We report the results of sensitivity, robustness, heterozygous peak height ratio, precision, concordance, caseworks, and mixture interpretations. We tested sensitivity, using serial dilutions of control DNA.The minimum amount of input DNA resulting in a full profile was 125 pg. Inhibition, inducted by urea, showed a progressively fragmentation of DNA and a full profile was obtained until 1M of inhibitor factor. To test the profile quality, casework samples were extracted with different extraction methods: Chelex®100, QIAmp DNA Micro Kit and Phenol-Chloroform extraction. The results demonstrated that extraction chemistries do not have affect on amplification performance. Concordance check was performed by typing some casework samples and comparing the typing results with those obtained with other available kits. Thus, concordance was expected and supported by the data.Reliable DNA typing results can be obtained using this new kit, demonstrating its effectiveness and utility in forensic analysis. This six-color system allows co-amplification and fluorescent detection of the 18 autosomal loci in the expanded CODIS core loci  as well as Amelogenin and DYS391 for gender determination. The Penta D, Penta E, D22S1045, TPOX, SE33 loci and the two rapidly mutating Y-STRs, DYS570 and DYS576 loci, are also included to increase discrimination, satisfying both CODIS and European Standard Set (ESS) recommendations  database consisting of 13 core short tandem repeat (STR) loci  has been successful for matching suspects with evidence. However some situations require inclusion of more loci and increased discrimination, such as mixture interpretation and degradation   was serially diluted in 2-fold increments to give the following amounts when added at 25 µL per PCR: 1.0 ng, 500 pg, 250 pg, 125 pg, 62.5 pg, and 31.25 pg total per reaction. Two replicate PCRs were prepared and PCR amplifications were performed according to manufacturer’s protocol. For allele designation and peak height ratio determination a 175 RFU threshold was used for data.One model of PCR inhibition, urea, was used to represent inhibitory compounds; the inhibitor stock solution was prepared as follows: urea  10M in water. PCR samples were prepared with 1.0 ng of male 2800M control DNA containing a range of inhibitor concentrations: 0M (no inhibition), 1M, 2M, and 5M of urea. Each condition was tested in duplicate PCRs.® Fusion 6C System. To test the performance of this kit with DNA mixture samples, a series of mixtures of two different human genomic DNAs  was made in different mixing ratios. Mixtures were formulated to always give 1.0 ng of total DNA per 25 µL volume, as manufacturer’s recommended, with ratios of minor to major DNAs being 1:1, 1:5 and 1:10 and vice versa. For example, the 1:5 mixture contained 167 pg minor and 833 pg major DNA per reaction. Each mixture samples was tested in two PCRs and amplified according to manufacturer’s protocol.In forensic caseworks the evidence often contains a mixture DNA from more than one individual. These mixtures can be very challenging to analyze and interpret. For improved mixture interpretation, two rapidly-mutating Y-STR loci, DYS570 and DYS576, are included in PowerPlex® Fusion 6C System. The consistency of typing results was evaluated considering previously typing through already validated kits such as AmpFlSTR® Identifiler Plus , PowerPlex® 16 HS System    and PhenCA, USA) .The detection was performed on an Applied Biosystems® 3500 Series Genetic Analyzer and data were analyzed with GeneMapper® ID-X software.® 3500 Genetic Analyzer. The size of each allele was determined with GeneMapper® ID-X v1.4 using the local Southern method. The size was retrieved from the genotypes’ tables and introduced into Excel spreadsheets to calculate the average and a standard deviation for each allele was plotted against the allele size.Sizing precision is fundamental to an accurate genotyping. The injection of the internal lane standard is the basis of an STR-typing system’s precision and essential to ensure genotyping accuracy. Sizing precision of the WEN ILS 500 size standard was evaluated by comparing the size of every allele in the allelic ladder over the course of 16 ladder injections during a run on Applied BiosystemsFollowing thermal cycling, PCR products were prepared for capillary electrophoresis (CE) by combining 9.5 µL of Hi-DiTM Formamide, 0.5 µL of WEN ILS 500 and 1.0 µL of sample and performed on the 3500 Genetic Analyzer according to manufacturer’s manual. Genotyping was performed with GeneMapper® ID-X v1.4 software, using the provided panel and bin files. For allele designation and peak height ratio determination a 175 RFU threshold was used. In a forensic setting peaks are considered valid for use in statistical analysis only above this threshold and it is coherent with our observation of peak height ratios across multiple loci in dilution series of DNA amplified in replicate.® Fusion 6C System using serially dilution from 1 ng to 31.25 pg of 2800M Control DNA. Full allele profiles were obtained for 2800M at and above level of 125 pg, below which stochastic dropout of alleles was observed and partial profiles could be observed even at 62.5 pg and 31.25 pg of template DNA. For 2800M, the mean allele counts for the lowest DNA level tested were 17 alleles out of 47 possible, corresponding to the 36.2% of a full profile . For all samples amplified with PowerPlex® Fusion 6C System, concordance among the systems was observed and the typing reliability of the new primer sets has been validated. While not an indication of discordance, the PowerPlex® Fusion 6C System kit resulted in more loci designation. This new kit includes 27 STR markers and increases discrimination, especially in the tested mixture samples, where a higher number of allele was detected.To test the reliability of PowerPlexlections . Further®100, QIAamp DNA Micro Kit and Phenol-Chloroform extraction), provided profiles in which quality was comparable, confirming that different extraction chemistries do not adversely affect the amplification.Tested samples extracted with different analytical methods  for separation and detection of amplified fragments are accurate sizing of fragment over the fractionation range of the STR multiplex amplification system, single-base resolution over the fractionation range, and high run-to-run precision to enable comparison of mobility of alleles in the allelic ladder to sample’s alleles. Size precision of the WEN ILS 500 size standard was evaluated by comparing the size of each allele in the allelic ladder over the course of 16 ladder injections during a run on the Applied Biosystems® 3500 Genetic Analyzer. The size of each allele was determined with GeneMapper® 3500 Genetic Analyzer. Further, we did not observe statistically significant differences in the sizing precision among different loci and with a confidence interval of 99.7%, the WEN ILS 500 demonstrated the accuracy of sizing over the entire fractionation range for genotyping. These results showed that the precision throughout the assay is adequate to size and distinguish alleles which differ by as little as one base. This high level of precision provides a measure of reliability.The sizes were averaged, and a standard deviation for each allele was plotted against the allele size . The sta® Fusion 6C System was designed to allow human identification applications including forensic analysis, relationship testing and research use. The system combines common and informative loci to overcome typical challenges faced by laboratories, including low levels of input DNA, sample inhibitors, degradation and mixtures. The PowerPlex® Fusion 6C System includes the 23 loci in the expanded CODIS core loci panel (including SE33), as well as Penta D and Penta E loci for greater discriminatory power, delivering more information in demanding forensic, paternity and relationship-testing cases. Three Y-STRs, two of which are rapidly mutating, were also included to improve mixture interpretation. This evaluation study examined many aspects of kit performance, following SWGDAM validation guidelines. The results of the performed validation studies demonstrated the robustness and reliability of the kit. The amplification chemistry of PowerPlex® Fusion 6C System provided accurate profiles and a wide range of input target DNA. Good results were also obtained from artificially inhibited samples and we demonstrated the potential limitations and the consistency verifying the accuracy of sizing, without statistically significant differences in the sizing precision among different loci. Therefore, profiles typed with this new kit would be of sufficient quality to be uploaded into DNA database and to do new population genetics studies. Comparison with other available amplification kits proves concordance and the reliability of the new primer set has been validated. PowerPlex® Fusion 6C improves the robustness and efficiency of the multiplex STRs system, such as minimizing sample consumption and maximizing informative data output. The greater number of alleles creates greater specificity and generates more data with the same amount of DNA, when compared to previous STR typing kits. Moreover the possibility to obtain Y-STRs profiles in low ratio male to female mixtures is of particular practical case application value. According to the guideline of quality assurance standards for forensic DNA testing laboratories, we carried out an internal validation and demonstrated reliability of PowerPlex® Fusion 6C system. The use of this amplification kit is suitable for the use in future casework samples. Additionally the short amplification time of this kit combined with the automatic CE detection system make it practical for a high-throughput workflow that generates good profile and great output data in less processing time.The 27-plex PowerPlex


Diagnoses of human immunodeficiency virus (HIV) infection among women declined 17% during 2011–2015, and a total of 7,498 women received a diagnosis of HIV infection in 2015 Among 3,020,068 CDC-funded HIV tests provided in 2015, a total of 1,454,499 (48%) were provided to women. The highest percentages of tests were provided to women who were aged 20–29 years (41%), black (49%), living in the South (62%), living in medium and high prevalence jurisdictions (97%), and who received testing in health care facilities (83%) .Overall, 4,749 women had positive tests for HIV infection in 2015; among these, 2,951 (62%) had previously received a diagnosis of HIV infection, and 1,798 (38%) received a new diagnosis. Compared with women aged 20–29 years, those aged 13–19 years were less likely to receive a new diagnosis , whereas prevalence was higher among women aged 30–39 years (aPR = 1.69), 40–49 years (2.58), and ≥50 years (2.53). Black women accounted for 58% of the new diagnoses of HIV infection and were more likely to receive a new diagnosis than were white women (aPR = 1.31). Compared with tests performed in the South, tests performed in in the Northeast, Midwest, West, and Puerto Rico and the U.S. Virgin Islands were more likely to yield new diagnoses . Compared with tests performed in high prevalence jurisdictions, tests performed in medium and medium-low/low prevalence jurisdictions were less likely to yield new diagnoses . Tests performed in health care facilities were less likely to yield new diagnoses than tests performed in non–health care facilities (aPR = 0.51) .Among the 1,798 women with newly diagnosed HIV infection, 1,104 (61%) were linked to HIV medical care within 90 days of diagnosis, and 1,096 (61%) were interviewed for partner services. The percentages of women with newly diagnosed HIV infection who were linked to care were higher in the Northeast  and Puerto Rico and the U.S. Virgin Islands  than in the South (59%) .Among the 2,951 women with a previously diagnosed infection, 2,554 (87%) were not in HIV medical care at the time of testing ; among tHIV testing and partner services are essential strategies for diagnosing and rapidly linking women living with HIV infection and their infected partners to medical care so they can achieve viral suppression. Findings from this report underscore the importance of HIV testing not only to identify new infections, but also to identify women who have previously received a diagnosis but are not receiving medical care, because either they were never linked to care or they stopped receiving care. A high percentage of women tested had already received an HIV diagnosis (62%) before the current test; however, 87% of those women were not receiving HIV medical care. Willingness of women with previously diagnosed HIV infection to take another HIV test might signal a desire or willingness to receive the additional support they need to be linked to care, representing an opportunity for an important public health intervention; however, in this analysis, only 58% of women with previously diagnosed HIV infection were linked to care within 90 days of the current test.Black women accounted for 72% of women with a previous HIV diagnosis, although they were significantly less likely than were white women to be linked to HIV medical care within 90 days of the current test. Disparities in socioecological factors such as poverty, health literacy, and health care coverage might contribute to lower linkage for black women  infection in the United States, 60% of whom were black, although black women accounted for only 12% of the female population. HIV testing, identification of HIV infections, and early linkage to HIV medical care are critical for ensuring that HIV-positive women receive the care they need to achieve viral suppression and improved health outcomes, and to reduce transmission to others. Providing partner services can further support these prevention goals.Analysis of 2015 data on CDC-funded HIV tests and HIV prevention services from 61 health departments and 123 community-based organizations indicated that among women identified as having HIV infection, 62% had received a diagnosis of HIV infection before the current test, and 87% of those women were not in HIV medical care at the time of the test. Rates for linkage to medical care within 90 days of the current test date were 61% and 58% for women with newly diagnosed and previously diagnosed HIV infection, respectively. Among women with previously diagnosed HIV infection, 57% of black women and 65% of white women were linked to HIV medical care.Enhanced efforts to test and identify women with HIV infection and promptly link them to HIV medical care, as well as to identify women with previously diagnosed HIV infection who are not in care, especially black women, and link them to care will improve health outcomes, increase rates of viral suppression, and reduce transmission of HIV to others.


