Bacillus based probiotics with gut microbial community members in cage free laying chickens both in rearing and production phases of life. In the current study, we investigated the changes in the gut microbiome of free range hens in the field after Bacillus based probiotic supplementation.The chicken gut microbiota passes through different stages of maturation; therefore, strengthening it with well characterised probiotics increases its resilience required for optimum gut health and wellbeing. However, there is limited information on the interaction of Bacillus based probiotic in gut health. Core differences in the beta diversity suggest that probiotic supplementation decreased microbial compositionality. The non-significant difference in alpha diversity between the probiotic and control chickens showed that the composition of community structure did not change. No Salmonella spp. were isolated from the probiotic supplemented birds. Egg internal quality was significantly higher, while egg production and body weight did not differ. Functional prediction data showed that probiotic supplementation enriched metabolic pathways, such as vitamin B6 metabolism, phenylpropanoid biosynthesis, monobactam biosynthesis, RNA degradation, retinol metabolism, pantothenate and CoA biosynthesis, phosphonate and phosphinate metabolism, AMPK signaling pathway, cationic antimicrobial peptide (CAMP) resistance and tyrosine metabolism.Overall, at phylum level, probiotic supplementation increased the populations of Bacteroidetes and Proteobacteria mainly at the expense of Firmicutes. The population of Bacteroidetes significantly increased during the production as compared to the rearing phase, and its higher population in the probiotic-supplemented chickens reflects the positive role of Bacillus based probiotic for optimum gut health and production.Overall, age was the main factor affecting the composition and diversity of gut microbiota, where probiotic supplementation improved the abundance of many useful candidates in the gut microbial communities. The generated baseline data in the current study highlights the importance of the continuous use of The online version contains supplementary material available at 10.1186/s42523-021-00112-5. In a healthy state, the assemblage of viruses, bacteria, archaea, and fungi forms a host microbiome that can mediate biological functions ranging from energy metabolism to immune response biomolecules production –4. A staSalmonella and Campylobacter. For example, an increased abundance of Faecalibacterium resulted in clearing Salmonella Typhimurium from the gut [Lactobacillus, and Lactococcus lactis and Bacillus thermoamylovorans were significantly higher in abundance in low body-weight laying hens, suggesting the role of these bacteria in body composition of the host [In layer chickens, gut microbiota influences performance and resistance to pathogens, such as  the gut . Higher  the gut . Gut hea the gut , rearing the gut , 18 and  the gut –21. Affethe host . Assessithe host . A compathe host . This suLactobacillus, Bacillus, Bifidobacterium and Streptococcus are used for probiotics formulation that are commercially available for poultry industry. Currently, there are limited reports on the development and maturation of gut microbiota in cage free/free-range laying chickens in the field [Bacillus based probiotic plays a role in improving the diversity and composition of gut microbiota in free-range laying chickens will help in devising strategies for improving gut health for product safety. The optimised probiotic can be used for the control of food safety pathogens, such as Listeria, Clostridium, Salmonella and Campylobacter. Therefore, we hypothesised that if used from the day of a hatch in the field, Bacillus based probiotic will improve the composition of gut microbiota, enhance egg quality and reduce gut pathogen colonisation in cage free hens in the field. The chosen Bacillus based probiotic is commercially available as a premix for poultry feed. The main objective of this study was to test the effects of Bacillus based probiotic on gut health and layer performance in free-range production system.One way to improve the diversity and composition of gut microbial communities is to supplement the diet with probiotics . Probiothe field , 28, 29.he field . FindingThe experimental work was approved by the Animal Ethics Committee at The University of Adelaide under approval number S-2019-109. Faecal swab collections were performed as per standard operative procedures approved by the Animal Ethics Committee.n = 40) were collected from empty rearing sheds (labelled as control and probiotic supplemented) before and after the clean-up procedures to determine the contamination level of Salmonella spp. (if any). The experimental flock (Hyline Brown) was distributed into 2 rearing sheds on a pullet-rearing farm from day 1 of placement with 10,860 and 10,457 chicks in the control and probiotic supplemented sheds, respectively. The stocking density was 30 birds/m2. One shed acted as a control, while chicks in the other shed received a premix of Bacillus based probiotic at a rate of 1 g/kg of feed from day 1 until the termination of the experiment (week 36 of flock age). The commercially available probiotic was composed of Bacillus subtilis DSM 32324, Bacillus subtilis DSM 32325 and Bacillus amyloliquifaciens DSM 25840. This probiotic was chosen as its continuous supplementation in a pen trial resulted in the overall lower shedding level of Salmonella Typhimurium [n = 40) to determine the contamination level of Salmonella spp. (if any). The pullets were shifted to free-range production sheds (control and probiotic supplemented) at 16-week of flock age. The distance between the rearing and production farms is approximately 30 km. Fresh faeces (n = 20) for DNA extraction, faecal swabs (n = 20) and environmental dust swabs (n = 10) for Salmonella isolation from each treatment group at each sampling time point were collected at day 1 (meconium samples), 5, 21, week 6, 12, 16 (Day 1 and 5 after shifting), 18, 24, 30 and 36 of flock age. Therefore, for 11 sampling time-points, a total of 440 faecal DNA samples, 440 faecal swab samples and 220 environmental dust swab samples were processed during this study. Once in lay, eggs (n = 30) from each treatment group were collected at week 24, 30 and 36 of flock age and processed for egg quality measurements. Faecal swab samples were collected in 4 mL buffered peptone water , while environmental swabs  were soaked in 20 mL BPW and individual swabs were dragged to cover at least 1 m2 area in the shed including exhaust fans and covering boards of nest boxes. Shoe covers from each shed were soaked in 150 mL BPW and processed for Salmonella isolation.A commercial farm was selected based on the willingness of the farm manager and the farm set up that was appropriate for this study. Prior to placing the chicks, environmental swabs  method was used to semi-quantify the load of Salmonella from the confirmed positive samples. Confirmed Salmonella isolates were further processed for traditional PCR using an invasion (invA) and TSR3 genes specific primers.The collected samples were processed for the isolation of n = 440; 20 samples per treatment group at each sampling for a total of 11 time-points) were sequenced by the Ramaciotti Centre for Genomics  for 16S rRNA metagenome sequencing and subsequent data analysis for the generation of operational taxonomic units (OTUs) table. For microbial profiling, the hypervariable region (V3-V4) of the 16S rRNA gene was sequenced using a barcoded primer pair .Total DNA from the collected faeces was extracted following a modified protocol of QIAamp FAST DNA Mini Kit , 9.5 μL of PCR grade water, 1 μL of each of the barcoded forward and reverse primers and 1 μL of faecal DNA template. The cycling conditions in SimpliAmp Thermal Cycler (Applied Biosystems) were: initial denaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 30 s for a total of 35 cycles. A final elongation at 72 °C for 5 min was included at the end of the cycles. The PCR amplicons were normalised and pooled using the SequalPrep Normalization Plate Kit  as per the manufacturer’s guidelines. The PCR library was purified using AxyPrep Mag PCR Clean-Up Kit  as per the manufacturer’s protocol. The quality and concentration of the pooled library were assessed by Qubit, and the library size was estimated on an Agilent 2200 TapeStation . From the pooled library, primer-dimer was removed by the Agencourt AMPure XP Bead Clean-up kit. The pooled library was sequenced on Illumina MiSeq using the MiSeq Reagent Kit v3 with a 2 × 300 bp run format as per the manufacturer’s protocol. For the MiSeq runs, custom primers were added to the reagent cartridge for Read1, Index and Read2.For individual faecal DNA samples, the n = 11,092). Sequencing error was assessed using the NO SEQ ERROR TEXT as a control in each run.Raw sequencing reads (Fastq files) were processed with the OTUreporter v1.0.1-beta (5576d57) pipeline base on mothur (v1.39.5) , 31. Bri2 relative abundance to account for the non-normal distribution of taxonomic counts data. In the CSS normalisation, raw counts are divided by the cumulative sum of counts up to a percentile determined using a data-driven approach [The OTU data were analysed in Calypso v 8.84 that functions based on R packages vegan  for micrapproach . Genus/Pq value > 0.05, leaving only the significant features to be visualised. In STAMP, Benjamini-Hochberg FDR was used for multiple test correction.The workflow of Tax4Fun2  was usedThe collected eggs were processed for measuring egg weight, shell weight, shell thickness, albumen height, Haugh Unit and yolk colour following a previously described method . Briefly10 (HT - 1.7 * EW^0.37 + 7.6); where HT is albumen height (mm) and EW is egg weight (g).HU = 100 * logIn addition, at the farm, egg production was recorded daily, while body weight of 100 birds from each shed was recorded weekly. Where appropriate, egg quality data were analysed in StatView v5.0.1.0 with one- or two- way ANOVA. Level of significance was established at protected least significant difference (PLSD) < 0.05. Flock performance data  were visualised in Excel Workbook.The sequence data assessment showed that out of 440 faecal DNA samples, 8 samples (1.81%) generated less than 10,000 reads per sample after passing the QC and chimera removal, and were therefore discarded from the downstream analysis  decreased the overall abundance levels (%) of Firmicutes and Spirochaetes and increased (P < 0.0001) Elusimicrobia  significantly different between the probiotic supplemented and control flocks were further investigated to understand the effects of probiotic in a