P = 0.001) and DFS (P < 0.001) in the CEA-high group, while, in the CEA-normal group, the only independent prognostic factor for OS (P = 0.031) and DFS (P = 0.043) was CA50. CA50 and CA724 could supplement CEA in monitoring recurrence and metastasis. Accordingly, nomograms based on CEA, CA50, CA724 and other clinical-pathological factors could improve prognosis prediction for colorectal cancer patients.A wide range of serum tumor biomarkers, including CA19-9, CA242, CA72-4, CA50, and CA125, has been studied in association with colorectal cancer (CRC). However, few previous studies have comprehensively considered the above tumor biomarkers to assess their clinical significance in predicting prognosis. Data from Fudan University Shanghai Cancer Center (FUSCC) between January 1, 2007 and December 30, 2012 was retrospectively analyzed. Univariate and multivariate analyses were performed to assess the association between prognostic factors and survival outcomes. Nomograms were established based on multivariate Cox regression model analysis for overall survival (OS) and disease free survival (DFS), and c-indexes were 0.772 (95% CI: 0.724-0.820) and 0.715 (95% CI: 0.678–0.752), respectively. Subgroup analyses according to CEA status  suggested that CA724 was the only independent prognostic factor for OS ( Colorectal cancer (CRC) is one of the most prevalent types of cancer, ranking as the third most common malignancy and the fourth leading cause of cancer deaths worldwideet al. demonstrated that the combined analysis of CEA, CA19-9, and CA242 could improve the accuracy of prognostic prediction in surgically treated CRC patientsSeven serological tumor biomarkers  are routinely adopted in clinical practice to assist diagnoses and determine prognosis in gastroenterological cancers at our institute. The most common and best-studied biomarker for CRC is CEA, which can be used for early diagnosis, recurrence detection and prediction of survival outcomes4The development of nomograms has led to their successful application in oncology prognostics in many studies. However, nomograms for predicting survival outcomes of CRC with serological tumor markers are scarce. In the present study, we evaluated the association between preoperative levels of tumor biomarkers and other baseline characteristics and developed nomograms to determine the value of tumor biomarkers for predicting the 5-year survival of patients with CRC who underwent radical resection.The descriptive characteristics of eligible patients are displayed in P < 0.05; P < 0.001), N stage (P < 0.001), CEA (P = 0.034), CA50 (P = 0.010), and CA724 (P < 0.001) were independent prognostic factors for OS , N stage (P < 0.001), CEA (P = 0.017), CA50 (P = 0.020), and CA724 (P = 0.001) were independent prognostic factors for DFS . In contrast, in the CEA-normal group, tumor biomarker CA50 was shown to be an independent risk factor for both OS  and DFS  = 2.853, 95% CI : 1.769–4.603, Distant metastases and local recurrences remain main concerns in CRC patients after surgical resection810Due to its simplicity and graphical representation, the nomogram has been widely adopted as a practical model for predicting prognosis. Each variable is assigned different points according to its weight, which is shown at the top of the scale. The total points of all included variables generate a numerical prediction of the 5-year probability of death or recurrence/metastasis for a patient, which is indicated on the lowest scale. The predictive accuracy of nomograms has been validated as favorable compared with the traditional TNM staging system for many malignancies. Nomograms can generate an individualized prediction of survival for patients, making it a practical tool for clinicians to identify patients at high risk for intensive follow-up.The nomogram is a simple and visual statistical prediction model that produces a numerical probability of a survival event. By adding the points assigned to each factor, a predicted 5-year probability of death and recurrence or metastasis for a patient can be calculated and shown in the lowest scale13In the present study, we performed a retrospective study in patients with CRC who had all six tumor biomarkers examined preoperatively and found that CEA, CA50, and CA724 were independently associated with DFS and OS in patients with CRC after surgical resection. Then, we developed nomograms including tumor biomarker predictors and found that the nomograms could improve the prognosis prediction of CRC patients. Elevated serum CEA, CA50, and CA724 were associated with pathological stage and unfavorable clinicopathological characteristics such as venous invasion, tumor length, and tumor differentiation. Another finding of the present study the different prognostic models concerning tumor markers in CEA-high and CEA-normal CRC.CA50 is a glycolipid antigen that plays an important role in cell growth and differentiation, indicating that tumor cells expressing this antigen may possess increased proliferative activity156et al. showed that there were statistically significant differences in CA724 levels between patients with and without distant metastasesCA724 is a glycoprotein, with higher levels in gastric17Tumor-related indicators in all individuals cannot be detected systematically and comprehensively due to limited economics and techniques7Patients with histologically confirmed colorectal adenocarcinoma who received radical resection at Fudan University Shanghai Cancer Center (FUSCC) between January 1, 2007 and December 30, 2012 were included in the study. The database of FUSCC was built prospectively, and all information of CRC patients was recorded since January 20061323Preoperative blood samples were drawn in the morning from all patients. Plasma was separated from the blood cells by centrifuging the blood sample at 1000 g for 10 minutes. Magnetic particle enzyme immunoassay using the UniCel DxI 800 Access immunoassay system  was performed to detect CEA, CA125, CA19-9, CA50, CA242, and CA72-4. According to the manufacturer’s instructions, 5.2 ng/mL, 35 U/mL, 27 U/mL, 25 U/mL, 20 U/mL, and 6.90 U/mL were chosen as cut-off values for serum CEA, CA125, CA19-9, CA50, CA242 and CA72-4, respectively.Clinicopathological variables, such as age, sex, grade, histologic type, T and N stage, treatment type, regional lymph node harvest, and number of positive regional lymph nodes were retrieved from the FUSCC database. All patients were restaged according to the American Joint Committee on Cancer (AJCC) TNM Staging Classification for CRC . The primary study endpoints were overall survival (OS), which was calculated from the date of diagnosis to the date of death from any cause, and disease-free survival (DFS), which was defined as the time from diagnosis to the first recurrence. Patients who were alive at the last follow-up were censored for analyses.This retrospective study was approved by the FUSCC Ethical Committee and Institutional Review Board. All methods were carried out in accordance with the relevant guidelines. All patients identified gave written informed consent for participation.P < 0.05 was considered statistically significant. Confidence intervals (CIs) at the 95% confidence level were used in this study.The relationship between clinicopathological features and tumor markers was tested by cross-tab analysis. The Kaplan-Meier estimator was used to calculate the 5-year overall survival (OS) and disease-free survival (DFS), with the difference between variables compared by log-rank tests. All prognostic predictors that were significantly associated with OS and DFS in univariate analysis were included in multivariate Cox proportional hazards analysis. Nomograms for prognostic parameters that were independently associated with OS and DFS were developed using R 3.1.2 software . Model performance for predicting survival probability was evaluated by Harrell’s concordance index (c-index). All statistical analyses were performed using SPSS 22.0 .How to cite this article: Li, Q. et al. Nomograms for predicting prognostic value of serological tumor biomarkers in colorectal cancer patients after radical resection.. Sci. Rep.7, 46345; doi: 10.1038/srep46345 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pyropia haitanensis. To understand the molecular mechanisms underlying high temperature stress in a high temperature tolerance strain Z-61, the iTRAQ technique was employed to reveal the global proteomic response of Z-61 under different durations of high temperature stress. We identified 151 differentially expressed proteins and classified them into 11 functional categories. The 4 major categories of these are protein synthesis and degradation, photosynthesis, defense response, and energy and carbohydrate metabolism. These findings indicated that photosynthesis, protein synthesis, and secondary metabolism are inhibited by heat to limit damage to a repairable level. As time progresses, misfolded proteins and ROS accumulate and lead to the up-regulation of molecular chaperones, proteases, and antioxidant systems. Furthermore, to cope with cells injured by heat, PCD works to remove them. Additionally, sulfur assimilation and cytoskeletons play essential roles in maintaining cellular and redox homeostasis. These processes are based on signal transduction in the phosphoinositide pathway and multiple ways to supply energy. Conclusively, Z-61 establishes a new steady-state balance of metabolic processes and survives under higher temperature stress.Global warming increases sea temperature and leads to high temperature stress, which affects the yield and quality of  Pyropia is an economically important marine red alga used for food, fertilizer, medicine, and chemicals. The farming and processing of Pyropia has become one of the largest seaweed industries in East Asian countries, including China, Japan, and South Korea12Pyropia haitanensis, a typical warm temperate zone species originally found south of China, has been extensively cultured in the Fujian, Zhejiang, and Guangdong Provinces for more than 50 years, and its output accounts for about 75% of the total production of Pyropia in ChinaP. haitanensis. Under natural conditions, P. haitanensis is cultured from September to March of the next year, and the optimal seawater temperature is 14 °C to 26 °C. However, in the early seeding period (September to October in south China), P. haitanensis farms often suffer from consistently high temperatures or the well-known “temperature rebound” as temperature increases following a dramatic drop to temperatures suitable for conchospore release and germinationP. haitanensis blades by inhibiting the survival and division of these conchospores. In addition, germlings become prone to disease, premature senility, and eventual decay, leading to a substantial reduction in yieldP. haitanensis cultivation and reduced its yieldPyropia cultivation, several high temperature tolerance strains  of P. haitanensis have been selected and widely cultivated in South China5Pyropia.Temperature is the major environmental factor that affects the development, metabolism, productivity, and quality of P. haitanensis high temperature stress responses, Zhang et al. compared the physiological response of a high temperature tolerance strain (Z-61) to that of a wild-type strain, and found that Z-61 induces its antioxidant and osmoregulation systems, whereas the wild-type strain only induces its osmoregulation system during high temperature stressPyropia. Transcriptomic work in Z-61 revealed that its high temperature response is characterized by rapid reprogramming of gene expression machinery leading to up- and down-regulation of thousands of genes within