Introduction. Neuroendocrine carcinoma (NEC) of pancreas is a rare tumor with aggressive progression and poor prognosis. Its coexistence with adenocarcinoma poses significant clinical problems and has not been addressed in the literature. Methods. We describe a case of a 51-year-old male who underwent pancreatoduodenectomy due to pancreatic head tumor 1.5 × 1 × 1.4 cm. Histological examination of the specimen revealed a mixed neoplasm: (1) a well differentiated adenocarcinoma, neoplastic blasts of which are extended focally to the submucosa without invading the muscular layer, and (2) a low differentiated NEC consisting of solid clusters and pagetoid formations. All 18 lymph nodes of the specimen were free of neoplastic disease and the surgical margins of the specimen were tumor-free. No adjuvant treatment was administered and two months after the operation the patient developed liver metastasis. FNA cytology of the hepatic lesions revealed low grade carcinoma with neuroendocrine characteristics. Five lines of chemotherapy were administered: VP + CDDP, paclitaxel + ifosfamide + Mesna + CDDP, Folfox + Avastin, Folfiri + Avastin, and CAV. During his treatment he revealed PD and succumbed to his disease 13 months after the operation. Conclusion. Coexistence of NEC with adenocarcinoma of the pancreas is a very rare entity presenting significant challenges regarding its adjuvant treatment and the treatment of distant relapse. Neuroendocrine tumors are considered rare neoplasms with an annual incidence of 2.5–5 per 100.000 [γGT, and ALP). With the diagnosis of cholangitis the patient underwent ERCP which revealed biliary gallstones which were removed after sphincterotomy as well as abnormal tissue surrounding the papilla of Vater. Biopsy revealed an adenoma with moderate-to-high grade dysplasia where the possibility of infiltrative development could not be definitely excluded. Tumor markers  were within normal levels. On May 2011 a CT scan of the abdomen was conducted and revealed a compact homogenous tumor 1.5 × 1 × 1.4 cm invading the lumen of the 2nd section of the duodenum. Subsequently, the patient underwent pancreatoduodenectomy. Due to a superficial surgical site infection the hospitalization of the patient was prolonged until the 17th postoperative day. Histological examination of the specimen revealed a mixed neoplasm consisting of a part with histological characteristics of a well differentiated adenocarcinoma ,2 and D2 CDDP 80 mg/m2. After 2 months, CT of the abdomen revealed progressive disease (PD) with 3.5 cm maximum lesion and received 2nd-line chemotherapy with paclitaxel, ifosfamide, Mesna, and CDDP . Three months later due to PD (with the largest lesion being 3.7 cm) the patient received 3rd-line treatment with Folfox and Avastin . Two months later due to PD (with the largest lesion being 4.2 cm and appearance of new hepatic lesion as well) the patient received 4th-line chemotherapy with Folfiri and Avastin . Octreoscan revealed a region with moderate scintillation in the left hepatic lobe. Four months later the patient had PD  and received 5th-line treatment with CAV: D1 Endoxan 1000 mg/m2, D1 Doxorubicin 40 mg/m2, and D1 Oncovin 1 mg/m2. In December 2012 the patient presented to the emergency department due to worsening general condition, malaise, weakness, and anemia. He was transfused with 3 blood units. A new CT scan of the abdomen revealed PD with multiple liver metastatic lesions with the largest one having 9.5 cm diameter. The patient experienced grade IV myelotoxicity without evidence of any response to chemotherapy and denied further treatment. He was discharged from the hospital and succumbed to his disease 13 months after the operation.Four months after the operation the patient developed liver metastasis. The CT scan showed 4 hypodense lesions in the liver segments V, VI, and VIII with respective maximum diameter of 13 mm, 2.5 cm, and 13 cm and hypodense lesion at the level of the hepatic vein ramification. FNA cytology of the hepatic lesions revealed low grade carcinoma with neuroendocrine characteristics and the patients received 1st-line chemotherapy treatment with VP and CDD: D1–D3 VP 100 mg/mThe gastrointestinal NEC are considered to include those arising from the endocrine cells scattered among the epithelial cells (diffuse endocrine system) of the gastrointestinal tract and those derived from the endocrine cells that appear with the differentiation of adenoma and adenocarcinoma cells. Traditionally it was speculated that gastrointestinal endocrine tumors arose from (a) prior general adenocarcinomas, (b) prior carcinoid tumors, (c) nonneoplastic pluripotent stem cells, and (d) nonneoplastic immature endocrine cells. However, the structures of lesions and the results of genetic analysis have led to the current belief that mainly the lump-like growth of a highly proliferative, neoplastic endocrine cell clone appearing in the deep portion of the gland tubule of prior well and moderately differentiated, intramucosal, tubular adenocarcinomas results in the formation of gastrointestinal endocrine tumors via endocrine carcinomas .Pancreatic neuroendocrine tumors are different from exocrine tumors of the pancreas (pancreatic adenocarcinoma), which account for about 95 percent of all pancreatic cancers . PancreaConcerning some more information according to the concurrent coexistence of adenocarcinoma and NET and the relationship between these two pathological entities it is rare to be found since these two entities appear together extremely rarely. In the literature very few cases have been described concerning NET with adenocarcinoma of the pancreas. Most published cases have described NET of the colon coexisting with adenocarcinoma of the sigmoid colon  or even The natural history of islet cell and carcinoid tumors tends to be favorable as compared with pancreatic adenocarcinoma. For example, the median survival duration from the time of diagnosis for patients with nonfunctioning metastatic islet cell tumors approaches five years. The diagnosis of islet cell tumors is aided by the different abnormal biochemical profiles that they may present, which often leads to radiographic means to try and locate the tumor. It would be a mistake to generalize too much about attempts to locate these tumors. But, generally, dynamic CT scans with radiocontrast dye, octreotide scintigraphy, transabdominal ultrasound, and selective visceral angiography are all methods employed to elicit radiographic information about the cancer, depending on individual circumstance. Although they arise from similar cells, these different types of neuroendocrine cancers all behave somewhat differently. The standard treatments tend to be tumor type specific, but some general observations can be made. Immediate treatment of the symptomatic conditions created by the oversecretion of the hormone(s) may be appropriate . The treatment of choice for localized islet cell tumors is generally curative surgery. The treatment of metastatic islet cell cancer disease, depending on the tumor type, will often include chemotherapy involving such agents as streptozocin, everolimus, sunitinib, temozolomide, capecitabine, 5-FU, Doxorubicin, dacarbazine, and octreotide. Recently, promising studies of aggressive surgery benefiting select cases of metastatic neuroendocrine tumors have been published in the medical literature.Therefore while there is a massive increase of metastatic pancreatic tumors and there is poor prognosis, we should be more aware as far as the treatment is concerned which should be chemotherapy or even palliative therapy when needed. We ought to offer a targeted and customized therapy to each patient.