longitudinal manner. The data showed that the probiotic supplementation affected the composition of gut microbiota with significant effects observed from Day 21 until Week 30 of flock age at least for one or more microbial communities Fig. . The abulla Fig.  a-d, and_ge Fig.  e, f werSphaerochaeta and Rikenella  in the community structure of individual samples collected from the probiotic and the control flocks  evenly distributed  and how evenly the taxa are distributed (evenness). Alpha diversity measures variation in the structure of microbial community within individual samples. Measured by the Shannon index and Richness, overall there was no significant variation P > 0.0 in the cP < 8.1e− 126) with flock age both in the probiotic supplemented and control flocks  and control (P = 1.7e− 78) flocks, the alpha diversity of individual samples within each treatment group significantly varied from day 21 and onwards of flock age. Alpha diversity (measured by Shannon index) both in the control and probiotic supplemented groups increased from day 5 to week 16 before the flocks were moved to a free range farm for egg production. The probiotic supplemented flock showed a significantly lower alpha diversity on week 16.5 just after transportation to the production farm and on week 36 of flock age. This indicates that immediately after transportation, the alpha microbial diversity increased in the control group. For assessing the effects of feed on the alpha diversity of gut microbiota, the data were analysed on the basis of feed category phases, such as starter, grower, pre-lay and peak-lay. As expected, the overall alpha diversity (measured by Shannon index) showed significantly lower values (P = 7.2e− 62) in the starter phase of the feeding regimen  in the control flock  in the probiotic supplemented  at week 16 in the control flock  than the control flock  the beta diversity in the production phase of the chickens  the beta diversity in both phases  when the flocks were on prelay and peak lay diets  diversity post-transportation  different  with flock age both in the probiotic supplemented and control flocks  on it  in composition with a change in diet regimen  was performed. The community structure of the probiotic and control flocks overlapped with each other but was significantly .The effects of the probiotic on gut microbial metabolic pathways were assessed by performing functional predictions analysis in Tax4Fun2 using the KEGG orthologies (KOs) database and STAMP for Welch’s t-test for calculating the level of significance between the treatment groups. The data were mapped to 345 functional metabolic pathways  was higher for the probiotic supplemented compared with the control flock  improved egg internal quality but not shell quality  measured on 100 birds at weekly intervals was not different between the control and probiotic supplemented flocks , no Salmonella was isolated from the probiotic supplemented shed. Measured through the mMPN method, a load of Salmonella in the faecal swab, environmental swab and shoe cover was 7.357, 11.170 and 15.06 per mL of BPW, respectively.No Salmonella Typhimurium, while one each of the environmental and shoe cover samples collected at 36 weeks of flock age from the control shed was non Typhimurium serotypes . The increased abundance of Bacteroidetes in the gut at the production stage compared with the rearing both in the control and probiotic flocks shows age driven effects. Interestingly, the probiotic effects on the abundance level of Bacteroidetes were higher in the production compared with the rearing stage. The decreased abundance of Firmicutes after week 16 of flock age might explain the increase in Bacteroidetes that contains both succinate and propionate producing microbial communities.At the phylum level, the composition of gut microbiota dominated by Bacteroidetes, Firmicutes and Proteobacteria show the important role of these bacteria in gut health. These data also show Proteobacteria is the third dominant phylum in the gut of chickens. In a previous study, Bacteroidetes, Firmicutes and Proteobacteria have been shown as the main dominant phyla in the caecal content of laying chickens . A 3.07%P = 0.001). Interestingly, assessing the effects of transportation of birds from rearing to production sheds on the beta diversity of gut microbiota showed that variation in the community structure increased in the probiotic supplemented flock post-transportation.The non-significant difference in alpha diversity (measured by Shannon index) between the probiotic supplemented and control flocks showed that microbial communities were more evenly distributed in the rearing phase. Probiotic supplementation did not significantly affect the alpha diversity (measured by Shannon index) of most of the faecal samples collected at different time-points. An overall significantly higher alpha diversity of microbial communities in production compared with the rearing phase highlights the role of rearing conditions. A steady increase in the alpha diversity index of the probiotic supplemented and control flocks with age shows that age had profound effect on community structure within chickens. However, beta diversity showed a reduction trend with flock age, suggesting that as the chickens increased in age, the variation in the gut microbial communities between birds reduced. Beta diversity was also lower for the probiotic supplemented flock  supplemented diet [Lactobacillus casei supplemented diet [Paraprevotella is involved in polysaccharide degradation, and propionate and butyrate production through the expression of xylose isomerase, cobalamin-binding methylmalonyl-CoA mutase and/or methylmalonyl-CoA epimerase and acetyl-CoA acetyltransferase, respectively [Megasphaera preferably produces butyrate through the production of acetyl-CoA acetyltransferase [Elusimicrobium is a member of the phylum Elusimicrobia, which was the least abundant both in the control and probiotic supplemented flocks. Elusimicrobium minutum is the only identified specie in the Elusimicrobia and is an obligatory anaerobic ultramicrobacterium that ferments glucose [Succinatimonas is a strict anaerobe that produces succinate and acetate from carbohydrates [B. licheniformis alleviated the adverse influence of heat on egg production and gut health [Bacillus subtilis strain DSM29784 has been shown to selectively increase the abundance of gut microbial genera, such as Bifidobacterium, Lactobacillus and Alistipes in different growing stages of layers [Bacillus based probiotic supplementation in the diet of layers.The intestinal microbiota is involved in the fermentation process of complex carbohydrates, such as indigestible dietary fibers and sugars to generate SCFAs that help in maintaining a healthy gut environment . In the tabolism . In our chickens . Parasutted diet , and in ted diet . In layiectively . Megasphnsferase . Elusimi glucose . Succinahydrates . Membershydrates . In 60-wt health . Bacilluf layers . The curIn the functional prediction data, the significant enrichment of metabolic pathways, such as vitamin B6 metabolism, cyanoamino acid metabolism, retinol metabolism, lipopolysaccharide metabolism and biosynthesis of secondary metabolites show that probiotic supplementation improved gut functions. Vitamin B6 contributes to intestinal immune regulation through the metabolism of lipid mediator sphingosine 1-phosphate. Microbial vitamin B6 is synthesized as pyridoxal phosphate from d-ribulose 5-phosphate and glyceraldehyde-3-phosphate or 4-phosphohydroxy-L-threonine and deoxyxylulose 5-phosphate . SeveralBacillus based probiotics in layers. In laying hens, diet supplemented with Bacillus based probiotics has shown to improve egg production and overall egg quality. For example, supplementation of Bacillus subtilis but not Bacillus licheniformis in layer diet resulted in improved egg internal quality and egg production [Bacillus licheniformis to a 28-week old layers flock for up to 8 weeks resulted in increased egg production, shell thickness and better intestinal epithelial cell morphology [Bacillus subtilis supplementation for up to 8 weeks resulted in improved FCR and reduced serum cholesterol level [Bacillus based probiotic on egg internal quality parameters; however, it is worth mentioning that the egg quality was measured only at week 24, 30 and 36 weeks of flock age. The positive effects of probiotics could be due to improved nutrient absorption in the gut [Different strains of probiotics vary in their mechanistic actions; therefore, we have only compared the findings of the current study with studies that used oduction . Feedingrphology . In 24-wol level . In the  the gut , 62 or t the gut , 64. EggSalmonella detected prior to the placement of day-old chicks on rearing and 16-week pullets on production farms demonstrates that the decontamination procedures performed were appropriate. Effective cleaning and disinfection of poultry sheds reduce the levels of Salmonella contamination; however, the recovery of Salmonella spp. from surfaces such as dropping boards and floors in cleaned and disinfected sheds is variable [Salmonella serotypes such as, Salmonella Mbandaka and Salmonella Typhimurium were isolated from a free-range layer production system [Salmonella isolation from the probiotic supplemented shed could be attributed to the beneficial effects of probiotics; however, a conclusive statement could not be made as the flock was not followed beyond 36 week of age. Moreover, the number of Salmonella positive samples were low (n = 3); therefore, it is hard to estimate the true effects of the Bacillus based probiotic on the flock Salmonella spp. status. In a pen trial, feeding of Bacillus based probiotic reduced the overall load of Salmonella in faeces but not all of the birds turned negative for Salmonella in the 12 week period of sampling [No variable . In a prn system . In the Bacillus based probiotic on the gut. The improvement in egg internal quality parameters suggests that the Bacillus based probiotic could be used continuously in a cage-free poultry production system. Exploring suitable alternative multidisciplinary programs that encompass efficient management as well as optimize nutrition and disease management is of great interest to the poultry industry. Findings from this study can play a significant role in creating an antibiotic-free production environment.In this field study, the most abundant bacterial phyla were Bacteroidetes, Firmicutes, and Proteobacteria in the gut microbiota. The probiotic supplementation from day-old significantly decreased overall abundance levels (%) of Firmicutes and Spirochaetes and increased Elusimicrobia. At the genus level, the higher abundance of