minutes of exposure to high temperature stressThe mechanisms of responding to high temperature stress have been investigated in a great deal of higher plants, especially in model plants and economic crops, and various mechanisms have been summarized. These include membrane stability maintenance, Reactive oxygen species (ROS) scavenging, antioxidants production, the accumulation and adjustment of compatible solutes, the induction of mitogen-activated protein kinase (MAPK) and calcium-dependent protein kinase (CDPK) cascades, and, most importantly, chaperone signaling and transcriptional activation8910strain Z-1 to thatPyropia and reveal the global proteomic response of the Z-61 stains of P. haitanensis under high temperature stress.Proteins represent biochemical machinery and the functional processes of how an organism responds to a changing environment. Meanwhile, proteomics presents an effective approach to pursue a systems-based perspective of how proteins change, and thus how organisms adapt to various abiotic environments1616A total of 269614 spectra were obtained from the iTRAQ-LC-MS/MS proteomic analysis of all Z-61 samples. After data filtering to eliminate low-scoring spectra, a total of 97072 unique spectra that met the strict confidence criteria for identification were matched to 1895 unique proteins  and S2.To assess the reproducibility of the iTRAQ data, PCA was performed on every 2 replicates at each time point . ResultsP < 0.05) in abundance, 151 differentially expressed proteins (DEPs) were chosen by a ratio >1.50 or <0.67 at least in 1 time point under high temperature stress , which are made of phycobiliproteins (PBPs) and linker polypeptidesThe first step of photosynthesis is light absorption. In contrast to higher plants, P. haitanensis, but not in PSI, and the PSI-driven cyclic electron flow allows Pyropia to survive in stress conditions39PSII is more thermolabile than photosystem I (PSI), and has been long believed to be a prominent heat sensitive component of photosynthesis as its activity can be greatly reduced or even partially stopped under high temperature stressP. haitanensis.Fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase (SBPase), fructose bisphosphate aldolase, ribose-5-phosphate isomerase, and transketolase, which are involved in the Calvin cycle2-concentrating mechanism (CCM) in algaePhαCA1, PhβCA2, PhβCA3, and PhγCA1) in P. haitanensis were found up-regulated during high temperature stressP. haitanensis, and the higher the temperature, the lower the utilization2 and HCO3− during stress. In addition to function in CCM, CA also shows antioxidant activity and plays a role in the hypersensitive defense responseOsCA1) and CA activity were up-regulated by some environmental stresses, and a transgenic Arabidopsis overexpressing OsCA1 had a greater salt tolerance at the seedling stage than wild-type plantsCapsosiphon fulvescens showed that OEE is also an excellent antioxidantAlthough most photosynthesis-related proteins down-regulated under heat stress, it is worth mentioning that carbonic anhydrase (CA) and oxygen-evolving enhancer protein 1 (OEE) were up-regulated . CA playP. haitanensis limited damage to a repairable level by reducing photosynthesis under high temperature stress.Based on the expression of proteins related to photosynthesis, we conclude that Twenty-seven identified DEPs were functionally characterized as being involved in the defense response , which wP. haitanensis. Calnexin is an endoplasmic reticulum-localized molecular chaperone protein involved in the folding and quality control of proteinsP. haitanensis.An essential component of acquired thermotolerance in plants and other organisms is the induction and synthesis of molecular chaperones or heat shock proteins (HSPs)P. haitanensis blades when under high temperature stress.High temperature stress may disturb cellular redox homeostasis and promote the production of reactive oxygen species (ROS). Excessive ROS could damage cellular components. To cope with oxidative stress, cells have developed a wide range of antioxidant systems555657P. haitanensis. Accumulating evidence shows that PCD is triggered by different environmental factors such as high light, ultraviolet radiation, drought, salt, heat, cold, or flooding. Upon reaching certain thresholds with these changes, plant cells can no longer maintain proper metabolic processes and PCD is inducedProgrammed cell death (PCD) in plants is a crucial component of development and defense mechanismsOther stress-related proteins such as polyamine oxidase, stress-induced protein, and aldehyde dehydrogenase, were up-regulated to deal with high temperature stress . Several64P. haitanensis under high temperature stress is complicated and involves molecular chaperones, antioxidant systems, PCD, and other stress-related proteins. These proteins work together to maintain cellular and redox homeostasis.In conclusion, the defense response of Plants respond to stress conditions by triggering a network of events linked to energy and carbohydrate metabolismP. haitanensis require high energy levels to fuel defense mechanisms and repair damage under high temperature stress.Glycolysis is a metabolic pathway that oxidizes glucose to generate ATP, reductant, and pyruvateSecondary metabolites of plants often refer to compounds that have no fundamental role in the maintenance of life processes but are important for interaction with the environment for adaptation and defenseP. haitanensis to adapt to heat stress.Sulfur is a ubiquitous and essential element for plants71P. haitanensis when under high temperature stress.The process by which plant cells sense stress signals and transmit them to cellular machinery to activate adaptive responses is referred to as signal transduction75P. haitanensis increased energy metabolism to maintain physiological balanceP. haitanensis can also use lipids as an energy source.Lipids are linked to many cellular functions including storage for energy generation and membrane synthesisP. haitanensis from abiotic stress.DNA methylation is an important mode of epigenetic modification in plants. It plays vital roles in regulating growth and development in plants, and is involved in responses to a variety of stresses. Environmental stimuli adjust gene networks associated with the stress response by changing the level and pattern of DNA methylation, thereby enhancing adaptation to various stressesP. haitanensis cellular homeostasis.The cytoskeleton in plant cells plays important roles in controlling cell shape and mediating intracellular signaling, and can undergo profound changes when under abiotic stressP. haitanensis using the iTRAQ technique. The expression of these proteins shows that there is a clear response to high temperature in Z-61  with 5 blades each at 29 °C (high temperature), and were illuminated by 50–60 mol m−2s−1 photons  for 0, 6, 12, 24, 48, and 144 hours. Each sample had 2 biological replicates. The cultured medium was natural seawater enriched with MES medium.The Z-61 strain of Blade proteins were extracted according to a previously published methodFor digestion, the 300-μg protein pellet from the previous step was resuspended in digestion buffer (100 mM triethylammonium bicarbonate (TEAB), 0.05% w/v sodium dodecyl sulfate (SDS)) to a final concentration of 1 mg/mL ). Equal aliquots (500 μg) from each lysate were then digested with trypsin  overnight at 37 °C in a sealed tube. The tryptic peptides were then lyophilized and dissolved in the 50% TEAB buffer, and the trypsin digested samples were analyzed using MALDI-TOF/TOF to ensure complete digestion.The iTRAQ labeling of peptide samples derived from each time point of high temperature stress was performed using iTRAQ Reagent-8plex Multiplex Kit  according to the manufacturer’s protocol. For each time point , 12 samples  were iTRAQ labeled. Peptides were labeled with respective isobaric tags , incubated for 2 h, and vacuum centrifuged to dryness.−1. Retained peptides were eluted using Buffer A (10 mM KH2PO4 in an aqueous solution of 25% acetonitrile acidified to a pH of 3.0 with H3PO4) and Buffer B (Buffer A with 500 mM KCl). The following chromatographic gradients were applied: 0–25 min 100% Buffer A; 25–32 min 90% Buffer A and 10% Buffer B; 32–42 min 80% Buffer A and 20% Buffer B; 42–47 min 55% Buffer A and 45% Buffer B; 47–60 min 100% Buffer B; 60–75 min 100% Buffer A. Chromatograms were recorded at 214 nm. The collected fractions were desalted with Sep-Pak Vac C18 cartridges , concentrated to dryness using vacuum centrifuge, and reconstituted in 0.1% formic acid for LC-MS/MS analysis.Labeled peptides were then fractionated using Polysulfoethyl A SCX columns  using an HPLC system  at a flow rate of 1.0 mL minProtein digestion and iTRAQ labeling was conducted in 2 technical replicates.The SCX peptide fractions were pooled to obtain 10 final fractions and reduce the number of samples and collection time. A 10-μL sample from each fraction was injected 3 times to the Proxeon Easy Nano-LC system. Peptides were separated on a C18 analytical reverse phase column  at a flow rate of 250 nL/min and a linear LC gradient profile was used to elute peptides from the column. We then analyzed the fractions using a Q Exactive mass spectrometer . Data were acquired using a data-dependent acquisition mode in which, for each cycle, the 10 most abundant multiply-charged peptides (2+ to 4+) with an m/z between 300 and 1800 were selected for MS/MS with 15 s dynamic exclusion setting. The fragment intensity multiplier was set to 20 and the maximum accumulation time was 2 s. Peak areas of the iTRAQ reporter ions reflected the relative abundance of proteins in the samples.P. haitanensis UniGene database, which was obtained from whole transcriptome sequencingp value. The resulting data set was auto bias-corrected to remove any variations due to unequal mixing during combining differently labeled samples. The average value of 2 biological replicates and 2 technical replicates were used to indicate the final protein abundances at a given time point. Proteins with a 1.5-fold change between samples and a p value less than 0.05 were determined as differentially expressed proteins.For iTRAQ/Shotgun protein identification, the raw mass data were processed with Proteome Discover 1.3 (Thermo Fisher Scientific) and searched with in-house MASCOT software 2.3.02  against the http://www.omicshare.com/tools/index.php/Home/Soft/venn).Principal component analysis (PCA) analysis was performed using the prcomp package, calculation was based on a singular value decomposition, and Differently expressed proteins were functionally classified according to MapMan ontologyHow to cite this article: Shi, J. et al. Differential Proteomic Analysis by iTRAQ Reveals the Mechanism of Pyropia haitanensis Responding to High Temperature Stress. Sci. Rep.7, 44734; doi: 10.1038/srep44734 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Tick infestation is the major problem for animal health that causes substantial economic losses, particularly in tropical and subtropical countries. To better understand the spatial distribution of tick species and risk factors associated with tick prevalence in livestock in Pakistan, ticks were counted and collected from 471 animals, including 179 cattle, 194 buffaloes, 80 goats and 18 sheep, on 108 livestock farms in nine districts, covering both semi-arid and arid agro-ecological zones.Hyalomma anatolicum , Rhipicephalus microplus (n = 715), Hyalomma dromedarii (n = 41) and Rhipicephalus turanicus (n = 30). The latter species is reported for the first time from the study area. Rhipicephalus microplus was the predominant species in the semi-arid zone, whereas