In addition, microscopic examination of urine sediment is also widely used to diagnose urinary tract diseases by identifying various types of cells, casts, and crystals in a urine sample. However, manual microscopic examination is a time-consuming procedure and requires expertise to maintain consistency of the result interpretation. Recently, different types of automated urine sediment analysis systems have been introduced. Among them, the iQ200  is an automated digital imaging-based system that uses flow morphology analysis to classify particles in a urine sample based on multiple parameters including size, shape, contrast, and texture. This instrument has exhibited satisfactory analytical performance for the quantitation of red blood cells (RBCs), white blood cells (WBCs), and epithelial cells compared with other automated sediment analysis systems and manual microscopic methodsHere, we evaluated an association between the results of urinalysis obtained by the iRICELL system including the iQ200 automated urine sediment analysis instrument with results of concurrent urine cultures. We also aimed to construct a simple but practical prediction model for the positive urine culture with the results of urinalysis including automated urine sediment analysis.Escherichia coli  followed by Enterococcus faecalis , Klebsiella pneumoniae , and Enterococcus faecium  (Supplementary Table P < 0.0001) and lower proportion of males  than the urine culture-no growth or contamination group (Table The median (1st–3rd quartiles) age of the 42,713 patients was 56 (24–69), and 38.7%  of the patients were included in the high-risk age group (Table P < 0.0001 for each) (Table P = 0.0007), nitrite , and LE  were independent risk factors for positive urine culture. Among the parameters of automated sediment analysis, the numbers of RBCs , WBCs , epithelial cells , and bacteria  showed significant associations with positive results of the urine culture (P < 0.0001 for each).The results of test strip and sediment analyses according to the urine culture results are summarized in Table h) Table . Among t6/L increase), and the number of bacteria  were selected considering the effect size of each variable by multivariable binary logistic regression in the training dataset with 21,522 patients , otherwise x1 = 0x2 = 1 for female; x2 = 0 for malex3 = grade of nitrite by test strip analysis (0.5 when the result is trace or weak positive)x4 = grade of LE by test strip analysis (0.5 when the result is trace or weak positive)x5 = number of WBCs by digital flow morphology analysis (1 × 106/L)x6 = number of bacteria by digital flow morphology analysis (1 × 106/L).Among the variables exhibiting significant associations with positive urine culture, six variables including age of higher risk , female , nitrite , LE , the number of WBCs , which is significantly higher than that of nitrite , LE , the number of bacteria , and the number of WBCs  , and negative predictive value (NPV) were 0.661, 0.862, 0.473, and 0.931, respectively , which was first introduced as UriSed 10, takes 15 microscopic images per urine sample prepared in cuvettes that mimic glass slides used in manual microscopic examination, and the result images are analyzed by particle recognition software11. The iQ200 investigated in this study is an automated digital imaging-based system that uses flow morphology analysis. In previous studies, the iQ200 system showed reliable performance in counting RBCs, WBCs, and epithelial cells in terms of imprecision and linearity and showed good correlation with manual microscopic sediment analysis and other automated analyzers13.Microscopic examination of urine particle is a useful tool for diagnosing UTI, although the gold standard for diagnosis is urine culture. To date, three different types of automated urine sediment analyzers have been introduced. Sysmex UF-1000i  utilizes the flow cytometric method. This analyzer measures numbers of cells, bacteria, and casts by electrical impedance per flow sample volume, sizes the components by forward light-scatter, and nuclear and cytoplasmic characteristics using fluorescent dye14. The number of bacteria in urine specimens obtained by Accuri C6  showed good correlation with the results of urine culture when a cutoff value for urine culture positive was ≥ 105 CFU/mL15. A recent interlaboratory study exhibited that the absence of microorganisms in the iQ200 screen was the strongest solitary predictor for a negative culture result with a sensitivity of 90.5%, and higher sensitivity (95.2%) could be obtained by the algorithm based on the presence of microorganisms and the number of WBCs16. Another study with the iQ200 system exhibited an acceptable NPV of 97.7% and approximately 50% reduction of urine culture when using WBC ≥ 4/HPF as a cutoff in predicting urine culture results, but the PPV was only 24.5% in the same study17. The scoring system suggested by Foudraine et al., which was composed of clinical symptoms