Elusimicrobium, Megasphaera, Parasutterella, Desulfovibrionaceae_unclassified, Paraprevotella, Succinatimonas, bacteria_unclassified, and Muribaculaceae_ge in the probiotic supplemented flock shows its positive effects on gut health. The functional enrichment of metabolic pathways including cationic antimicrobial peptide (CAMP) resistance, vitamin B6 metabolism, AMPK signaling pathway, monobactam biosynthesis, RNA degradation, and tyrosine metabolism highlights the positive effects of the Additional file 1. Layer chicken diet composition, isolation of Salmonella from swabs, faecal DNA extraction and traditional PCR.Additional file 2. Quality of reads generated in the study.Additional file 3 Fig. S1. Rarefaction curve analysis of OTUs depicting the quality of the 16S rRNA reads generated from DNA obtained from laying chickens faeces. a) Rarefaction curve analysis of the reads based on the probiotic supplemented and control cohorts. b) Rarefaction curve analysis of the reads based on samples collected at Day (D) and Week (W) post-hatch from the probiotic supplemented (P) and control (C) groups. The flatten curves towards right show that the underlying microbial communities were well covered by the sequenced data.Additional file 4. OTU Table containing details of taxa of faecal samples collected from the probiotic supplemented and control groups at different sampling time-points. (SUMMARY 1369 kb)Additional file 5. Metadata file required for the OTU Table for further analysis and data visualisation. (CSV 52 kb)Additional file 6 Fig. S2. Overall gut microbiota composition at phylum level in both the probiotic supplemented and control flocks pooled together. Microbial abundance (%) in the probiotic supplemented group. For percent calculation of microbial abundance at phylum level, total sum scaling normalised but untransformed data obtained from Calypso software were visualised in Excel 2016 and panel graphs were prepared in Graphpad prism v. 8.0.0.Additional file 7 Fig. S3. Abundance of gut microbiota at genus level affected by probiotic supplementation at multiple sampling periods. a). Bacteroides. b). Rikenellaceae_RC9_gut_group. c). Lactobacillus. d). Alistipes. In each panel of the Supplementary Fig. S3, the letters “D” and “W” refer to day and week post-hatch, while the letters “C” and “P” refer to control and probiotic supplemented groups, respectively.Additional file 8 Fig. S4. Abundance of gut microbiota at genus level affected by probiotic supplementation at multiple sampling periods. a). EscherichiaShigella. b). Enterococcus. c). Clostridium sensu stricto. d). Enterococcaceae_unclassified. In each panel of the Supplementary Fig. S4, the letters “D” and “W” refer to day and week post-hatch, while the letters “C” and “P” refer to control and probiotic supplemented groups, respectively.Additional file 9 Fig. S5. Alpha diversity of gut microbiota of laying chickens affected by probiotic supplementation, rearing and laying conditions. a). Alpha diversity (Shannon index) of gut microbiota affected by feeding regimen. b). Richness of gut microbiota affected by feeding regimen. c). Evenness of gut microbiota affected by feeding regimen. d). Alpha diversity (Shannon index) of gut microbiota affected by transport stress. e). Richness of gut microbiota affected by transport stress. f). Evenness of gut microbiota affected by transport stress. Within each treatment group, “C” refers to control, while “P” refers to the probiotic supplemented cohort. Alpha diversity was measured at genus level by Shannon index, Richness and Evenness in Calypso software. Within each treatment group at specific sampling period, asterisk (*) shows a significant difference at P < 0.01, while asterisks (**) show P < 0.001.Additional file 10. Details of metabolic pathways included in the functional prediction analysis. (XLS 3419 kb)Additional file 11 Fig. S6. Metabolic pathways of the gut microbiota affected by probiotic supplementation. To understand the effect of Bacillus based probiotic on the differential abundance of metabolic pathways of the gut microbiota, the functional prediction data obtained through Tax4Fun2 were analysed in STAMP by using Welch’s t test with 99% confidence interval. For multiple test correction in STAMP, Benjamini-Hochberg was used with q value filter > 0.05 that resulted only in the features that were significantly different between the two treatment groups.Additional file 12 Fig. S7. Metabolic pathways of the gut microbiota affected by probiotic supplementation in the rearing phase of laying flock. To understand the effect of Bacillus based probiotic on the differential abundance of metabolic pathways of the gut microbiota, the functional prediction data obtained through Tax4Fun2 were analysed in STAMP by using Welch’s t test with 99% confidence interval. For multiple test correction in STAMP, Benjamini-Hochberg was used with q value filter > 0.05 that resulted only in the features that were significantly different between the two treatment groups. In the figure, titles Rear_C and Rear_P refer to faecal samples collected from the control and probiotic supplemented cohorts at rearing phase of flock age, respectively.Additional file 13 Fig. S8. Metabolic pathways of the gut microbiota affected by probiotic supplementation in the rearing phase of laying flock. To understand the effect of Bacillus based probiotic on the differential abundance of metabolic pathways of the gut microbiota, the functional prediction data obtained through Tax4Fun2 were analysed in STAMP by using Welch’s t test with 99% confidence interval. For multiple test correction in STAMP, Benjamini-Hochberg was used with q value filter > 0.05 that resulted only in the features that were significantly different between the two treatment groups. In the figure, titles Prod_C and Prod_P refer to faecal samples collected from the control and probiotic supplemented cohorts at production phase of flock age, respectively.Additional file 14 Fig. S9. Feed conversion ratio of the probiotic supplemented and control flocks. Weekly (W) feed conversion ratio was calculated from feed intake and flock body weight.Additional file 15 Fig. S10. Lay rate and egg weight of probiotic supplemented and control flocks.Additional file 16 Fig. S11. Body weight of probiotic supplemented and control flocks. Body weight of 100 randomly selected chickens was taken weekly (W) from each of the probiotic supplemented and control flocks.Additional file 17 Fig. S12. Egg quality parameters of eggs collected from probiotic supplemented and control cohorts. a) Egg weight (g). b) Shell weight (g). c) Shell thickness (mm). d) Albumen height (mm). e) Haugh Unit. f) Yolk colour. Freshly laid eggs were collected at week 24, 30 and 36 week of flock age and immediately analysed for egg shell and internal quality parameters.Additional file 18 Fig. S13. PCR products ofSalmonellapositive samples visualised on 2% agarose gel. L) Ladder; 1)Salmonella Typhimurium isolated from faecal swab at week 18 of flock age from the control shed. 2)Salmonella spp. isolated from environmental swab isolated at week 36 of flock age from the control shed; 3)Salmonella spp. isolated from environmental swab isolated at week 36 of flock age from the control shed. 4)Salmonella Typhimurium as positive control. For Salmonella spp. typing primer pair (605 bp) amplifying the fragment of invA gene were used, while for Salmonella Typhimurium confirmation, primer pair (303 bp) amplifying the fragment of TSR3 gene were used.


This work evaluated the efficacy of pomegranate byproducts, specifically peel powder, as valid preservatives for food quality. Ready-to-cook cod sticks breaded with pomegranate peel powder were prepared. Shelf-life tests were conducted on breaded cod sticks during refrigerated storage (17 days) at 4 °C, monitoring the pH, microbiological and sensory quality. In addition, the nutritional quality of both the breaded and control samples was assessed. The results highlighted that active samples showed higher phenol and flavonoid content and higher antioxidant activity compared to the control fish, suggesting that pomegranate peel powder was responsible for a significant increase in cod stick nutritional quality. Furthermore, the cod stick active breading led to a delay in microbial growth without affecting the sensory properties; rather, it helped slow down the sensory attribute decline during the refrigerated storage. The data suggest that using pomegranate byproducts in breaded cod stick was effective in prolonging its shelf life, as well as improving its nutritional quality. Therefore, pomegranate peel powder can be considered as a potential resource as natural food preservative. The growing interest to extend the shelf life of fresh fish has led to numerous studies on the optimization of handling, refrigeration and transport, packaging practices and nonthermal methods to maintain the high quality and product safety of fish goods . Fresh fFood applications and potential health benefits of pomegranate and its derivatives have been abundantly discussed . Li et aIn this context, this study tested pomegranate peel powder on breaded cod sticks. To this aim, the breading was prepared with and without adding pomegranate peel powder. During refrigerated storage, vital cell loads, pH and sensory quality were monitored to assess whether the addition of the active ingredient to the classic breading could extend the shelf life of the packaged food product. In addition, differences in phenolic compounds, flavonoids and antioxidant activity between the control and the active samples were also assessed to evaluate the nutritional quality of the tested samples.The nutritional quality of breaded cod sticks added with pomegranate peel powder was evaluated in terms of total phenols (mg GAE/g dw), flavonoids (mg QE/g dw) and antioxidant activity ; the data obtained in this study are shown in Pseudomonas spp. (PSE), Shewanella putrefaciens (HSPB) and Photobacterium phosphoreum (PHAB). The TMB and TPB evolution of the investigated breaded cod sticks is shown in 6 cfu/g) after about 6 days of storage.The microbial quality decay of the investigated breaded cod sticks was determined by monitoring the viable cell concentration of total mesophilic and psychrotrophic bacteria , 2S off-odors and off-flavors, which in turn are produced by specific microbial groups [Pseudomonas spp. (PSE), hydrogen sulphide-producing bacteria (HSPB—Shewanella putrefaciens) and psychrotolerant and heat labile aerobic bacteria (PHAB—Photobacterium phosphoreum) were also monitored.The active samples showed a slower increase in mesophilic counts compared to the control. In particular, at the end of the storage period A, B and C samples reached values of 5.12, 4.39 and 4.13 