The aim of this study was to compare and analyze Ewing test, heart rate variability (HRV), and heart rate turbulence (HRT) in the diagnosis of cardiac autonomic neuropathy (CAN) in diabetic patients.Ninety diabetic patients (age 18–78) and 20 nondiabetic control subjects were studied. Standard Ewing test and 24-hour Holter was performed in all participants to evaluate CAN. Patients with Ewing score ≥2 were classified as CAN+.P < .05). Using the HRV analysis and HRT analysis, rates of CAN+ in diabetic patients were 56.67% (51/90) and 52.22% (47/90), respectively. SD of all normal-to-normal (NN) intervals (SDNN), SD of the average NN intervals calculated over 5-minute periods of the entire recording (SDANN), low frequency power (LF), and turbulence slope (TS) were significantly correlated negatively with Ewing scores. TS  and SDNN  had the strongest correlation with Ewing scores among relevant factors. Combining TS with SDNN as diagnostic criteria for CAN, the diagnostic sensitivity can be increased to 98%.The rate of CAN+ in diabetic patients [44.4% (40/90)] was higher than that in the controls [5% (1/20)] (Parameters used for evaluating parasympathetic functions in Ewing test, HR variability, and HR turbulence were found to significantly decrease in CAN+ group. The combination of SDNN and TS showed greater diagnostic value than Ewing test, HRV analysis, or HRT analysis alone. However, the diagnostic screening for CAN is not widely available due to its occult onset and its different diagnostic criteria. Ewing test is recommended as the gold standard for clinical testing, but the operation is too cumbersome. Heart rate variability (HRV) analysis is another CAN detection method recommended by the ADA, but it can be influenced by many factors.–8 Heart rate turbulence (HRT) is a relatively new noninvasive cardiac electrophysiological method and a reliable indicator of baroreceptor sensitivity, which can reflect the regulatory function of the autonomic nervous system in the heart.–11 At present, there is still a lack of research on the diagnostic value of these 3 methods of evaluating CAN in the diabetic patients. The objective of this study was to compare and analyze the values of these diagnostic methods for the clinical assessment of CAN in patients with DM. We hypothesized that a combination of certain parameters of both HRV analysis and HRT analysis would show higher sensitivity and specificity for the diagnosis of CAN in DM patients.Cardiac autonomic neuropathy (CAN) is an important chronic complication of diabetes mellitus (DM), which can significantly increase the mortality of patients with diabetes. Early diagnosis of CAN can reduce the risks of the painless myocardial ischemia, myocardial infarction, and sudden cardiac death associated with CAN.22.1Patients with DM age 18 to 78 years were studied. Any subject who was pregnant, or who has been taking beta receptor blockers for the past 2 weeks, or showed signs of illnesses  was excluded. One hundred eight patients with DM  hospitalized in the endocrinology department of The First Affiliated Hospital of Shantou University Medical College (SUMC) from November 2015 to October 2016 were recruited. Twenty-eight nondiabetic control subjects were recruited from The Health Center of SUMC during the same period. Among them, 18 diabetes patients and 8 control subjects were excluded because there were less than 2 ventricular premature beats on their 24-hour Holter recordings that would make it difficult to evaluate HR turbulence. Consequently, 90 patients and 20 nondiabetic control subjects were enrolled in the study. All participants had signed the informed consent and all procedures conformed to the Declaration of Helsinki. Patients’ blood pressure, body weight, and heart rate were recorded. Laboratory measurements including glycosylated hemoglobin (HbA1c), serum lipids including high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglyceride (TG) were investigated as well. CAN was assessed by 3 methods in each diabetic patient, including Ewing test, HRV analysis, and HRT analysis.2.2 Three of these tests assess parasympathetic function: heart rate responses to deep breathing (E/I ratio), to standing (30 s/15 s ratio), and to the Valsalva maneuver . The other 2 tests assess sympathetic function: blood pressure responses to lying to standing and to a sustained handgrip. Each of these 5 tests was assigned a score of 0 for normal, 0.5 for borderline, and 1 for abnormal results, and the sum of these 5 scores made up the Ewing score, which was used to assess severity of CAN. Patients having Ewing score ≥ 2 formed the CAN+ group and patients who had Ewing score less than 2 formed the CAN− group.CAN was assessed by 5 standard cardiovascular reflex tests proposed by Ewing et al.2.32) and high frequency power . CAN+ requires at least 2 of the following 6 abnormal parameters: SDNN <50 ms, SDANN <40 ms, RMSSD <15 ms, PNN50 <0.75%, LF <300 ms2, HF <300 ms2.,14Twenty-four hour Holter was performed in all participants to evaluate the HRV. Time-domain HRV and frequency-domain HRV indexes were analyzed. Time-domain HRV indexes include the SD of all normal-to-normal (NN) intervals (SDNN), the SD of the average NN intervals calculated over 5-minute periods of the entire recording (SDANN), percentage of adjacent RR intervals with a difference of duration greater 50 ms , and the root mean square successive difference of the RR interval . Frequency-domain HRV indexes include low frequency power  and turbulence slope (TS). TO denotes the early acceleration phase of sinus rhythm, while TS quantitatively analyzes the late deceleration phase of sinus rhythm after premature ventricular complexes (PVCs). When T0 is greater than 0 or TS is less than 2.5 ms/RR, it is considered as CAN+.2.52 test. Differences for continuous variables among the study groups were assessed by analysis of variance (ANOVA) test. Spearman rank correlation method was used to determine correlation between Ewing score and Holter variables.All the statistical analyses were performed using SPSS 19.0 . Data were expressed as mean ± standard deviation (SD) for continuous variables, and as percentages for categorical variables. Differences in categorical variable distribution between groups were assessed by χ33.1P < .001). Both DM duration and age were significantly higher in CAN+ group than in CAN− group (both P < .01).Totally 90 diabetic patients (83 type 2 diabetes and 7 type 1 diabetes) and 20 nondiabetic control subjects were studied. Demographic and clinical characteristics of the study population are summarized in Table 3.2Holter data are presented in Table 3.3r = −0.68, P < .05) and SDNN  had the strongest correlation with Ewing scores among relevant factors.Correlation analyses among individuals with diabetes showed a significantly negative correlation between total Ewing scores and SDNN, SDANN, LF, and TS Table . TS , of which parameters that reflect functions of parasympathetic nerves decreased significantly. HRT analysis had a similar sensitivity as HRV analysis, but the former had a higher specificity in the assessment of CAN. The most important finding was that TS and SDNN were tightly correlated with Ewing score. Significant increases in the sensitivity and the Yoden index were observed when combining SDNN and TS. Therefore, a combination of SDNN and TS showed a greater diagnostic value than using HRV analysis or HRT analysis alone, which can be a better diagnostic screening method for CAN in diabetic patients clinically. found out that the detection rate of CAN+ when combining Ewing test and HRV indexes including RMSSD, HF, and LF was higher than that of using the HRV analysis or HRT analysis alone. It is the first time that such a clinical research was carried out on Chinese population for evaluating and analyzing CAN in DM by using Ewing test, HRV analysis, and HRT analysis. The result showed that the rate of CAN+ was significantly higher in diabetic group than the control group by using Ewing test (44.4%), HRV analysis (44.4%), or HRT analysis (52.22%). Such a relatively high rate of CAN+ in diabetic patients is of relevant clinical interest. In this study, there was also a positive correlation between CAN+ and DM duration, which was consistent with the other research findings.,18CAN is an independent risk factor for cardiovascular and cerebrovascular diseases that accelerate the mortality rate in diabetic patients. ADA recommended that for diabetic patients, CAN should be routinely screened while taking measures to limit the associated factors. However, complicated diagnostic tests with various criteria and a lack of relevant studies make CAN become one of the diabetic complications that is most likely to be neglected. Until now, there still lacked studies concerning the diagnostic values of different analytic methods for evaluating CAN in diabetic patients. Interestingly, Weimer ,20 HRV analysis is composed of time-domain and frequency-domain parts. Time-domain HRV parameters SDNN and SDANN assess parasympathetic functions, while RMSSD and PNN50 are for sympathetic functions. Frequency-domain HRV parameter HF assesses the sympathetic functions, while LF represents both sympathetic and parasympathetic activities.,11 Parameters of Ewing test and HRV analysis in diabetic group were significantly greater than that of control group. Of interest was the finding that parameters, including Valsalva index, E/I, SDNN, and LF of Ewing test, HRV and HRV analysis that were used for evaluating parasympathetic functions presented significant differences between CAN+ and CAN− groups. It indicated that parasympathetic dysfunctions play a major role in the development of CAN in diabetic patients. In addition, Ewing test and HRV analysis showed consistency in the autonomic function assessment.Ewing test and HRV analysis are comprehensive diagnostic methods for CAN, which are recommended by ADA. For Ewing test, Valsalva index, E/I and 30 s/50 s mainly represent the parasympathetic functions, while the difference between blood pressure responses to standing and supine position can assess the sympathetic functions. found that HRT analysis was an independent predictor of CAN in type 2 diabetic patients. Our study turned out that TS significantly decreased in diabetic patients, while TO did not change markedly. The reason accounting for this phenomenon is that TO mainly reflects sympathetic activation, which prevails the parasympathetic inhibition after PVCs. On the contrary, TS is a parameter driven by parasympathetic effect, hence, influenced by CAN.HRT analysis has become increasingly concerned in terms of the CAN diagnosis. Balcıoğlu and Müderrisoğlu  HRV analysis, a much convenient, objective, and accurate method, can quantitatively analyze autonomic functions, and demonstrate HRV time regularity and balanced state of sympathetic and vagal effects. Nevertheless, lots of influencing factors diminish its specificity and stability for assessing cardiac autonomic functions and subsequently reduce its diagnostic and practical values. It is now held that HRT analysis is much more beneficial to predict the prognosis of cardiovascular diseases than the traditional methods. HRT is a responding result to a weak intrinsic stimulus after a PVC. On the basis of domestic and other researches, HRT can not only reflect the sensitivity of baroreflex but also the tension of autonomic nerves. It is of high sensitivity and specificity, correspondingly target at certain organs and systems. In spite of these advantages, HRT analysis can only be utilized when sinus rhythm is present and PVCs index reaches a certain value. The sensitivity and specificity of HRT analysis were higher than those of HRV analysis, as shown in our results. In addition, TS and SDNN had the tightest correlation with Ewing scores.Respective merits and faults of Ewing test, HRV analysis, and HRT analysis were discussed as follows. The most classical Ewing test is of high repeatability with inexpensive assisting devices. Limitations of HRV analysis are listed: it detects only few cardiac cycles; do not necessarily reflect the actual condition of patients; has a complicated procedure; is influenced by the patient's cooperative capacity and operator's subjective factors; is analyzed semi-quantitatively; is affected by respiratory diseases and hypotensive medications, etc. We tried to combine certain parameters of HRV analysis and HRT analysis for assessment. Most remarkably, the combination of TS and SDNN significantly increased the sensitivity and the Youden index to 0.61, revealing that the screening for CAN is highly effective by using combination of TS and SDNN.Some researchers considered that the relationship between SDNN, SDANN and T0, TS is correlative as well as independent. Both HRT analysis and HRV analysis can provide the quantitative information of cardiac autonomic nerves, but the physiologic contents they cover are different. Compared with HRV analysis, which mainly measures a series of physiologic reflexes to external stimulus, HRT analysis has a particular emphasis on the reflexive modulation caused by autonomic nerves following the baroreceptor activation.There were few relevant researches that focus on CAN evaluation in diabetic patients through using these 3 diagnostic tests simultaneously. This research comprehensively analyzed and compared the clinical values of Ewing test, HRV analysis, and HRT analysis for assessing CAN. We figured out that TS and SDNN showed the strongest correlation with CAN+. Combining TS and SDNN together as diagnostic criteria can greatly cover the shortage, that is, low sensitivity of Ewing test. To a certain degree, this combination is beneficial to early screening for CAN in diabetic patients, providing a new clinical strategy. Nevertheless, due to a relatively small number of samples were involved in this clinical study, our hypothesis needs to be further confirmed by enlarging the sample size.The authors thank Dr Guoshu Yin from the Department of Medicine and UIC Cancer Center, University of Illinois at Chicago, for helpful suggestions and criticisms, and all the participants for agreeing to be in the study.