H. anatolicum was the most abundant species in the arid zone. The overall proportion of tick-infested ruminants was 78.3% (369/471). It was highest in cattle (89.9%), followed by buffaloes (81.4%), goats (60.0%) and sheep (11.1%). The median tick burden significantly differed among animal species and was highest in cattle (median 58), followed by buffaloes (median 38), goats (median 19) and sheep (median 4.5). Female animals had significantly higher tick burdens than males and, in large ruminants, older animals carried more ticks than younger animals. The intensity of infestation was significantly lower in indigenous animals compared to exotic and crossbred cows. Analysis of questionnaire data revealed that the absence of rural poultry, not using any acaricides, traditional rural housing systems and grazing were potential risk factors associated with a higher tick prevalence in livestock farms.In total, 3,807 ticks representing four species were collected: R. turanicus in the Punjab Province of Pakistan for the first time. The outcomes of this study will be useful in the planning of integrated control strategies for ticks and tick-borne diseases in Pakistan.Absence of rural poultry, not performing acaricide treatments, traditional rural housing systems and grazing were important risk factors associated with higher tick prevalence in livestock farms. Age, gender, breed and animal species significantly affected the intensity of tick infestation. This report also describes the presence of The online version of this article (doi:10.1186/s13071-017-2138-0) contains supplementary material, which is available to authorized users. Bos indicus and Bos taurus) population is 41.2 million, and there are 35.6 million buffaloes , 68.4 million goats (Capra hircus) and 29.4 million sheep (Ovis aries) (figures are based on inter-census growth rate of Livestock Census 1996 & 2006) [Ticks cause substantial economic losses to resource-poor farming communities, especially in tropical and subtropical regions, where approximately 80% of the world’s cattle population is raised . Tick inMany risk factors are associated with tick infestation in farm animals , 7, whicSo far, many aspects of the epidemiology of ticks and TBDs in Pakistan have not been elucidated. To limit the damage to livestock and humans caused by TBDs, it is necessary to devise an evidence-based tick control program for Pakistan’s livestock sector. To date, only a few studies have investigated the risk factors associated with tick infestation on livestock farms in Pakistan , 13.Although Pakistan’s climatic conditions are suitable for the rapid development of various tick species, there is still a lack of systematic work to investigate the frequency and distribution of tick species infesting ruminants. Many of the previously conducted studies were confined to a small area and did not consider production systems and sampling strategies, which are all factors that can affect prevalence estimates of ticks and TBDs . MoreoveThe aims of this study, therefore, were to (i) estimate the tick prevalence among farm animals in the semi-arid and arid agro-ecological zone; (ii) identify tick species infesting farm animals in Pakistan; and (iii) identify potential risk factors associated with high tick infestation facilitating the development of effective interventions for tick control.Pakistan was divided into five agro-ecological zones based on aridity data obtained from the Consultative Group for International Agricultural Research - Consortium for Spatial Information Global-Aridity and Global-PET Database . Of the A cross-sectional study was designed to investigate the distribution of ticks infesting ruminants on livestock farms. The sample size was calculated for large populations assuming a 50% prevalence with 95% confidence level and 10% desired precision, resulting in at least 97 livestock farms to be sampled . This waThe livestock farms were selected in nine districts, namely Attock (ATK), Gujranwala (GJW), Kasur (KSR) in the semi-arid zone and Okara (OKR), Khanewal (KNW), Multan (MTN), Vehari (VHR), Bahawalpur (BWP) and Rahim Yar Khan (RYK) from the arid zone Fig. . All disAll tick specimens were collected from September to November 2013 except for MTN district, where the samples were collected in June of the following year. Ticks were collected from buffaloes, cattle, goats and sheep after informed consent from the farm owners had been obtained. To estimate the tick prevalence within livestock herds, two animals from each ruminant species varying in age and sex were randomly selected based on the assumption that at least 50% of the animals on that farm would be infested if ticks are present on the farm . Thus, aThe following predilection sites were examined for the presence of ticks: (i) ears; (ii) brisket (dewlap in the case of cattle); (iii) withers; (iv) knees; (v) udder in the case of females and testes in males along with the perineum region; and (vi) tail . The totAn effort was made to collect a representative proportion of all different types of ticks from predilection sites using a tick removal tool , according to the manufacturer’s instructions. A blunt steel forceps was used for large sized ticks. Ticks were transferred to Safe-Lock Eppendorf® tubes containing 70% ethanol, labelled with a unique sample ID, comprising the farm ID, host species and body location. A separate tube was used for each predilection site. Information regarding each specimen and host related factors including species, breed, gender and age were recorded on predesigned forms.To identify risk factors associated with tick prevalence, a questionnaire containing 21 closed and ten open-ended questions was used. The questionnaire was divided into three parts: (A) farm-related information, (B) tick-related information and (C) herd management-related information. The questionnaire was pilot tested by five farmers in Urdu and Punjabi to ensure that they understood all the questions. The data were collected with the help of local veterinarians.Tick samples were shipped to the Institute of Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Germany for further morphological identification using taxonomic keys covered in Multikey 2.1 software  as well 2, 2.5 μl of 2 mM dNTPs, 1 μl (10 μM) of each primer, 0.25 μl (2U/μl) of Maxima™ Hot Start Taq DNA Polymerase  and 2.5 μl of template DNA in a C1000™ Thermal Cycler . The cycling conditions comprised a 5 min denaturation and polymerase activation step at 95 °C, 40 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 50 s and a final extension step for 5 min at 72 °C. A negative control was used to authenticate the PCR reaction. PCR amplicons were visualised by gel electrophoresis, purified using the Zymoclean™ Gel DNA Recovery Kit  according to the manufacturer’s instructions and sequenced . The resulting sequences were subjected to a Basic Local Alignment Search Tool (BLAST) analysis (www.ncbi.nlm.nih.gov/blast/). Following confirmation of tick species identity by the ITS2 gene, three R. microplus specimens  were further characterised as it was known that different clades exist in this species. A 1,592 base pair fragment of the cytochrome c oxidase subunit 1 (cox1) gene from these three R. microplus specimens were amplified and sequenced using primers Rhcox1 F (5′-CCG CCT AAA CTT CAG CCA TT-3′) and Rhcox1-R (5′-GTC TGA AAA TG(C T)TA ATT GAG ATC AAG-3′) with identical PCR conditions except for the extension time, i.e. 100 s, as described for the amplification of the ITS2 gene.To confirm results of the morphological identification, a fragment of about 750 nt from the second internal transcribed spacer gene (ITS2) of 19 randomly selected tick specimens was amplified and sequenced. Prior to DNA extraction, ticks were washed with distilled water and subsequently homogenised using sterilised pestles in 1.5 ml Safe-Lock Eppendorf® tubes. DNA extraction was performed using the NucleoSpin® Tissue Kit , according to the manufacturer’s recommendations for the purification of genomic DNA from insects. Purified DNA was quantified using an Epoch Spectrophotometer . The following primers were used: Metastriata ITS-F (5′-AGG ACA CAC TGA GCA CTG ATT C-3′) and Metastriata ITS-R (5′-ACT GCG AAG CAC TTR GAC CG-3′). Polymerase chain reaction (PCR) amplification was performed in a total reaction volume of 25 μl containing 2.5 μl of 10× Maxima Hot Start Taq Buffer, 2.5 μl of 1.5 mM MgClhttp://www.cgiar-csi.org/data/global-aridity-and-pet-database). The Aridity Index (AI) values reported within the Global-Aridity dataset had been multiplied by a factor of 10,000 to derive and distribute the data as integers . This multiplier had been used to increase the precision of the variable values without using decimals . Therefore, Global-Aridity values were divided by 10,000 to retrieve the values in the correct units. The data relevant to Pakistan was extracted by a mask of a polygon indicating the borders of Pakistan (Pakistan Admin). The raster map (PakAridityMap) was converted to a polygon. Subsequently, the aridity polygon features for Punjab Province (PunjabAridity) were extracted from “PakAridityMap”. A higher AI and darker colours represent more humid conditions, while low AI Index and lighter colours represent higher aridity.The Global-Aridity dataset was downloaded as one grid layer representing the annual average for years 1950–2000  for proportions were estimated in R using the package ‘binom’  with theP < 0.2, which were included with an additive mode of interaction in the multivariable model [P-value for that specific variable was less than 0.05. Potential confounding effects were evaluated by assessing the change in any remaining parameter estimate. If this change was greater than 20% as compared to the full model, the variable was considered a confounder. Farms were categorised at a dichotomous level . The ultimate model was fitted with the farm category (low or high infested) as the response variable. Deviance residuals were examined for homoscedasticity and normal distribution. Pearson goodness-of-fit statistic (χ2) was applied to assess model fit. Additionally, Akaike information criterion (AIC) values were also utilised to assess the quality of the model. The multivariable model was run using the glm function. The link function “logit” was used in the model to report the coefficient, the ratio of the coefficient to its standard error and the P-value. Odds ratios (OR) along with 95% CI were calculated using the exp function. The software records an OR of 1 for the reference variable. When the upper limit of the CI of the OR of the examined variable was below than 1, it was considered as a protective factor, and if the lower limit of the CI was above 1, it was considered as a risk factor. OR are presented for the independent variables that showed statistical significance in the multivariable analysis. All statistical analyses were performed with R for Windows software  and RStudio as an interface .To measure the effect of various determinants on tick prevalence, a multivariable logistic regression model was built. In the first step, a univariable analysis (Fisher’s exact test) was performed to select the variables (predictors) with le model . SubsequMaps were produced in the ArcMap software environment version 10.3 . The base map for Pakistan was obtained from the database of Global Administrative Areas and was set for Gujrat, Gujranwala, Narowal and Okara districts using the ‘dissolved function’.n = 139) and arid zone (n = 332). The majority (66%) of the animals were female. The median herd size was 10 (Q1-Q3: 8–15) animals. The median age of the animals was 2.5 years (Q1-Q3: 1.0–4.5). The median age in large