including dysuria and urgency and the number of WBCs obtained by the iQ200 analyzer, gave good diagnostic performance with a high AUC value of 0.950 for predicting positive blood cultures18. However, the diagnostic performance of a test could vary according to the characteristics and composition of cases and controls included in each study, thus it would be difficult to directly compare diagnostic performance among the studies. In addition, the definition of significant growth in urine culture in each study was different, thus it is also difficult to generalize the diagnostic performance of a test in the literature.There have been several studies to evaluate possible associations between the results of microscopic urine sediment examinations and those of urine culture. A meta-analysis for predicting positive urine culture by the results from the Sysmex UF-1000i or UF-100 systems showed good sensitivity, using the number of WBCs  and bacteria  counted by flow cytometry as indicators19. By simply adding these two risk factors as variables of the prediction algorithm, the UTOPIA value exhibited better diagnostic performance than the other models those are consisted of only the variables from urinalysis , the NPV was 0.978 (95% CI = 0.973–0.982) when applying a cutoff for the UTOPIA value of > 5.72, and 20.0% of total culture cases was estimated to be reduced at the expense of 2.2% of false negative results, i.e. 1—NPV based on the UTOPIA value . To make an accurate prediction model for positive urine culture, the results of urine cultures were categorized into “Positive” and “No growth or contamination”.The results of test strip analysis and sediment analysis by digital flow morphology analysis were retrieved from the electronic medical records. Automated urinalysis were performed using the iRICELL system , which consisted of the iChem VELOCITY urine chemistry analyzer and the iQ200 SPRINT urine microscopy analyzer, following the manufacturer’s instructions. For the iQ200 instrument, approximately 1.3 mL of urine passes through a flow cell, and a digital camera captures 500 images of magnified sample. Then, the Auto-particle Recognition (APR) software (current version 7.1.4) interprets the captured images. The flow morphology interpretation with flags for suspicious errors or abnormal results by the APR software were reviewed with on-screen images by operators. Based on comprehensive consideration with on-screen images, previous urinalysis results of the same patient, and the test strip results concurrently obtained by iChem, cases with discrepant interpretations between operators and the analyzing software were subjected to manual microscopic sediment examination. If needed, the results for these cases were corrected as the number of cells per high-power field by manual microscopic examination, and were excluded from our study due to inaccurate quantitative values for RBCs, WBCs, and epithelial cells by the iQ200 analyzer in those cases. During the study period, three quality control materials for the iChem VELOCITY including IRISpec CA, CB, and CC (Beckman Coulter Inc.) and two materials for the iQ200 including iQ positive and negative controls (Beckman Coulter Inc.) were run every eight hours.20 and positive rates of urine culture according to age in our data. A positive urine culture was determined when a single uropathogen  or two uropathogens  were recovered. Uropathogens include Gram-negative bacilli, Staphylococcus aureus, Candida species, Enterococcus species, and Aerococcus urinae, as previously described21. Cases with more than three species recovered from urine culture were considered as contamination regardless of the quantity of bacterial growth21.The high-risk age group for UTI was defined as patients younger than 2 years or older than 69 years considering high positive rates of urine culture according to national surveillance studyP value was < 0.05.All statistical analyses were performed by Analyse-it for Microsoft Excel Method Evaluation Edition version 5.65.3  and IBM SPSS Statistics 25 . Patient characteristics and the results of urinalysis according to the groups classified by the urine culture results were compared with chi-square tests for categorical variables and Mann–Whitney U tests for continuous variables. Binary logistic regression with the results of urine culture as the dependent variable and those of urinalysis and patients’ characteristics as the multivariate independent variables was performed to determine the coefficient for each independent variable in the regression model. With the regression model equation, the UTOPIA value for each case in the test dataset was calculated to predict the probability for positive urine culture, and diagnostic performance of the UTOPIA value for the prediction of urine culture results was evaluated by calculating the area under the curve (AUC) value. All statistical analyses in this study were considered significant when the Supplementary Tables.