Climate effects and human impacts, that is, nutrient enrichment, simultaneously drive spatial biodiversity patterns. However, there is little consensus about their independent effects on biodiversity. Here we manipulate nutrient enrichment in aquatic microcosms in subtropical and subarctic regions  to show clear segregation of bacterial species along temperature gradients, and decreasing alpha and gamma diversity toward higher nutrients. The temperature dependence of species richness is greatest at extreme nutrient levels, whereas the nutrient dependence of species richness is strongest at intermediate temperatures. For species turnover rates, temperature effects are strongest at intermediate and two extreme ends of nutrient gradients in subtropical and subarctic regions, respectively. Species turnover rates caused by nutrients do not increase toward higher temperatures. These findings illustrate direct effects of temperature and nutrients on biodiversity, and indirect effects via primary productivity, thus providing insights into how nutrient enrichment could alter biodiversity under future climate scenarios. et al. conduct field experiments at two mountain elevation gradients to show that temperature and nutrients have independent and interactive effects on microbial diversity.Increased temperature and nutrient pollution are key features of anthropogenic change, but their dual effects on biodiversity remain unclear. Here Wang  Spatial patterns of biodiversity are a core topic in ecology; however, the mechanisms driving these patterns remain unclear. Climatic factors, especially temperature, are regarded as the main drivers underlying diversity gradients over broad spatial scales. For instance, the positive relationships between temperature and species richness prevail along gradients in elevation and latitude, which are explained by numerous hypotheses, including the metabolic theory of ecology (MTE)12167In addition, human impacts, such as nutrient enrichment, have been identified as one of the main drivers of biodiversity loss in recent decades1418et al.A promising approach to exploring climatic effects is the use of macroecological experiments  on mountainsides. This approach integrates elevational gradients with experimental manipulations of nutrient enrichment to explore the independent effects of climate and human impacts on biodiversity20Here we conducted comparative field experiments on two mountainsides—in Norway and China—to examine the independent effects of temperature and nutrient enrichment on aquatic bacterial richness and community composition . Along n2326We considered three components of bacterial biodiversity: alpha, beta and gamma diversity30P<0.05, F-test) fitted for most of the relationships of temperature-primary productivity, as represented by Chlorophyll a (Chl a)  , which s (Chl a) . This fi (Chl a) . Nutrien (Chl a) . Chl a aevations .r2=0.332, P<0.01) and elevation  based on a permutational multivariate analysis of variance (PERMANOVA)  . Communianalyses , Mantel analyses  and canoanalyses ).Interestingly, the bacterial communities at the higher elevations in China were more similar to the communities in Norway than those at lower elevations in China (lower panel of 32n=300) and experimental site (n=10), respectively, were 1.97 times higher in China than in Norway  of each sample  linear and quadratic models were fitted in 15 and 7 cases, respectively (10(S)∝E × (1/kT), where S is species richness, k is Boltzman's constant 8.62 × 10−5 eV K−1, T is absolute temperature in Kelvin and E is the slope or ‘activation energy' in eV characterizing the temperature dependence of species richnessE  more negative in Norway than in China , significant , −1 TN intermediate nutrient enrichments used in our experiment, though with a high spatial heterogeneity  with nutrient enrichment only at intermediate elevations  at all nutrient levels (−1) with lower turnover rates at intermediate nutrient levels  along temperature gradients and then tested how these turnover rates varied with the nutrient gradients. More negative DDR slopes indicate higher species turnover rates. In both regions, the temperature DDRs were significant  (P<0.05) toward high elevations  in China, but did not decrease significantly (P=0.159) in Norway. This pattern indicates that the species turnover rate resulting from nutrient enrichment did not increase at higher temperatures. Our results therefore differ from the findings for other organisms, such as vascular plantsFourth, we examined how species turnover rates on the nutrient gradient, quantified with nutrient DDR slopes, varied with the temperature gradient. In both regions, the nutrient DDRs of each elevation were typically significant  . The sigFinally, to synthesize all the findings, we conducted partial least squares path modelling (PLS-PM)2To further elucidate the interactive effects of temperature and nutrient enrichment on biodiversity, future studies are encouraged to consider different taxonomic groups, various habitats, and even more advanced experimental designs. For instance, relevant comparison of communities of micro- and macroorganisms3511Collectively, we answered five specific questions regarding the effects of temperature and nutrient enrichment on bacterial biodiversity. By conducting experiments along climatic gradients, we have presented the first empirical evidence of the patterns and pathways of the effects of temperature and nutrient enrichment on biodiversity in subtropical and subarctic regions. For over two centuries, ecologists have documented the relationships between biodiversity and temperature123212454819355658First, our results highlight the fact that macroecological experiments along environmental gradients  are an important tool in ecological research because they allow for the disentangling the effects of individual environmental drivers on biodiversity, the independent effects of which are not be easily separated due to their covariance in nature. The current findings using microbes as model organisms offer strong examples of the importance of the study of global changes using integrating experiments and natural environmental gradients, and illustrate an emerging approach which can be distributed globally to advance our predictive understanding of ecological trends and responses. Second, temperature and nutrients play pivotal roles in maintaining elevational biodiversity patterns such that the temperature dependence of species richness is strongest at very low and high nutrient enrichment, while the effect of nutrients on species richness is strongest at intermediate temperatures. We found clear segregation of bacterial species along temperature gradients (or climatic zones), and decreasing alpha and gamma diversity toward higher nutrient levels. We documented the direct effects of temperature and nutrient enrichment on biodiversity, and also showed that both factors indirectly affected communities through primary productivity. Thus, we fill the knowledge gaps in how well we understand the direct and indirect effects of climate change and human impacts on the spatial patterns of biodiversity, and provide thoughtful insights into how nutrient enrichment may alter biodiversity under future climate scenarios.The parallel field experiments were conducted in a subarctic region, Balggesvarri Mountain in Norway , and in a subtropical region, Laojun Mountain in China oC for 30 min, and the following salts were added: CaCl2 7.55 g l−1, MgSO4·7H2O 6.78 g l−1 and NHCO3 3.53 g l−1. To facilitate the initial colonization of heterogenetic microbes, 0.91 g l−1 glucose was added. KNO3 was added at rates of 0.00, 0.45, 1.80, 4.05, 7.65, 11.25, 15.75, 21.60, 28.80 and 36.00 mg N l−1 to generate ten nutrient levels including the control of 0.00 mg N l−1. To compensate for the nitrate additions, KH2PO4 was added so that the N/P ratio of the overlying water was 14.93, which was similar to the annual average ratio in Taihu Lake during 2007 (14.49). The nutrient concentrations for the experiments were selected according to the nutrient levels of the eutrophic Lake Taihu in China, and the highest nitrate concentration was based on the maximum TN of Taihu in 2007 (20.79 mg N l−1).Along the side of each mountain, we selected unshaded locations at five different elevations. At each elevation, we set up 30 1.5 l bottles, which included ten nutrient levels and three replicates of each level . The eleThe bottles were left in the field for 28 and 31 days, respectively, to allow airborne organisms  to colonize the water and sediments of microcosms. The field setups were completed in 3 days. To keep the species dispersal events as natural as possible, we did not cover the experimental set-ups in case of rainfall. We checked the experimental set-ups twice during each experimental period, and added sterilized MilliQ water to obtain a final volume of approximately 1.2 l. Filling to a volume of 1.2 l with artificial freshwater into the 1.5 l bottles ensured that the water would not overflow due to rain or splash out in the heavy rains during the experimental periods.To avoid the effects of daily temperature variation, we measured the water temperature and pH within 2 h before noon at all elevations in the day before the final sample collection. At the end of the experimental period, we aseptically sampled the water and sediments of each bottle. The samples were frozen at −20 °C after sampling until chemical and molecular analyses.It should be noted that we analysed the sediment bacteria, but not the water column bacteria. We did not use any specific natural aquatic bacterial communities from ponds or lakes in the current experiments, but established new communities via post-dispersal effects. More details on the experimental design are provided in the 4+), nitrate (NO3−), nitrite (NO2−) and dissolved inorganic phosphorus (PO43−) were measured with a flow injection analyser . Sediment Chl a was extracted with 90% acetone28Water ammonium  was based on the community Sørensen similarity, which is a popular beta diversity metric used in ecological studies for DDRs31We used linear and quadratic models to explore the relationships between alpha and gamma diversity with elevation and nutrient. The more appropriate model was selected based on a lower value of Akaike's information criterionkT, in which k is Boltzmann's constant and T is absolute temperature in K. The slope of TRR was defined as the activation energy (E), indicating the temperature dependence of species richness. Furthermore, the slopes of the temperature DDR, based on Sørensen similarity, were used to explore the turnover rates of species composition across temperature gradients. Finally, the slopes of TRR and temperature DDR were related to nutrient enrichment and the relationships were explored with linear and quadratic models. The better model was selected based on lower value of Akaike's information criterion. For NRR, the slope of the linear regression was used to represent changes in species richness with nutrient enrichment (log10). The slopes of nutrient DDR were used to investigate the turnover rates of species composition across nutrient gradients. The significance of DDR slopes was tested with Mantel test .Water temperature was highly correlated with elevation, and was among the strongest factors related to species richness and community composition ; therefoa and pH), diversity (species richness), and composition (the first axis of nMDS). We used pH as a proxy for primary productivity because of its positive correlations with Chl a th Chl a . Observeth Chl a . We ran The amplicon sequences were deposited in MG-RAST under accession number 17710. Other relevant data in this study are available from the authors.How to cite this article: Wang, J. et al. Nutrient enrichment modifies temperature-biodiversity relationships in large-scale field experiments. Nat. Commun.7, 13960 doi: 10.1038/ncomms13960 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Methods and Supplementary References.