log(cfu/g), respectively; whereas the Con sample reached 8.09 log(cfu/g). These results suggest that pomegranate peel powder was effective in slowing down the microbial growth. A similar trend has also been observed on the psychrotrophic counts in all the investigated samples b. The rePseudomonas spp. viable cell concentration of active samples was observed if compared to the control sample, which reached the microbiological acceptability limit (106 cfu/g) on the ninth day, whereas all the active samples never reached the limit during the 17 days of monitoring.As can be observed from data shown in Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.The abovementioned results suggest that adding pomegranate peel powder delayed microbial growth. In fact, during the observation period a substantial difference between active samples and Con of about 1 log cycle was observed. Similar results were reported by Khan and Hanee , who shoShewanella viable cell concentration in the investigated samples during refrigerated storage. A similarity can be observed between the evolution of Shewanella viable cell concentration and that of aerobic mesophilic bacteria. In particular, HSPB counts for all investigated samples were initially low, about 2.0 log (cfu/g), suggesting a good initial quality of cod fish. During refrigerated storage, a significant microbial growth inhibition was evident in all active samples compared to the control sample, even though during the observation period no samples exceed the microbiological acceptability limit (106 cfu/g). A low viable cell concentration was also recorded for P. phosphoreum, where the cell load was always below the detection level for all investigated samples (data not shown). Instead, for Enterobacteriaceae, there was a substantial difference between control and all active samples. The Con sample began to increase from the third day of storage (3.46 log(cfu/g)), with a steady trend along the entire observation period (data not shown). To sum up, the control fish became unacceptable for excessive TMB and TPB proliferation around the sixth day of storage, whereas all the investigated active fish samples remained acceptable during the entire observation period (17 days) with cell loads that never reached microbial thresholds.Another important physical-chemical characteristic is the pH, used to establish the quality and durability of a given food product. The pH evolution during refrigerated storage of the investigated samples is reported in As reported beforehand, a panel of expert judges was used to assess the breaded cod stick sensory properties in terms of odor, color, texture and general appearance. The mean values of sample overall quality were reported in The latter result could be related to the growth of off-odor produced by microbial proliferation. In fact, as discussed beforehand, pomegranate peel powder has been proven to be effective in slowing down the growth of microorganisms responsible for off-odors development. A similar trend was also found in the study by Khodanazar , where a−1) and rinsed. The pomegranates were cut manually to separate the seeds/arils, the pulp and the peel. Pomegranate juice was extracted using a fruit extractor  and stored at −18 °C until it was used. Pomegranate peel and pulp were separated, cut into small pieces with a sharp knife and dried in a food dehydrator  at 38 °C for 48 h. The dried product was reduced to powder in a laboratory blender and then sieved using a 500-μm sieve. Fine peel and pulp powders were stored separately in plastic bags at 4 °C in an environment protected from light. Other ingredients used to prepare the cod stick breaded fish, such as breading, spices, potato flakes and fresh milk, were purchased at a local market .Refrigerated salted cod fillets and pomegranate fruits were purchased at local market . Pomegranates were carefully washed with tap water to remove residues, immersed for 1 min in chlorinated water (20 mL Lw/w water and about one percent w/w NaCl) were cut into sticks of about 12 g. Two mixtures were prepared: no-active mixture (no-active mix) containing breading with fish spices and potato flakes in a ratio of 1:1 and active mixture (active mix) prepared with pomegranate peel powder and non-active mix in a ratio of 1:1. Four sample types were prepared . The control sample (Ctrl) was obtained as follows: after dipping in a solution of water and milk (1:1), the sample was breaded in the no-active mix by repeating the passage twice; then, it was manually compacted, placed above a food tray with a pad and packaged in air using a high-barrier film bag (multilayer film Nylon/Polyethylene) with a thickness of 150 µm, provided by Biochemia  and kept under refrigeration (4 ± 1 °C). All active samples were prepared using the same procedure as for the control sample but using different breading: active sample A (A) was breaded twice in the active mix, active sample B (B) was first breaded with pomegranate peel powder and then with the no-active mix and active sample C (C) was first breaded with pomegranate peel powder and then with the active mix. All the samples were stored at 4 ± 1 °C for 17 days. Uncooked samples were used to determine the pH, sensory and microbiological quality during the entire storage period. The nutritional quality of both cooked and uncooked samples was also assessed. Samples were cooked at 200 °C for 15 min in an electric oven . Two replicates for each treatment were evaluated to ensure repeatability.Cod fillets were coarsely desalted, soaked and stored at 4 ± 1 °C for five days, changing the water every day. On the sixth day, cod fillets were drained to remove excess water for about half an hour, and the skin was removed. Then, fillets (about 67% 2S2O8), Trolox , aluminum chloride (AlCl3), sodium nitrite (NaNO2), sodium hydroxide solution (NaOH) and quercetin were supplied from Sigma-Aldrich . Anhydrous sodium carbonate (Na2CO3) was supplied from Carlo Erba . All reagents were of analytical grade.Folin–Ciocalteu reagent, gallic acid monohydrate, ethanol, ABTS diammonium salt), potassium persulfate (Kv/v) in Erlenmeyer flasks and maintained for 30 min in a water bath  at 60 °C under agitation. The extracts were filtered to obtain clear supernatants. Triplicate extractions were made for each sample.For chemical analyses, both raw  and cooked samples  were first subjected to drying at 35 °C in a ventilated stove , milled to obtain a powder and then subjected to extraction, as reported by Cedola et al. . Briefly2 = 0.9934). Total flavonoid content was determined using the aluminum chloride colorimetric method, according to Cedola et al. [2 = 0.994). The antioxidant activity of breaded cod sticks was assessed using the ABTS  assay method. The test is based on the ability of the antioxidants to interact with the radical cation 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), inhibiting its absorption at 734 nm. The analysis was conducted according to the method used by Cedola et al. [2 = 0.9976), and the antioxidant activity was expressed as milligrams of Trolox equivalents/gram of dry weight (dw). All tests were conducted in triplicate.All the chemical analyses were performed the same day the samples were prepared. Total phenols were determined according to the Folin–Ciocalteu method, as reported by Cedola et al. . The cola et al. . The meaa et al. . A calibPseudomonas spp., hydrogen sulfide-producing bacteria (HSPB), psychrotolerant and heat-labile aerobic bacteria (PHAB) and mesophilic and psychrotrophic bacteria, Enterobacteriaceae, according to Danza et al. [6 cfu/g for total viable mesophilic and psychrotrophic bacteria, 106 cfu/g for Pseudomonas spp. and Shewanella 107 cfu/g for Photobacterium [Control and active samples (20 g each) were aseptically weighed into a sterile stomacher bag, diluted with peptone water (dilution 1:10) and homogenized for 90 s with a Stomacher LAB Blender 400 . Serial dilutions were plated onto specific media in Petri dishes to enumerate a et al. . The conThe measurement of pH was performed in triplicate on the first homogenized dilution of fish samples, using a pH meter . Two samples were used for each measurement. Microbiological analyses and pH were analyzed at the initial time and after 3, 6, 10, 13, and 17 days of refrigerated storage at 4 °C.The quantitative descriptive analysis (QDA) was used for a sample comparison, according to the guidelines of the Codex Alimentarius Commission. To this aim, breaded cod sticks were submitted to a panel of five trained judges. The panelists had familiar eating habits with fish and fish products and had experience in the evaluation of burgers and fillets based on fish. They were retrained for two days (2-h sessions) to establish the appropriate attributes for sensory evaluation and to minimize individual differences and ensure repeatability of the results. The panelists were asked to give judgments on odor, color, appearance, texture, and overall quality using a nine-point scale. In the scale, 9 corresponded to excellent, 8 to very good, 7 to good, 6 to reasonable, 5 to not good (acceptable limit), 4 to dislike, 3 to bad, 2 to very bad and 1 to completely unacceptable . Before p < 0.05), was performed to determine significant differences among the samples. STATISTICA 7.1 for Windows  was used.Experimental data were compared by one-way analysis of variance (ANOVA). A Duncan’s multiple range test, with the option of homogeneous groups (In this work, pomegranate byproducts were included in cod stick breading to improve the final product quality from a nutritional perspective, as well as to prolong its shelf life by about three times. The obtained results showed also a significant improvement in the nutritional quality of fortified samples after cooking. In fact, final cooked cod sticks breaded with pomegranate peel powder were characterized by phenolic compounds from three to five-fold higher than the cooked control, flavonoids from two to seven-fold higher than the cooked control and antioxidant activity from two to four-fold higher than the cooked control fish. Furthermore, the results underlined that the addition of pomegranate peel powder also slowed down the microbial growth during refrigerated storage, without negatively altering its sensory characteristics. Rather, as deduced from the results found, the addition of pomegranate peel powder to the cod stick breading considerably reduced the sensory decline during refrigerated storage compared to the control sample. Therefore, it can be concluded that the reuse of pomegranate by-products in fresh food industry could be a sustainable way to reduce environmental impact and costs associated with byproducts disposal, with great advantages to the product quality and its shelf life.