Verbal episodic memory and mood were assessed pre consumption and 2 h post consumption. An ANOVA analysis showed that the DC was associated with better verbal memory performance for several outcome measures of the Rey Auditory Verbal Learning Test relative to the WC, however, there were no effects on mood. These findings lend support to the notion that everyday available portions of dark chocolate can confer benefits to the brain in healthy consumers.There is good evidence that cocoa flavonoids can acutely improve cognitive function in humans, possibly via mechanisms such as increased cerebral blood flow. To date, much of the evidence is based on measures of executive function with extracts and cocoa-based interventions with a high flavonoid content. The aim of the present study was to explore whether benefits to episodic verbal memory and mood are observed two hours post consumption of a commercially available dark chocolate (DC) bar relative to a 35 g white chocolate bar (WC). Ninety-eight healthy young adults ( Dark chocolate contains a group of phytochemicals known as flavonoids, which naturally occur in many fruits and vegetables. The consumption of flavonoids and flavonoid-rich foods has been associated with numerous health benefits, and reviews of the literature indicate that cocoa flavonoids are associated with benefits to cardiovascular health  and cognhttp://phenol-explorer.eu/contents/food/439 accessed 15/08/2019) collating the analysis from multiple peer reviewed studies of dark chocolate using chromatography methods indicates a total flavonoid content of 237 mg/100 g which would equate to an 83 mg flavonoid dose for the DC treatment here. White chocolate contains no flavonoids [A parallel groups design was implemented with two treatments: dark chocolate (DC) and white chocolate (WC). Participants were assigned to treatments on arrival by the experimenter according to a predetermined alternating order generated by the principle investigator who did not collect any data . Outcome measures were assessed at two test sessions: pre consumption (Baseline) and two hours post consumption. Both treatments were matched for calories  and weight (35 g) and were commercially available 35 g organic products . The 35 g serving was chosen on the basis that this is a portion of chocolate typically consumed. The macronutrient content of the DC and WC treatments (respectively) was fat 15 g and 13 g; saturated fat 8.8 g and 8 g; carbohydrate 13 g and 18 g; sugars 10 g and 18 g; protein 3.2 g and 2.8 g; fibre 3.5 g and 0.01 g; salt 0.03 and 0.09 g. The DC was 70% cocoa content. The specific flavonoid content was not assessed. A database  were recruited (n = 49 DC and n = 49 WC) via opportunistic sampling from the University of Reading student population using posters and online advertisements. Exclusion criteria were any mental or physical illness, currently taking medication or supplements and smoking. Inclusion criteria were aged between 18–24, English speaker, and currently enrolled in a university degree programme. The latter enabled control over educational attainment, which is a predictor of episodic memory performance. p > 0.05).Ninety-eight healthy young adults   is a welSubjective mood was assessed with two questionnaires: the Positive and Negative Affect Schedule (PANAS)  and the t-tests comparing the two treatments at each time point, with a Bonferroni corrected alpha adjustment for type 1 error.All testing took place at the University of Reading School of Psychology in individual cubicles. Interested participants were checked for inclusion and exclusion criteria by email, phone call, or in person prior to attending the test day. Two hours prior to arrival on the test day, participants were required to fast from all food and drink except water. Informed consent was attained on arrival, at which point demographic measures were collected by a self-report questionnaire. Height and weight were measured by the experimenters. First, the Bond Lader was completed followed by PANAS and the AVLT. Participants then consumed the assigned treatment. Session 1 lasted approximately 30 min. Participants then left the laboratory and were required to remain fasted (expect water) until returning two hours later for Session 2. Session 2 followed the same procedure as Session 1, without the demographic measure assessment. Session 1 commenced anytime between 9 a.m. and 1 p.m. All data was collected between November 2018 and February 2019. Laboratory conditions, such as humidity, illuminance, and ambient temperature, were not assessed but remained constant throughout each test day. The experimenters were not blinded to the treatment on the test day, however, the mood and