The sources of modern dust aerosols and their emission magnitudes are fundamental for linking dust with climate and environment. Using field sample data, wind tunnel experiments and statistical analysis, we determined the contributions of wadis, gobi (stony desert), lakebeds, riverbeds, and interdunes to modern dust aerosol availability in the three important potential dust sources including the Tarim Basin, Qaidam Basin, and Ala Shan Plateau of China. The results show that riverbeds are the dominant landscape for modern dust aerosol availabilities in the Qaidam Basin, while wadis, gobi, and interdunes are the main landscapes over the Ala Shan Plateau and Tarim Basin. The Ala Shan Plateau and Tarim Basin are potential dust sources in northwestern China, while the Qaidam Basin is not a major source of the modern dust aerosols nowadays, and it is not acting in a significant way to the Loess Plateau presently. Moreover, most of modern dust aerosol emissions from China originated from aeolian processes with low intensities rather than from major dust events. On global scale, annual intensities of 0.1–10 µm fraction of dust vary between 981 and 4313 Tg16, or approximately 1654 Tg yr−1 17 or 1490 Tg yr−1 18, and the 0.1–6 µm fractions range from 1604 to 1960 Tg yr−1 20. Without considering the contributions of human activities, modern dust aerosols emissions originating from natural sources are 1840 Tg yr−1 21. These large differences may result in uncertainties in reconstructing and predicting climate and environmental changes of the past and present, determining current air pollution controls, and combating desertification.Modern dust aerosols generated by aeolian processes−1 of modern dust aerosols, or more than 50% of the global total, originate from China such as from the Qaidam Basin, Tarim Basin and Ala Shan Plateau26 37. However, the heterogeneity of underlying surfaces may result in large differences in aeolian process intensities39, leading to uncertainties for estimating modern dust aerosol emission quantities and identifying the potential modern dust aerosol source areas in the region.In Asia, approximately 400 to 1100 Tg yr40, which is important for understanding the intensity of dust aerosol loading in the region41. Although there have been direct measurements of aerosol loadings in Chinese desert regions42, the absence of direct measurements of modern dust aerosol availabilities in different landscapes gives rise to several uncertainties (S1), especially when 1) simulating global and regional modern dust aerosol emission intensities; 2) assessing the impacts of modern dust aerosols on global climate, carbon and biogeochemical cycles; 3) identifying the source of aeolian sediments that have been employed as proxies in climate reconstructions in the sediment areas44. To address this gap, we use field investigations, sampling, wind tunnel experiments, and particle size measurements as well as the statistical analysis to determine modern dust aerosol availabilities of different landscapes over the Tarim Basin, Qaidam Basin and Ala Shan Plateau. The results improve our understanding of modern dust aerosol availabilities and aeolian sediment sources in China.The amount of fine materials  transported by aeolian processes is taken as the dust aerosol availability45. Landscapes with higher modern dust aerosol availabilities in the Qaidam Basin include riverbeds, wadis and lakebeds , which indicates that the mobile sand dunes are not the dominant landscape for modern dust aerosol availability. However, in the other regions such as Sahara Desert and western Queensland, aeolian abrasion among sand grains breaks the clay coatings on grain surfaces of the dune sands, which act as the main source of particulate matter less than 10 microns (PM10)47.The Qaidam Basin is a potential source of the Loess Plateau during Pleistoceneeds Fig. . In the S2). After combining the areas of different landscapes, the results show that more than 60% of PM2.5 to PM50 fractions in the Qaidam Basin is emitted from riverbeds, while the gobi provides only 18–28% in the region . The annual modern dust aerosol availabilities calculated from dust storm events over the three regions for PM2.5, PM5, PM10 and PM50 are approximately 18, 36, 56, and 105 Tg, respectively (S3).Therefore, assuming that the magnitude of modern dust aerosol emissions is not overestimated by current dust emission models, the modern dust aerosol emissions in China are related to low-intensity aeolian processes that are below the minimum criteria of the WMO for classification as dust storm events.Finally, more than 80% of modern dust aerosol availabilities in China are from the Ala Shan Plateau, Tarim Basin, and Qaidam BasinAlthough wadis, gobi, lakebeds, riverbeds, and interdunes are potential landscapes for modern dust aerosol emissions over the Tarim Basin, Qaidam Basin, and the Ala Shan Plateau, their roles in modern dust aerosol availabilities are different. In the Qaidam Basin, riverbeds play a major role in modern dust aerosol availabilities, while for the Ala Shan Plateau and Tarim Basin, wadis, gobi, and interdunes are the dominant landscapes for modern dust aerosol availability. In addition, the Qaidam Basin is not a major potential source of dust for sediment in the Loess Plateau and for modern dust aerosols transported to East Asia and North Pacific regions, at least in the context of modern aeolian processes.2, respectively, and are potential sources of modern dust aerosol emission in northwestern China , the gobi and interdune39 that have high modern dust aerosol emissions, most water systems in these regions are temporarily developed, causing the riverbeds and lakebeds to dry up, providing abundant fine particles for emission via aeolian processes38.The sampling areas include the Tarim Basin, Ala Shan Plateau and Qaidam Basin with an area of 5,300,000, 2,700,000, and 121,000 km62 to provide sources for modern dust aerosol emission. In China, these loose and fine particles are typically generated via water erosion, aeolian abrasion and weathering56 and loosely accumulate on land surfaces such as wadis, gobi, lakebeds, interdunes and riverbeds39. Because only surface materials with approximately 1–10 cm depth are affected by aeolian processes64, we collected samples within 10 cm of the surface during field sampling in 2013 and 2014. For each landscape, five representative samples were collected, except for the gobi on the Ala Shan Plateau where 15 samples were collected. In total, ninety-five samples were employed for further wind tunnel experiments. The sampling criteria were as follows: geomorphologic characteristics of the sampling sites are typical landscapes within an area of 10 × 10 km2, the underlying surfaces have no vegetation cover, no biological or physical soil crusts are present, and no human disturbance is detected. At each site, the samples were collected in horizontal intervals of approximately 100 to 200 m. The particle size characteristic of each landscape is shown in Table Only loose and fine particles on the surface  can be transported by aeolian processes68, and the wind tunnel set-up is shown in Fig. Wind tunnel experiments were performed at the Key Laboratory of Desert and Desertification in the Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, China. The size and operating characteristics of the wind tunnel were described in detail in previous papersBefore the wind tunnel experiments, all samples were fully crushed and disaggregated. Each sample was spread over an area of 150 × 30 cm in the working section of the wind tunnel, with the surface at the same level as the bottom of the wind tunnel. At a distance of 30 cm downwind from the sample, a trap with 30 cm width and 30 cm height was used to collect windblown material.−1. Wind events with velocities of 5–10 m·s−1 occupied about 89–100% of those with velocities higher than the threshold wind velocity (5 m·s−1). During our preliminary experiments in November 2009, we found that, no erodible particles existed on the sample surfaces after approximately 360 seconds under a wind velocity of 8 m·s−1, and under a wind velocity of 22 m s−1, no erodible particles existed after 60 seconds. Hence, the free-stream wind velocity at the axis of the wind tunnel were set to 8, 10, 12, 14, 16, 18, 20, and 22 m·s−1. For each free-stream wind velocity, we simultaneously measured the wind velocity at 10 different heights  above the floor of the wind tunnel using a wind profile sampler 6 on an ultrasonic vibrator for 10 min to facilitate dispersion before measuring the particle size.After wind tunnel experiments have been finished, the transported and surface materials collected in the field were analyzed for particle size using a Mastersizer 2000 . Before conducting the particle size measurements, we immersed the sediments in 10% HRw is the weighted mean of different particle sizes, Ri is the raw data for the contents (%) of different particle sizes at predefined wind velocities (8 to 22 m·s−1), and Mi is the corresponding contents of the raw transported material at the same velocities. Combining the results of particle size analysis, the availabilities of PM2.5, PM5.0, PM10 and PM50 were acquired.During the wind tunnel experiments with increasing wind velocities, the erodible particles were gradually depleted. Therefore, the raw data acquired during the wind tunnel experiments and during the measuring procedures must be re-processed using statistical methods. Stronger winds transport not only coarse materials but also materials that would be transported at lower velocities. Therefore, we used the weighted average method to determine the transports at the certain wind speed:1, may result in lower contents of loose and fine particles on intact surfaces than on disturbed samples. Therefore, the modern dust aerosol availabilities from the wind tunnel experiments may be higher than those in the field. In addition, meteorological records indicate that the wind velocity rarely exceeds 22 m s−1 during severe dust events. Therefore, we took the transport acquired at a velocity of 22 m·s−1 as the maximum modern dust aerosol availability in the study regions. Additional explanations and discussions are provided in S2.In natural conditions, the bonding abilities of particles, the cover of coarse fractions on the fine particles, and the differences in airflow structuresSupplementary Information