Background: Walking fatigability is prevalent in MS and can be measured by a percentage distance decline during a 6-min walking test. Walking is characterized by an accurate and consistent interlimb antiphase coordination pattern. A decline in coordination each minute during a 6-min walking test is observed in persons with MS (pwMS). Measuring coordination during a 6-min seated coordination task with minimized balance and strength requirements, is assumed to examine a more fundamental interlimb antiphase coordination pattern in pwMS. This research aimed to answer the following research question: How does interlimb antiphase coordination pattern change during a seated coordination task in pwMS with walking fatigability (WF), non-walking fatigability (NWF) and Healthy Controls (HC)?Methods: Thirty-five pwMS and 13 HC participated. Interlimb coordination was assessed by a seated 6-min coordination task (6MCT) with the instruction to perform antiphase lower leg movements as fast as possible. Outcomes were Phase Coordination Index (PCI) and movement parameters .Results: Mixed models revealed a significant effect of time for the the variability of generating interlimb movements, with a difference in mean values between WF and HC. A significant group∗time interaction effect was found for movement amplitude, represented by a significant decrease in movement amplitude in the WF group from minute 1 to the end of the task.Conclusion: The higher variability in interlimb coordination and decrease in movement amplitude over time during the 6MCT in the WF group could be an indicator of decreased control of fundamental antiphase coordination pattern in pwMS with walking fatigability.Clinical Trial Registration:www.clinicaltrials.gov, identifier NCT04142853  and NCT03938558 . Fatigue is highly reported in people with MS (pwMS), with a prevalence up to 80%, showing a high impact on quality of life . Recent In recent years the focus of the assessment of motor fatigability have been changed from body function level of ICF to walking at ICF activity level. Walking-related fatigability has been quantified in pwMS by comparing walking velocity during different paradigms , and morWalking requires accurate and consistent antiphase interlimb coordination pattern, to ensure safe ambulatory functions. Interlimb coordination involves a distributed network at both supraspinal and spinal level . It has The aim of this study was consequently to examine more fundamental antiphase interlimb coordination pattern while seated in pwMS, with and without walking fatigability in comparison to healthy controls.www.clinicaltrials.gov: NCT04142853  and NCT03938558 (MS n = 18). Seven pwMS participated in both studies and therefore, data of their first participation (NCT03938558) was included. PwMS and healthy controls (HC) were tested at the REVAL Rehabilitation Research Centre of Hasselt University, and recruited via REVAL, Rehabilitation, the MS Centre Overpelt and the non-for-profit organization Move To Sport. The ethical committee of Hasselt University, MS Centre Overpelt and Hospitalo-Facultaire Universitaire de Liège approved the protocols (B707201835771 and B9115201836892) and written informed consent was obtained from all participants. Participants were aged between 18 and 70 years, and had to be able to walk for 6 min with or without support . PwMS were diagnosed according to the McDonald criteria , Paced Auditory Serial Addition test (PASAT), Single Digit Modalities test (SDMT), Modified Fatigue Impact Scale (MFIS), and Multiple Sclerosis Walking Scale (MSWS-12), were collected in pwMS.6–1) (∗100. Before the start of the test (baseline), and after each minute of the 6MWT and 6-min coordination test (6MCT), participants were asked to rate their subjective perception of fatigue on a VAS scale, where 0 is no fatigue and 10 is extremely fatigued. Change in perceived fatigue was calculated as followed: (perceived fatigue VAS score in minute 6 - perceived fatigue VAS score at baseline) × 10.To divide patients into groups with or without walking fatigability, subjects performed the 6-min walking test (6MWT) according to the script of 6–1)  was used6–1) : . The accuracy in generating antiphase left-right leg movements is expressed by absolute error ABS(φ) = ((mean |φi−180|)/180) × 100. The coefficient of variation (CV φ) was used to express the consistency in generating antiphase left-right leg movements CV(φ) = (Standard deviation φ/mean φ) × 100.Originally, the phase coordination index (PCI) measures the consistency and accuracy in generating antiphase left-right leg movements while walking . The PCIThe PCI is the sum of the absolute error (ABSφ)and coefficient of variation (CV) of the relative phase (φ), expressed as a percentage. A lower PCI indicates a higher phase control and coordination.The movement amplitude, seen as the spatial measure, consists of the peak-to-peak amplitude for each individual cycle . Movemen® Pro 14.10. . Linear mixed models were used to analyze the effect of group and time on interlimb coordination and spatiotemporal parameters, after checking the conditional residuals plots. To assess asymmetry between legs, mean values of movement amplitude and movement frequency of left and right leg were compared. Comparison of the mean values over 6MCT between left and right legs revealed no significant difference in any groups. Therefore, the analysis for spatiotemporal parameters during the 6MCT was performed with the mean value of both legs, per minute. Fixed effects were included in the mixed model which were group , time  and the interaction group∗time, using a Bonferroni correction for significance (p = 0.05/3). Participants were included as random factor. To compare factors between groups  a ANOVA was used, after normality was confirmed by Shapiro-Wilk test. For non-normal distributed data, the non-parametric Wilcoxon/Kruskal-Wallis test (Rank sums) was used. Two-sided p-values were set at alpha level of 0.05. If a significant effect existed, multiple comparison Tukey HSD was used for normal distributed data and Steel-Dwass method for non-normal distributed data. The Chi2 test was used to compare groups for nominal data.Data analysis was performed using SAS JMPIn total, 35 pwMS and 13 HC could be included in data analysis of 6MCT. Data of the 6MWT of one HC was excluded from descriptive analysis because the participant started to jog. In our study sample, 13 pwMS showed walking fatigability (WF = 37.15%). Descriptive and demographic outcomes are shown in t(23.44) = 2.23; p = 0.036]. Additionally, a significant difference was found between WF-NWF  for the change in perceived fatigue for the 6MCT.F = 3.20; p < 0.01]. After post hoc multiple comparison significant differences were seen in CV φ from minute 2–5  and 2–6  . The mean values of most interlimb coordination parameters differed significantly between HC and PwMS with WF  = 4.57; p < 0.001]. Post-hoc multiple comparison showed that pwMS with WF decreased their movement amplitude significantly from minute 1 to both minute 5  and 6 . In contrast to the movement amplitude, no significant main time effect, nor an interaction effect was observed for movement frequency, suggesting that no group significantly changed their movement frequency over the 6MCT. This is the first study that investigates an antiphase coordination pattern of the lower limbs during a seated 6 min coordination task (6MCT) in pwMS and changes over time. The seated 6MCT was designed to examine more fundamental interlimb coordination of the lower limbs, assuming minimized balance and strength demands, which are required during walking. Our main hypothesis was that a change in coordination pattern over time, due to performance fatigability, would be observed during seated 6MCT in pwMS with WF, by means of change in antiphase coordination movements , and decrease in spatial and temporal movement parameters . We hypothesized this change, as a similar trend of deterioration in coordination pattern over each minute have been observed in pwMS while performing a 6-min walking task .The exploratory results of this study showed that interlimb coordination changes over time, observed by an increase in their variability in generating antiphase left-right movement CV(φ), toward the end of the task. As expected, this change in variability was greatest in pwMS with WF. Besides the observed change over time, the mean values of CV(φ) and PCI differed significantly between HC and pwMS with WF, with worse values in pwMS with WF. Noteworthy is that ABS(φ) in antiphase generation did not differ between group and did not changed significantly over time. These results suggest that pwMS with WF have more difficulties in the control of seated antiphase coordination of the lower limbs compared to HC.Regarding the temporal and spatial movement parameters during the 6MCT, we expected to observe a similar trend of decline in performance in movement frequency or amplitude in pwMS with WF, as they show a decrease in walking distance during the 6MWT. As the 6MWT requires not only a consistent and accurate antiphase coordination pattern, but also balance and strength, the 6MCT was designed to minimalize balance requirements or major muscular effort, and therefore in theory stresses more central neural mechanism. This 6MCT was based on a similar test in previous research to measure phase coordination deficits, in stroke, elderly and Parkinson . We founIn the WF group, the change in perceived state fatigue during the 6MCT was lower than during the 6MWT, indicating that the seated task was perceived less fatiguing than walking. However, despite low levels of perceived state fatigue, the test detected differences in antiphase interlimb coordination pattern between groups. These results suggest that the 6MCT can be used as an alternative for patients who are not able to perform the 6MWT due to extreme fatigue or having severe motor disabilities. This assumption should, however, be further investigated, as the 6MCT cannot be directly compared to walking, as explained further. We note that, although pwMS without walking fatigability did show walking impairments assessed by the T25FWT and 6MWT, no significant differences were found in seated interlimb coordination performance, nor in change in perceived state fatigue, between pwMS in the NWF group and HC. Besides, we note that abnormal trait fatigue was present in many pwMS with WF . The patients/participants provided their written informed consent to participate in this study.FV was responsible for study conceptualization, data collection, data processing, and writing of the manuscript. MGo was responsible for data analyzing, data interpretation, and writing of the manuscript. MGe was responsible for data processing. RM was responsible for equipment conceptualization, data processing, and data interpretation. LT and LM were responsible for data interpretation and writing of the manuscript. PF was responsible for study conceptualization, data interpretation, and writing of the manuscript. All authors contributed to the article and approved the submitted version.PF was steering committee member of Neurocompass, participated to advisory board meetings of BIOGEN IDEC, and received teaching honoraria for EXCEMED and PARADIGMS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vaccination efforts to eradicate polio currently focus on children under 5 years of age, among whom most cases of poliomyelitis still occur. However, in the Democratic Republic of the Congo (DRC), an outbreak of wild poliovirus type 1 occurred in 2010–2011 in which 16% of cases occurred among adults; in a related outbreak in the neighboring Republic of Congo, 75% of cases occurred among the same adult age-group. Given that infected adults may transmit poliovirus, this study was designed to assess adult immunity against polioviruses.We assessed poliovirus seroprevalence using dried blood spots from 5,526 adults aged 15–59 years from the 2013–2014 Demographic and Health Survey in the DRC.Among adults in the DRC, 74%, 72%, and 57% were seropositive for neutralizing antibodies for poliovirus types 1, 2, and 3, respectively. For all three serotypes, seroprevalence tended to be higher among older age groups, those living in households with more children, and among women.Protection against poliovirus is generally low among adults in the DRC, particularly for type 3 poliovirus. The lack of acquired immunity in adults suggests a potentially limited poliovirus circulation over the lifetime of those surveyed (spanning 1954 through 2014) and transmission of vaccine-derived poliovirus in this age group while underscoring the risk of these outbreaks among adults in the DRC.The online version