cognitive assessments were undertaken in a cubicle without the presence of the experimenter, and the responses were collected and subsequently coded by a computer to allow blinding for the analysis, which was undertaken by the principal investigator. All outcome variables were assessed with a 2 × 2 (Treatment*Session) mixed ANOVA. Significant interactions were clarified with post hoc independent t-test showing that the DC was associated with benefits to episodic memory relative to WC at 2 h, whilst at the pre-consumption baseline there were no differences between the treatments.As shown in p < 0.05; main effect of Session).Specifically, DC benefits were seen for immediate word span, words learnt, final acquisition, total acquisition, delayed recall, and total recall. The findings indicate that the consumption of a standard 35 g 70% cocoa dark chocolate bar can benefit verbal episodic memory relative to an equicaloric 35 g white chocolate bar two hours post consumption in healthy young adults. Previously, acute cognitive benefits in healthy young and middle-aged adults following cocoa consumption have been seen for measures of executive function and processing speed ,12,13, sThe present data shows that recall was enhanced at all trials during the RAVLT following the DC relative to WC, indicating an increased capacity to learn words at initial presentation and throughout the task, including the short-delayed recall. Interestingly, there were no effects of interference, which suggests that the DC did not reduce the process of forgetting caused by the learning of new words or reduce the effect of old learning on new learning. The benefit of increasing one’s capacity to learn and recall words, with few effects on interference, is also seen in the blueberry flavonoid literature . It woulIn summary, this research shows that 70% cocoa dark chocolate consumption can benefit verbal episodic memory two hours post consumption in healthy young adults relative to a white chocolate control. These findings support the notion that everyday available portions (35 g) of dark chocolate can confer benefits to the brain in healthy consumers.


The aim of this study is to investigate the stress distributions of different restoration options for class II mesio-occluso-distal (MOD) cavities. A class II MOD cavity with proximal box gingival floor 1 mm below cementoenamel junction was designed in a mandibular first molar tooth model. 3D finite-element analysis (FEA) and 3D-CAD modelling were used to examine the occlusal stresses distributed to the remaining buccal enamel (RBE), remaining lingual enamel (RLE), adhesive surfaces, and restorative materials by direct and indirect materials resulting from a 600 N of static occlusal load stimulating foodstuff. von Mises (VM) and maximum principal (Pmax) stresses were evaluated for two CAD/CAM materials and three direct materials. CAD/CAM materials exerted less stress than the direct restorative materials. Significant von Mises and Pmax stress value differences were seen among all restoration models on RBE. Reducing RLE and including it into the cavity would be a more effective option for this model in this scenario. As VM and Pmax stresses of PIHC CAD/CAM material for RBE and dentin were significantly lower than other tested materials, it may be the choice of material for indirect MOD restorations. Functional and parafunctional forces in the mouth can cause stress on the sound teeth, on supporting bone, on soft tissues, and on adhesively restored teeth after dental therapy . DetermiFirst permanent molars are the most affected teeth with dental caries due to the time of eruption, morphology, and position of the tooth in the oral cavity . Class IIn class II cavities, if all margins of the cavity are in enamel, the success rate of the restoration increases; on the contrary, it decreases when the cavity margins are in dentin or cement. Nowadays, different restorative materials are used alone or together to reduce the negative effects of the stresses on the tooth. Amalgams and composites are still being used; on the other hand, there are some other new options like glass carbomer cement (GCC) and computer-aided design/manufacturing (CAD/CAM) materials , 15. NowFinite element (FE) stress analysis is a popular technique that helps to better understand the dental biomechanics of any given geometry consisting of a mesh of elements with given mechanical properties –20. The The aim of this study was to examine the stress distribution under a simulated occlusal loading condition in tooth tissues and different restorative materials applied with different techniques on mandibular first molar tooth having a class II mesial-occlusal-distal (MOD) cavity with