In an attempt to increase profits and sustainability in the swine industry, the gut microbiome has become a focus of much research. In this study, we performed a comparative analysis of the gut microbiome in the ileum, cecum, and colon of Duroc × (Landrace × Yorkshire) (DLY) pigs showing two extreme feed conversion ratios (FCRs) using 16S rRNA gene sequencing. The results revealed that the microbial community in the cecum and colon had significantly higher alpha diversity than the ileum. We further identified 11, 55, and 55 operational taxonomic units (OTUs) with significantly different relative abundances between the high and low FCR pigs among the three gut locations, respectively. These OTUs were mainly associated with bacteria that participate in the metabolism of dietary polysaccharides and proteins. We then identified two and nine metabolic pathways that were enriched in the cecum and colon of the high FCR pigs, respectively. The results suggested that the short chain fatty acids and indolic compounds produced by microbial fermentation might influence porcine feed efficiency. These results should improve our understanding of microbiota compositions in the different gut locations of commercial pigs and provide important insights into the effect of gut microbiota on porcine FCRs. Pork is a major meat source for humans, and the Duroc × (Landrace × Yorkshire) (DLY) cross is the most widely bred pig in the swine industry. In swine production, the feed conversion ratio (FCR) is an important economic metric that is used to measure feed efficiency. Low FCR values result in relatively high pork prices as a result of elevated feeding costs. Increasing the FCR of commercial pigs thus represents an important strategy for minimizing swine production costs.3. In addition to these factors, increasingly studies have demonstrated that the intestinal microbiota in pigs play a crucial role in nutrient processing and energy harvesting5, which, in turn, leads to variation in feed efficiency. Ramayo-Caldas et al. showed that porcine gut microbial composition was significantly associated with body weight and average daily gain6. Moreover, many studies have also shown that intestinal microbiota can provide an extensive array of enzymes and substrates for the host, resulting in improved host energy and nutrient supply8. For example, Turnbaugh et al. and Looft et al. discovered that many metabolic enzymes encoded by microbial genomes can degrade plant cell wall components that otherwise cannot be digested by the host into short chain fatty acids (SCFAs) in the swine intestine10. Delzenne et al. identified numerous microbial genes related to the synthesis of essential amino acids and vitamins in the porcine large intestine11.Previous studies have revealed that many factors influence feed efficiency, including animal genetics, diet, disease, and production managementet al. reported the relationship between fecal microbiota with feed efficiency in Duroc pigs12. Tan et al. detailed the relationship between cecal microbiota and feed efficiency in Landrace pigs13, while McCormack et al. analyzed the association of ileum and cecal microbiota with feed efficiency in Large White × Landrace pigs14. However, to our knowledge, few studies have analyzed the association between the microbiotal composition and functional capacity of the ileum, cecum, and colon with regards to feed efficiency in DLY pigs.Furthermore, Yang 15. Generally, pigs with a high FCR are capable of obtaining more energy and nutrients, thus gaining more weight, from the same amount of feed than pigs with a low FCR16. However, pigs with higher caloric gain will be more likely to deposit body fat, especially when the caloric gain from the feed exceeds caloric utilization17. Consequently, given the same quantity of feed intake, high FCR pigs are also prone to becoming obese17, which can be linked to non-genetic diseases associated with obesity in humans. Many previous studies that investigated the gut microbiotal composition of pigs were performed using feces19. The microbial community in the fecal matter is likely to represent only a discrete population of the intestinal microbiota, rather than the total composition of the gut microbiome. Conversely, the direct sampling of specific gut locations has been shown to provide information relating to the differential composition of gut microbial communities9.Pigs are recognized as important animal models in gastrointestinal tract studies due to their nutritional similarities with humans in terms of nutrient absorption and utilization20. In the present study, we performed 16S rRNA gene sequencing analysis of the gut microbiome at three different gut locations in 12 pigs with high FCRs and 12 pigs with low FCRs. Our aim was to characterize the differences in gut microbiome composition between the high and low FCR pigs and evaluate the underlying association of the gut microbiota with feed efficiency.The 16S rRNA gene is a phylogenetic marker that can be used to classify microbial taxa, allowing for the analysis of microbial community composition in intestinal content samplesvs. 2.65 ± 0.07, P = 7.4e−07) on day 140 , cecum < colon  . Additionally, the colon had a significantly higher Chao1 index than the cecum (P < 0.05). However, no significant differences in the Shannon index were observed between the cecum and colon  plot, based on the weighed Unifrac distance matrices, showed that the gut bacteria composition differed significantly at the different gut locations. The microbiotal composition in the ileum samples was significantly different from that of the cecum and colon, while the cecum and colon samples were more similar to each other Fig. . FurtherEscherichia-Shigella (23.1%), Terrisporobacter (17.9%), Romboutsia (13.7%), and Clostridium sensustricto1 (12.9%) were most prevalent in the ileum. Alloprevotella (7.2%), Lactobacillus (5.0%), and Prevotellaceae NK3B31group (4.4%) were the three most prevalent genera in the cecum. Streptococcus (10.4%), Lactobacillus (8.8%), and Clostridium (8.0%) were the three most prevalent genera in the colon  and Verrucomicrobia (1.06%) in the cecum, and Spirochaetes (2.80%) in the colon Fig. . At the lon Fig. . These rEscherichia-Shigella, Terrisporobacter, Romboutsia, Lawsonia, and Actinobacillus. Three OTUs, including OTU10 (Leeia), OTU28 (Prevotella) and OTU63 (Alloprevotella) were relatively more abundant in the cecum than in the other two gut locations. In the colon, five other OTUs exhibited higher relative abundance compared to the other gut locations, and two of these OTUs were represented by the genus Lactobacillus  on the taxa that exhibited linear discriminant analysis (LDA) scores greater than four. Figure lus Fig. .21. The results indicated that 11 pathways, including neurodegenerative diseases, enzyme families, environmental adaptation, cellular processes and signaling, infectious diseases, poorly characterized, membrane transport, cell motility, signal transduction, xenobiotics biodegradation and metabolism, and transcription were overrepresented in the ileum in comparison to the cecum and colon. Ten pathways, including metabolism of other amino acids, signaling molecules and interaction, metabolism of cofactors and vitamins, cancers, metabolism, digestive system, metabolism of terpenoids and polyketides, glycan biosynthesis and metabolism, folding sorting and degradation, and immune system were overrepresented in the cecum than the ileum and colon. Two pathways, including carbohydrate metabolism and lipid metabolism, were overrepresented in the colon than the ileum and cecum. In summary, the most important predicted functional capacities of the intestinal microbiota were enzymatic digestion in the ileum, glycan biosynthesis, and multi-metabolic capabilities, which included protein metabolism, vitamin metabolism, terpenoid metabolism and polyketide metabolism in the cecum, and lipid and carbohydrate fermentation in the colon  were then compared. A significantly higher Shannon index was observed in the high FCR pigs compared with the low FCR pigs in the cecum and colon (SHA-109) showed significantly higher relative abundance in high FCR pigs than in the low FCR pigs , OTU132 (Christensenellaceae R-7 group), and OTU367 (Ruminococcaceae UCG-010) were dominant in the high FCR pigs, while OTU118 (Prevotellaceae NK3B31 group), OUT164 (Rikenellaceae RC9 gut group), and OTU1347 (Roseburia) were dominant in the low FCR pigs  contribute to lactic acid production35. Another study suggested that the enzymatic digestion and absorption of starch constitute the predominant functions of the small intestine, while the large intestine mainly functions to ferment non-starch polysaccharides via bacteria and produce SCFAs36, which serve as important nutrients for the epithelium and body tissues. Therefore, the functional capacities of the prevalent bacteria were quite similar to the physiological functions of the pig intestine. This suggests that in each gut location, the prevalent bacteria play an important role in the physiological functions of the particular location. In the ileum of the high FCR pigs, most of the dominant OTUs belonged to Streptococcus, Ruminococcaceae, and Christensenellaceae. The OTUs associated with Streptococcus were linked to lactic acid-producing bacteria35, and the OTUs associated with Ruminococcaceae were capable of producing SCFAs by fermenting dietary polysaccharides37. The OTUs associated with Christensenellaceae can produce volatile fatty acids as the end products of fermentation38. Moreover, previous studies revealed that the phyla of SHA-109 were associated with carbohydrate degradation40. Interestingly, the ileum of the low FCR pigs contained more OTUs that were associated with the Lachnospiraceae, Prevotellaceae, and Rikenellaceae families. Many members of Lachnospiraceae are capable of producing butyric acid through the fermentation of various substrates41. Prevotellaceae and the Rikenellaceae can facilitate the breakdown of proteins and carbohydrates in feed43. The dominant OTUs in the ileum of the high and low FCR pigs have similar metabolic functions, and these results were consistent with the result of the PICRUSt analysis. The experimental pigs in this study were fed a formula diet that included fiber-enriched corn and high-protein soybean. Therefore, we hypothesized that the cecum or colon microbiome of the high FCR pigs might have a greater ability to utilize the crude protein or dietary indigestible cellulose. The most dominant OTUs in the cecum belonged to the Rikenellaceae, Prevotellaceae, Lachnospiraceae, and Ruminococcaceae families. Many members of these families show a high potential for fermenting various polysaccharides and dietary proteins45. Excluding the four families mentioned above, the colon