contains supplementary material available at 10.1186/s12879-021-06951-6. While no cases of wild poliovirus (WPV) have been detected in the Democratic Republic of the Congo (DRC) since 2011 . FurtherPrior to the initiation of routine and supplemental immunization programs, high rates of endemic infection among children resulted in infrequent paralytic poliomyelitis cases among adults in developing countries . HoweverSerological protection among adults is also related to transmissibility of wild and vaccine viruses , 11. WhiAmong adults who were not vaccinated, serological protection must be due to exposure to WPV, cVDPV, or through exposure to the Sabin live, attenuated oral poliovirus shed by a vaccine recipient. Individuals with serum immunity against polio are protected from developing paralysis; however, as intestinal immunity to poliovirus wanes over time, exposure to the virus may result in infection of the gut, resulting in shedding and exposure to others , 13. ThiGiven the important role adults may play in the transmission of poliovirus and the global effort to eradicate polio, understanding adult immunity against polioviruses could be useful in identifying high risk populations in the DRC . In thisThe second Demographic and Health Survey (DHS) took place in the DRC from November 2013 to February 2014, at which time trivalent OPV  was being utilized as a part of routine immunization (RI) activities in the DRC under WHO guidelines (inactivated poliovirus vaccine (IPV) and bivalent OPV [types 1 and 3] are currently used in the DRC as of 2020). Using a 2-stage stratified cluster design, the survey generates nationally representative data on population health and social indices, with GPS-identified cluster sites. Details on the sampling design and data collection procedures are described elsewhere . During To ensure sufficient power for analysis, DBS samples were divided into two subsets: polio, presented in this paper, and measles, mumps, varicella zoster, rubella and tetanus (MMVRT) from multiplex testing. This sub-setting was done to maintain two representative samples of adult participants and of the population from which they were drawn. Within each sampling cluster of the DRC DHS survey design, available samples were sorted by sex and age. The youngest two individuals of the same sex were assigned to a unique pair identification number, then the next two, and so on. Where an odd number of males or females appeared in the cluster, the oldest person was treated as a singleton. Within sample pairs, each individual was assigned a random number between 0 and 1; the participant that was assigned 0 was tested for neutralizing antibodies against polio at the CDC, while the remaining individuals were assigned to be assessed for antibody response against MMVRT at the UCLA-DRC laboratory at the National Institute for Biomedical Research (INRB) in Kinshasa, DRC. Lastly, singletons were assigned with a 0.5 probability to either the polio or MMVRT testing groups. Among the available 17,420 DBS samples from those selected and eligible for HIV testing (850 participants did not have a corresponding DBS sample obtained), 8713 were sent to CDC, while 8706 were retained at the INRB. Assessment of demographics across sample subsets showed comparable distribution across province, age and sex  was used as the threshold of seropositivity [Following the DHS protocol, DBS samples were collected in the field from participating adults, transported at room temperature to the DRC’s National AIDS Control Program national reference laboratory in Kinshasa, and stored at – 80 °C until testing. One spot (up to three 6 mm punches) per subject was shared with the UCLA-DRC program at the INRB for testing of VPDs as described above. DBS were shipped at − 20 °C to CDC to test for neutralizing antibodies against poliovirus serotypes 1, 2, and 3. As described elsewhere, we used the modified poliovirus microneutralization assay which measures the ability of antibodies eluted from DBS punches to block the infectivity of poliovirus in an in vitro cell culture system , 19. Upositivity .Among the 8713 adult samples received by CDC, 2941 (33.8%) could not be evaluated due to insufficient sample volume (at least three 6 mm punches) and 60 were indicated as missing during randomization. Of the 5712 DBS samples assessed for neutralizing antibodies against poliovirus types 1, 2, and 3, 5526 (96.7%) were successfully merged with DHS sociodemographic survey data (2974 women and 2552 men), among whom 5,381 (97.4%) had results for all three serotypes  , residence , and province. For serologic analyses, neutralization titers were dichotomized as follows: titers < 3.0 logStudy methods were carried out in accordance with relevant guidelines and regulations. All subjects provided informed consent prior to enrollment, survey completion, and biological sampling. For minors, informed consent was obtained from parents or legal guardians. Ethical approval for this study was obtained from Institutional Review Boards at the UCLA Fielding School of Public Health, the Kinshasa School of Public Health, and the US Centers for Disease Control and Prevention (CDC).Among adults participating in the 2013–2014 DRC-DHS, 74%, 72%, and 57% were seropositive for neutralizing antibodies for type 1, type 2 and type 3, respectively , seroprevalence varied widely across provinces Fig. . Bas-UelAs seroprevalence was higher among women overall, and this trend generally persisted within age groups as well Fig. . Among aThis study provides the first nationally representative estimates of neutralizing antibodies against poliovirus type 1, 2, and 3 in adults of the DRC. Among adults represented in the survey (men ages 15 to 59 years and women ages 15 to 49 years), an estimated 26%, 28% and 43% remain susceptible to poliovirus type 1, 2, and 3, respectively. As only 9% were seronegative for all three, the vast majority of adults exhibit some level of protection against one or more serotypes . Seropositivity was generally higher among women, those in households with more children, and among older age groups, and also varied by province.Overall, we observed relatively low seroprevalence among adults across the country. In some areas, the low seroprevalence, particularly to type 3 poliovirus 57% overall), may indicate a limited viral circulation and/or possibly weak poliovirus transmissibility in the population, which permits individuals to escape infection through adulthood. In addition, this result may be influenced by the lower overall immunogenicity of poliovirus type 3 administered by trivalent OPV vaccine as compared to that from the type 1 and 2 components . The bui% overallIn the present study, type 1 had the highest seroprevalence across all age and sex groups followed by types 2 and 3. Although the assay does not distinguish between neutralizing antibodies induced by exposure to WPV, OPV, or VDPV, this finding may reflect the atypical outbreak of wild poliovirus type 1 that occurred from 2010 to 2011 [Similar to the serosurvey findings of 2013–2014 DRC DHS child participants aged 6–59 months , we alsoWhile this study provides the first nationally representative estimates of poliovirus immunity among adults in the DRC, there are several limitations. As in the 2013–2014 child polio serosurvey, the adult serosurvey was carried out in conjunction with the household-based DHS, thus mobile or remote, difficult-to-reach populations may not be well represented in the sampling frame; indeed, there were no selected clusters near the previous cVDPV2 outbreak areas . DespiteOur finding of high susceptibility to poliovirus among adults in the DRC is of concern, given the potential for importation of WPV1 or cVDPV. These results may have been influenced by several factors. While we found evidence for increased seropositivity with household size, and number of children in the household, secondary spread of vaccine viruses was not sufficient to achieve high immunity for all serotypes. Second, low seropositivity suggests more limited historical circulation of virus, potentially due to population distribution and rurality. Lastly, while it is generally believed that polio immunity is lifelong once acquired, waning immunity below the limit of detection in adulthood may be possible. Given the global effort to eradicate polio, this study provides a first step in understanding adult immunity against polioviruses in a setting with continued clusters of outbreaks; this may influence future vaccination policies countrywide.Additional file 1: Table S1. Comparison of DRC 2013–2014 adult DHS respondent demographics across sample split. Table S2. To test for intra-household correlation in seropositivity, we merged the adult and child datasets, and assessed whether any child and/or any adult in the household was seropositive for each type of polio. The table below summarizes the results across households. Using Pearson’s Chi-squared test, we find that sero-status has a mild positive association for Type 2 (p = 0.01), but there is little evidence for within household correlation for Types 1 and 3. Figure S1. Study design flowchart for dried blood spot (DBS) sampling, testing, and analysis. From a total of 8713 DBS collected in the field, 5526 (63.4%) were successfully processed and merged to DHS questionnaire data for inclusion in this analysis.Additional file 2.  R code for data analysis.Additional file 3.  Serology data for poliovirus among adults surveyed and sampled in the 2013-2014 DHS in the Democratic Republic of Congo.


Background: Multiple sclerosis (MS) patients have a wide spectrum of severity and responses to therapy; the personalization of treatment relies on sensitive and specific biomarkers. Previous studies have suggested that susceptibility contrast in demyelinated plaques is associated with iron-related pathology in multiple sclerosis which may indicate clinical severity. The aims of this study were to characterize the spatial distribution of MS lesions with different iron patterns by using quantitative susceptibility mapping and to explore neuroradiological findings that correlate with poor clinical outcome.Methods: Twenty-six patients with relapsing–remitting MS  were included in this study. Differences in lesion number, T2 volume, and susceptibility were compared among lesions subcategorized by location and by the presence or absence of a hyperintense rim on quantitative susceptibility mapping. Associations between these imaging features and clinical outcomes including Expanded Disability Status Scale scores and annual relapse rates were investigated.Results: A total of 811 unifocal MS lesions were included, and their QSM patterns were nodular hyperintensity with no rim  or with a hyperintense rim on the edge  and nodular isointensity . Rim+ lesions had significantly larger volume  and lower susceptibility  than rim– lesions. More rim+ lesions were found in periventricular areas , whereas a larger proportion of rim– lesions were distributed in juxtacortical  and deep white matter  areas. The annual relapse rate was positively correlated with the proportion of periventricular rim+ lesions  and the proportion of subtentorial rim+ lesions . Additionally, a significant association was found between the burden of periventricular rim+ lesions  and the burden of subtentorial rim– lesions .Conclusions: A high number or lesion burden of periventricular rim+ lesions or subtentorial lesions is associated with frequent clinical relapses. Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that is characterized by diffuse demyelination with neurodegeneration, vasculitis of brain venules, and meningeal inflammation –3. The win vivo iron assessment via iron-sensitive imaging   years; age range, 21–52 years]. The severity of the clinical disability of the patients was quantified by using the Expanded Disability Status Scale (EDSS) within 48 h of the MR scan. The times of the clinical relapse since the patients began disease-modifying therapy in our institution were retrospectively reviewed to estimate the annual relapse rate (ARR). All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration. All patients gave written informed consent.