ruminants was 3.0 years  and 1.5 years in small ruminants. The median age of the infested animals was 3.0 years (Q1-Q3: 1.0–5.0) while it was 1.5 years (Q1-Q3: 1.1–2.5) in the non-infested group.A total of 471 ruminants  were examined on 108 livestock farms in two different agro-ecological zones: semi-arid zone  was the most common species, followed by R. microplus , H. dromedarii , and R. turanicus . Rhipicephalus microplus was predominant in the semi-arid zone, while H. anatolicum was the most common tick species in the arid zone. Hyalomma dromedarii and R. turanicus were only present in the arid zone. Hyalomma anatolicum was found in all the districts of the province, while R. microplus was absent in MTN, BWP, and RYK. In all the districts, multiple tick species were found except in MTN district, where only H. anatolicum was detected. Rhipicephalus microplus and H. dromedarii were mainly found in cattle, whereas R. turanicus was mainly found on goats. Hyalomma anatolicum infested all ruminant species.In total, 3,807 ixodid ticks  were collected from 108 livestock farms (Table H. anatolicum showed 100% identity to H. anatolicum strains from China (HQ005303) and Iran (FJ593703). Two R. microplus samples had 100% identity to a R. microplus isolate from Laos (KC503276). The sequence of H. dromedarii showed 99% identity to a registered sequence of H. dromedarii from a dromedary camel in India (JQ733570), whereas the sequence of R. turanicus was 99% identical to the R. turanicus isolate 80-T-He4 (accession no. KF958417). In addition, the BLAST results of the cox1 gene sequences from three R. microplus ticks revealed the highest identity (95.5%) with a Chinese R. microplus isolate from Guizhou, China (KC503259). Representative ITS2 sequences for each tick species and cox1 (only for R. microplus) were submitted to GenBank and under the following accession numbers: H. anatolicum , R. microplus , H. dromedarii (KY373258), R. turanicus (KY373259).The morphological identification was supported by sequencing a partial fragment of the ITS2 gene from 19 randomly selected samples followed by a BLAST analysis. Of these 19 sequences, 15 samples previously identified as n = 265) were infested with H. anatolicum, 17.0% (n = 59) with R. microplus, 1.4% (n = 5) with H. dromedarii, 1.1% (n = 4) with R. turanicus and 9.5% (n = 36) were found to have a mixed infestation with more than one tick species.All livestock herds, irrespective of their geographic location, were found infested with one or multiple tick species. Within the herds, the tick prevalence varied from 20 to 100% . The overall proportion of tick-infested ruminants was 78.3% (369/471); this was highest in cattle (89.9%), followed by buffaloes (81.4%), goats (60.0%) and sheep (11.1%) . It was highest in cattle (median 58), followed by buffaloes (median 38), goats (median 19) and sheep (median 4.5) , and between bovines, the infestation was higher in cattle (P < 0.001) than in buffaloes. Tick burden on livestock farms was significantly lower  in the semi-arid zone as compared to the arid-zone  was significantly different among the examined animal species , where indigenous animals had lower tick burdens as compared to exotic (P < 0.001) and crossbred cows (P < 0.001), while the difference was not statistically significant between crossbred and exotic cattle (P = 0.11)  Fig. . In otheOnly a small proportion of farms  raised a single ruminant species, while the majority  held more than one species. Cattle were present on 87.0% (95% CI: 79.2–92.7) farms, buffaloes on 92.6% (95% CI: 85.9–96.8), goats on 39.8% (95% CI: 30.5–49.7) and sheep on 10.2% (95% CI: 5.2–17.5). Additional file When farm owners were asked about the months when ticks occur on their farm, most  reported the period from March to November, while 15.7% (95% CI: 9.4–24.0) observed ticks throughout the year, and the remaining 19.4% (95% CI: 12.5–28.2) did not know. Regarding the months of highest tick infestation, 83.3% (95% CI: 74.9–89.8) of the farmers reported the period from June to September and 16.7% (95% CI: 10.2–25.1) did not know.Eruca sativa) on the body of the animals, while only a few farmers  stated that they had not used anything against ticks. It was a common practice that farmers  offered only green roughages to their animals, except for milking animals. Only five farmers  fed all their animals with green roughages plus concentrate (energy-rich feed).Almost one-third of the total farmers  used acaricides to control ticks. When they were asked about the names of the acaricides used, 16  out of 31 farmers did not know, whereas the remaining farmers  answered that they used ivermectin (injectable) (19.4%), trichlorfon 97.1%  (12.9%) or both (16.1%). Most the farmers  used at least one tick control method like acaricides, hand picking, keeping rural poultry, topical application of “Taramira oil” (n = 106) or when artificial insemination (n = 65) or vaccination (n = 24) was needed.Almost half of the farmers  reported a behavioural change in tick-infested animals, e.g. restlessness and scratching. More than half of the farmers  had noticed a drop-in milk production of the tick-infested animals, while the others had not observed a change in performance  or did not know (28.7% 95% CI: 20.4–38.2). Only two farmers reported visits of para-veterinary or veterinary staff daily, while the others reported that these staff visited the farm only when animals became sick . The presence of rural poultry on farm significantly (P = 0.006) affected the tick prevalence, and the odds of having higher tick prevalence on farms where rural poultry was absent were 4.4 times as high as on farms with rural poultry. The tick prevalence was significantly lower (P < 0.001) on farms where acaricides had been used. The housing type also had a significant effect on the tick prevalence (P = 0.007), and the chances of getting higher tick prevalence on farms with traditional rural housing system were almost 13 times as high as on farms with open houses. The feeding method was also an important variable associated with the tick prevalence and farms, where grazing was practised, had a higher prevalence (P = 0.003) as compared to farms with a stall-feeding system.The initial main effect model included eight variables, which had yielded To our knowledge, this is the first report from Pakistan in which the identified tick species are confirmed using molecular techniques, which can be utilised to identify and discriminate different species and subspecies of ticks in cases where the morphological identification is doubtful or ambiguous . PreviouH. anatolicum was the most abundant tick species found in this study, which agrees with previous studies from Pakistan [Hyalomma anatolicum is a potential vector responsible for the transmission of Theileria annulata and Theileria lestoquardi in Pakistan [Overall, Pakistan , 31 and Pakistan , 33. It Pakistan . FurtherPakistan  and may Rhipicephalus microplus was the second most prevalent tick species, which also infested all animal species in both the agro-ecological zones. Our finding that R. microplus was the dominant tick species in the semi-arid zone (northern part of the province), is in accordance with those of a local study conducted on two governmental farms [R. microplus. The species was absent in the southern part of the province , which is a drier region. The geographical distribution and abundance of this tick species have been greatly promoted by the retaining water capacity of the underlying layer of the soil and the increased relative humidity. It was recently reported that R. microplus is, in fact, a species complex, which consists of at least five taxa: R. annulatus, R. australis, R. microplus clade A, R. microplus clade B and R. microplus clade C [R. microplus, whose cox1 gene we sequenced, showed the highest identity to a R. microplus cox1 sequence from Malaysia (KM246868), referred to as haplotype 17 in [R. microplus complex, which also comprises ticks from India and Malaysia.al farms , where t clade C , 36. TheH. dromedarii was confined to the BWP district in the arid zone, from where it has already been reported, but always in small numbers [H. dromedarii is specialised to feed on camels. Moreover, the finding that the presence of H. dromedarii appeared to be confined to this district is perhaps explicable by the influence of the low relative humidity in the area on this desert-adapted tick species [The ecological niche for  numbers . The maj species .R. turanicus in Punjab Province. Only limited numbers (n = 30) were found, and the tick was confined to the BWP and RYK districts of the arid zone. The tick species was only found on water buffaloes and goats. Although some studies have been conducted in the past, R. turanicus has never been reported from Punjab Province; however, it has been reported from Sindh Province, but quite a long time ago [R. turanicus in Pakistan, but the author considered it as a subspecies of R. sanguineus due to the previous work of Pervomaisky [Rhipicephalus sp. III from Pakistan and India were morphologically and genetically similar to, but still different from, R. turanicus [R. turanicus has been there all the time but was misreported as R. sanguineus, as the two are morphologically very difficult to distinguish and molecular techniques were previously not utilised for confirmation.Here we report for the first time the occurrence of time ago . McCarthtime ago  also fouvomaisky , 39. Nevvomaisky , India [vomaisky , Bangladvomaisky  and Chinvomaisky . A reporuranicus . After cHyalomma anatolicum has been reported as the principal tick vector for Crimean-Congo haemorrhagic fever (CCHF) [Rhipicephalus microplus and Hyalomma spp. are potential vectors of Anaplasma centrale and A. marginale [Theileria annulata and Babesia bovis in buffalo and cattle in Pakistan [R. turanicus may still be of medical as well as veterinary importance as it is known to harbour Coxiella burnetii, the causative agent of Q fever, which has recently been recorded in Pakistan [These tick species infest a broad range of host species and transmit several important pathogens including viruses, bacteria and protozoa of medical and veterinary importance. r (CCHF) . After sr (CCHF)  and the r (CCHF) . Rhipicearginale , the cauarginale . FurtherPakistan , 26. AltPakistan .It is evident from the results that the tick prevalence significantly differed between animal hosts, which concurs with previous studies , 48. TheLimited information is available about tick prevalence in small ruminants in Pakistan, as only a few studies have focused on these animals. In general, the tick prevalence observed in the present study was higher in goats  as compared to sheep , which is also in agreement with a local study . HoweverThe present study revealed that all the livestock herds were found infested with one or multiple tick species. Within-herd tick prevalence ranged from 20 to 100% (Mean ± SD:  80 ± 20%). The tick prevalence on an animal level and median tick burden were lower in the semi-arid zone as compared to the arid zone. The semi-arid zone is located at a higher elevation and observes low annual mean temperatures (minimum and maximum) as compared to the arid zone. Previous reports from Pakistan did not consider the agro-ecological zones and were based on administrative units only . A studyR. microplus ticks, the second most common tick species in our samples. A plausible explanation could be that the thick skin of the buffalo reduced the ability of these ticks to attach because of their short hypostome. Additionally, the immune system of the buffalo showed an increased sensitivity to tick proteins than that of cattle [Like the tick