had a greater abundance of OTUs that represented the genera Anaerotruncus and Hydrogenoanaerobacterium, which are involved in sugar fermentation with the concomitant production of formate and acetate as major metabolites46. Consistent with the results of the PICRUSt analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to carbohydrate and lipid metabolism were enriched in the cecum of the high FCR pigs, and the pathways related to protein metabolism were enriched in the colon of the low FCR pigs. Previous studies suggested that the fermentation of dietary polysaccharides could result in the production of SCFAs, which can improve the absorptive capacity of the intestine and increase porcine feed efficiency47. Gut microbiota are able to degrade proteins into diverse metabolites, including indolic compounds that maintain the epithelial barrier function of the intestinal tract48. However, the direct relationship between indole compounds and animal growth requires further investigation. Upon analysis of the microbiotal abundance, variability, and metabolic pathways in the different FCR pigs, we discovered that high FCR pigs generally exhibited a higher abundance of microbes that are beneficial for feed digestion and fermentation than low FCR pigs. Moreover, microbial metabolites, such as SCFAs, can improve the feed efficiency of swine.Consistent with previous reportsWe detected 11, 55, and 55 OTUs that were potentially associated with swine FCR in the ileum, cecum, and colon, respectively. Our results suggested that the OTUs in the cecum and colon of the high FCR pigs might have a greater ability to utilize dietary polysaccharides and dietary protein compared to low FCR pigs, and the SCFAs and indolic compounds produced by microbial fermentation might improve porcine feed efficiency and promote intestinal health. However, the exact mechanisms remain unclear, thus warranting further investigation. These findings should improve our understanding of the microbiotal compositions in the different gut locations of commercial pigs and provide important insights into the effect of gut microbiota on porcine FCRs.The experimental procedures used in this study met the guidelines of the Animal Care and Use Committee of the South China Agricultural University (SCAU) . All animal experiments in this study were approved by the Animal Care and Use Committee (ACUC) of the SCAU .ad libitum from nipple drinkers. All animals were healthy and did not receive any antibiotic treatment in the two months prior to slaughter. The feed intake and body weight of each pig was measured by Osborne’s FIRE (Feed Intake Recording Equipment) System daily. All pigs were sorted according to their FCR value (feed intake divided by the weight gained) at 140 days of age. The 12 pigs with the highest FCR and the 12 pigs with the lowest were selected for this study. The selected pigs were slaughtered at 141–142 days of age, after overnight fasting. The analyzed pigs were from different pens, and the luminal content samples were collected at the same site for each gut location. Briefly, the luminal contents were separately gathered from the middle section of the colon and the ileum. Cecum luminal samples were collected from the bottom section of the cecum. The experimental platform was disinfected before each sample was collected to avoid cross-contamination between samples. All samples were harvested within 30 min of slaughtering and transferred immediately to liquid nitrogen for temporary storage. Samples were then sent to the laboratory where they were stored at −80 °C until analysis.A total of 226 normal weaning (28-day-old) female DLY pigs were raised in a fattening house comprised of 30 pens, each housing 6–8 pigs. All pigs in this study were selected from populations with similar genetic backgrounds and husbandry practices. All of the pigs were fed twice a day with customized corn-soybean feed (free of probiotics and antibiotics) containing 16% crude protein, 3100 kJ of digestible energy, and 0.78% lysine. Water was available TM DNA Isolation kit , according to the manufacturer instructions. DNA concentration and quality were measured using UV-Vis spectrophotometry  and agarose gel electrophoresis. The DNA obtained from each sample was diluted to 1 ng/μL with sterile water. Amplification of the V4–V5 hypervariable region of the bacterial 16S rRNA gene was performed using universal primers, where the reverse primer contained a 6-bp error-correcting barcode unique to each sample . Amplification was performed using an initial denaturation at 98 °C for 1 min followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s, and a final step of 72 °C for 5 min. All PCR reactions were carried out using Phusion® High-Fidelity PCR Master Mix . PCR products were run in an electrophoresis chamber on a 2% agarose gel to confirm the successful amplification of the target gene. DNA bands of 400–450 bp, corresponding to the 16S rRNA amplicon, were excised and purified using the GeneJET Gel Extraction Kit  according to the manufacturer’s instructions. Purified amplicons were used for library preparation and pyrosequencing. Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina , following the manufacturer’s recommendations, and index codes were added. A Qubit@ 2.0 Fluorometer  and Agilent Bioanalyzer 2100 system  were used to assess the quality of the library. Pyrosequencing was performed on the Illumina HiSeq. 2 × 250 platform  according to the manufacturer’s instructions.Total bacterial DNA from the luminal samples was extracted using a Soil Genome49. Quality filtering of the raw tags was performed under specific filtering conditions to generate high-quality clean tags according to the QIIME (v1.7.0) quality controlled process50. In order to generate effective tags, the chimeric sequences were removed from the clean tags using the UCHIME algorithm51 based on the reference database (Gold database)52. After selecting representative species for each operational taxonomic unit (OTU), each of the remaining sequences was assigned to an OTU when at least 97% threshold identity was obtained using UPARSE software (v7.0.1001)53. The taxonomy of each OTU representative sequence was assigned for further annotation using the Ribosomal Database Project (RDP) classifier (v2.2)54 based on the GreenGenes Database55. Subsequently, the information pertaining to OTU abundance was normalized using a standard sequence number corresponding to the sample with the least number of sequences. Subsequent analyses were performed based on the normalized data. Alpha diversity analysis is usually used to study the complexity of species diversity in samples56. In this study, we evaluated the alpha diversity of the samples using the Chao1 and Shannon indices58, which were calculated by QIIME (v1.7.0)50. Moreover, we evaluated the sample size and the sequencing depth using Good’s coverage index59. Beta diversity analysis was used to evaluate the differences in microbial community composition in the samples60. In this study, the weighted UniFrac distance matrixes were calculated by QIIME (v1.7.0) to evaluate the differences in the microbiotal community compositions of all the test samples from the three gut locations61, and the results were visualized by PCoA and dendrograms using R software (v2.15.3) and FastTree (v1.9.0)62, respectively. Finally, in order to evaluate the differences in phylogenetic composition between the groups, multi-response permutation procedure (MRPP) analysis was performed.Sequencing reads were assigned to each sample based on unique barcodes, and truncated by cutting off the barcode and primer sequence. The original DNA fragments were merged into tags using FLASH (v1.2.7)63. Briefly, the algorithm first used the non-parametric factorial Kruskal-Wallis (KW) sum-rank test to detect the taxa with significantly different abundances, followed by pairwise Wilcoxon tests to detect biological consistency between the two groups. Finally, an LDA score was used to estimate the effect size of each differentially abundant feature. The functional capacity of the gut microbiome was estimated by inferring metabolic functionality from the 16S rRNA gene sequencing data using the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) v1.0 software21. Z-scores were calculated to construct a heatmap to demonstrate the relative abundance of the pathways in each group with the formula z = (x − μ)/σ, where x is the relative abundance of the pathways in each group, μ is the mean value of the relative abundances of the pathways in all groups, and σ is the standard deviation of the relative abundances. The Wilcoxon Signed-Ranks test was also used to determine the significance of the OTU number, alpha diversity, gene pathways, and OTU relative abundance between the sample groups.Linear discriminant analysis coupled with the effect size (LEfSe) algorithm was used to identify the OTUs that differed significantly among the three groups  based on the OTU relative abundance valuesAll 16S rRNA gene sequencing data were submitted to the NCBI’s Sequence Read Archive (SRA) database under the accession ID PRJNA391047.Supplementary Figures and Tables