proximal box gingival floor within dentin, by the 3D finite element stress analysis method.This 3-dimensional (3D) FE study was performed using Rhinoceros 4.0 3D modeling software , VR Mesh studio meshing software , and Algor Fempro analysis programme . A 3D finite element model of the permanent mandibular first molar was built according to the standard anatomy described in Wheeler's atlas . After the models were created geometrically with the VRMesh software, they were transferred to the Algor Fempro  software in stl format to make them ready for analysis. The stl format is of universal value for 3D modeling programs. As the coordinate information of the nodes are stored in the stl format, there is no information loss when transferring between programs. After the model is compatible with the Algor software, it is necessary to introduce the materials and the tooth structures, as well as the created model is mandible to the software. The physical properties of materials (elasticity modulus and Poisson ratio) are defined for each of the structures that make up the models.The final modeling and scaling of the tooth were done by using the Rhinoceros software. The intact lower first molar tooth (IT) was modeled as the control group . A thickClass II MOD cavity was modeled in Rhinoceros, and the restored models were obtained by using Boolean operations between the cavity, enamel, and dentin surfaces . An MOD μm [μm for indirect materials (models 4 and 5). Five restoration models were generated:The cavity was then restored with different direct and indirect materials. The adhesive surface was modeled as 30 μm  for the Model 1 (M1): the cavity surface was covered with 30 μm adhesive layer, and the rest of the cavity was restored with amalgam.Model 2 (M2): the cavity surface was covered with 30 μm adhesive layer, and the rest of the cavity was restored with glass carbomer cement (GCC).Model 3 (M3): 1 mm resin-modified glass ionomer cement (RMGIC) base which covered the dentin floor and axial walls of the cavity was modeled. Subsequently, a 30 μm adhesive layer was placed on the surface of RMGIC, and the remaining surfaces of the cavity were restored with resin composite.Model 4 (M4): after the cavity surface was covered with 100 μm dual-cure resin cement layer, the rest of the cavity was restored with nanoceramic resin CAD/CAM material.Model 5 (M5): After the cavity surface was covered with 100 μm dual-cure resin cement layer, the rest of the cavity was restored with polymer-infiltrated hybrid ceramic (PIHC) CAD/CAM material.The mechanical properties of oral tissues and dental materials including Young's modulus and Poisson's ratio used for the analysis were presented in Bricks and tetrahedral solid elements with different numbers of elements and nodes were prepared to generate the models .The surface of the food bolus in touch with the occlusal surface of the tooth was modeled as a copy of the occlusal tooth surface . A 600 NIntermediate von Mises (54.86 MPa) and Pmax (12.81 MPa) stress values were observed on the sound tooth model on the enamel. In the sound tooth model, von Mises and Pmax stresses were highly observed in the cervical region (67.95 MPa) of the enamel with 67.95 MPa and 7.28 MPa values, respectively.The Pmax values of all restoration models and the von Mises values of M3, M4, and M5 were greater in the RLE than in RBE. M1, M2, M4, and M5 exhibited similar VM stresses to RLE, whereas M3 transferred the least stress . ConsideSignificant von Mises and Pmax stress value differences were seen among all restoration models on RBE. The minimum values were observed in M5 followed by M4, M3, M1, and M2 as seen in In case of dentin, minimum von Mises and Pmax stress values were seen on indirect restoration models (M5 followed by M4), and maximum values were observed on M2 among the tested restoration models . Intact Maximum von Mises stress accumulation 79.46 MPa) occurred in PIHC CAD/CAM material and maximum Pmax stresses accumulated in amalgam and glass carbomer cement (79.16 MPa and 75.62 MPa), respectively. Nanoceramic resin CAD/CAM material showed the lowest von Mises and Pmax stress accumulation. Intermediate stress values occurred on resin composite. Amalgam had a similar von Mises stress value with resin composite . Glass c MPa occuThe decreasing order of von Mises and Pmax stress values for adhesive surfaces of direct materials were as follows: M3 ~ M2 > M1 . MinimumThere are several studies investigating the occlusal stress loading of direct and indirect restorations conducted for class II MOD cavities differently restored in mandibular first molars , 8, 12. μm-thick cement layer was simulated in our model parallel with other studies as it is