prevalence, tick burden  was also highest in cattle followed by buffaloes, goats and sheep. Our results agree with a previous study from Pakistan . These af cattle .Our finding that tick burdens were higher in female animals as compared to males is consistent with previous studies carried out in ruminants , 14. MalA significantly lower tick burden in calves as compared to older animals agrees with previous studies . A previBos taurus taurus) followed by crossbred and indigenous cows (Zebu cattle or Bos taurus indicus), which is in line with previous reports from Pakistan [R. microplus is related to the proportion of zebu genes in the breed [Bos indicus and their crosses, the resistance against different tick species has been found a highly heritable trait [Our results showed that tick infestation was highest in exotic cattle  compared to European breeds (which have been selected for a high production).Hyalomma nymphs and adults [Results of the multivariable logistic regression model showed that traditional rural housing was positively associated with higher tick prevalence and the odds of acquiring higher tick prevalence on farms with traditional housing type was almost 13 times as high as the farms with open housing system. In the traditional rural housing, a farm has an uncovered and covered area. The latter consists of almost completely closed animal shed without proper ventilation and a simple roof structure and are used for protection from cold weather during the winter season, while the roof structure along with trees is used for protection during the summer and the monsoon season. The walls of the buildings are made of hard or soft bricks with mud as a seal, whereas the roof is made of bricks placed on wood or iron rods with a thin layer of mud on top. A previous study has shown that the animals were more prone to tick infestation in a closed-type of housing, which is quite similar to the traditional housing system, as compared to open-type housing . In addid adults . Caulkind adults .We found that farms, where grazing was practised, had a higher tick prevalence as compared to farms with stall feeding system (OR = 12.6), which is likely to be caused by a decreased exposure to questing ticks in stall-fed animals compared to animals in pastures , 40. A nEruca sativa), to control tick infestation. In Punjab Province, Taramira is used in different ways against ticks, e.g. as “Taramira oil” topically on the animal body, whereas others prepare a mixture with salt and water and drench tick-infested animals. This formula is not only believed to decrease tick burdens, but it is also considered as a milk booster. However, there is no published report confirming the acaricidal activity of Taramira oil.Although the use of acaricides was significantly associated (OR = 7.5) with tick prevalence and the farms, where acaricides were used, had a low tick prevalence, ticks were found on all livestock farms, suggesting that acaricide resistance might occur. So far, no study from Pakistan evaluated acaricidal resistance, but it has been reported from other countries . ApplicaAnother important risk factor associated with higher tick prevalence on the farm was the absence of rural poultry. Conversely, the farms that reared rural poultry by integrated farming with ruminants had a significantly lower tick prevalence. The OR suggested that the chances of having higher tick prevalence on farms, where rural poultry was absent, were approximately four times as high as on farms where poultry was reared. Rearing chicken on livestock farms reduces tick burden as chickens act as a natural pest control by picking ticks from animal bodies as well as from their surroundings.In our study, flooring showed a statistically significant association with high tick infestation in univariable analysis and the farms with soft floors had a higher tick prevalence, but it appeared insignificant when included in the multivariable analysis when confounding effects were accounted for. Previous studies from Pakistan reported non-cemented (soft and mixed type) flooring as a risk factor, and the chances of being infested with ticks were two times as high as in animals kept on cemented floors .H. anatolicum and R. microplus. Hyalomma dromedarii and R. turanicus were also found, whereby the latter species had not been reported from the study area before. Animal species, gender, age and breed were important host characteristics that determined the intensity of tick infestation. Several risk factors were found to be associated with higher tick prevalence on livestock farms, including the absence of rural poultry, not performing acaricide treatments, traditional rural housing systems and grazing. The outcomes of this study will be useful in the planning of integrated control strategies for ticks and TBDs in Pakistan.The most common tick species infesting ruminants in Punjab, Pakistan were Additional file 1: Table S1.Weather data for Punjab as recorded by the Pakistan Meteorological Department. (DOCX 14 kb)Additional file 2: Table S2.Table S3. Survey of livestock farms in Punjab Province (2013): Summary of numeric variables included in the questionnaire. (DOCX 17 kb)Survey of livestock farms in Punjab Province (2013): Summary of categorical variables included in the questionnaire. Additional file 3: Table S4.Survey of livestock farms in Punjab Province (2013): Summary of variables included initially in the multivariable logistic regression model. (DOCX 21 kb)


Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of bone callus and its possible use as autograft material for patients needing bone grafts. Histology, immunohistochemistry, micro-computed tomography, and biomechanics were performed to examine osteogenic cells, osteoinductive factors, and the osteoconductive structure of bone callus. Alkaline phosphatase-positive osteoblasts, osteoinductive factors , and a porous structure were found in bone callus. Early-stage callus (within 3 months after fracture) presented significantly improved osteogenic properties compared to medium- (3–9 months) and late-stage (longer than 9 months) callus. The results revealed that bone callus induced new bone formation in a nude mouse model. Early-stage callus showed better performance to medium- and late-stage callus in the induction of new bone formation at both 8 and 12 weeks. These findings indicated that bone callus, especially early-stage callus, possesses osteogenic potential and can potentially serve as an alternative source of material for bone grafts. Bone callus is commonly observed at fracture sites. To ensure the accuracy of reduction and fixation during surgical procedures for fractures or nonunions, bone callus is commonly removed and discarded, especially in secondary surgery in nonunion patients245There is significant interest among orthopaedic surgeons to learn whether bone callus can be used as autograft material. Bone callus forms along with fracture healing, which is a process involving a complex interplay of cells, growth factors, and extracellular matrix789911129131491313in situ grafting of excised bone callus, followed by external fixation, achieved a satisfactory therapeutic effect for the treatment of nonunionsNakase and colleagues reported that Bone calluses were observed at fracture sites by anteroposterior and lateral radiographs . The graOsteoblasts were identified according to their morphology and physiological localization in the bone callus, which lines the edge of the trabecular bone in a single layer . In the The activity of osteoblasts was examined by alkaline phosphatase (ALP) staining. Positive particles, stained brown-black, were localized in the osteoblasts . In the Areas positively stained for the osteoinductive factors BMP2, FGF2, TGFB1, and IGF1 were observed in the bone callus at all stages, as well as in CI . In the Similar to BMP2, FGF2, TGFB1, and IGF1 were expressed at higher levels in bone calluses at 3 months post-fracture and decreased thereafter. These results indicated that bone callus contained plentiful osteoinductive factors at 3 months post-fracture, which decreased remarkably thereafter.Histological examination and micro-CT scanning revealed the porous and internal cross-linked structure of bone callus. Histological examination showed regular morphology of trabecular or woven bone in the EC group, but mature lamellar bone in the MC and LC groups . QuantitStructural changes of bone callus were also evident by micro-CT scanning. Porosity in the EC group was 71.2% ± 6.9%, which was comparable to 71.5% ± 3.2% in the control CI group  or Masson’s trichrome, as well as by micro-CT scanning. H&E staining revealed plentiful osteoid and irregular trabecular bone formation in the EC group 8 weeks after implantation. In contrast, only fibrous tissue and sparse osteoid formation were evident in the CI group. There was less osteoid formation in the MC and LC groups than in the EC group . After 1The Tb.N of new bone in the EC group at 8 and 12 weeks were significantly higher (P < 0.05) than those in the CI group, whereas these parameters in the MC and LC groups were lower than in the CI group. The values for Tb.Sp of new bone in the EC group at 8 and 12 weeks were significantly lower (P < 0.05) than those of the CI group, whereas these parameters were higher in the MC and LC groups than in the CI group. There were no significant differences in the Tb. Th of new bone among the four groups at 8 and 12 weeks , bone callus was shown to have significant osteogenic potential. However, the formation of both osteoblasts and osteogenic growth factors decreased with time, strongly indicating a limit to the osteogenic capability of bone callus. We believe that this downward trend is related to fracture healing and bone callus formation. When fracture occurs, the local injury stimulates chemotaxis and growth factor release, resulting in the rapid formation of numerous osteoblasts20212324As bone callus formation progresses, osteoblasts differentiate into osteocytes, undergo apoptosis, or convert to bone-lining cells (inactive osteoblasts), thereby decreasing the number of active osteoblasts2828As a potential graft material, bone callus can be harvested from fracture sites, avoiding the donor-site complications of harvesting a traditional autologous bone graft, such as pain, bleeding, infection, and soft-tissue injury3032et al., new bone formation is stained green and part of the degenerative bone matrix is stained red in Masson stainingThere were several limitations in this study. CI was used as the sole control for bone callus, and bone graft alternatives such as allogeneic bone and inorganic materials were not included. To demonstrate the internal structure of the callus, HE staining and micro-CT were performed . AlthougTo study the osteogenic potential of bone callus at different stages of callus formation, samples harvested from patients were divided into three groups: the EC group (within 3 months), the MC group (from 3 to 9 months), and the LC group (more than 9 months). CI was chosen as the control bone graft material.To investigate osteogenesis of bone callus, the callus was stained with H&E, and Gomori for ALP. To evaluate the osteoinductivity of bone callus, samples were assessed by immunohistochemistry for the BMP2, FGF2, TGFB1, VEGF, and IGF1 proteins. To evaluate the osteoconductivity of bone callus, samples were analysed by H&E staining and micro-CT scanning. To assess new bone formation, human bone callus was implanted subcutaneously in nude mice. The formation of new bone was detected by H&E staining, Masson staining, and micro-CT scanning.The project was approved by the Ethics Committee of Shaoxing People’s Hospital (No. 