*-weighted multi-echo spoiled GRE sequence with the following parameters: first echo time (TE) in ms/TE spacing in ms/number of echoes = 3.4/3.5/14, repetition time (TR) = 52.5 ms, flip angle = 20°, field of view = 180 × 180 mm2, matrix = 256 × 256, in-plane resolution = 0.7 × 0.7 mm2, thickness = 1 mm, and gap between slices = 0. T2-weighted images (T2WI) were acquired with the following parameters: voxel size = 0.7 × 0.7 × 1 mm3, field of view = 180 × 180 mm2, matrix = 256 × 256, flip angle = 111°, thickness = 1 mm, and no gap between slices. Conventional MR sequences included T2 fluid-attenuated inversion recovery (T2-FLAIR)  and T1-FLAIR , each with a slice thickness of 5 mm and no gap between slices.All images in the study were acquired on a 3.0-T MRI scanner  equipped with an eight-channel head coil. Gradient-echo images (GRE) for QSM reconstruction were acquired with a three-dimensional T2QSM images were reconstructed using the STI Suite package . iHARPERImaging data were reviewed to (1) record the distributional areas, number, and QSM patterns of the unifocal MS lesions (ovoid-like shapes with discrete borders rather than confluent borders) and (2) quantify the susceptibility values of hyperintense lesions on QSM and their lesion volume on T2WI. Confluent MS lesions were included only in the measurement of individual T2 burden (sum of lesion volume).An MS lesion was defined as a T2-hyperintense lesion with an axis length of at least 3 mm. Distributional areas included juxtacortical (a maximum distance of 3 mm from the corticomedullary junction), periventricular , deep , and subtentorial white matter areas , 22. TheThe susceptibility value and T2 volume of lesions were estimated by using a region of interest (ROI) semiautomatic tool in Analyze 12.0 . A neuroradiologist (Z.G.) outlined the lesions on the involved slices and revised the outlines manually to exclude veins. Lesion ROIs were drawn on T2WI and QSM separately. The susceptibility of a lesion was defined as the mean of all voxels in the ROI and calculated relative to the susceptibility of central cerebral spinal fluid (CSF). Lateral ventricles were semiautomatically segmented on the first echo of GRE images, and the choroid plexus regions were manually excluded. The ROIs were copied to the QSM to obtain CSF susceptibility. The susceptibility of NAWM relative to CSF was also measured.p < 0.05. The following analysis focused on QSM hyperintense lesions.Statistical analysis was conducted using SPSS 21.0. The criterion for statistical significance was set at U-test at the lesion level in terms of lesion volume and susceptibility. To investigate the distributional tendency for rim+ and rim– lesions, we used the Friedman test to determine whether the numbers of lesions in four distributional areas were significantly different from each other within subjects; a stepwise step-down test was used for multiple comparisons.Rim+ and rim– lesions were compared by the Mann–Whitney To compare the differences in T2 volume and susceptibility in juxtacortical, deep, and periventricular and subtentorial white matter areas, the Kruskal–Wallis test was used with the Bonferroni correction for multiple comparisons.A mixed-effect regression model was implemented with susceptibility at the lesion level as a response variable and age, sex, individual T2 burden, ARR, and EDSS as covariates. The model also accounted for random factors including intrasubject correlations and intersubject variability in the number of lesions. The model was implemented to assess the associations between the covariates and lesion susceptibility while controlling for rim+ or rim– status as well as lesion locations.p < 0.10 in the univariable analysis were included in the multivariable models. Age and gender were entered as covariates in the models.Correlation analysis at the individual level was also performed. Multivariable linear regression was used to evaluate the correlations of individual lesion burden and average lesion susceptibility with clinical outcomes (EDSS and ARR). Candidate variables associated with clinical outcomes at a level of U-test. The correlation of lesion portion in various locations with clinical outcomes was evaluated by using Spearman's correlation test.Additionally, we divided patients into subgroups by EDSS score (< or ≥4) and ARR (< or ≥1) and evaluated the differences between groups in terms of lesion burden and average lesion susceptibility using the Mann–Whitney Clinical characteristics of the MS patients are summarized in A total of 840 white matter lesions were detected, with 29 lesions excluded because of poor QSM quality. The QSM contrast patterns of the remaining 811 lesions were nodular hyperintensity with no rim  or with a hyperintense rim on the edge  and nodular isointensity  . In partp < 0.001) than rim+ or isointense lesions. The mean susceptibility of QSM hyperintense lesions was 7.9 ± 13.4 ppb, which was on average 35.4 ± 11.3 ppb higher than that of NAWM (−27.5 ± 11.1 ppb). The mean individual lesion burden was 12,775 ± 11,043 mm3.In our cohort of MS patients, 23 (88%) patients had more rim– hyperintense lesions (p < 0.05) and increased T2 volumes . The numbers of rim– lesions in juxtacortical  and deep white matter  areas were higher than the numbers in periventricular  and subtentorial  areas.Compared with rim– lesions, rim+ lesions had significantly reduced susceptibility values , and lesion susceptibility tended to decrease as the distance from the ventricle decreased (6 ± 16 ppb in deep white matter and 4 ± 16 ppb in the periventricular area). This gradient was not found in NAWM. The average susceptibility was −32 ± 4, −23 ± 2, and −27 ± 1 ppb in juxtacortical, deep, and periventricular NAWM, respectively. The mean volume of periventricular lesions was significantly larger than that of the other supratentorial lesions (adjusted p < 0.001)  (adjusted < 0.001) . No signp < 0.001). More rim lesions were located in the deep white matter area  than in the periventricular  or subtentorial  areas (p < 0.05). Additionally, larger proportions of rim lesions were found in the juxtacortical white matter  than in the periventricular and subtentorial areas, but the results of multiple comparison testing were not significant. Furthermore, more rim+ lesions were detected in the periventricular area  than in the juxtacortical area  or deep white matter , although the differences between groups were not significant.More rim+ lesions were found in periventricular  and juxtacortical  areas than in deep white matter , whereas only three lesions (2%) were found in subtentorial areas. Sixteen patients had subtentorial rim lesions, while only three patients had rim+ subtentorial lesions. At the individual level, our study also demonstrated significant differences in rim– lesion numbers in four distributional areas and rim+ lesion numbers (both p = 0.02), while there was no significant association found with age, sex, EDSS score, or ARR. The mixed-effects model also confirmed significant differences in susceptibility values among rim+ and rim– lesions in various locations  and the proportion of subtentorial rim+ lesions  (p < 0.05) and rim– (p < 0.005) lesions than patients with low ARR. No significant difference was found in the comparison of lesion burden or susceptibility between patients with EDSS scores ≥4 and <4.We found positive correlations of the annual relapse rate with the proportion of periventricular rim+ lesions ( = 0.40) . No signp < 0.001) and subtentorial rim– lesions  remained in the final model (R2 = 0.59) . When ARR was entered as a response, univariable analysis yielded the following candidate variables: the lesion burden of rim– lesions in the juxtacortical, periventricular, and subcortical areas and the lesion burden of rim+ lesions in the periventricular area. In multivariate linear regression analysis with stepwise selection, the burden of periventricular rim+ lesions  . The regThe current study reveals high heterogeneity in distribution and susceptibility contrast among MS lesions, which might contribute to the breadth of the clinical spectrum in RRMS patients. A high number or lesion burden of periventricular rim+ lesions or subtentorial lesions is associated with frequent clinical relapses.In this study, lesions with a hyperintense rim accounted for only 21% of lesions visible on QSM, similar to the figure reported in a previous study . AlthougInterestingly, the current study found different distributional preferences between rim+ and rim– lesions at the individual level: more rim– lesions were located in juxtacortical and deep white matter areas, whereas a larger proportion of rim+ lesions were distributed in periventricular areas. Considering the distinct pathological characteristics of rim+ and rim– subgroups, these distributional features may facilitate the presentation of large and confluent periventricular lesions over time. Both the current study and a previous study confirmed larger lesion volumes in periventricular areas than in other regions . Our finLongitudinal MRI studies demonstrate that the volume difference between rim+ and rim– lesions is significant even at their first appearance , 31, witSusceptibility contrast variations in demyelinated plaques are associated with MS pathology. In the early weeks of active demyelination, myelin debris is degraded by macrophages. The breakdown of myelin results in a rapid, slight increase in tissue susceptibility because of the loss of chemical bonds within diamagnetic myelin lipid layers , 32, 33.MS, especially RRMS, features a wide spectrum of symptoms, severity, and response to therapy. Personalized therapy is important for disease modification, and the personalization of treatment relies on biomarkers correlated with clinical outcomes. Consistent evidence indicates that rim+ lesions are associated with poor clinical consequences , 27, 35.Prior observations suggest that iron content, as measured by susceptibility, is higher in rim+ lesions than in rim– lesions , 30, 31,However, the current study did not show a significant association between clinical outcomes and average susceptibility of lesions after controlling for location. This result may be explained in part by the contribution of myelin loss, which may increase the susceptibility of MS lesions and compromise the linear relationship between iron and susceptibility. Another explanation is that the annual relapse rate  and the susceptibility  were obtained in different stages of disease, although susceptibility in the chronic stage has been proven to be stable for several years. In contrast to an earlier longitudinal study showing that susceptibility values in rim+ lesions were higher at each time point than those in rim– lesions, our results demonstrated a lower average susceptibility in the rim+ subgroup of lesions, which may suggest lower iron content. However, in a previous study, only 32 lesions were included in their longitudinal observation, and lesion locations, which may have played a role in susceptibility, were not controlled in the analysis. On the other hand, susceptibility in our study was derived cross-sectionally from MS lesions at different ages. Previous data have indicated that the susceptibility of lesions declines over time and that the average variation range of susceptibility is as large as 20 ppb . TherefoIn subtentorial areas, we found a positive correlation between ARR and the burden of rim– lesions, although their clinical consequences, based on supratentorial comparison with rim+ lesions, were modest. The infratentorial brain consists of abundant nuclei and compact fiber tracts ascending or descending to functionally connect the forebrain and spinal cord . In our 3+) ions are released from the ferritin of oligodendrocytes and myelin sheaths and are exposed in the extracellular space, where they are converted to unbound ferrous (Fe2+) ions  with increasing distance from the lateral ventricles and proximity to the cortex, suggesting a theoretically more pronounced susceptibility increase caused by periventricular myelin loss. In addition, we suggest that regional heterogeneity of iron accumulation in WM lesions should be considered in future studies. The clinical and pathological implications of high iron levels in juxtacortical lesions should be determined.The results of this study suggest that MS lesion susceptibility is not uniform throughout the white matter. Juxtacortical lesions, whether in the rim+ or rim– subgroups, carry the highest susceptibility. This high susceptibility may result from large amounts of iron being liberated from myelin degradation products during demyelination. In support of this view, oligodendrocytes in the juxtacortical regions of normal or MS brains are known to be richer in iron than those in other regions . At the 2+) ions . The late tissue . Thus, oThis study has limitations. First, the size of our patient cohort was small, and our findings are somewhat preliminary and warrant large-sample, longitudinal studies for confirmation. Second, our inference concerning the pathologic basis of QSM patterns was hypothesized based on prior evidence from studies correlating histopathology and imaging; the current study did not include its own histopathologic validation. Contrast-enhanced or longitudinal images were not collected to evaluate the possible pathologic status of the lesions. In addition, positive susceptibility values with respect to water are suggested to be a valid criterion to identify rims that contain real iron deposition . This prOur study confirms the differences between the rim+ and rim– subgroups in terms of distributional patterns, susceptibility, extent of tissue damage, and clinical relevance. Lesion distribution holds important prognostic value in MS. A high number or lesion burden of periventricular rim+ lesions or subtentorial lesions is associated with frequent clinical relapses.