clinically realistic [The finite element analysis (FEA) method was chosen as a tool to indicate the stress distributions in different direct and indirect adhesive materials used for class II MOD cavity restorations. The polymerization shrinkage of the resin composite, resin-modified glass ionomer cement, luting cement, and adhesives were not taken into consideration in this study. Although Ausiello et al.  neglecteealistic , 29. Genealistic . von Misealistic , 30–33. ealistic . The maxExcept the von Mises stress transferred by PIHC CAD/CAM material and nanoceramic CAD/CAM material to RBE, the stress analysis of the intact tooth model showed significantly lower stress values for both enamel and dentin for all the tested restoration models. Furthermore, the minimum VM and Pmax stress values were found in the intact tooth model for RLE. On the contrary, Pmax stresses in the intact tooth were higher than the two CAD/CAM materials and resin composite for RBE but lower than those with all the tested materials for RLE. In case of dentin, except Pmax stresses of CAD/CAM materials, all the tested materials transferred higher VM and Pmax stresses when compared to the intact tooth. It was reported by previous studies that class II MOD cavities decreased the fracture resistance of intact teeth for about 59-76% –36. AccoEakle et al.  reportedAdditionally, there were significant differences between the von Mises and Pmax stress values and intact tooth. This resulted us to comment that buccal cusp reduction may also be needed in addition to lingual cusp coverage for GCC restorative material.Although there are many materials providing many options to the clinicians, it can be challenging to choose the best one for a given situation . VariousThere are different adhesive thicknesses in the literature: 2, 5, 10, and 30 micrometers. In the Ausiello et al.  study, aArola et al.  studied Glass carbomer cement is a newly developed material containing nanosized hydroxyapatite-fluorapatite particles in powder form . In a fiWith one of the newest developments in dentistry, CAD/CAM technology, it has been possible to design and fabricate restorations with mechanized, computer-aided techniques , 43. WitYamanel et al.  reportedOn the contrary, all restoration materials with different elasticity moduli transferred high VM and Pmax stresses to RLE. Considering the class II MOD inlay cavity design with proximal gingival margins 1 mm below the CEJ in the mandibular first molar tooth, reducing the remaining lingual enamel and including it into the cavity would be a more effective option.μm and the luting cement layer as 100 μm. Parallel with the Ausiello et al. [Ausiello et al.  reportedo et al.  study sio et al.  reportedo et al.  study beMicroleakage is still a concern for class II cavities having gingival margins in dentin. Uludag et al.  investigThe 3D-FEA analysis is an engineering tool applied to biology, medicine, and dentistry, from orthodontics to implantology, is able to investigate the mechanical behaviour of differently structured systems in vitro by a mathematical analysis and simulation. The goal consists of creating a model as close as possible to the real one. The obtained outputs are applicable and practical, have clinical significance, and give direction to experimental and clinical research. In this study, the information obtained for suitable restorative materials for MOD cavity had gingival margins ended in dentin tissue. However, restorations have other problems such as microleakage, polymerization shrinkage of resin containing materials, and postoperative sensitivity that should be investigated. As oral conditions cannot be completely imitated by in vitro studies, further in vivo studies are needed to determine the effectiveness and durability of materials for class II MOD cavities.As the lingual cusp of the mandibular first molar with MOD cavity was found to be more susceptible to damage than the buccal cusp, the cuspal coverage can be recommended in designing a class II MOD inlay cavityμm adhesive and 100 μm cement layer) and dental tissues, indicating that the failure initiation between the luting cement/adhesive and enamel has the least likelihoodVita Enamic, the PIHC CAD/CAM material, transferred a minimum amount of stress to the adhesive materials (30 Within the limitations of this study, the following statements can be drawn for the restoration of class II MOD cavity with proximal box gingival floor 1 mm below the CEJ in mandibular first molar:For this reason, this material may be a better choice to restore class II MOD inlay cavity with proximal box gingival floor 1 mm below CEJ when it is desirable to minimize stress concentrations in the mandibular first molars.