080) and the protocol was carried out in accordance with approved guidelines. Preoperative informed consent was obtained from each patient. From January 2010 to June 2014, bone callus samples were obtained from patients undergoing surgical treatment in the Department of Orthopedics of Shaoxing People’s Hospital. EC was collected from patients who required surgical treatment for failure of skeletal traction in four cases, fracture angulation or displacement after conservative treatment with plaster in the primary treatment in five cases, and after external fixation temporary for open fractures with severe soft tissue injury in two cases and multiple trauma in one case. MC was collected from secondary surgeries after loosened or broken plates or screws in eight cases, and bent or broken intramedullary nails in four cases. LC was collected from secondary surgeries for hypertrophic nonunion in 16 cases. CI was collected from patients who needed self-ilium grafts during the surgery. These patients exhibited healing post-surgery during the follow-up period.Inclusion criteria were as follows: (1) surgeries after failure of conservative treatment or external fixation were applied temporarily before open reduction and plate fixation for long bone fractures; (2) secondary surgeries after failure of internal fixation, including loosened or broken plates or screws, bent or broken intramedullary nails, and fracture angulation and aversion abnormalities; and (3) secondary surgeries for hypertrophic nonunion. Exclusion criteria were as follows: (1) fracture complicated with microbial infection; (2) fracture complicated with brain injury; (3) bone tumours; (4) systemic bone-related diseases; and (5) patients treated with hormones, steroids, vitamin D, or calcium. The general characteristics of the patients are presented in Radiographs of the anteroposterior and lateral views of the fracture sites were taken before bone callus samples were harvested. The default acquisition parameters included a source image distance of 120 cm, 55 kV, 5 mAs, and no added tube filtration. According to the scan protocol, image post-processing was performed at a set window width of 3845 and window level of 2031. The gray value of bone callus was judged with a Picture Archiving and Communication System , using Image-Pro Plus (IPP) 6.0 software .et al.Bone samples harvested from patients, and implants from mice, were fixed and decalcified in 10% ethylenediaminetetraacetic acid at pH 7.4 for 1 month. The maximum cross-sectional area of the calluses was selected as the embedding surface, which was parallel to the bottom of the embedding box. Thus, the maximum cross-sectional area of the calluses could be included in the sections. To ensure that the mid part of the regenerating callus was included in histological sections, rough trimming with sectioning was performed until the maximum cross-sectional area of callus tissue was nearly exposed in the sections, and serial sectioning (5 μm) was then started. In total, 60 sections per sample were collected. The sections were stained with H&E, Gomori, immunohistochemistry, and Masson’s trichrome. The number of osteoblasts was quantitated in five nonconsecutive sections per sample after H&E staining. Values for the BV/TV, Tb.Th, Tb.N, and Tb.Sp were calculated by H&E staining, as described by Zhang 3) cubes at the centre of the calluses. Porosity, Tb.Th, Tb.N, and Tb.Sp were then measured using data analysis software . The precise borders of the implants were poorly defined at 8 weeks and 12 weeks post-implantation. To study new bone formation within the implants, the region of interest was limited to 3 mm × 3 mm × 3 mm at the centre of the implants. ΔBV/TV, ΔTb.Th, ΔTb.N, and ΔTb.Sp were calculated as the differences between the parameters post- (8 or 12 weeks) and pre-implantation.The structure of bone callus harvested from patients, and the implants harvested from mice, were analysed using a μCT100 micro-CT scanner . Scanning was performed at 70 kV and 200 μA, with a resolution of 10.0 μm per pixel. Image reconstruction software (μCT V6.1) and three-dimensional model visualization software (μCT Ray V4.0) were used for further analyses. The volume of interest (VOI) was drawn in 3 mm × 3 mm × 3 mm . Samples were cut into 5 mm × 5 mm × 5 mm cubes, fixed with bone cement, and exposed to a cross-load at 2 mm/min, using a 2-mm diameter Kirschner wire. The displacement and applied force were recorded during the test, and the ultimate load and elastic modulus were calculated from the stress-strain curve.All procedures involving animals were approved by the Ethics Committee of Shaoxing People’s Hospital (No. 079) and conformed to its guidelines. Six-week-old male nude mice  were assigned randomly into the EC group, MC group, LC group, and CI groups (4 mice per group) for each time point. Anaesthesia was induced with intraperitoneal pentobarbital (50 mg/kg). All surgeries were performed under aseptic conditions. Two longitudinal incisions (approximately 1 cm) were made over the iliac crest and 5 mm bilaterally from the posterior midline. The volume and shape of the calluses or ilium were highly variable among the patients. Suitable samples (large enough) were selected for subcutaneous implantation and biomechanics testing, and the remaining samples were used in histological analysis, immunohistochemistry and micro-CT scanning. Two bone callus or ilium samples (5 mm × 5 mm × 5 mm each) were then implanted subcutaneously into each mouse. The incisions were closed with sutures, and mice were subsequently scanned with CT along the axial plane. Four animals per group per time point were used, and two bone implant samples were implanted subcutaneously into each animal. Thus, eight samples were implanted per group per time point.Data are expressed as means ± standard deviation, and statistical analyses were performed using SPSS software, version 16.0 . Student-Newman-Keuls multiple comparisons test was performed to analyse donor characteristics. Statistical analyses of radiographic score, histologic examination, micro-CT results, and biomechanical testing were performed by one-way analysis of variance and Least Significance Difference (LSD) test post hoc. P values of < 0.05 were considered statistically significant.How to cite this article: Han, W. et al. The osteogenic potential of human bone callus. Sci. Rep.6, 36330; doi: 10.1038/srep36330 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations.


Streptococcus mutans Cid/Lrg system represents an ideal model to study how this organism withstands various stressors encountered in the oral cavity. Mutation of lrgAB renders S. mutans more sensitive to oxidative, heat, and vancomycin stresses. Here, we have performed a comprehensive proteomics experiment using label-free quantitative mass spectrometry to compare the proteome changes of wild type UA159 and lrgAB mutant strains in response to these same stresses. Importantly, many of identified proteins showed either a strikingly large fold-change, or were completely suppressed or newly induced in response to a particular stress condition. Notable stress proteome changes occurred in a variety of functional categories, including amino acid biosynthesis, energy metabolism, protein synthesis, transport/binding, and transcriptional/response regulators. In the non-stressed growth condition, mutation of lrgAB significantly altered the abundance of 76 proteins  and several of these matched the stress proteome of the wild type strain. Interestingly, the statistical correlation between the proteome changes and corresponding RNA-seq transcriptomic studies was relatively low (rho(ρ) <0.16), suggesting that adaptation to a new environment may require radical proteome turnover or metabolic remodeling. Collectively, this study reinforces the importance of LrgAB to the S. mutans stress response.The  Streptococcus mutans, a major etiological agent of human dental caries1, is intimately linked to its ability to efficiently and rapidly adjust to the adverse environment of the oral cavity, thus improving its survival and persistence in dental plaque biofilm3. In this regard, considerable work has focused on the S. mutans Cid/Lrg system, consisting of two dicistronic operons lrgAB (SMU.575c/574c) and cidAB (SMU.1701c/1700c)7. Expression of cid and lrg is highly responsive to complex and unfavorable environmental signals that integrate into the regulatory networks modulating S. mutans virulence. For example, lrg and cid genes display opposite patterns of expression in response to growth phase, glucose concentration, oxygenation, and blood plasma8 and are transcriptionally cross-regulated6. Regulation of the lrg and cid operons is further complicated by involvement of multiple major regulators, including CcpA (carbon catabolite protein A) and the TCSs  LytST and VicKR6. Consequently, the Cid/Lrg system affects comprehensive virulence traits such as autolysis, biofilm development, oxidative and heat stress response, antibiotic resistance and genetic competence6 required for successful colonization in the oral cavity. For these reasons, the Cid/Lrg system has potential as an attractive target for development of anti-caries treatments.The pathogenic potential of lrgA and cidA encode membrane proteins with predicted similarity to bacteriophage holin: antiholin proteins that modulate cell lysis. Holins are known to control the timing and onset of host cell lysis during bacteriophage lytic infection, and thus it has been hypothesized that Cid and Lrg may contribute to inducing death and/or lysis in a subpopulation of the bacterial community for the altruistic benefit of the entire population, as a survival strategy for adapting to environmental conditions11. Conclusive data proving that the Cid/Lrg systems of S. mutans and S. aureus are functionally analogous to holins/antiholin pairs is still lacking. However, published S. mutans data strongly suggest that Cid and Lrg proteins are functionally interrelated7. For example, we recently found that the lack of either CidB or LrgAB results in very similar phenotypes with respect to ultrasensitivity to aeration, heat, and vancomycin stress7. More interestingly, our recent RNA-seq data has also demonstrated almost identical transcriptome changes between cidB and lrgAB mutants, compared to wild type, underlining likely functional and mechanistic similarities between CidB and LrgAB7. However, molecular mechanisms for how these encoded proteins modulate S. mutans virulence and its stress response are still not well understood. As well, clues to their specific cellular functionality are relatively scarce due in part to the inherent difficulties associated with studying membrane proteins.Another value in studying the Cid/Lrg system is that lrgAB mutants under anaerobic (control) and stress-inducing culture conditions , as a way to connect known stress-sensitive phenotypic aspects to information regarding specific changes in gene expression7. These environmental stresses and lrgAB mutation both influenced the transcriptome in S. mutans, with implications for bacterial cell death/lysis, adaptation and virulence. Nevertheless, understanding transcriptomic changes alone may be insufficient for defining the exact dynamic cellular changes of S. mutans in response to environmental stresses.