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics committee of the Third Affiliated Hospital of Sun Yat-sen university. The patients/participants provided their written informed consent to participate in this study.SC and WQ: study concepts and design and manuscript revision. ZG and LL: data analysis, statistical analysis, and manuscript preparation. TL and YS: clinical data acquisition and analysis and manuscript review. LZ, KZ, and LF: imaging data acquisition and manuscript review. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To investigate the clinical effect of concentrated growth factors (CGF) combined with deproteinized bovine bone mineral (DBBM) on Alveolar ridge preservation during implantology.A total of 38 patients were selected and randomly divided into 2 groups, with 19 cases in each group. The extraction sockets were filled with DBBM with or without CGF. Visual analogue scale (VAS) pain score was recorded within1 week and Landry wound healing index (LWHI) was recorded at 1, 2 and 3 weeks after operation. CBCT was taken preoperatively and 3 and 6 months postoperatively to measure and compare the changes of vertical height, width and gray value of alveolar bone at extraction site. The changes of alveolar bone contour were observed clinically and compared between the two groups.P < 0.05). The LWHI of CGF group was higher than control group 1 week after operation (P < 0.05). The absorption of the labial and palatal plates height and the width in the CGF group was significantly less than the control group at 3 months (P < 0.05). The gray value of alveolar bone in CGF group was significantly higher than control group at 3 months (P < 0.05). There was no significant difference in new bone contour between the two groups (P > 0.05). 94.7% cases in CGF group did not undergo bone grafting, which was significantly higher than control group (78.9%).The VAS score of CGF group was lower than control group on the 1st and 3rd day after operation (The use of CGF combined with DBBM can help to reduce postoperative pain at the early stage of healing, form sufficient keratinized gingival tissue, effectively maintain the height and width of alveolar bone in the three-dimensional direction and provide good conditions for implant repair in the future. Implant restoration has been gradually accepted by more and more patients, because there is no damage to the adjacent teeth. Besides, implants have the same function as natural teeth which can bear and transmit masticatory force well. However, the absorption of alveolar bone after tooth extraction limits the application of implant. Alveolar bone can be absorbed to different degrees after tooth extraction, especially after 3–6 months. The alveolar bone can be absorbed to varying degrees 3–6 months after tooth extraction, and most of dimensional change can be occurred within 2 weeks . The alvThe concept of Alveolar ridge preservation (ARP) was first proposed in 1994. It refers to the protective intervention on the sites that need delayed implant restoration while extracting the tooth , allograIn recent years, concentrated platelets have been used in wound healing because of their high growth factor content. Among the preparations, CGF, platelet-rich plasma (PRP), platelet-derived growth factor, transforming growth factor beta and platelet-rich fibrin (PRF) currently used for the regeneration and reconstruction of bone and connective tissues are. CGF is a new generation of plasma extract prepared from patients' own venous blood by special centrifugation. CGF was first proposed by Sacco in 2006, which contains high concentrations of a variety of growth factors and fibrin . The preHowever, the researchs on the combined application of CGF with DBBM in ARP were limited. Therefore, the aim of this study was to perform a clinical and radiographic evaluation of the ARP technique using CGF and DBBM, to observe the conditions of the alveolar ridge bone after tooth extraction and evaluate the application value of CGF combined with Bio-oss bone powder in alveolar bone increment.38 patients who underwent incisor, lateral incisor and canine single tooth extraction at the Outpatient department of Stomatology from October 2020 to May 2021 were collected. 38 extraction sites were randomly divided into CGF group and control group, 19 cases in each. All patients were informed about the potential benefits and risks of surgery, as well as alternative treatment options, and volunteered to participate in and signed an informed consent. The protocol of this study was consistent with the ethical guidelines of the Declaration of Helsinki and was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University . All patients voluntarily participated in the study and signed informed consent.Inclusion criteria were as follows (1) Patients over 18 and under 35; (2) Teeth cannot be retained due to severe caries, chronic periapical periodontitis or trauma, and need implant restoration; (3) All extraction sites had adjacent teeth with healthy periodontal tissue or only mild periodontal disease (plaque index and bleeding score less than 15%); (4) At least 2 or more bone plates exists at the extraction site; (5) Without serious systemic diseases, psychosis and epilepsy; (6) Non smoking and good compliance.Exclusion criteria were as follows (1) Patients with acute periodontal or periapical infection; (2) Both of the buccal and lingual alveolar bone resorption exceeds 25% of root length; (3) With severe hypertension, diabetes, kidney and liver diseases, especially patients taking anticoagulants; (4) Pregnant patients; (5) Patients with a history of radiotherapy and chemotherapy.Preoperative examination including general conditions, routine blood tests, oral hygiene, occlusal relationship, etc. CBCT was taken to measure the height and width of alveolar bone. Fig. . We adjuPreparation of CGF Venous blood was collected from each patients in CGF group using sterile vacuum tubes , without additive. Then the tubes with whole blood (4 mL in each) were immediately centrifuged by Medifuge  at fixed temperature. After centrifugation, CGF gel represented as the buffy coat in the middle layer and was carefully isolated from the red blood cell clots , and filled into the extraction socket in layers to make it about 2 mm higher than the crest of the surrounding alveolar ridge, the wound was completely covered with CGF membrane and then Collagen membrane . Finally, the flap was repositioned coronally and sutured tightly with non-resorbable sutures. In the control group, Bio-Oss was filled into the extraction socket to make it about 2 mm higher than the crest of the surrounding alveolar ridge, the wound was completely covered with Collagen membrane and the flap was sutured Fig. .Fig. 4SuAfter the grafting surgery, systemic antibiotics were prescribed to all patients 1 day before operation for 5 days. Visual analogue scale (VAS) was used to measure the degree of postoperative pain on day 1, 3, and 7. The sutures were removed after 1 week. According to the color of the gingiva, the presence or absence of granulation tissue, bleeding, pyorrhea and epithelial formation, the Landry wound healing index (LWHI) was recorded at 1, 2 and 3 weeks.  CBCT wasMeasurements were recorded in a spreadsheet in Excel 2013  and were then analyzed using SPSS 20.0 . Data are given as mean ± SD and were evaluated via a Shapiro–Wilk test to assess distribution normality. Normally distributed data were compared via t-test or analysis of variance (ANOVA), while non-normally distributed data were compared via Mann–Whitney U test. A significance level of α = 0.05 was used for all analyses.A total of 38 patients who met the inclusion criteria were identified in this study, 19 cases in each group, 23 females and 15 males, with a mean age of (28.89 ± 2.7) years, all of them completed the experimental protocols and subsequent implant restoration. All sockets healed uneventfully, and no adverse reactions such as wound dehiscence and acute infection were observed during the 6-month clinical healing period. The contour of alveolar bone in the CGF group was more conducive to implant implantation. There was no significant differences in average patient age between the two groups.P < 0.05) (Table P < 0.05).The VAS score of CGF group was lower than control group on the 1st day after operation (5) Table . The LWHP < 0.05). The gray value of alveolar bone in CGF group was significantly higher than control group at 3 months (P < 0.05). .The alveolar ridge resorption and soft tissue shrinkage after tooth extraction are the main problems affecting the aesthetics and function of alveolar ridge. In order to achieve successful implantation and long-term effect, sufficient bone volume, keratinized gingiva and appropriate occlusal relationship should be obtained. Therefore, alveolar bone should be preserved as much as possible during tooth extraction to reduce edentulous ridge resorption rate and promote the bone remodeling of alveolar bone . CompareMost studies on the dimensional changes of soft and hard tissues after tooth extraction suggested that the ideal bone graft material should not only have osteoconductive properties but also promote osteoinduction and osteogenesis . Only auP < 0. 05). Histological studies have found that the rate of new bone formation is about 26.0% ± 23.7% when DBBM was used alone, and it can even reach 48.3% [DBBM has strong hydrophilicity, high biocompatibility and good plasticity, it can promote the adhesion of osteoblasts to bone, plays important role as a scaffold, and has a very low risk of causing host allergy, inflammation and immune reactions. Twenty-seven patients were randomized into two treatment groups following single tooth extraction in the incisor, canine and premolar area. In the test group, the alveolar socket was grafted with Straumann Bone Ceramic (SBC), while in the control group, Bio-Oss was applied. 8 months later, they found that both materials preserved the mesio-distal bone height of the ridge . In anotch 48.3% . Althougch 48.3% , DBBM lach 48.3% . The usech 48.3% . TherefoCGF is a new generation of platelet concentrate after PRP and PRF. The production of CGF requires variable speeds to separate blood cells from fibrin-rich blocks, which are denser and contain a higher concentration of growth factors than PRF . This reIn this study, none of the patients experienced rejection or wound infections around the grafting region, which indicated that the deproteinized bovine bone and CGF were safe and biocompatible. Due to the dispersive nature and small particles of DBBM, leakage often occurs, so it is necessary to cover the wound with a collagen membrane, and the gingival flap is tightly sutured to prevent the leakage of bone powder. In this study, CGF was cut into small pieces and fully mixed with DBBM, which was easy to mold. In the meantime, because of the elasticity and adhesion of CGF membrane, leakage rarely occurs even if the tooth extraction wound cannot be closed tightly.The vertical bone loss of labial buccal bone wall was more obvious than the lingual bone wall due to its thin thickness after tooth extraction . SimilarSufficient alveolar ridge dimensions is important for implant placement, it is also necessary that the regenerated bone is of good quality. It is pointed out that the quality and quantity of regenerated bone influence the initial stability of implant and can determine the success of dental implant osseointegration , 39. TheRelaying on the strong soft tissue induction ability of CGF membrane , a full Although long-term studies with large samples are still needed, the following conclusions can be drawn. The use of CGF combined with DBBM in the anterior tooth region can help to reduce postoperative pain at the early stage of healing, form sufficient keratinized gingival tissue and effectively maintain the bone mass of alveolar bone in the three-dimensional direction. Meanwhile, it can provide good conditions for implant repair in the future, and reduce the need for bone grafting before implantation.