The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host  DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects. In veterinary and human medicine, diagnostic polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) for identification of microorganisms are routinely carried out on a broad range of sample types. Furthermore, veterinary samples specifically are collected from a wide range of animal species. Fresh or frozen tissue samples are ideal for DNA extraction, amplification, and various downstream diagnostic and experimental analyses. However, diagnoses using samples, such as swabs, which may contain insufficient amounts of sample material or autolytic tissues due to delayed sampling and transport or storage under suboptimal conditions, is sometimes unavoidable. Additionally, retrospective analyses are often restricted to formalin-fixed and paraffin-embedded (FFPE) archived material. Unfortunately, DNA extracted from FFPE samples has an increased degree of degradation compared to samples generated from fresh material, resulting in reduced amplification of DNA targets exceeding 200 base pairs (bp) .The goal of this study was to evaluate samples collected during routine pathological examination and/or Chlamydiaceae family-specific qPCR analysis for the presence of the eukaryotic 18S ribosomal RNA (rRNA) gene, with the aim of determining the presence of PCR-amplifiable host DNA as an indicator of sufficient sample quality for qPCR analysis. The primary intended application of this type of analysis is the differentiation of samples with DNA quality/quantity sufficient for microbial identification from those samples with degraded DNA or insufficient sample size/content. This can serve to help identify false negative samples in diagnostic and research settings and/or to screen samples for appropriate DNA quantity/quality before subsequent evaluation. Another possible application for the described 18S rRNA qPCR is preliminary screening of archived samples prior to acceptance for biobanking projects.Samples were collected immediately after necropsy and fixed in 10% formalin  for 24 to 48 hours. Formalin-fixed liver samples were embedded in paraffin and processed as previously described [DNA was manually extracted from fresh tissues and swabs using a DNA extraction kit with spin-column format  or automated equipment and associated extraction kit, which uses paramagnetic bead separation , per manufacturer's instructions.μm sections of samples were deparaffinized in xylene and centrifuged at 13,500 ×g for 5 min. Subsequently, xylene was removed by ethanol extraction followed by the removal of ethanol. The resulting deparaffinized pellet was treated with proteinase K and incubated overnight on a thermomixer set to 55°C and agitated at 550 rpm. The DNA was then extracted manually or using the automated instrument/kit.For FFPE samples, 20–30 6 cells/mL were suspended in sterile phosphate buffered saline prior to subsequent manual and automated DNA extraction. 300 μL cell suspension and elution volumes were used for automated extraction and 200 μL cell suspension and elution volumes for manual extraction, in accordance with manufacturer's recommendations, to yield DNA corresponding to 4 × 103 cells/μL of undiluted eluate.To generate DNA from HeLa cells , 4 × 10DNA content and OD 260/280 ratios were determined using a spectrophotometer  per manufacturer's instructions.23S rDNA gene-based Chlamydiaceae family-specific qPCR was carried out as previously described . In brieThe internal amplification control (177-bp product), which was included concomitantly with all samples, consisted of primers EGFP-1-F (5′-GACCACTACCAGCAGAACAC-3′) and EGFP-10-R (3′-CTTGTACAGCTCGTCCATGC-5′) and probe EGFP-HEX (HEX-AGCACCCAGTCCGCCCTGAGCA-BHQ1)  , 5.μL reaction were as follows: 2.5 μL of extracted DNA, 12.5 μL of Mastermix , no AmpErase UNG, Life Technologies, Carlsbad, CA), and a final concentration of 5 pmol/μL of each primer and the probe . The cycling program was as follows: initial denaturation  followed by 45 cycles of amplification , with an automatically calculated cycle threshold value. A Ct value of >38, in the case of Chlamydiaceae assay, was considered to be a negative result. A Ct value of >32 was considered to be negative for the internal amplification control target [Quantities per 25 μL reaction were as follows: 12.5 μL 2x Mastermix, 1.25 μL 20x 18S probe/primers, and 2.5 μL sample. The threshold value was automatically calculated and Ct values of <38 were considered positive. This positivity cutoff was determined based on serial dilutions generated from HeLa cell DNA extracted via both described methods.A commercially available endogenous control for eukaryotic 18S rRNA was used ). Analysis was carried out using the above-described PCR Mastermix, as recommended for our qPCR instrument, using the denaturation and amplification conditions and number of cycles as described above for Chlamydiaceae diagnostic qPCR. Volumes per 25 Chlamydiaceae diagnostic samples representative of the range of animals and sample types evaluated in our laboratory were chosen for DNA extraction. In detail, they consisted of material collected from turtle , guinea pig , alpaca , pig , and pig . Additionally, DNA extracts were generated from chinchilla, chicken, cat, dog, mouse, guinea pig, and turtle liver FFPE material.The first set of samples evaluated in this study see  consisteμL, corresponding to at least 31.25 ng/reaction. All OD 260/280 ratios were between 1.6 and 1.9 with an average of 1.8 . All samples tested, regardless of animal species, tissue type, or swab sample, with the exception of a single swine rectal swab, showed 18S rRNA gene amplification by less than 23 cycles.For this study, a commercially available endogenous control for eukaryotic 18S rRNA, typically employed to allow quantitation of relative gene expression in complementary DNA samples, was used to determine the presence of amplifiable 18S rRNA gene in these various sample types. A positivity cutoff was determined based on serial dilutions made from HeLa cell DNA extracted via both automated and manual methods, wherein samples corresponding to DNA extracted (by either method) from at least 1–10 cells were considered positive, and samples corresponding to diluted DNA representative of <1 cell were considered negative see . The samThe representative range of animals, tissues, and swab samples commonly submitted to our laboratory for diagnosis of Chlamydiaceae was selected specifically to confirm that the described 18S rRNA gene qPCR is appropriate/sufficient to confirm that Chlamydiaceae negative samples contain amplifiable host DNA for a wide range of animal species. The DNA from the majority of these samples was extracted using automated equipment and the associated extraction kit. The less commonly encountered turtle sample was included to confirm that a nonmammalian 18S rRNA gene is sufficiently conserved to allow amplification. Additionally, the turtle sample was an FFPE sample and the DNA was extracted manually with an extraction kit commonly used for research purposes. Our findings confirm that the 18S rRNA gene is an appropriate target for demonstration of eukaryotic DNA content in a variety of sample species, tissues, and alternative sample types (swabs), as expected based on the wide-ranging conservation of 18S rRNA gene in eukaryotes.Spectrophotometry  alone can ensure that nucleic acids are present and has value to quickly show if technical errors have resulted in insufficient DNA extraction and if DNA is relatively pure, but it cannot be used to differentiate species of DNA, single stranded DNA, or single nucleotides from more intact genomic DNA. Because the amplicon size generated in this assay is 187 base pairs (per manufacturer's information), the assay gives a relatively rapid confirmation, compared to gel electrophoresis analysis, that excessive DNA fragmentation of a sample has not occurred, at least for the purposes of the described Chlamydiaceae diagnostic qPCR , which rμL DNA, effectively eliminating the possibility that DNA was not extracted due to technical error. Rectal swabs are commonly contaminated with feces, which contain various compounds and molecules, such as complex polysaccharides, known to inhibit PCR. Feces or feces-contaminated samples, as such, are considered to be potentially difficult to utilize for DNA extraction and PCR analysis [The swine rectal swab sample, though 18S rRNA gene-negative, contained 130.8 ng/analysis . BecauseWe expect that this swine rectal swab sample contained relatively high levels of bacterial DNA and/or degraded swine DNA. This might occur in a sample likely to contain substantial fecal contamination, as feces harbor very high bacterial loads, and bacterial DNases may degrade DNA . For theAlthough the single FFPE sample included in the above evaluation proved to be positive for the eukaryotic 18S rRNA gene, FFPE specimens in general are well known to be less desirable for DNA extraction and analysis due to DNA fragmentation [μL, corresponding to at least 22.25 ng/reaction. Notably, the automated DNA extraction method OD 260/280 ratios, ranging from 1.7 to 1.8 with an average of 1.7 , were consistently lower than those of the manual DNA extraction method, which ranged from 1.7 to 2.0 with an average of 1.9 . All samples tested, regardless of animal species or extraction method, showed 18S rRNA gene amplification by less than 29 cycles.This second set of samples see  consisteThe automated DNA extraction method frequently resulted in greater DNA yield from equivalent samples, and in all cases, regardless of DNA yield, resulted in lower mean cycle thresholds than the corresponding manual DNA extraction method. Interestingly, this was true despite the association of the automated DNA extraction method with lower OD260/280 ratios. However, for all samples examined herein, both extraction methods are sufficient to allow 18S rRNA gene amplification and may be regarded as equivalent in the context of the Ct <38 positivity cutoff. The analysis of turtle and chicken samples again verifies that the 18S rRNA gene target is appropriate for analysis of samples from a wide range of animals that might be evaluated for the presence of Chlamydiaceae. Furthermore, we demonstrate here that FFPE samples from various animals processed by standard methods and submitted to frequently used DNA extraction methods consistently yield DNA of sufficient fragment length/quality to allow demonstration of presence of the 18S rRNA gene by qPCR. In cases for which DNA is extracted with Gram-positive bacteria as a target, a bead-beating step is routinely recommended in the DNA extraction process to break down the bacterial wall. We did not evaluate this extraction method in the current study but would not expect this step to interfere with the successful extraction of host 18S DNA. Specific extraction methods should be evaluated, depending on target DNA .The need for quality control measures in molecular diagnostics is well accepted, and for veterinary applications the amplification of reference genes for such controls is complicated by the wide range of animal species that may be encountered. A previous report evaluated the proliferative cell nuclear antigen gene for use as such a control and was successful in amplification from a wide range of fresh and FFPE mammalian tissues . The curDue not only to the high level of conservation of the 18S rRNA gene amongst eukaryotes  but alsohttps://www.genomeweb.com/pcrsample-prep/betting-bank). FFPE specimens, extensively archived in pathology and histology departments and commonly used in all areas of biomedical research, have been generated and stored for many years [Another application for the described 18S rRNA qPCR could be the preliminary screening of archived samples prior to acceptance for biobanking, the need for which is highlighted by the fact that biobank projects have received sets of samples of which only a small fraction was actually suitable for the intended research (ny years , 13.