“-Omics” technologies have provided a powerful means of broadly and globally assessing the cellular responses and adaptation of bacteria to stresses. We recently performed a comprehensive RNA-seq experiment to assess the transcriptomic changes of wild-type and isogenic S. mutans wild-type and lrgAB mutant samples used in our recently published RNA-seq study7. To this end, mass spectrometry-based label-free quantitative proteomics15, a technology known to enable comprehensive identification and quantification of complete bacterial proteomes, was adapted to study changes in S. mutans intracellular protein levels in response to aeration, heat, and vancomycin stress. Furthermore, the degree of correlation between S. mutans protein abundance profiles and gene expression changes was determined. Further understanding of these combined “-omics” data at the cellular or molecular level will enhance our knowledge of Cid/Lrg-mediated cellular responses of S. mutans to adverse environments. Furthermore, these “-omics” data will serve as a valuable resource that can be mined to help clarify the role of the S. mutans stress response and physiological activity to its dynamic survival in the oral cavity.This present study investigates the changes in proteome profiles that occurred in the same set of S. mutans UA159, a serotype c strain16, and a previously published isogenic lrgAB mutant6, which was created using the PCR ligation mutagenesis technique17. Both S. mutans strains were grown and maintained on Brain Heart Infusion (BHI) agar plates , at 37 °C and 5% CO2.The bacterial strains used in this study were 7 were also used in this present study. Briefly, wild type strain UA159  and ΔlrgAB  overnight cultures were each diluted 1:50 in 50 ml sterile BHI broth and grown to mid-exponential growth phase (OD600 = 0.4) under four different environmental conditions ; aerobic, (a); heat stress, (h); and vancomycin, (v) stress), as follows: For anaerobic growth, including heat and vancomycin stress conditions, sterile mineral oil was placed on top of the cultures. For aerobic growth, each culture was grown in a 250-ml conical flask at a 1:5 volume to flask ratio, and incubated at 115 rpm and 37 °C. For heat stress growth, cultures were incubated at 40 °C. For vancomycin growth, cultures were supplemented with 1 µg/ml vancomycin and incubated at 37 °C. An aliquot (5 ml) of each culture was previously used for the RNA-seq experiment7, and the rest (45 ml) of the culture was subjected to the mass spectrometry experiments in this study. Growth curves of these strains under each condition can be found in our previous publication7.The identical set of bacterial cultures analyzed in our recently-published RNA-seq workn = 5 replicates per S. mutans strain per growth condition, including the n = 3 replicates analyzed previously by RNA-seq.7) of each S. mutans culture were processed for protein extraction according to Fujiki et al.18 with the following modifications: Samples were ground into fine powder in liquid nitrogen and agitated in extraction buffer  with an equal volume of phenol (pH 8.0) for 2 hours at room temperature. The phenol phase was precipitated by adding five volumes of 0.1 M ammonium acetate in methanol. After washing twice with 0.1 M ammonium acetate in methanol and twice with 80% acetone, the dried pellet was dissolved with 50 mM ammonium bicarbonate buffer. The mixtures were incubated on ice before centrifugation at 4 °C for 20 minutes at 12,000 × g, separating the proteins into soluble and insoluble phases. The insoluble phase was incubated with protein buffer ) for at least one hour with occasional vortexing. The proteins were quantified using an EZQ Protein Quantitation Kit  and SoftMax Pro Software v5.3 .Biological pentaplicates (i.e. m/z) resolution of 70 000 and five MS/MS experiments . Dynamic exclusion for 10 s was used to prevent repeated analysis of the same peptides, and a lock mass of m/z 445.12003 (polysiloxane ion) was used for real-time internal calibration. The MS system was interfaced with an automated Easy-nLC 1000 system . Each sample fraction was loaded onto an Acclaim Pepmap 100 precolumn  and separated on an Easy-Spray analytical column  at a flow rate at 300 nL/min during a linear gradient from solvent A (0.1% formic acid (v/v)) to 25% solvent B (0.1% formic acid (v/v), 99.9% acetonitrile (v/v)) for 280 min, followed by ramping up to 98% solvent B for an additional 20 min. Peptides were sprayed into the orifice of the mass spectrometer, which was operated in an information-dependent data acquisition mode.Proteins were precipitated in 25 mM ammonium bicarbonate, pH 8.0 with Amicon Ultra-0.5 ml Centrifugal filters . For each sample, a total of 30 μg of protein was reduced with 40 mM DTT, alkylated with 100 mM of iodoacetamide, and trypsin-digested. Trypsin-digested peptides were then desalted with C18-solid phase extraction. A hybrid quadrupole Orbitrap (Q Exactive Plus) mass spectrometry (MS) system  was used with high energy collision dissociation (HCD) in each MS and MS/MS cycle. The instrument was run in data-dependent mode with a full MS  using Scaffold Q + S  and MASCOT 2.4 . The following parameters were used for all the searching: Peptide tolerance at 10 ppm, tandem MS tolerance at ± 0.01 Da, peptide charges of 2+ to 5+ , trypsin as the enzyme, allowing one missed cleavage, Carbamidomethyl (C) as fixed modifications, and oxidation (M) and phosphorylation  as variable modifications. Peptide and protein were filtered using Scaffold Q + S with strict peptide and protein probabilities, 0.9 and 0.95, respectively. Peptide probability was applied to filter peptide assignments obtained from MS/MS database searching results using predictable false identification error rate. Protein probability was used to filter proteins with the null hypothesis that the database matching is random and consideration of the peptide probability for all the peptides apportioned to that protein. For protein quantification, only MS/MS spectra were normalized by spectral abundance factor (NSAF). To avoid analyzing abundance differences that were unlikely to be biologically relevant, proteins with extremely low abundance  were excluded from further analysis. Differentially expressed proteins were identified by student t-test, and a protein should be quantified with at least three of the biological pentaplicate, and a fold change >1.4 or <0.6 with p-value < 0.05. Protein annotation/functional categories presented in all Tables and Supplemental Files were obtained using UniProt19, the Los Alamos S. mutans genome data base  and/or the NCBI S. mutans UA159 reference genome (NC_004350.2). To maximize the number of identified proteins in this analysis, we extensively annotated the detected peptides using the combined outputs of multiple search engines at a stringent FDR.Raw MS/MS data files were processed using a thorough database search, considering biological modification and amino acid substitution against a nonredundant 20 partner repository with the dataset identifier PXD006735 and 10.6019/PXD006735.The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE7, the Reads Per Kilobase of transcript per Million mapped reads (RPKM) were used for all RNA-seq data sets. The n = 3 proteomic replicates and triplicate RNA-seq replicates used in the correlation analysis were derived from the same n = 3 biological samples. The programming language R was used for all statistical analyses. First, to determine the correlation of the RNA-seq and proteomic datasets, Pearson (linear) correlation was calculated for each two matched samples from RNA-seq and proteomic data using all the genes shared between the two datasets. The expression values were log 2 transformed. The significantly correlated samples were called at thresholds of a false discovery rate (FDR) <0.1. Second, to determine the correlation of the genes between RNA-seq and proteomic datasets across all samples, the Spearman correlation between RNA-seq and proteomic datasets for each gene was calculated using all the samples. The significantly correlated genes were called at thresholds of FDR <0.05 and Spearman’s rho >0.5.To perform correlation analysis with our recently published transcriptomic dataS. mutans to efficiently and rapidly adjust to the ever-changing oral cavity environment, consequently contributing to many key virulence traits of S. mutans7. Since stress responses are closely integrated into bacterial physiology, we also envision that studying the Cid/Lrg system may provide novel insights into the mechanisms that regulate S. mutans cell homeostasis. The present study provides a valuable opportunity to detect proteomic changes in the same biological set of S. mutans wild-type and lrgAB cultures as those used in our previous transcriptome study7, using a label-free mass spectrometry-based quantitative proteomics. The rationale behind this study is that modulating the degradation and/or regulation of proteins may allow the cell to more rapidly and specifically adjust to stress, compared to transcriptomic changes. This more rapid proteome-based response may function to conserve resources by favoring only those cellular processes required for each particular stress condition21.We have started to develop an understanding of how Cid and Lrg membrane proteins are related to the ability of p-value < 0.05) in response to aeration, heat, or vancomycin stress, compared to the wild-type control samples grown anaerobically  of proteins identified were differentially accumulated  in response to the same conditions (Table lrgAB mutant strains. Tables S. mutans wild type (A through W) and lrgAB mutant (I through XXIII) strains, respectively, in response to aeration (a), heat (h) and vancomycin (v) stress, relative to control (anaerobic), showing a holistic view for how the proteomes of the organisms shift in response to each stressor or between the stressors. To observe global patterns in the protein abundance data, principal component analysis (PCA) of the complete (both wild-type and lrgAB mutant) protein dataset was performed. As shown in Supplemental Fig. lrgAB. This analysis also suggested that the overall abundance trends in lrgAB samples obtained from the aerobic and vancomycin growth conditions were most similar to each other, while the heat lrgAB sample cluster was relatively separated from the other lrgAB samples. In the wild type strain, each stress sample was greatly separated from the control anaerobic sample cluster. Overall, these data indicate that S. mutans reorganizes its proteome composition upon exposure to different environmental stressors and possibly through the involvement of LrgAB.By using LC-MS/MS and a spectral counting based label-free approach, a total of 1049 proteins in the wild type UA159 were identified at a 95% confidence level, and 535 (51%) of proteins identified were differentially accumulated  were differentially accumulated in response to vancomycin stress  or heat (n = 234), reflecting that roughly 22–36% of the identified S. mutans proteome was abundantly reorganized to cope with these stressors. A high percentage of the proteins were largely associated with amino acid biosynthesis, energy metabolism, protein synthesis, and transport/binding  in abundance in response to all three stressors were depicted as Venn diagrams  was upregulated in response to both aeration and heat, but downregulated to vancomycin stress. Thus, unlike the response to aeration and vancomycin, response to heat may involve the contribution of additional or different physiological mechanisms in the wild type strain. The common 64 proteins were assigned to 16 different functional categories and alterations in protein abundance were enriched in the categories of amino acid biosynthesis (n = 10) and transport/binding proteins (n = 9) , heat (h) or vancomycin stress (v)), and to better understand the general and specific proteomic responses of the wild-type strain (WT) to each environmental stress, proteins that increased and decreased significantly  and CopZ (putative copper chaperone), which