A prevalent PEDV strain, designated FJLY06, was isolated from Fujian, China. The four of structural genes sequences of PEDV obtained were analyzed to determine their phylogenetic relationships and homology respectively, revealing that FJLY06 was highly homologous to virulent PEDV strains. The four structural genes all differed genetically from the vaccine strain CV777. The sequence alignment results further showed that N, M and E genes of Chinese PEDV strains is highly conserved. Compared with the vaccine strain CV777, 8 mutations were detected in COE of FJLY06 S gene. The recombination analysis revealed FJLY06 is similar to other pandemic strains in China with a variant S gene, and maybe a reason for recent vaccination failures. PEDV has four major structural proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins . Since tThe sample was obtained from sucking piglets showing watery diarrhea and dehydration, which could not be cured with any commercial available antibiotic. The E, M, N and S genes of this PEDV strain was cloned and sequenced to investigate the genetic characteristics and phylogeny of PEDV circulating in Fujian during this period. These data will provide a basis for further analysis of the genetic evolution of PEDV in China, and should help to develop a novel vaccine of PEDV for more effective prevention piglets from PEDV.The FJLY06 was isolated from a sucking piglet collected in the Fujian province of China. After the piglet was necropsied, tissue samples of the intestinal, feces and intestinal contents from piglet were homogenized and 10% (w/v) suspensions were made in sterile Dulbecco’s phosphate-buffered saline . RNA was extracted from the sample using RNAiso Plus . Reverse transcription was carried out and PCR was performed  using the gene-specific primers. The amplified PCR products were subjected to gel electrophoresis, excised from the agarose gel, and purified using an Agarose Gel DNA Purification Kit (TaKaRa). The clones were sent to Shanghai Sangon Biological Engineering Technology and Services Co., Ltd.  to be sequenced. The S, E, M and N gene sequences of the FJLY06 were edited, aligned and analyzed with the DNAMAN software. The nucleotides sequences were assembled into a multiple sequence alignment. Phylogenetic trees derived from the nucleotides sequences were constructed using the program MEGA version 5.2 [Compared with the nucleotides sequences of 51 strains used in our study, the N gene of FJLY06 showed nucleotide identities of 94.3% to the vaccine strain CV777 used in China. Sequence comparison with the CV777 revealed that E gene of FJLY06 had nucleotide sequence identities of 96.5%. The M gene had 98.7% identity to the CV777 vaccine strain. The nucleotide sequence homology results of S gene showed FJLY06 had the low DNA sequence identity to the CV777, which is 92.2%.The phylogenetic analysis found that the evolutionary relationship of N, M, E and S genes of the FJLY06 from our study were more closely with Chinese strains isolated after 2010, and belong to the PEDV strain variants. Meanwhile, the results showed that the FJLY06 from our study differed genetically from the vaccine strain (CV777) and other earlier PEDV strains found in China, South Korea and Belgium, as well as some classical strains . There was no difference compared to those identified in Chinese strains isolated after 2010. In terms of amino acid sequence of M genes, the G2 including the FJLY06 and other prevalent variant strains had the same three unique mutations as CV777, , and no difference was found in the M gene of the FJLY06 when compared to the other prevalent variant strains. It also had a unique point mutation in E gene of the FJLY06 compared with CV777 (R → Q at 65), which was similar to the other strains of G2 group.The S gene of PEDV contains four domains that can induce neutralizing antibodies, COE 499–638 aa), SS2 (748–755 aa), SS6 (764–771 aa), and 2C10 (1368–1374 aa) [99–638 aaNucleotide sequence similarity were assessed by SimPlotv.3.5.1, and showed that the FJLY06 is very similar to other sequences in G2 group (Fig.