were more than 8-fold and 4-fold upregulated, respectively, and have been previously reported to be important in protection of S. mutans against oxidative stress29. Particularly, CopZ has also been shown to be important in modulating a variety of virulence traits, including biofilm formation, stress tolerance, genetic competence, and competitiveness against commensal streptococci30. In contrast, the production of two putative ABC transporters (SMU_238c and SMU_370) and possible transmembrane efflux protein (SMU_1605) was completely suppressed in response to aeration  biosynthesis31. Next, it is noteworthy that proteins uniquely responding to heat in the wild type, including multiple transcriptional  and global (CcpA and FruR) regulators, suggesting that response to heat stress may require a tighter coordination of both gene and protein expression and/or modification, probably to avoid unwanted cellular and physiological changes  increased abundance. Major functional categories that were uniquely upregulated in response to heat stress included energy metabolism and PTSs, whereas DNA metabolism and protein synthesis-related proteins showed significantly decreased abundance, suggesting possible metabolic rewiring for production of extra energy (ATP) to cope with this stress33. It is also notable that among the heat-shock related molecular chaperones and proteases, only DnaK and Clp-like proteases (SMU_956 and SMU_2029) showed increased abundance uniquely in response to heat stress, suggesting that these proteins may be especially important for S. mutans cells living in environments with continuous heat. Interestingly, MazF, the predicted toxin component of the MazEF toxin/antitoxin (TA) module, was newly produced and uniquely responded to heat stress. This likely contributes to cell vialbility during heat stress, as overexpression of MazF presumably results in growth inhibition34. In contrast, the production of Cas9 (SMU_1405c), a CRISPR-associated endonuclease, was completely suppressed , DacA and PtsH.We also identified protein signatures representing unique responses to each stress in the wild type (n = 125) were observed between aeration and vancomycin proteome changes, while the smallest overlapped proteins (n = 18) were observed between aeration and heat proteome changes. Overall, it is notable that the proteome changes of wild-type were quite different from its transcriptome changes7 in response to the three stressors 7, compared to its corresponding proteomic changes (n = 234). Aeration moderately influenced both the proteome and transcriptome of the organism. Therefore, these data suggest that proteome adjustment may provide an alternative way to respond to stress as a counterpoint to transcriptional modulation, and be intertwined with expression of virulence-related molecular elements.Finally, Fig. ors Fig. . In partlrgAB mutant were most affected by heat (n = 335), and least by vancomycin stress (n = 147)   Fig. , top. 17lly Fig. , top. Ov9) Table . Notably9) Table . Anotherp < 0.05) changes in common under all three stressors. Unlike the wild type, the lrgAB mutant showed relatively similar protein abundance patterns in response to all three stressors, with the exception of 7 proteins , SMU_1287  and SMU_769. Particualrly, SMU_769 is one of the late competence genes regulated by ComX (or SigX), an alternative sigma factor of S. mutans.38. Other notable downregulated proteins included AtlA, SerB, DexA, amd SMU_1661c . Interestingly, two putative cell wall hydrolases, SMU_609 and AtlA, responded to all three stresses in an opposite manner in the lrgAB strain  and energy metabolism (n = 6).As shown in a Venn diagram displaying the degree of overlap in protein abundance among all ABx/AB Fig. ; bottom,lrgAB mutant, the greatest number of proteins (n = 182) uniquely responded to heat  showed a significant decrease in abundance  and vancomycin stress (n = 55), which are shown in Supplemental Tables lrgAB, was completely suppressed in the lrgAB strain uniquely in response to aeration and forms a three gene-operon with two putative ferrous iron transport proteins . Given that iron plays a role in protection against oxidative stress45, SMU_571 may contribute to enhanced sensitivity of lrgAB to aeration, possiblly as a signaling peptide associated with FeoA/B transporters. It is also noteworthy that MvaK, a putative mevalonate kinase, was newly produced only when the lrgAB mutant responded to aeration  in the lrgAB mutant was moderate, while downregulated proteins showed greater fold-change or were not accumulated  in response to each stressor with a focus on the lrgAB-specific proteome changes, using Venn diagrams  showed an opposite abundance pattern between the wild type and lrgAB mutant in response to aeration. 89 proteins responded to aeration only in the lrgAB strain, one or more of which may be responsible for the growth defect of the strain under the aerobic condition  abundance changes common to both WTh/WT and ABh/AB proteome comparisons , and SMU_521 (uncharacterized protein). Particularly, CpsY, a conserved transcriptional regulator, is known to activate transcription of genes involved in methionine biosynthesis and uptake in S. mutans53 and S. agalactiae54. CpsY has been also characterized as as virulence determinant that regulates amino acid metabolism54, methionine transport55, and cell wall modifications necessary for systemic infection in S. iniae58. Therefore, CpsY may play a role in the Cid/Lrg system that relates the environmental/stress status of S. mutans to the control of growth and expression of virulence traits. A great number of proteins (n = 213) differentially responded to heat in the lrgAB mutant only  showed a significantly (p < 0.05) decreased abundance, and among them 82 proteins showed a fold change by more than 5-fold or were not accumulated at all. SloR, a DtxR family metalloregulator, known to modulate S. mutans metal ion homeostasis, biofilm formation, oxidative stress tolerance, and antibiotic gene regulation64, was uniquely suppressed in the lrgAB mutant in response to heat stress. SloR was also recently shown to crosstalk with VicKR and GcrR, major regulatory systems controlling critical S. mutans cellular processes and virulence69, through manganese availability70. Since VicKR was shown to regulate expression of lrgAB5, SloR may be one of the intimate regulators collaborating with LrgAB. Lastly, 84 proteins demonstrated significant (p < 0.05) changes common to both WTv/WT and ABv/AB proteomes  than upregulated (n = 20), and among them twelve proteins showed an opposite abundance pattern between wild type and lrgAB mutant in response to vancomycin stress. Interestingly, one uncharacterized protein (SMU_843) was newly induced in the wild type, and not accumulated in the lrgAB strain. This protein is predicted to play a role in cell wall/envelope biosynthesis as a putative poly-gamma-glutamate biosynthesis protein. In response to vancomycin stress, less proteome abundance changes were uniquely elicited in the lrgAB strain (n = 63) than wild-type (n = 297)  and ams Fig. . In respams Fig. , represeion Fig. . 122 proons Fig. . 20 protnly Fig. . More prmes Fig. . More pr97) Fig. . One intlrgAB mutant in an optimal growth condition , loss of LrgAB significantly (p < 0.05) altered the expression of 76 proteins  with 17 downregulated and 59 upregulated , GtfC, and GlgC, which were newly induced or had a fold-change by more than 4-fold. These data suggest that mutation of lrgAB may impose inherent stress on S. mutans cells, as well as shows potential functional partners of LrgAB and related molecular processes.When we directly compared the proteome of wild-type and 7. As the transcriptome datasets contain three biological replicates per condition, only the three matched samples from each condition were used. In other words, correlation analysis was performed on a total of 24 samples from all conditions for RNA-seq and proteomic datasets, respectively. First, Pearson correlation was performed for the 24 matched samples to assess sample level correlation using all the shared genes/proteins for the matched RNA-seq and proteomic datasets. All of the 24 samples had a relative low positive correlation coefficient (rho(ρ) <0.16) between the RNA-seq and proteome datasets  with q-values  less than 10%  correlation analysis across all samples. At thresholds of q-value 0.05 and rho >0.5, a total of 46 genes (~6%) out of 776 tested genes showed significant (p < 0.05) correlation. Of these, 42 represent significantly differentially-expressed proteins  in 707 genes/proteins but none is statistically significant . In contrast, when used the 24 biological samples across all the conditions, we identified 46 significantly (p < 0.05) correlated genes/proteins. Therefore, it is possible that with more biological samples, more significantly correlated genes can be potentially identified. Finally, from a technical standpoint, the approach using mass spectrometry would not completely cover the identification of rather hydrophobic proteins, although several cell membrane and surface proteins were identified here. Also, the current experimental approach did not not allow the quantitative analysis of secreted proteins, consequently generating a less representative proteome than its corresponsing transcriptome.We attempted to determine the relationship between the proteomic profiles and the gene expression profiles, recently obtained by high-throughput RNA-seq technology on corresponding aliquots of the same cultures used for this proteome experimentationS. mutans under multiple stress conditions. The data suggest that S. mutans adjusts its stress response through substantial and dynamic remodeling of the cellular proteome, and LrgAB has an important influence on this reorganization. This study also provided a valuable opportunity to compare transcriptomic and proteomic data generated on corresponding aliquots of the same cultures. It is possible that the overall low correlation between two ‘-omics’ datasets may be due to potential analytical and technical limitations, as well as unknown cellular mechanisms in the presence of rapid or continuous stress shock. These two integrative omics studies have provided a large number of transcripts and proteins that could be used in targeted studies on the molecular mechanisms triggering the persistence phenotype of S. mutans against unfavorable stresses. The data may also give an integrative view of a specific physiological state in coping with an external stress. More detailed biological interpretation of the altered proteins will be an interesting future direction. Given that within biofilms, oral bacteria become more resistant to adverse environments, including antibiotic challenge, compared to planktonic growth, it would be interesting to evaluate the proteomic changes elicited by these environmental stresses on S. mutans biofilms. This approach could provide more ecological insights into how this bacterium accommodates the heterogeneous microenvironments that develop in biofilm, as well as accelerate phenotypic and genotypic diversity within the population. Furthermore, studying proteomic shifts in multispecies oral biofilms by S. mutans in response to other types of environmental challenges, such as nutrient limitation and toxic metabolites, could be another interesting follow-up. Consequently, all these data will improve our integrated models and understanding of how bacterial cells adjust to a new challenging environment, an initial step in initiation of disease.In summary, to our knowledge, this is the first comparative study on the intracellular proteome of Supplemental FiguresSupplemental Dataset 1